TW201131168A - Methods and compositions for identifying modulators of G protein-coupled receptors - Google Patents

Abstract

The subject invention provides methods for making ligand upregulatable G-protein coupled receptors (GPCRs). Ligand upregulatable GPCRs contain a modified TM5-IC3-TM6 segment, which provides for increased levels of the ligand upregulatable GPCR I a host cell in the presence of a ligand as compared to the absence of the ligand. The subject invention also provides assays for screening test compounds for a ligand of a GPCR using a ligand upregulatable GPCR. In these assays, a ligand upregulatable GPCR is contacted with a test compound, and an increase in the detectable amount of the ligand upregulatable GPCR in the host cell indicates that the test compound is a ligand of the GPCR. Said indicated ligands encompass modulators of the GPCR. Said modulators of the GPCR are particularly useful in treating GPCR-related conditions.

Description

201131168 VI. Description of the Invention: [Technical Field] The present invention relates to a method and a chemical composition for identifying whether a test compound is a G protein coupled receptor (GPCR) ligand. The GPCR ligands identified by the above methods also comprise modulators of GPCRs. [Prior Art] Although there are several types of neurotransmitter receptors in the human body, the G-protein coupled receptor (GPCR)-class has the largest and most drug-related effects so far. It is estimated that there are about 3 in human genomes. Up to 40,000 genes, of which about 2 are. /. The part is about the gene code of the GPCR. GPCR-associated nerve conduction plays a decisive role in many physiological phenomena, including at least metabolism, inflammation, neurological function, and cardiac function. For example, 'GPCRs may include at least receptors for biogenic amines such as dopamine, epinephrine, histamine, glutamic acid, acetylcholine, serotonin; mouth scorpions like receptors for ADP and ATP; nicotine Acid; inflammatory reactive lipid mediators like prostaglandins, lipoxins, platelet activating factors, and leukotrienes; peptides like hormones such as calcitonin, follicle stimulating hormone, gonadotropin releasing hormone, stomach Endogenous hormones, motilin, neurokinin, and oxytocin; or non-hormone peptides such as beta-endorphin, dynorphin A, leu-enkephalin, egg enkephalin; or hormones of non-peptides are fading Black hormone; or multi-peptides like C5a anaphylatoxins and chemokines; or peptones like coagulation protein plum, tensin 晦 ' and factor a, or sensory message transduction, such as reticular sensitization Pigment and olfactory stimulating molecules. GPCR has also shown an important position in the development of pharmaceutical products; the city's 156362.doc 201131168 face about 60% of all prescription drugs, developed from 20 of the 100 known GPCRs. For example, in 1999, the top 1 prescription drugs were applied to GPCRs (in the brackets, the drug is treating the disease or disorder): Claritin® (allergic), Prozac® (melancholy) Vasotec® ( Hypertension), Paxil®, Zoloft® Zyprexa®, Cozaar®, Imitrex®, Zantac®, Propulsid® Risperdal® (schizophrenia), Serevent® (peech)' Pepcid® (countercurrent), Gaster® (ulcer), Atrovent® (bronchospasm), Effexor® (depression), Depkote® (epilepsy), Cardura® (care) Glandular hypertrophy), Allegra® (allergic), Lupron® (prostate cancer), Zoladex® (prostate cancer) 'Diprivan® (numbness), BuSpar® (anxiety), Ventolin® (bronchospasm), Hytrin® ( Hypertension), Wellbutrin®, Zyrtec®, Plavix®, myocardial infarction/stroke, Toprol-XL®, Tenormin®, Xalatan®, Singulair® ), Diovan® (hypertension) and Harnal® (prostate) Health). (According to Med Ad News 1999) The structure of GPCR has a common design with seven segments consisting of 22 to 24 hydrophobic amino acids and forming a seven-segment alpha helix, which individually crosses the membrane (each segment has Numbered, for example, inter-membrane zone-1 (ΤΜ1), inter-membrane zone-2 (TM2), etc.). The "outside" between TM2 and TM3, TM4 and TM5, TM6 and TM7, or "extracellular" are connected by a group of amino acids, called "extracellular part" 1, 2, 3, etc. (EC1) , EC2 and EC3). Inter-membrane helix TM1 and TM2, TM3 and TM4, TM5 and TM6 156362.doc 201131168 "inside" or "intracellular" are also connected by a period of amino acid, respectively called "intracellular part" 1 , 2, 3, etc. (ici, IC2 and IC3). The "carboxy" (carbon) end of the receptor is located in the intracellular space inside the cell, and the "amine" (nitrogen) end of the receptor is located in the extracellular space outside the cell. In general, when a ligand binds to a receptor (generally referred to as a receptor, the conformation of the "activated j"' receptor changes to promote the coupling of the intracellular portion to an intracellular "G protein." Studies have shown that the gPCR response to g protein is not differentiated, that is, a GPCR can interact with more than one G protein. This can be seen in the 1095 Life Sciences (Kenakin, τ., 43 Life Sciences 1095 ( 1988)). Although there are other G proteins, Gq, Gs, Gi, Gz and Go are currently recognized. A lighter match with Gq results in an increase in intracellular IP3 concentration and an increase in intracellular Ca2+ concentration. And (1), G〇 and Gz depletion can lead to an increase in the concentration of intracellular cAMP. A GPCR that is activated by a ligand and activated by a ligand initiates a process of transmitting a signal (so-called "cell signaling"). In general, the transmission of a message ultimately leads to cell activation or cell suppression. Under certain physiological conditions, GPCRs on the membrane are in equilibrium between two different forms: one is "inactive" and the other is "activated". Receptors in an inactive state cannot be linked to intracellular signaling processes to produce a biological response. Changing the shape of the receptor to an activated state allows the signal-transport process to communicate (through the action of the G protein) to produce a biological response. The receptor can exist in an activated state by a compound such as a ligand or a drug. Alternatively, the effect of coupling to the receptor can also be achieved by altering the amino acid sequence of the receptor and mimicking the ligand 156362.doc -6 · 201131168. The stability obtained in such a manner other than the ligand is called "essential receptor activation", and this receptor is called "essential activation". As stated in the previous section, compounds that bind to specific GPCRs and alter their conformation will be the focus of research, and therefore methods and chemical compositions that recognize such compounds are also needed. There is a particular need for methods for screening test compounds that are widely available, effective, high yield, and sensitive enough to target GPCRs. The present invention not only meets the above requirements, but also meets other needs, and has achieved remarkable results. The literature discusses the following literature: Ramsay et al., Detection of receptor ligans by monitoring selective stabilization of a Renilla luc if erase-tagged, constitutively active mutant, G-protein-coupled receptor. Br J Pharmaol. 2001 133:315-23 Stevens et al" Resolution of inverse agonist-induced up-regulation from constitutive activity of mutants of the alpha lb-adrenoreceptor. Mol Pharmacol 2000 58:438-48 ; McLean et al., Visualizing differences in ligand regualation of wild-type and Mol Pharmacol. 1999 56:1182-91 ; McLean et al., Generation and analysis of constitutively active and physically destabilized mutants of the human beta( 1)-adrenoceptor Mol Pharmacol. 2002 62: 747-55; Samama et al., Ligand-inducded overexpression of a constitutively active beta2-adrenergicc receptor: 156362.doc 201131168 pharmacological creation of a phenotype in transgenic mice. Proc Natl Acad Sci 1997 94:137-41; Samaam et al., negative antagonists promote an inactive coformation of the beta 2-adenergic receptor. Mol Pharmacol. 1994 45:390-4;

Ren et al., Constitutively active mutants of the alpha 2-adrenergic receptor. J Biol Chem. 1993 268:16483-7;

Lefkowitz et al_, Constitutive activity of receptors coupled to guanine nucleotide regulatory proteins. Trends Pharmacol Sci. 1993 14:303-7; Milligan et al., Inverse agonism at adrenergic and opioid receptors: studies with wild type and constitutively active mutant receptors. Receptors Channels. 1997 5:209-13; Pei et al. A constitutively active mutant beta 2-adrenergic receptor is constitutively desensitized and phosphorylated. Proc Natl Acad Sci 1994 91:2699-702 ; Samama et al., A mutation-induced activated state Ext. The ternary complex model. J Biol Chem. 1993 268:4625-36 ; Parnot et al., Lessons from constitutively active mutants of G protein-coupled receptors. Trends Endocrinol Metab. 2002 13:336 -43; Teitler et al., Constitutive activity of G-protein coupled receptors: emphasis on serotonin receptors. Curr Top Med Chem. 2002 2:529-38; Itoh et al, Free fatty acids regulate insulin secretion from pancreativePcells through GRP40. Na Ture 2003 422:173-6 ; Stevens et al., 156362.doc 201131168

Mol Pharmacol (2000) 58: 438-48; PCT publication WO 02/44362, US Patent No. 6221660; PCT publication WO 98/46995 » Vassilatis et al, The G protein-coupled receptor repertoires of human and mouse. Proc Natl Acad Sci 2003 100:4903-8 » Cello et al, Chemical synthesis of poliovirus cCNA: generation of infectious virus in the absence of natural template. Science 2002 297:1016-8. [Invention] The present invention proposes that the ligand can be adjusted upwards. A method for the production of a G protein coupled receptor (GPCR). This "glycan-upregulated GPCR" contains at least one modified TM5-IC3-TM6' such that the degree of activation of the GPCR increases as the ligand appears compared to the absence of a ligand. The present invention also proposes an isolated polynucleotide comprising at least a nucleotide coding sequence of a GPCR which can be up-regulated by a ligand, and related compounds. The present invention further proposes a method for recognizing whether or not a test compound is a GPCR ligand, and contacting the compound to be tested with a GPCR which can be adjusted by a ligand. The GPCR ligand recognized by the above method contains a modulator of GPCR. The screening method proposed by the present invention can detect GPCR ligands including GPCR regulators, which are significantly less expensive and require fewer steps than conventional secondary signaling subtests. In most embodiments, this test is not radioactive and has a high sensitivity and is sufficient for presentation of a large amount of output. First, the present invention proposes a method for producing a GPCR which can be up-regulated by a ligand by modifying the TM5-IC3-TM6 fragment of the parental GPCR by 156362.doc 201131168 to make the segment TM5-IC3-TM6 contain Amino acid sequence substitution of the GPCR uplift (GURC). In certain embodiments, the above method further comprises (a) making a modified GPCR in a host cell; and (b) the degree of activation of the so-called substituted GPCR in the presence of a ligand compared to the absence of a ligand. Significantly improved; wherein, in the presence of ligands in the host cells, the degree of activation of the GPCR is higher, and the GPCR is a GPCR that can be up-regulated by the ligand. In certain embodiments, the ligand used in step (b) above is a native ligand of a parental GPCR. In certain embodiments, the ligand used in step (b) above is a non-native ligand of a parental GPCR. In certain embodiments, the ligand for step (b) above is located on the surface of the so-called host cell. In certain embodiments, the parental GPCR is not a β2 adrenergic receptor or a mutant thereof. In certain embodiments, the parental GPCR is not an adrenergic receptor or a mutant thereof. The so-called alpha adrenergic receptors (αΙΑ, αΙΒ or alC adrenergic receptors) and the β adrenergic receptors (βΐ, β2 or β3 adrenergic receptors) can be found in Singh et al., J. Cell Phys. 189: Discussion of 257-265, 2001. In some embodiments, the so-called comparison is quantitative. In certain embodiments, the parental GPCR is a native GPCR or a modified native GPCR. In certain embodiments, the parental GPCR is a known GPCR or a modified known GPCR. 156362.doc -10- 201131168 In certain embodiments, the parental GPCR is an orphan ligand GPCR or an altered orphan ligand GPCR. In certain embodiments, the parental GPCR is an orphan ligand GPCR that can be coupled to a ligand or an altered orphan ligand GPCR that can be coupled to a ligand. In certain embodiments, the parental GPCR can be selected from the following list (native or modified): purine receptor, vitamin receptor, lipid receptor, peptide hormone receptor, non-hormone peptide The body, the non-peptide hormone receptor, the multi-peptide receptor, the protease receptor, the sensory signal mediator, and the essential amino acid receptor that is not the β2 adrenergic receptor. In certain embodiments, the so-called essential amino acid receptor is not an adrenergic receptor. In certain embodiments, the "GPCR riser" comprising the sequence of SEQ ID:1 is at least approximately one carbon end of the GPCR uplift. In certain embodiments, the nitrogen terminus of the substituted GURC amino acid sequence is ligated to the first of the nine amino acid residues of the TM5 proline end of the parental GPCR or has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 residue residues at the carbon or nitrogen end; and the carbon terminal amino acid sequence is ligated to the second segment of the TM6 proline at the end of the second GPCR with its parental GPCR The amino acid residues are identical or have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 carbon or nitrogen terminal residues. In certain embodiments, the nitrogen terminus of the substituted GURC amino acid sequence is ligated to the first amino acid residue of the TM5 proline end of the parental GPCR; and the carbon terminal amino acid sequence A residue fragment of the twelve amino acids attached to the second terminal of the TM6 proline of the parental GPCR. In certain embodiments, the parental GPCR is flanked by a nucleoside acid at the nitrogen end 156362.doc • 11 - 201131168. The two amino acids are mutated to Gly-Thr (GT). In certain embodiments, the two amino acids of the parental GPCR flanked by the substituted GURC at the nitrogen end are mutated to Glu-Phe (EF). In certain embodiments, at least 70% of the GURC is said to be identical to the sequence of SEQ ID NO: 2 and comprises at least 10 consecutive amino acids of SEQ ID NO: 2, or SEQ ID NO The amino acid sequence of :2 or a variant thereof. In certain embodiments, the GPCR that is said to be ligand-regulated further comprises a reporter protein region, and the so-called assay step is a reporter gene assay. In certain embodiments, the so-called substituted GPCR is fused to luciferase and the so-called assay step comprises measuring the activity of luciferase, ie, the activity of the native species or modified jellyfish luciferase. . In certain embodiments, the so-called luciferase is fused downstream of the so-called substituted Gpcr. In certain embodiments, the so-called assay step (b) utilizes an immunodetection technique to utilize an antibody that binds to a ligand-upregulated GPCR. In certain embodiments, the parental GPCR is a (native or altered) mammalian (e.g., human) protein. In certain embodiments, the parental GPCR is a choline receptor (primary or altered), muscarinic 3; melanin-concentrating hormone receptor 2; biliary receptor, muscarinic 4; Acid receptor; histamine receptor; gastric hormone receptor; CXCR3 chemokine receptor; motilin receptor; serotonin (melatonin) receptor 2A; serotonin (melatonin) Body 2B; 5_hydroxychrome 156362.doc 12 201131168 Amine (melatonin) receptor 2C; dopamine receptor D3; dopamine receptor D4; dopamine receptor D1; histamine receptor H2; histamine receptor H3 Galanin receptor 1; neuropeptide Y receptor Y1; angiotensin receptor 1; neurokinin receptor 1; melanocortin 4 receptor; peptide 1 receptor of glycoside; gland The picric acid A1 receptor; the cannabin receptor 1; the melanin-concentrated hormone receptor gpr 40; and GPCR2. In a second aspect, the invention provides a nucleic acid sequence corresponding to a GPCR which is referred to in the first aspect as being upregulated by a ligand. Briefly, this method recognizes a nucleic acid coding sequence of a modified GPCR by replacing the TM5-IC3-TM6-encoding nucleic acid fragment of the nucleic acid encoded by the __ parental GPCR, such that the TM5-IC3-TM6 fragment comprises Substitution of the nuclear bitter acid fragment of the GPCR uplift. In an embodiment, the method further comprises (a) making a so-called substituted GPCR in a host cell; and then, (b) comparing the substituted GPCR in the presence of a ligand and no ligand The degree of activity measured, wherein 'GPCR with a higher degree of activation in the presence of a ligand in the primary cell than in the absence of a ligand, in this case a substitution 01> (: 11 encoded nucleic acid is a ligand The nucleic acid sequence corresponding to the up-regulated GPCR encodes the second aspect. The present invention is a purified and isolated polynucleotide comprising the method of the above-mentioned first part and the related composition, which can be adjusted upward by the ligand. The nucleotide sequence of the GPCR is encoded. In a fourth aspect, the present invention provides a method for identifying whether a test compound is a ligand for a parental GPCR. Briefly, the method includes: ^) GPCRs that are up-regulated by ligand 156362.doc 201131168 are made in accordance with the method set forth in the first aspect; and then (b) the GPCRs that are up-regulated by the ligand are compared 'in the presence of the test compound and the untested compound The degree of activation under the condition of the substance; if the degree of activation of the former is higher than the degree of activation of the latter, it indicates that the test compound is one of the ligands of this parental GPCR. In certain embodiments, the so-called up-regulated GPCR package contains a reporter protein and the test step (b) is a reporter gene assay. In certain embodiments 'the so-called substituted GPCR is fused to luciferase' and the so-called assay step comprises measuring the activity of luciferase, ie the activity of the native species or modified jellyfish luciferase . In certain embodiments, the so-called assay step (b) employs immunodetection techniques using antibodies that are coupled to a ligand-upregulated GPCR. In certain embodiments, the so-called identified ligand is one of the modulators of the parental Gpcr. In certain embodiments, the parental GpCR modulator is an agonist, a reverse agonist, Antagonist or partial agonist.

In certain embodiments, the so-called parental gpcr modulator is a selective regulator of this parental CJPCR. In certain embodiments, the parental gpcr modulator is a representational vector comprising a polynucleotide that is upregulated by a ligand. The fifth aspect of the invention provides a method for preparing a compound which comprises identifying a ligand of a GPCR and blending it with a carrier, and the ligand referred to in the oxime is identified according to the method proposed in the fourth aspect. A sixth aspect of the invention provides a method for modulating a GPCR, the method 156362.doc 14 201131168 comprising contacting a ligand identified by the method according to the fourth aspect with a corresponding GPCR^S, wherein the ligand is A GPCR regulator. In a seventh aspect, the present invention provides a method for treating a GPCR-associated disease in a patient'. The method comprises administering an effective dose of a gPCR ligand to the so-called patient, wherein the ligand is identified according to the method set forth in the fourth aspect. And the so-called ligand is - GPCR regulator. [Noun Definitions] Before the present invention is further described in detail, it is to be understood that the invention is not limited to the illustrated embodiments. It is to be understood that the terminology set forth herein is intended to describe a particular embodiment of the invention and is not intended to limit the scope of the application. Unless otherwise stated, the technical or scientific terms used in this application are the same as those familiar to those skilled in the art. If a value in a range is mentioned in the text, it means that each value between the lower limit and the upper limit is 'accurate to one tenth of the unit used (if the other standard is not clearly indicated in the text), or In the scope of the specification: any particular reference or intermediate value thereof is considered to be in accordance with the invention. The upper and lower limits of these ranges may also be individually included in the proposed small range and are included in the present invention, as well as the subject matter of any proposed exclusion range in this range. Regardless of whether the proposed range of values includes one or two thresholds, the scope of the one or two thresholds is also included in this application, and many publications, patents, and publications are mentioned. Patent application (4). These publications, which are hereby incorporated by reference in its entirety, are hereby incorporated by reference. The publications, patents, published patent applications, etc. referred to by the Applicant herein do not mean that the Applicant has considered such publications, patents, published patent applications, etc. to be prior art. It must be pointed out that the “one” and “one” counties used in this paper and the corresponding patent application scope can be inferred from the plural (four) sentence of the plural. For example, "an agent" encompasses the plural form of the agent, and the description of the GPCR also includes equivalents to one or more of those skilled in the art. Moreover, it is necessary to know that the patent application may be written in a relatively simple manner without listing possible additional units. At this time, the statement will be used for further exclusionary terms such as “only” and “unique”, or as a “negative” restriction. "G-protein coupled receptor" or "GPCR" refers to a group of multi-peptides with the same structural morphology - having seven segments consisting of 22 to 24 hydrophobic amino acids and forming a seven-segment alpha helix, individually crossing Membrane [each segment is numbered, for example, inter-membrane region_1 (ΤΜ1), inter-membrane region-2 (τμ2), etc.] inter-membrane region-2 and inter-membrane region_3, inter-membrane region_4 The "outside" between the intermembrane region _ 5, the intermembrane region -6 and the intermembrane region·7, or the "extracellular" are connected by a length of amino acid [referred to as "extracellular portion" i, respectively). 2 3 and so on (EC1, EC2 and £C3)]. The "inside" between the inter-membrane inter-membrane region and the inter-membrane region-2, the inter-membrane region_3 and the inter-membrane region_4, the inter-membrane region and the inter-membrane region-6, or the "intracellular" Connected with a length of amino acid [referred to as "intracellular portion" 1, 2 '3, etc. (IC1, iC2, and IC3), respectively]. The "slow-base" ("carbon") end of the sensation is located in the intracellular space of the cell 156362.doc •16·201131168, and the “amine” (“nitrogen”) end of the receptor is located in the extracellular space outside the cell. . The structure and classification of GPCRs is well known in the art. For further discussion of GPCRs, see Probst, DNA Cell Biol. 1992 11:1-20, Marchese et al Genomics 23: 609-618, 1994 〇 or refer to 歹丨J Books: Jtirgen Wess (Ed) Structure-Function Analysis of G Protein-Coupled /iecepior·?, published by Wiley-Liss (Oct. 15, 1999); Kevin R., published by Wiley & Sons (March, 1988). Lynch (Ed) Identification and Expression of G Protein - Tatsuya Haga (Ed), G Protein. Coupled Receptors, published by CRC Press (Sep. 24, 1999) \ Steve Watson, published by Academic Press (1st ed, 1994) (Ed) G-Proiek. A typical GPCR construction diagram can be found in the first figure. "Golden upregulatable GPCR" is a modified GPCR that allows an increase in the degree of coupling to a ligand in the cell or on the cell membrane. This term means that a modified GPCR can be raised in the presence of a ligand compared to the absence of a ligand, ie, the number of modified GPCRs detected can be increased (eg Reporters fused with this GPCR were used as detection markers). "Native GPCR" refers to a GPCR produced by a mammal or its pathogen (such as a virus), which may also be a GPCR-related gene generated by an allelic variant of a GPCR. A detailed description of native GPCRs can be found in the Human Montessori Genetics Database on the National Center of Biotechnology Information (NCBI) website 156362.doc 17 201131168 (On-line Mendelian Inheritance in Man) . An in-depth discussion of native GPCRs can also be found at www.primalinc.com. If the GPCR is implicated in a GPCR-associated disease, then the native GPCR will be the subject of drug treatment. "known GPCR" refers to a native GPCR, and its unique native ligand has also been identified. "Orphan GPCR" refers to a native GPCR, and its unique native ligand has not been identified or known. "Gated (ligated-ophan GPCR)" refers to an orphan ligand GPCR, but has found its unique non-native ligand. "Parental GPCR" refers to a native or altered native GPCR. (For example, a native GPCR is either ligated to other proteins or has been substituted, inserted, deleted, etc. in its amino acid sequence, but this GPCR still has the same activity as a parental GPCR, like Ability to couple with ligands, etc.). A parental GPCR may be substituted in the TM5-IC3-TM6 section with a "GPCR-up" amino acid sequence to form a one-substituted GPCR. A "substituted GPCR" may be a ligand-upregulated GPCR, and the coupling performance of this substituted GPCR with a ligand may be measured if it is measured intracellularly or on the membrane. As described above, a parental GPCR, a one-substitution GPCR, and a ligand-upregulated GPCR are all related to and evolved from a native GPCR at the level of their amino acid sequence. 156362.doc •18· 201131168 The “TM5-IC3-TM6” GPCR is an amino acid sequence fragment defined by TM5, IC3 and TM6 on a GPCR, as detailed below. If the TM5-IC3-TM6 segment is changed, it means that this segment has been partially changed. A "GPCR-upregulating cassette" or "GURC" can be used to modify a parental GPCR to make it a ligand-up GPCR. The term "upregulating" appears here for convenience only and does not imply any mechanism that would cause a GPCR to become ligand-accelerated. The term "ligand" refers to a molecule that is coupled to a particular GPCR. For example, a ligand may be a multi-peptide, a lipid, a small molecule compound, an antibody, and the like. A "native ligand" refers to an endogenous, natural ligand of a native GPCR. A ligand may be an "antagonist", "agonist", "partial agonist" or "reverse agonist" of a GPCR, or a similar effect. "Modulator" refers to a ligand that, when contacted (i.e., coupled to) a GPCR that is expressed on a cell, can enhance or slow the intracellular response of a GPCR. An "agonist" refers to a ligand that, when coupled to a GPCR, elicits an intracellular response to the GPCR. "Partial agonist" refers to a ligand that, when coupled to a GPCR, elicits an intracellular response to this GPCR, but is much less effective than an agonist. "antagonist" refers to a ligand that competes with the agonist for the same GPCR coupling position, but it does not cause the activation state of this GPCR. 156362.doc -19- 201131168 will not promote cell Internal reaction. In general, antagonists inhibit the intracellular response elicited by agonists and partial agonists. Antagonists generally do not attenuate the reference intracellular response of this GPCR when no agonist or partial agonist is present. "Reverse agonist" refers to a ligand that, when coupled to a GPCR, inhibits the reference intracellular response of the GPCR when no agonist or partial agonist is present. In most embodiments, the inverse agonist inhibits the intracellular response baseline by at least 30%, further by 50%, and further up to 70% compared to the absence of a reverse agonist. %. Here, "GPCR-related condition" and "GPCR-related disorder" are alternately used to mean that any disease or symptom is caused by a change in specific GPCR activity or can be treated. The GPCR-related status may be related to the abnormal activity of a GPCR, or may be caused by the abnormal activity of a GPCR, which may be a mutation of a GPCR, resulting in excessive activation, sustained activation, or insufficient activation of the GPCR. For example, some diseases are not related to the abnormal activity of GPCRs, but they can be treated by modulating the activity of GPCRs. This term also includes some modifications to the appearance of the appearance, although it is not a life-critical but is loved. Such GPCR-related states are, for example, allergies, hypertension, esophageal reflux, asthma, bronchial cramps, prostate, ulcers, epilepsy, angina, rhinitis, cancers such as prostate cancer, glaucoma, and stroke. . More GPCR-related status can be found in the online version of the Human Montessori Genetics Database on the NCBI website. 156362.doc •20- 201131168 "Phenomenon associated with aberrant GPCR activity" refers to the structural, molecular level or functional characteristics associated with GPCR abnormal activity, especially A measurable feature in a human or animal model. These features may include, at a minimum, subsequent events at the molecular level after activation of the .GPCR, as well as phenotypes or symptoms' such as sneezing, runny nose, acid reflux, emotion, asthma, pain, height, and the like. "Deletion" is defined herein as a modification of an amino acid or nucleotide sequence that is 'eliminated one or the other against the amino acid sequence or nucleotide sequence of the parental GPCR polypeptide or its nucleic acid. More than one amino acid or a nuclear I residue. For a GP C R or a portion thereof, the deletion may include 2, 5, 10, or up to 2, or up to 3 or up to % or more amino acids. Moreover, a GPCR or a portion thereof may have more than one deletion fragment. "Insert" or "addition" is defined herein as an alteration of an amino acid or nucleotide sequence to the amino acid sequence or nucleotide sequence of the parental GpcR polypeptide or its nucleic acid. In comparison, one or more amino acid or nuclear acid residues are added. "Insert" generally refers to the insertion of one or more amino acids into the amino acid sequence of a multi-peptide, and "addition" may mean insertion or reference at the carbon end, the nitrogen end, or It is the amino acid sequence added to both ends. For a GPCR or a portion thereof, 'this insertion or addition may include i, 3, 5, 1 、, or up to 20, or up to 30 or up to 50 or more amino acids. . Moreover, a GPCR or a portion thereof may have more than one insert. 156362.doc -21- 201131168 "substitution" is the comparison of one or more amino acids with the amino acid sequence or nucleotide sequence of the parental GPCR polypeptide or its nucleic acid Nucleotides are substituted with different kinds of amino acids or nucleotides. It is to be understood that a GPCR or a fragment thereof may be substituted with a conservative amino acid, that is, it does not substantially affect the activity of the GPCR. This conservative substitution is based on the following combinations; gly, ala; val, ile, leu; asp, glu, asn, gln; ser, thr; lys, arg; and phe, tyr. "Biologically active" GPCR refers to a GPCR having the structural and biochemical functions of a native GPCR. Here, the "determination", "measuring", "assessing", and "assaying" of the stomach are mutually versatile and include the determination of quality and quantity. If the text refers to the “quantity” of a GPCR, unless otherwise stated, it may indicate quantitative or qualitative measurements, and does not necessarily refer to a quantity of measures. Here, "polypeptide" and "protein" are used interchangeably to refer to an amino acid polymer of any length, including encoded and uncoded amino acids, chemically or biochemically. Modified or derivatized amino acids, as well as multi-peptides containing modified peptide backbones. The term contains a fusion protein comprising at least a protein fused to a heterogeneous amino acid sequence, fused to a heterologous and homologous leader sequence, whether or not fused to a nitrogen-terminal thiohistidine residue; with an immunological marker a protein; a fusion protein fused to a measurable unit, such as fluorescent protein, β-galactosidase, luciferase, or the like; or the like. Here, "nucleic acid molecule" and "multinuclear 156362.doc -22- 201131168 polynucleotide" can be used interchangeably, and refer to a nucleotide polymer of any length, whether it is a deoxyribose nucleus. Glycosylates are also composed of ribonucleotides, or other similar components. Polynucleotides may have a three-dimensional structure and may have many known or unknown functions. The polynucleotide includes at least a gene, a gene fragment, an exon, an intron, a messenger RNA (mRNA), a transfer rNA, a nuclear RNA, a ribozyme, a cDNA, a recombinant polynucleotide, a branching polynucleotide, Plastid, vector, any piece of independent DNA sequence, control region, any piece of independent RNA sequence, nucleic acid probe, primer', etc. The nucleic acid molecule may be linear or circular. By "isolated" in this document is meant that the compound is already in an environment different from its self-generated environment. "Isolated" is used to describe a substantial increase in the amount of compound sought in sample ten or that the compound has been partially or substantially purified. Substantially purified as used in this document means that a compound has left its natural environment and that at least 60% or more, or more than 75% or even more than 9%, does not contain any other compounds that are naturally associated with it. A "coding sequence" or a multi-winning "encondmg" sequence refers to a transcribed (in the case of dna) or translated (in the case of mRNA) into a multi-peptide Nucleic acid molecules, for example, are located in a host cell under the control of appropriate regulatory sequences (also known as "control factors"). The boundaries of the coding sequence are usually determined by the start codon at the 5' end (amino terminus) and the stop codon at the 3' end (via the base). A coding sequence to > may include: _Α of the virus, and the dna sequence of the transcript. A transcription termination sequence may be located at the 3' end of the coding sequence. Other 156362.doc -23- 201131168 "control factor" also It may be associated with a coding sequence. The optimal expression of a DNA sequence encoding multiple traits in a selected cell may be based on the preferred codon of the selected cell' to represent a DNA copy of the polypeptide coding sequence. Encoded by)" refers to a nucleic acid sequence encoded by a multi-peptide sequence, wherein the multi-peptide or a portion of the multi-peptide encoded by the nucleic acid sequence comprises at least 3 to 5 amino acids, or曰 At least 8 to 1 amino acid 'more than at least 15 to 20 amino acids'. This also includes the multi-peptides that can be determined by immunological methods and compiled according to this sequence. "operably linked" means that the arrangement of the factors allows the components to perform their original functions. Therefore, the GPCRs that are substantially linked to GURC, that is, the TM5 IC3·TM6 fragments in the GPCR are matched. Amine Substituted by the acid sequence, the tone is the GURC as described above. If it is a promoter, the promoter will be related to the coding sequence, which will affect the expression of the coding sequence. The promoter or other control factors do not require a coding sequence. Connected as long as it can guide the function of the coding sequence (4). W said that the translated but not transcribed sequence can be placed between the promoter sequence and the coding sequence, and the promoter sequence is still sS and the coding sequence. "Substantially linked. "Nucleic acid (3) is a nucleic acid sequence that is created to contain one or more functional units that do not co-occur in nature. For example, this includes a ring, Linear, double-stranded extrachromosomal DNA knives (plastids) c cssmids (plastids with a COS sequence derived from human phage), viral genomes containing non-native nucleic acid sequences, etc. 156362.doc -24- 201131168 Vector can transfer a gene sequence into a host cell. In general, "vector construct", "expression vector" and "gene transfer vector" refer to The expression of the desired gene can be derived, and the gene sequence can be transferred to any nucleic acid construct of the host cell; this can be achieved by integrating all or part of the genome into the host gene, or by treating the vector as a chromosome. The factor is to maintain its heritability for a short or long period of time to achieve the above requirements. "Expression cassette" is a nucleic acid construct that derives the expression of the gene or coding sequence sought and the promoter of the expression cassette This expression can be added to a "vector", "vector composition", "expression vector" or "gene transfer vector" to transfer the expression locus to a host cell. Therefore, this term includes, in addition to viral vectors, transgenic and performance vehicles. Methods for "seqUence identity" of nucleic acids and amino acids are well known in the art. In general, this technique involves determining the nucleotide sequence of the mRNA of the gene or determining the amino acid sequence it has programmed and aligning the sequence with another nucleotide or amino acid sequence. Generally, "consistency" refers to the complete isochronism between nucleotides or amino acids, depending on whether two polynucleotides or two peptide sequences are compared. Two or more sequences (polynucleotides or amino acids) can be compared to each other to determine their "percent identity." The percent identity of the two sequences, whether referred to as a nucleic acid or an amino acid sequence, refers to the exact number between the two side-by-side sequences divided by the shorter sequence length and multiplied by 100. The approximate alignment of nucleic acid sequences can be performed using a local comparison algorithm, see

Smith and Waterman, Advances in Applied Mathematics 156362.doc •25- 201131168 2:482-489 (1981). This algorithm applies fractional arrays to the calculation of amino acid sequences. The fractional array can be found in Dayhoff, Sewewca and Structure^ MO Dayhoff ed., 5 suppl. 3:353-358, National Biomedical Research Foundation, Washington, DC, USA, and It is generalized below: Gribskov, Nucl. Acids Res. 14(6): 6745-6763 (1986). An example of the use of this algorithm to obtain a sequence of percent identity can be found in the "BestFit" instrument of the Genetics Gomputer Group (Madison, WI). The default parameters for this method can be found in the Wisconsin Sequence Analysis Package Program manuan, Version 8 (1995) (produced by Genetics Computer Group, Madison, WI). A preferred method for calculating the percent identity in the context of the present invention is to use the MPSRCH program, copyrighted by the University of Edingurgh > developed by John F. Collins and Shane S. Sturrok, and by IntelliGenetics, Inc. (Mountain View, CA) issued. In this set of software, the Smith-Waterman algorithm can use the preset parameters for the score table (for example, the gap open buckle is divided into 12, the gap extension buckle is divided into 1, and the gap is 6). Finding a "match" value from these data means "sequence consistency." Other programs well known in the art for calculating the percent agreement or the similarity of the two sequences also include BLAST, which is calculated at its preset value. For example, BLASTN and BLASTP can use the following parameters to preset values: Gene Code = Standard; Screen = None; Gene Strand = Yes; Threshold = 60; Expected Value = 10; Array = BLOSUM62; Description = 50 Sequences; High to low; database = not repeated; GenBank = EMBL + DDB J + PDB + GenBank CDS conversion + Swiss 156362.doc · 26 · 201131168 protein + Spupdate + PIR. Details of these applications can be found at http://www.ncbi.nlm.gov/cg-bin/BLAST. In addition, the determination of sequence identity can also hybridize polynucleotides in a specific environment to obtain stable replication products of corresponding regions, and then enzymatically digest them with a single nuclease, and then determine these hydrolyzed solutions. Sequence fragment. Two DNA or two peptide sequences, if referred to each other as "greatly identical", mean that the sequence identity of at least 8〇 to 85% within a certain length of the two sequence molecules is 'at least 85%. Up to 9〇%, and more precisely at least about 90% to 95%, and very precisely at least 95% to 98% of the sequence identity, is detected by the above method. In the following, roughly identical can also mean exactly the same as a particular DNA or multi-peptide sequence. The roughly identical DNA sequence can be determined using Southern blot analysis, and the stringency conditions are as defined by the experimental design. How to determine the appropriate hybridization conditions is the skill of this familiar artisan. See, for example, Sambr〇〇k al. 5 infra; DNA Cloning, supra; Nucleic Acid

Hybridization, supra· The meaning of “selectively hybridize” of two nucleic acid sequences is as follows. The percent identity between two nucleic acid molecules will affect the effect and strength of this hybrid. Partially identical nucleic acid sequences at least partially inhibit the hybridization of the fully identical sequence to the target molecule. The degree of inhibition of the hybridization of a completely identical sequence can be measured by well-known hybridization assays (such as, for example, Southern's ink collapse method, Northern ink collapse method, / Valley liquid hybrid method), etc., see Sambr. 〇〇k et al 广/ ni^g^ A Laboratory Manual, Second Edition, (1989) Cold 156362.doc •27· 201131168 ng Ha- NY). These tests can be performed with different rigor, which means that the strictness of the experimental setting conditions is from lowest to highest. If a low stringency setting is used, an unspecified coupling can be measured with a secondary probe that is partially inconsistent (for example, a probe with less than 30% sequence identity does not interact with the target molecule). Because of the lack of an unspecified constipation position, this secondary probe cannot hybridize to the target. Right-based hybridization-based assays must be selected—the nucleic acid probes are matched to the target nucleic acid sequence' and then selected for the strictness of the appropriate probe to allow the probe to selectively hybridize to the target sequence, or to allow the two to interact with each other. Get together and form a hybrid molecule. A nucleic acid molecule capable of selectively hybridizing to a target sequence under "moderate stringency" is typically set to a target nucleic acid sequence of at least 10 to 14 nucleotides in length and at least with the sequence of the selected nucleic acid probe About Mo. 70. /. Sequence-induced. Strict hybridization conditions are generally defined as nucleic acid sequences of at least 1G to 14 nucleotides in length and about 90% to 95% or more sequence-selective with the sequence of the selected nucleic acid probe. Suitable hybridization conditions for the probe/target hybridization reaction. There should be a degree of sequence identity between the probe and the target, which can be determined in a manner well known in the art (see Nucleic Acid Hybridization: A Practical Approach, editors BD). Hames and SJ. Higgins, (1985) 〇xford; Washingt〇n>DC; IRL Press). If strict hybridization conditions are employed, many equivalent conditions are known in the art for establishing a particular stringency, which can be achieved by changing a number of variables, such as 疋. The length and characteristics of the probe and target sequences are different. Sequence 156362.doc •28· 201131168 Basic composition 'The concentration of salts and other components in the hybridization buffer, whether there is a blocking agent in the hybridization solution (for example, guanamine, glucosyl sulfate) and polyethylene Glycol) 'hybridization reaction temperature and time, as well as different rinsing conditions. A particular combination of hybridization conditions can be selected from standard procedures well known in the industry (see Sambrook et al).

Cloning: A Laboratory Manual, Second Edition, (1989) Cold Spring Harbor, N.Y.). One example of stringent hybridization conditions is as follows: hybridization temperature above 50 °C and 0_lxSSC (15 mM sodium chloride / 15 mM sodium citrate). Another stringent hybridization condition is: overnight culture at 50 °C in 50% methyl-bronamine '5xSSC (15 mM sodium chloride / 15 mM sodium citrate), 50 mM sodium citrate (ph 7.6) '5x Denhardt's solution, 10% glucosyl sulfate' and 20 mg/ml denatured, cut salmon sperm DNA, and then rinsed in a filter at about 65 ° C. 1 XSSC solution. Strict hybridization conditions mean at least as indicated above. The hybridization conditions of the sexual configuration are considered to be at least 80% as they are generally strictly 'accurately at least 9〇% as strict as the above-mentioned particularly stringent conditions. There are still many strict hybridization conditions known in the art that can also be used to detect nucleic acids in particular embodiments of the invention. If the first polynucleic acid is "derived from" or "corresponds to" the second polynucleotide, then the first polypeptide must be associated with the second polynucleotide. Some regions have identical or approximately identical nucleoside nuclear sequences' whose cDNA also conforms to the above conditions; or the former and the latter exhibit sequence identity as written above. If the first multi-peptide is "derived from" or "corresponds to" the second multi-peptide, then the first multi-win 156362.doc -29· 201131168 must ( 1) encoded by the first nucleotide derived from the second polynucleotide, or (2) as shown above, which is sequenced with the second polypeptide. Treatment (treating, treat) or the like refers to achieving a desired pharmaceutical and/or physiological effect. This effect may be to completely or partially avoid the disease or condition and thus may be considered to have a full or partial effect on a disease and/or a bad influence caused by the disease. As used herein, "treatment" encompasses any disease with a mammal, especially a human, and includes: (4) avoidance - the disease occurs in an individual who is considered to be susceptible to the disease but has not yet been diagnosed; (b) inhibits the disease For example, to control it: (C) to alleviate the disease, that is to say, the disease retreats and/or relieves more than one symptom. The term "treatment" also includes the administration of a medicament to provide a remedy, even if the disease or condition is not already present. For example, "treatment" includes administration of a GPCR regulator that provides an improvement or desired effect on the individual (eg, 'reducing the number of pathogens, increasing the physiological index of the individual toward: increasing the direction of the disease', reducing the symptoms of the disease, etc. ). "Subject", "individuai", "host", and "patient" J are used interchangeably herein to refer to an animal, human or non-human, suspected or existing GPCR-related diseases and respond to the proposed treatment methods of the present invention. The test examples are as follows, at least there may be. Human primate primates 'mouse' rats, cattle, sheep, Shanxu 'dog', and horses Among them, the human protein is of interest. [Embodiment] The up-regulated G protein narnar coupling receptor comprises a method which can produce ligands (GPCRs) due to ligands. Increased quail eggs 156362.doc •30· 201131168 A modified TM5-IC3-TM6 fragment allows a G-protein coupled receptor that can be up-regulated by a ligand in a host cell to have a ligand The presence of a ligand increases its activity. The present invention also utilizes a ligand-upregulated GPCR to propose a test for screening a test compound. In this assay, a ligand is raised by a GPCR and a test compound. Contact, if measured in the host cell The increase in the number of GPCRs that are up-regulated by the ligand indicates that the chemical is the ligand for the GPCR. The ligands detected are involved in the regulation of this GPCR. The GPCR regulators indicated are in treatment. It is particularly useful when it comes to GPCR-related conditions. The following description of the invention will be more detailed than that provided in the summary, background and definition above. First, the method of manufacturing a GPCR can be upregulated by a ligand, and then how to apply one. The ligand is up-regulated by a GPCR to detect whether a test compound is a ligand for a GPCR. This is followed by a brief review and description of a representative application using the method of the present invention, as well as a complete set designed for the method of the present invention. Apparatus and Procedures. Method of Making Ligand Up-Up GPCRs In one aspect, the present invention provides a method of making a GPCR that can be up-regulated by a ligand. In general, a ligand-up GPCR is a modification of a natural biogenic GPCR. Transformations or variants thereof that differ in their biological activity, including modified TM5-IC3-TM6 fragments. In most embodiments, this modified TM5-IC3-TM6 fragment is modulated by a GPCR Provided in the pedestal. In most embodiments, this ligand-upregulated GPCR can be measured more intracellularly or on the cell membrane in the presence of a ligand than in the absence of a ligand. 156362.doc -31 · 201131168 In short, the method of the present invention is to identify TM5-IC3-TM6 fragments corresponding to the TM5, IC3, TM6 regions of the parental GPCR, and at least some of the above fragments. It is replaced by the amino acid sequence of a GPCR up-regulation and becomes a monosubstituted GPCR. The substituted GPCRs are typically expressed in a host cell and compared to the number of substituted GPCRs measured in the presence or absence of a ligand, respectively, to determine their upregulation. As will be appreciated by those skilled in the art, the process of determining a one-substituted GPCR may be performed in parallel or in sequence with the control of the control (e.g., the relative amount of GPCR is determined separately before and after the addition of the ligand). A substituted GPCR, if expressed in the presence of a ligand, is increased in the presence of a ligand than in the absence of a ligand, and is referred to as a ligand-upregulated GPCR. The number of substitutions for GPCRs can be determined in several ways, including the use of a coupling partner, such as an antibody. However, in many embodiments, in order to facilitate the determination of the number of substitution GPCR measurements, the substituted GPCR will be fused to a reporter multi-peptide, such as GFP, luciferase or the like. One example of a suitable multi-peptide to be reported is the luciferase. Therefore, the measured quantity of a substituted GPCR is usually based on the amount of reported activity of the reporter. If the activity of the reporter is higher than the activity of the reporter of a control group, the substituted GPCR is indicated. It is a GPCR that can be upgraded. In certain embodiments, the reported multi-peptide may be a native or modified species of jellyfish luciferase. In the following, the GPCR will be described first, followed by the TM5, IC3, and TM6 regions of the GPCR. Then, the GPCR is raised, and the skilled person can create a GPCR that can be upgraded according to the description. 156362.doc -32- 201131168 G protein-coupled receptors Any known GPCR can be used in the present invention as long as three regions of TM5, IC3, and TM6 can be found in the sequence. The paper published by Joost et al. mentions the sequence-generation relationship system of 277 GPCRs (Genome Biol. 2002 3: RESEARCH0063, all of which are incorporated herein by reference), so at least 277 GPCRs are applicable to The method proposed by the present invention. A recent paper by Vassilatis et al. refers to the sequence and genetic relationship system of 367 human GPCRs, Proc Natl Acad Sci 2003 100:4903-8, www.primalinc.com, which is listed as Reference), therefore, at least 367 humans and 392 mouse GPCRs are suitable for use in the method proposed by the present invention. The method proposed by the invention can be applied to: sputum receptor, vitamin receptor, lipid receptor, peptide hormone receptor, non-hormone peptide receptor, non-peptide hormone receptor, multi-peptide Body, protease receptors, sensory signal mediators, and essential amino acid receptors that are not beta 2 adrenergic receptors. In certain embodiments, the so-called essential amino acid receptor does not comprise an adrenergic receptor. Alpha-adrenergic receptors ((Χία, (Xib or ctic adrenergic receptors) and β-adrenergic receptors (β, β24β3 adrenergic receptors) can be found in Singh et al., J. Cell Phys. 189: Discussion of 257-265, 2001. Native and altered native GPCRs are known to be useful in the methods of the invention. Thus, in certain embodiments, a modified native GPCR (eg, a native GPCR is added, replaced, deleted) , inserting a sequence such as a reporter) is still coupled with its corresponding native GPCR and the same ligand. In the method of the present invention, the "parental" GPCR can be a native GPCR or a modified original 156362.doc -33 - 201131168 GPCRs are therefore produced. Therefore, the GPCRs listed below (primary or altered) are particularly useful in the methods of the invention for parental GPCRs: choline receptors, muscarinic 3; melanin-concentrated hormone receptor 2; choline receptors , muscarinic acid 4; nicotinic acid receptor; histamine receptor 4; gastric hormone receptor; CXCR3 chemokine receptor; motilin receptor; serotonin (melatonin) receptor 2A; - serotonin (melatonin) receptor 2B; serotonin (melatonin) 2C; dopamine receptor D3; dopamine receptor D4; dopamine receptor D1; histamine receptor H2; histamine receptor H3; galanin receptor 1; neuropeptide Y receptor Y1; angiotensin II receptor 1 ; neurokinin receptor 1 ; melanocortin 4 receptor; peptide 1 receptor of glycoside; adenylate A1 receptor; cannabinoid receptor 1; melanin-concentrated hormone receptor 1; GPR 40; and GPCR 2. TM5, IC3, and TM6 regions of GPCR In the method of the present invention, the TM5-IC3-TM6 fragments corresponding to the TM5, IC3, and TM6 regions of a parental GPCR usually need to be identified and The modification is such that it comprises an amino acid sequence of a GPCR upregulation. Thus, in most embodiments, the methods of the invention include how to identify the TM5, IC3, and TM6 regions of a GPCR. A typical structural diagram of a GPCR can be seen in the figure, and these regions can be clearly distinguished in the GPCR architecture. The IC3 region of the GPCR is located between the intermembrane regions TM5 and TM6, and the general length is 12 amino acids ( CXCR3 and GPR40) to 235 amino acids (choline receptor, muscarinic 3). Familiar with this technique For those who can use the experimental procedure such as detecting the inter-membrane region, the TM5 and TM6 regions 156362.doc •34-201131168 domain can be easily identified; once the TM5 and TM6 regions are recognized, IC3 will understand. TM5, IC3, And the TM6 region can also use methods such as pairing or multiple sequence comparison (for example, the University of Wisconsin GCG program like GAp or BESTFIT' or GLUSTAL sorting program], please see Higgins.

Gene. 1988 73: 237-44) 'Using a target GPCR, such as GPCRs of known structure, or adjustable gpcrs as shown in the fourth figure. Experimental procedures suitable for identifying inter-membrane regions include those mentioned in the book *M〇Uer (Bioinformatics, 17: 646-653, 2001). A particularly suitable term, TMHMM, is proposed by Krogh et al. (J〇urnal 〇f Molecular Biology, 305: 567-580, 2001). These programs, together with the user interface, can see the corresponding sequence of a GPCR or its fragments on this interface. Such programs are available on the Internet, such as the Center for Biological Sequence Analysis: cbs.dtu.dk/services/. The output formats of these programs may vary. 'But they usually use the amino acid localization of GPCRs to indicate the intermembrane region of this GPCR. For example, the human adrenergic receptor β2 is continuously 413 amino acids. The default value is calculated as follows, where the inter-membrane region ΤΜ is shown as Tmhelix, and the start (beg) and end (end) are shown as amino acid localization. #序列长度: 413 # Sequence expected inter-membrane area number: 7 # 序 薄膜 区域 区域 区域 区域 区域 区域 : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : 0.00400 # Sequence possible nitrogen terminal signal sequence: I56362.doc -35· 201131168 TM Beg end sequence TMHMM2.0 extracellular 1 35 sequence TMHMM2.0 intermembrane 36 58 sequence TMHMM2.0 intracellular 59 69 sequence TMHMM2.0 membrane 70 92 Sequence TMHMM2.0 Extracellular 93 106 Sequence TMHMM2.0 Membrane 107 129 Sequence TMHMM2.0 Intracellular 130 149 Sequence TMHMM2.0 Membrane 150 169 Sequence TMHMM2.0 Extracellular 170 200 Sequence TMHMM2.0 Membrane 201 223 Sequence TMHMM2.0 Intracellular 224 274 Sequence TMHMM2.0 Membrane 275 297 Sequence TMHMM2.0 Extracellular 298 306 Sequence TMHMM2.0 Membrane 307 326 Sequence TMHMM2.0 Intracellular 327 413 So, those skilled in the art can recognize this GPCR The TM5 region corresponds to the position 201-223 of the continuous amino acid, and the IC3 region of the GPCR corresponds to the position 224-274 of the continuous amino acid. The TM6 region of the GPCR corresponds to 275-297 of the continuous amino acid. position. TM5-IC3-TM6 is known to correspond to the 201-297 position of the continuous amino acid. All GPCRs can use this program to determine the appropriate predictions for the amino acid localization of the TM5, IC3, and TM6 regions of this GPCR. When we use the TMHMM program to find the intermembrane region of a GPCR, we usually get the following GPCR map: an extracellular nitrogen end, followed by a paragraph with seven inter-membrane regions, and then in the intracellular Carbon end. In this typical GPCR map, the inter-membrane region is numbered (TM1) from the nearest nitrogen end to the nearest carbon end (TM7). Sometimes, TMHMM will calculate six or eight inter-membrane areas. If it is 156362.doc -36- 201131168, the nitrogen end is considered to be located extracellularly and the carbon end is supposed to be located intracellularly. The number of TM is as follows: A) If TMHMM is calculated to have eight TM regions and nitrogen ends In the intracellular, for example, serotonin (melatonin) receptor 2C, then the TM closest to the nitrogen end is skipped, and thus the second tm starts numbering (TM1) until the closest to the carbon end ( Tm7). B) If TMHMM calculates that there are eight TM regions and the carbon ends are extracellular, then the tm closest to the carbon end is skipped 'from the nearest nitrogen end number (TM1) until the closest to the carbon end (TM7). C) If TMHMM calculates that there are eight TM regions and the nitrogen terminal is intracellular (for example, neurotensin receptor 1), then the highest possibility among the intracellular TM regions negated by TMHMM is accepted. The number closest to the nitrogen end (TM1) is the closest to the carbon end (TM7). Thus, in many embodiments, the amino acid localization of the TM5, IC3, TM6 regions of a GPCR can be calculated using a suitable method such as TMHMM. In general, once the TM5-IC3-TM6 fragment is determined, this amino acid sequence is suitably substituted with an amino acid sequence with a GPCR upregl. In many embodiments, this substitution region is positioned using a conserved or semi-conservative amino acid in the TM5 and TM6 intermembrane regions. In certain embodiments, the nitrogen-terminated GURC amino acid substitution sequence (eg, SEQ ID NO: 1 or a variant thereof) is identical to the nine amino acid residues at the carbon terminal of the TM5 proline of the parental GPCR or a residue fragment having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 carbon or nitrogen ends; and the substituted GURC amino acid sequence has its carbon end attached to the second segment and its pro The twelve amino acid residues at the nitrogen end of the TM6 proline of the GPCR are identical or have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 156362.doc -37·201131168 carbon end Or a residue fragment at the nitrogen end. In certain embodiments, the nitrogen terminus of the substituted guc amino acid sequence is ligated to the first half of the carbon-terminal nine amino acid residue fragment of the TM5 proline of its parental GPCR; this replaces the GURC amino acid The carbon terminus of the sequence is ligated to the second stretch of the residue of the twelve amino acids of the TM6 proline end of the parental GPCR. If the GPCR is not a conservative proline residue at TM5 and TM6, the position of the inserted GURC can be determined by the following two considerations: a) the sequence of this GPRC and other receptors of the same subclass (containing in TM5 or TM6) Corresponding comparison of conserved proline residues; b) If the receptor to be tested does not belong to GPCRs containing a conserved proline residue in TM5/TM6, then hydrophobicity analysis is used to determine TM5/TM6 The joint position. In certain embodiments, the parental GPCR mutates at the nitrogen end with two amino acids flanked by the substituted GURC, for example, to Gly-Thr (GT). In certain embodiments, the parental GPCR mutates at the nitrogen end with two amino acids flanked by the substituted GURC, e.g., to Glu-Phe (EF). A total of 28 peptide PCR sequences of the GPCRs were modified to be upregulated by the ligand, as shown in Figure 4, SEQ ID NOS: 10-37. For example, note that in the substituted nicotinic acid receptor SEQ ID NO: 13, the nitrogen-terminal arginine of the GURC1 amino acid sequence, and the carbon end of the niacin-receptor TM5 The amino acid residues are joined, and the carbon-terminal leucine of the GURC 1 amino acid sequence is joined to the 12 amino acid residues of the nitrogen end of the niacin in the nicotinic acid receptor TM6. This nicotinic acid receptor has been mutated to GT with a diamino acid flanked by a substituted GURC at the nitrogen end. This nicotinic acid receptor has been mutated to the EF 〇 156362.doc-38 - 201131168 by the substitution of the GURC at the carbon end. Another example is the substitution of the glycoside peptide 1 in the substitution. In the SEQ ID NO: 32, the nitrogen-terminal arginine of the GURC1 amino acid sequence is joined to the 19 amino acid residues of the carbon terminal of the proline in the glycoside-like peptide 1 receptor TM5, and the GURC1 The carbon-terminal leucine of the amino acid sequence is conjugated to the 12 amino acid residues at the nitrogen end of the proline in the glycoside-like peptide 1 receptor TM6. This glycosidic peptide 1 receptor has been mutated to GT with a biamino acid flanked by a substituted GURC at the nitrogen end. This nicotinic acid receptor has been mutated to EF with a diamino acid that is substituted at the carbon end of the substituted GURC. In addition to the above methods, those skilled in the art can also compare the sequences of the parental GPCRs using the pairing or multiple sorting method as shown in the fourth figure, and use the data to judge which amino acids to be substituted are those. For example, BLAST can be used to derive the closest parental GPCR, which is then paired with the closest parental GPCR for reordering comparison to obtain the substitution region, and then refer to the transformation of the GPCR that can be up-regulated by the ligand. Determine the area to be replaced. In certain embodiments, GPCRs that have been isolated in connection with the present invention may also be made by the methods set forth herein. GPCR Upstation The GPCR Upstation is an amino acid sequence that can be inserted or substituted for a parental GPCR to engineer this GPCR into a ligand-upregulated GPCR. In general, a GPCR uplift has a length of about 30-70 or more amino acids (eg, about 30, about 35, about 40, about 45, about 50, about 55). , about 60, about 65, about 35), and in certain embodiments, comprises the sequence of SEQ ID:1. SEQ ID ΝΟ:1, if present in the GPCR uplift, is usually located at the carbon-terminal portion of the 156362.doc •39-201131168 GPCR riser, and usually there will be 1, 2, 3, The nitrogen ends of the four '5' or 6, 7, and 8 amino acids are on the carbon end of the GPCR uplift. Examples of GPCR uplifts include SEq id n〇: 2 or one of them. Active variant. The variant of SEQ ID NO: 2 may contain any substitutions, deletions, additions, substitutions, etc., particularly substitutions of a conserved amino acid after comparison with the SEq ID n〇:2 sequence. When it comes to GPCR, it is biologically active to have the ability to turn a GPCR into a GPCR that can be up-regulated by a ligand. A GPCR upregulation can comprise at least 10 amino acids of SEQ ID NO: 2, or at least 15 amino acids, or at least 20 amino acids, or at least 25 amino acids, or at least 30 amine groups An acid, or at least 35 amino acids, or at least 4 amino acids or at least 45 amino acids, or at least 50 amino acids, and in certain embodiments includes the completeness of SEQ ID NO: sequence. In certain embodiments, a GPCR locus comprises a fragment that is at least 70% identical to SEQ ID NO: 2, or at least 75% identical, or at least 80. /. Consistency, or at least 85% consistency, or at least 905% consistency' or at least 95% consistency, the fragment may be about 10, or about 15 or about 20 Or about 25, or about 30 ' or about 35, or about 40, or about 45, or about 50 consecutive amino acids. An example of a GPCR uplift includes SEQ ID NO: 2. SEQ ID N: 2 corresponds to the 221_275 amino acid sequence fragment ' of the human native β2 adrenergic receptor and has four positions substituted at the end closest to the carbon end. 156362.doc •40· 201131168 SEQ ID ΝΟ: The result of the substitution of the sequence 1 in which Ser, Arg, the first Lys, and the second Ala are substituted into the human native β2 adrenergic receptor. A suitable GPCR riser can be found in the third figure. These GPCR uplifts are derived from a variety of non-human species of beta 2 adrenergic receptors, such as the following β2 GPCRs found in the GenBank database: AF192345 (cat), X94608 (dog), AJ459814 (guinea pig), X15643 (mouse), rat (NM_012492), sputum (L38905). In many embodiments, the GURCs of the invention comprise a sequence of SEQ ID ΝΟ:1. The sequence configuration of these adrenergic receptors does not alter the biological activity of GURCs after being substituted by these amino acids, and therefore, a large number of GURCs can be produced by the method of the present invention. The nucleic acid encoding the GPCR can be upregulated by the ligand

As described above, we can replace the TM5-IC3-TM6 fragment of a parental GPCR with a GPCR up-regulation to make a ligand-up GPCR. In some embodiments, standard DNA synthesis techniques can be employed (see Asuubel, et al, Short Protocols in Molecular Biology, 3rd ed., Wiley & Sons, 1995; Sambrook, et al., Molecular Cloning: A

Laboratory Manual, Second Edition, (1989) Cold Spring Harbor, NY) in order to replace, delete, and/or add a nucleic acid sequence encoding a parental GPCR to a parental GPCR-encoding nucleic acid. An encoding nucleic acid capable of being up-regulated by a GPCR by a ligand is produced.譬 For example, a site-directed mutagenesis can be performed by a site-directed mutagenesis using a site-directed mutagenesis method and a subsequencing method to introduce/delete/replace a nucleic acid encoding a polynucleotide of a parental GPCR. In other embodiments, PCR is used in 156362.doc -41 - 201131168. Recombinant techniques and gene codes for operating polynucleotides are known, and the amino acid sequences of GPCRs and GPCRs are also described above, and those skilled in the art can design and manufacture GPCRs that can be upregulated by ligands. Encoding nucleic acids. The encoded nucleic acid referred to as the GPCR designed and manufactured to be upregulated by the ligand includes a method of chemical synthesis entirely from the oligonucleotide. The method of combining oligonucleotides to produce a nucleic acid encoding a GPCR that can be up-regulated by a ligand is well understood by those skilled in the art [see Cello et al., Science (20〇2) 297:1016. -8; this article is based on its overall list]. An example of a nucleic acid encoding a GPCR that can be up-regulated by a ligand can be found in SEq ID NOS: 38-46'. The ligand-upregulated GPCRs programmed are SEq m NOS: 10-37, respectively. The present invention also proposes a vector (also referred to as "construct") carrying a GPCR-encoding nucleic acid which is said to be upregulated by a ligand. In many embodiments of the invention, a nucleic acid sequence encoded with a GPCR that is upregulated by a ligand will be expressed in a host cell upon substantial association with a expression control sequence (including, for example, a promoter). The so-called nucleic acid is also typically placed into a display vector so that it can be replicated in the host cell in a unified paragraph of the chromosomal or host chromosomal DNA. In general, the expression vector will carry the selected marker 'like tetracycline or neomycin to detect cells that have transferred these DNA sequences (see U.S. Patent No. 4,736,362, the disclosure of which is incorporated herein by reference. ). Whether it is a single expression vector or a co-expression vector is well known to those skilled in the art (see Asuubel, et ai, still 〇 ri Protocols in Molecular Biology^ 3rd ed., Wiley & Sons, 156362.doc • 42- 201131168 1995 Sambrook, et al., Molecular Cloning: A Laboratory Manual, Second Edition, (1989) Cold Spring Harbor, NY). Vectors suitable for use in the present invention include viral vectors, plastids, plastids, artificial chromosomes (artificial human chromosomes, artificial bacterial chromosomes, artificial yeast chromosomes, etc.), minichromosomes, and the like. Retroviral vectors, adenoviral vectors, and adenovirus-like vectors are also useful. There are also a number of performance vectors available in the industry for native and modified native GPCRs. An example of a suitable carrier is pCMV, which is used in some embodiments. This vector was included on October 13, 1998 at the American Type Culture Collection (ATCC) (10801 University Blvd., Mannassas, VA 20110-2209 USA), based on international certification for the preservation of microbial organisms in Budapest. agreement. This DNA is available for ATCC testing certification. The ATCC reserved for pCMV is ATCC #203351. The nucleic acid typically carries only one open reading frame for the alleged ligand up-regulation GPCR encoding, although in certain embodiments, the host cell expressing the ligand-upregulated GPCR may be a eukaryotic cell, such as a mammalian cell, Like human cells, this open reading frame may be interrupted by introns. The nucleic acid is usually a part of a transcription unit which, in addition to the nucleic acid, may contain 3' and 5' non-transcribed regions, which determine the stability of the RNA, or the efficiency at the time of transcription, and the like. The nucleic acid may be part of a phenotype, and in addition to the promoter, a promoter is included to determine the transcription and expression of a GPCR-encoding RNA by a ligand, and may also include a transcription terminator. 156362.doc -43- 201131168 The eukaryotic promoter can be any promoter that functions in a sputum-eukaryotic host cell, including viral promoters and eukaryotic genes obtained from eukaryotic cells. An example of a eukaryotic promoter is as follows: mouse metallothionein weeping sequence (Hamer et al., j. Mool. Appl ^ UR·);

The τκ promoter of Herpes virus (McKnight, Cell 3 1:355_365); the pre-SV40 promoter (B〇n〇ist et vomit, nature (L〇d〇n) 29〇: 3〇4_ 3U), 1981); yeast肸丨丨 gene sequence promoter (J〇hnst〇n to ai,

Proc. Natl. Acad. Sci. (USA) 81:5951-59SS, 1981); CMV promoter, EF-1 promoter, ecdysone-inducible promoter, tetracycline-inducible promoter, and the like. Virus promoters may be of particular interest because they are usually powerful promoters. In certain embodiments, the promoter used is the promoter of the target pathogen. The promoter selection criteria used in the present invention are such that they function in the selected cells (and/or in the animal) after implantation. In certain embodiments, the so-called promoter is the CMV promoter. As mentioned above, ligand-upregulated GPCRs can be produced by a parental GPCR that is substantially linked to a suitable reporter, such as beta-galactosidase, luciferase (such as jellyfish photozyme or photonis), beta-urine. Glycerylase, pneumomycin acetyltransferase, secreted embryonic alkaline phosphatase; proteins used in immunoassays are hormones and cytokines; proteins that promote selective cell growth are adenosine deaminating Enzyme, aminoglycoside phosphorylase (product of ne〇 gene), dihydrofolate reductase, tyrosine-phosphorylated enzyme, thymidine-active hormone (using HAT as medium) 'xanthine guanine phosphoribosyl transfer Enzyme (XGPRT), a protein deficiency that is lacking in auxotrophic bacteria, makes the cell grow weaker protein' can convert a non-toxic matrix into a toxic substance like thymus 156362.doc -44 - 201131168 pyrimidine active hormone enzyme (to contain bromination The deoxyuracil medium is used together), orotate nucleotide decarboxylase (combined with 5-fluoroorotic acid). The GPCR may be directly linked to a reporter, or there may be spacer residues between the two (preferably more than 12, of course, this number can be confirmed by those skilled in the art). A method of fusing a reporter to a GPCR, such as at the carbon or nitrogen end of a GPCR, is well known to those skilled in the art (e.g., McLean et al, Mol. Pharma. Mol Pharmacol. 1999 56: 1182-91; Ramsay et Al., Br. J. Pharmacology, 2001, 3 15-323) is not described here. It is generally desirable to have a ligand-up GPCR-starting with a parental GPCR and essentially connecting one of the aforementioned reporters. In certain embodiments, the reporter is said to be fused to the carbon end of the ligand-upregulated GPCR. In some embodiments the reporter is a wild or modified species of jellyfish luciferase. Nucleic acid fragments of the invention may also include restriction sites, multiple selection sites, primer coupling sites, recombination sites, and the like, to enhance the construction of modified ligand-upregulated GPCR-encoding nucleic acids. Identification of Ligand Up-GPCRs In some embodiments, the final step of making a ligand-up GPCR, a modified GPCR (eg, a parental GPCR has been raised in its TM5-IC3-TM6 fragment by a GPCR) The amino acid sequence substitution) is tested to determine if its amount in the host cell or on the cell membrane is increased by the presence of the ligand. In general, this test step involves the production of a GPCR in a cell, comparing the amount of the substituted GPCR measured in the presence and absence of a ligand, and confirming whether the substituted GPCR is a ligand. Increase the GPCR. In most embodiments, the nucleic acid encoding the replacement GPCR is transcribed into a cell of 156362.doc • 45- 201131168, and the cell is cultured under suitable conditions to produce a substituted GPCR. The quantity 'measured in the presence of a ligand and no ligand, respectively. In most of the examples, the number of substitution GPCRs in the absence of a ligand was first measured, the GPCR was added to the ligand, and the number of substituted GPCRs in the presence of the ligand was determined. It is often desirable to have the above measurement procedures be designed in parallel rather than sequentially. If there is more GPCR in the case of ligand than in the case of ligand, and there is more GPCR than in the absence of ligand, then the substituted GPCR is One ligand upregulates the GPCR. In most embodiments, if the substituted GPCR is a ligand-upregulated GPCR, the number of ligands must be at least increased by more than 丨〇%, or at least 30%, in the presence of the ligand compared to the absence of the ligand. Above, or at least 5% or more, or at least 7% or more, or at least 100. /. Above, or at least i 5〇% or more, or at least 200% or more, or at least 300% or more, or at least 400% or more, or at least 5% or more, or in certain In the embodiment, it can be more than 1000%. As briefly described above, a GPCR can be assayed by immunoassay, or can be substantially linked to an epitope, such as the Ha, V5, c-myC or FLAG markers for detection. However, in some embodiments the 'substitutional GPCR can be substantially linked to a reporter so that the amount of reporter can be measured to determine the amount of GPCR. In certain embodiments, the reporter is said to be a luciferase' such as a wild species or a modified species of jellyfish luciferase. Methods for introducing GPCR-encoding nucleic acids into cells are well known in the art. Suitable methods include electroporation method, particle grab technology, calcium phosphate coprecipitation technique 156362.doc -46 - 201131168, direct microinjection, etc. ^Select the s, the side to see the cell type to be converted and its The environment (in vitro, in vitro) depends on the environment. A general discussion of these methods can be found in Ausubel t - al > Short Protocols in Molecular Biology ^ 3rd ecj Wiley & s〇nc y & S〇ns, 1995. In some embodiments, the use of liposomes and the introduction of calcium-mediated nucleic acids into cells has also been well known in the art, and TV turbulence is seen as a transfection technique. Will ring

Ausubel's methods for measuring reporter activity, such as luciferase, GFp, etc., are also commonly known in the industry (eg Ramsay et al, Br j phar (4), 2001, 133: 315-323). This statement. Identification of ligands for GPCRs The present invention also proposes a method for screening a ligand for GpcR in a test compound. In many embodiments, these methods are performed in a test tube, and a cell with a ligand-upregulated GPCR is co-located with the test compound, and then the ligand is measured to increase GpCR& in the presence of the test compound. The amount is compared to the applicable control group. In many embodiments, a suitable control group is a ligand that does not contain a test compound to upregulate GpCR. Usually one ligand will result in a higher number of ligand-up GPCRs than in the control group. In many embodiments, a ligand will increase the ligand by 〇1 compared to the control (>: the measured number of 11 will increase by at least 10%, or at least 2%, or at least 40 % or more, or at least 6〇% or more, or at least ι〇〇% or more, or at least 150% or more, or at least 2% or more, or at least 300% or more, or At least 5% or more, or more than 1 〇 or more than 20 times. 156362.doc -47- 201131168 Taking an example, an encoding nucleic acid of a ligand-upregulated GPCR is introduced into an appropriate cell. 'This cell is then cultured under conditions that allow the ligand to upregulate GPCR performance. Detecting ligand-adjusted cell measurements of cells that produce ligand-upregulated GPCRs (one, or a population of such cells), respectively Measured under conditions of ligand and no ligand. In some embodiments, before contacting a test compound with a test compound, the amount of GPCR on the ligand is measured, and at least about 1 after contact with the test compound. 〇 minutes, or at least 3 〇 minutes, or at least about 1 hour' or at least about 2 hours, or The amount of ligand upregulated GPCR on the cells after contact with the test compound is measured for about 4 hours, or at least about 8 hours, or at least about 12 hours, or at least about 24 hours. In some embodiments, 'detection' The procedure is a parallel design rather than a sequence design. As described above, the detection of a ligand-upregulated GPCR can be carried out by any suitable method. In most embodiments, the detection method is to raise the ligand to the GPCR and the reporter (for example, Luciferase 'GFP, etc., is fused and then quantified by the reporter. In some embodiments, the luciferase is a wild or modified species of jellyfish luciferase. The above assay includes Let the ligand upregulate the GPCR in the appropriate host cell 'like eukaryotic cells, such as animal cells (such as insects, fish, amphibians, birds or mites), plant cells (such as corn or Arabian) Fen cells), yeast cells (yeast &

The cells are then tested for their GPCR content before the selected agent has been added. Any cells which are sufficient for the expression of the nucleic acid sequence of the ligand to upregulate the GPCR can be used as host cells. Usually, an animal cell is used, and the test examples are as follows: monkey kidney cells (COS cells); monkey kidney CVI 156362.doc • 48· 201131168 cells (COS-7, ATCC CRL 165 1); human embryos modified by SV40 Renal cells (1^1 <:-293, Graham et al. J. Gen Virol. 36:59 (1977)) * HEK-293T cells; young hamster kidney cells (BHK, ATCC CCL 10); Chinese voles ovary cells ( CHO, Urlaub and Chasin, Proc. Natl. Acad. Sci.

(USA) 77·.4216, (1980)); mouse 赛toli cells (TM4, Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CVI ATCC CCL 70); Africa Green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical cancer cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat kidney cells (BRL 3) A, ATCC CCL 75); human lung cells (hep G2, HB 8065); human hepatocytes (W138, ATCC CCL 75); mouse mammary tumor cells (MMT 060562, ATCC CCL 51); TRI cells (Mather et al. , Annals NY Acad. Sci 383:44-68 (1982)); NIH/3T3 cells (ATCC CRL-1658); mouse L cells (ATCC CCL-1). In some embodiments, melanocytes are used. Melanocytes are found on the epidermis of lower vertebrates. The related materials and methods thereof can be operated as disclosed in U.S. Patent No. 5,462,856 and U.S. Patent No. 6,051,386. These published patents are hereby incorporated by reference in their entirety. Many other cell lines are readily available to those skilled in the art, and a wide variety of cell lines are available to the American Type Culture Collection (10801 α·).

University Boulvard, Mannassas, Va. 20110-2209 USA). In most embodiments, the assay used is a high-performance output, such as a 96 or 384-well plate, using appropriate automated equipment (such as I56362.doc -49·201131168 automated dispensing device), and Available experimental instruments (96 or 384 grids of luminescence or fluorescence analyzers to read the reporter's activity). An example of the test is as follows. The measure of the activity of the reporter may be a Wallace 145〇.

Microbeta C0Unter (Perkin_Elmer). In some embodiments, a measure of the activity of the reporter may be used with a fluorometer equipped with a CCD camera. A large number of different kinds of compounds can be screened by the above method. The compound to be tested contains a wide variety of chemical agents, usually organic molecules, preferably small molecular organic compounds having a molecular weight greater than 50 and less than 2,500 dox. The molecule to be tested contains a functional group having a structural action of hydrazine and a protein, especially a hydrogen bond' and usually carries an -amino group, a few groups, a (four) or a stilbene group, preferably having two or more prosogenic groups. The compound to be tested also often has a structure of a ring carbon or a heterocyclic carbon and/or a structure of an aromatic ring or a polyaromatic ring and has at least one of the preceding substituents. The test compound is also often a biochemical molecule including a peptide, a saccharide 'fatty acid, a steroid', a valproate, or a variant, structural approximation, or a combination thereof. Other test compounds also include native ligands for this GPCR. The test compound can be obtained from a variety of different clamped β ^ U sources, including libraries of artificial or synthetic compounds. For example, there are several other wild-type methods that can be used to make arbitrary or unique determinations of organic compounds and biochemical molecules, including oligonucleotides and oligopeptides of arbitrary composition. In another way, the octagonal compound tributary library of the type of bacteria, mold, plant and animal extracts is easy to use or easy to prepare. In addition, Scorpio ..., or 50% of the compounds and their databases can be modified by general chemical, physical and biochemical methods. 156362.doc •50· 201131168 can create a comprehensive library of compounds. It is known that medicinal compounds can also be the target of any or a specified chemical adjustment, such as guanidation, alkylation, esterification, amination, etc., to achieve structural similarities. Compounds of particular interest to the present invention are like multipeptides, such as proteinaceous compounds. A particular class of polypeptide compounds of the invention are antibodies to GPCRs, or fragments thereof coupled to GPCRs. The antibody may be in the form of a single plant or a plurality of strains, or may be prepared by methods well known in the art. Other test compounds also include variants of this GPCR natural ligand, such as natural ligands with at least one amino acid substituted, deleted or added, or chemically modified. In certain embodiments, the test compound comprises an endogenous multi-peptide that is not known to be a GPCR ligand. The characteristics of the compounds to be tested mentioned above are merely examples, and are not intended to limit the scope of the invention. Method for Regulating GPCR The GPCR ligand recognized by the method of the present invention also includes a modulator having this GPCR. Whether a ligand is a regulator of a GPCR, and its judgment should be familiar to those skilled in the art. We will present a number of exemplary tests in the examples. Therefore, the test compound identified by the ligand up-regulation GPCR by the method of the present invention is also a ligand of a parental GPCR, and the so-called ligand is a regulator of the corresponding native GPCR, which can be used to regulate the native GPCR or its variation. body. The method of the present invention can be used to identify a GPCR ligand which is a GPCR regulator, such as an antagonist, an agonist, a partial agonist or a reverse 156362.doc-51 - 201131168 agent, etc., A ligand that is a regulator can increase or decrease the activity of a GPCR. GPCR activity can be measured by any suitable activity assay (GTP light test, etc., as will be detailed in the examples.) For example, if the [35s] GTPyS test is used, an agonist will increase. [35s] The amount of gtpys coupled on the membrane. Both the antagonist and the reverse agonist reduce the amount of [35s]gtpys on the membrane via the mediator of the corresponding agonist. The reverse agonist reduces the activity of a natively activated native GPCR or an essentially activated GpcR associated with an agonist, but the antagonist does not. If the modulator in the example increases GPCR activity, the GPCR will increase by at least about 1% of the activity of the regulatory body by at least about 20%, or at least about 20% of the appropriate control group to which the compound is not added. About 3%, or at least about 50% 'or at least about 8%, or at least about, or at least about 00°/. Or at least 10 times more. Good regulation can be achieved in the presence or absence of the natural ligand of GpCR. If the modulator in the embodiment reduces GPCR activity, the GPCR will be reduced by at least about 20%, or at least about 20%, of the activity of the regulatory body compared to an appropriate control group to which the compound is not added. 3〇%, or at least about 50%, or at least about 70%, or at least about 8%, or at least about 90%, or at least about 95% » The presence or absence of a natural ligand in this GPCR Under the existing conditions, good regulation can be obtained. In certain embodiments, these assays also include measuring the GPCR activity of a compound with or without both conditions. Measurement of activity may include detaching a separate cell membrane (eg, a cultured cell) from a cell, 156362.doc • 52 · 201131168 cell membrane '-cell extract' or isolated GPCR, with sufficient weight of GPCR regulation Body contact acts to modulate the activity of this GpcR. In other embodiments, a biopsy is used, including GPCRs in a chewing animal, such as primates (eg, humans, gorillas, etc.), rodents (mouse or rat) Etc.) or any other animal body that can be provided, a sufficient amount can be considered as this (3) regulatory ligands are administered to the mammal to adjust the Gp (:R activity of the mammal. (4) When performing this method' An effective dose of the active agent must be administered to the individual, wherein the term "effective dose" means a sufficient amount to achieve the desired effect, and the effect sought is that the desired modulation is to enhance and reduce at least one GPCR. In the case of the in vivo method of detection, the active compound is usually administered to the host by means of a physiologically acceptable transport carrier, for example, a form of the agent. The supply formula, dosage, route of the test, etc. of the test compound will be described in detail below. Formulations, Dosages, and Routes of Administration The present invention also provides formulations, including pharmaceutical formulations, one of which is to modulate the individual's GPOI activity. - Generally, the formulation contains at least some An effective dose of the medicinal agent can be used to modulate the individual's native GpcR activity. In many embodiments, the result is such that the phenotype is increased or decreased to bring the phenotype closer to the normal state than the control group. Formulations In the methods of the present invention, the 'active ingredient compound can be applied by any conventional method sufficient to achieve the desired GPCR regulation. Therefore, the compound can be used in combination with various formulas to 156362.doc -53-201131168 It is understood that the compounds of the present invention can be formulated into pharmaceutically acceptable compositions, and pharmaceutically acceptable carriers or solvents, and formulated in a solid, semi-solid, liquid or gaseous form, such as pills, Capsules, powders, small particles, oils, sputum, Ι Ι λ -tel / mixture suppositories, injections, inhalants or aerosols. The compounds used in the sputum sputum type may be based on their pharmacologically acceptable salts. Or the other may be applied alone or in combination with or in combination with other suitable pharmaceutically active ingredients. The methods and excipients described below are merely examples and are not intended to limit the invention. The compound may be administered alone or in combination with suitable excipients to form tablets, powders, granules, or capsules, for example, with conventional additives such as lactose, mannitol, corn powder or Yangmoudian powder; added with adhesive like crystalline cellulose, cellulose derivative, gum arabic, corn starch or gel; added with a bulk agent like corn house powder, artichoke powder or slow-shelled cellulose Adding a lubricant such as talc or magnesium stearate; or adding a diluent, buffer, moisturizer, preservative or flavoring agent as needed. The compound of the former can be formulated into a shot preparation, dissolved, or Emulsified in an aqueous or non-aqueous solvent, such as vegetable oil or other similar oils, synthetic fatty acid glycerides, higher fatty acids or glycerol sulphides; or additives that can be added as needed, such as co-solvents, Isotonic agents, suspending agents, emulsifiers, stabilizers and preservatives. The compounds of the preceding formula can be administered by spraying in the form of a spray. The compounds of the present invention may be formulated with a pressurized propellant such as dichlorodifluoromethane, alkane, nitrogen or the like. "°C 156362.doc •54· 201131168 Further Steps The compound of the former can be mixed with a variety of matrices to form a suppository, such as a sputum base or a water-soluble base f. The compound of the present invention can be administered to the rectum in the form of a test. The suppository may also contain a carrier such as cocoa butter, cab〇Xawes and polyvinyl alcohol, which can be dissolved at body temperature but can be coagulated orally or rectally at room temperature. The dose used each time is like a teaspoon, a two:: two-plug' can contain a known amount of a compound and include one or more inhibitors. Similarly, a single dose of a drug administered in a subcutaneous or intravenous form may include one or more inhibitors in its combination, such as a mixture dissolved in sterile water, normal saline or the like. A pharmaceutically acceptable carrier. The term "unh d〇sage f〇rm" as used herein refers to the application to humans. Or a unit dose administered by an animal, a physically distinguishable physical list: 'per unit contains a defined amount of a compound of the invention, calculated to produce the desired effect, and with - in a pharmaceutically acceptable dilution Agent transport agent or carrier. The new unit dosage form to be used in the compounds of the present invention will depend on the individual compound employed, as well as the efficacy to be achieved and the pharmacokinetics of the parent compound in the host. Other application forms may also be employed. For example, the compounds of the invention may be formulated as a suppository and, in some instances, as a spray or nasal stopper. In the case of suppositories, the t' carrier mixture may comprise conventional spirulina-like transport agents such as decyl glycol glycol oils, triglycerides. The thus prepared 156362.doc -55- 201131168 suppository may be composed of a mixture comprising from about 05% to about 〇% (w/w) of the active ingredient, more precisely from 1% to 20%. Formulations for nasal administration typically include carriers that do not cause nasal mucosal irritation and impede nasal cilia function. A diluent such as water, saline or other known substances can be used in combination with the compound of the present invention. Nasal suppositories may also contain preservatives such as, for example, tri-tert-butanol and vaporized methylammonium hydroxide. Surfactants can also be used to increase the extent to which the proteins of the invention are absorbed by the nasal cavity. The compounds of the invention may also be administered by injection. Usually, the injection formulation is made into a solution or suspension; it is also feasible to use a liquid carrier to be cooled into a solution or suspension prior to injection. This method can also be carried out by emulsifying or by coating the active ingredient with a liposome carrier. Suitable excipient carriers include, for example, water, physiological saline, glucose, glycerol, ethanol, and the like, or a mixture of the foregoing. In addition, the carrier may also contain minor amounts of adjuvants such as moisturizing emulsifiers or acid-base buffers, if desired. Methods of preparing the above various formulations are well known or understood by those skilled in the art. See Remingt〇n, s heart

Sciences, Mack Publishing Company, Easton, Pennsylvania, 17th edition, 1985, Remington: The Science and Practice Of Pharmacy, A.R. Gennaro, (2000) Lippincott, Williams & Wilkins. The composition or formulation administered, however, contains a certain amount of the agent to achieve the desired state of the subject. Pharmaceutically acceptable excipients such as vehicles, adjuvants, delivery agents or diluents are well known. Moreover, pharmaceutically acceptable auxiliary ingredients such as pH balance and buffer, tension regulator, stabilizer, moisturizer, etc., can also be obtained publicly. Dosage Although the dosage used will vary depending on the desired effect, a suitable dose of about 1 pg to 1000 pg, or about 10,000 Å, may reduce the symptoms of GPCR-related diseases in an individual. Those skilled in the art will recognize that the dosage will depend on the severity of the compound, the condition, and the strength of the side effects of the compound. The optimum dosage of a compound can be determined in a number of ways by those skilled in the art. Routes of Administration This is a pharmaceutically acceptable route of administration including intranasal absorption, intramuscular injection, intratracheal absorption, (four) injection, subcutaneous injection, intradermal injection, topical application, intravenous injection, rectal, nasal, oral and other means. A route of administration that is not administered orally. The route of administration may be combined if desired, or adjusted according to its formulation or/and the desired effect. The administration of this drug may be one dose or multiple doses. Drugs administered to the host may be taken at any route, including systemic or topical routes of administration, including inhalation. The general methods and medication routes that are easy to use. Generally speaking, this hair: intestine, parenteral, or parenteral route of inhalation of neighboring, ancient abuse, and swimming left (external example can be as follows: local skin penetration, subcutaneous, muscle, In the orbit, intracapsular, intrahepatic h, and intravascular, etc., _ contains intravertebral, chest, and wide, deducting any path of shovel without nutrition. Parenteral routes of administration can be used. Le sputum formula, preoccupied # '', 'first sputum or local delivery Μ. Right to get (4) secret material efficacy, usually 156362.doc •57- 201131168 will be invasive, or systemic absorption of local or mucosal pathway. The alleged agent can also be administered to an individual by parenteral administration. Routes of enteral administration include, but are not limited to, oral administration and administration of the rectum (e.g., using a suppository). The method of administering the compound of the present invention via the route of administration of the skin or mucosa may include, but is not limited to, the topical application of a suitable pharmaceutical formulation, transdermal, injection, and epidermal administration. 'The absorption enhancer and iontophoresis are all suitable methods. The iontophoresis transmission uses a commercially available "stick" to continuously permeate the product through the current pulse for several days or more. By treatment is meant to at least ameliorate the symptoms associated with the morbid condition of the host, where the so-called improvement is a general term that at least a certain parameter has a reduced intensity, such as a symptom associated with the symptoms of the condition being treated. In this context, the term treatment also includes certain situations in which the morbid condition or at least its associated symptoms are completely suppressed (eg, no longer occurs), or stopped (eg, aborted), leaving the host no longer affected by this The morbid condition or at least the characteristic symptoms of this morbid condition suffers. The use of the formulations and methods of the present invention to modulate GPCR activity can be found in a variety of therapeutic procedures. Generally, these procedures comprise administering to a subject suffering from a GPCR-related disease a sufficient amount of one or more compounds to modulate a GPCR to modulate gpcr in the host, thereby treating the subject with the condition. In certain embodiments, if one wants to reduce the activity of a certain GPCR, I56362.doc •58· 201131168 may be administered to one or more compounds to reduce the activity of a certain gpcr; The activity can be administered to one or more compounds to increase the activity of a certain GPCR, and a variety of individuals can be treated in accordance with the methods set forth herein. Usually these individuals are mammals, and these words are often used in the general term for mammals: including carnivores (such as dogs and cats), saliva (such as small salted rats), and primates ( For example, people, chimpanzees and jackals). In many embodiments, the so-called individual will be a human. The treatment of the present invention is usually applied to an individual having the aforementioned disease or to an individual who wants to avoid the aforementioned diseases. The present invention also encompasses the administration of a formulation containing a needle body to avoid or reduce the condition associated with GRCP. Tuning Kits The present invention also has kits that can actually use the methods previously described. The kit of the present invention to &&& v, v L includes one or more of the following: a nucleic acid-binding receptor up-regulator' or a ligand-upregulated GPCR, or can be made = up-regulated GPCR Cells == multiple selection sites, primer sites, etc., to facilitate integration with other =. Other optional accessories in this kit include 胄: limiting enzymes, nucleus:, buffer receptors or ligands (4) (iv) controll 2 buffers, cells, etc., to carry out the assay of the invention. The accessories in this month's kit may be that the accessories in the other container may be combined and collected in the same container t, as needed: in addition to the accessories proposed above, the kit of the present invention usually also contains 156362. Doc -59· 201131168 How to use the various parts to carry out the invention's instructions for the detection method. The original is a soil: it will be recorded on a suitable recording medium. Such as the description of the kit of the invention may be plastic or the like. And the sergeant ^ on the soil 'like the paper attached #, μ, 'the instructions of the kit of the invention may be packaged t

The attachment is on the label of the kit or its accessories, which is related to the package and the small package. In other embodiments, the description of the specification is electronic. Store the storage medium on the file format, for example, (3) RfW, Tian B, electric 4 such as CD-ROM, magnetic disk, and so on. In a further embodiment, the 'true description indication is not included in the kit, but the (4) of the book obtained from a distance (4), for example, through the Internet; the 7F of the example contains the website. The address can be read and/or downloaded to the required manual. As with the instructions, this method of obtaining the instructions is also printed on a suitable substrate. [Preferred Embodiments] The following examples will be disclosed to those skilled in the art to disclose and describe how to use the present invention 'is not intended to limit the inventions that the inventors believe, or to enumerate the following. The experiments are all or the only experiments performed. The description of the present invention is made with reference to certain specific embodiments, but those skilled in the art will appreciate that many variations and equivalents can be made without departing from the invention. The true meaning of the invention and its scope of application. In addition, adjustments may be made to special circumstances, materials, formulations, procedures, and operating procedures to suit the subject matter, true meaning, and scope of application of the present invention. All such improvements are considered to be within the scope of the invention. 156362.doc •60· 201131168 Materials and Methods Abbreviations: AR: adrenergic receptors Rlu: jellyfish luciferase. GPCR: G protein coupled receptor. NMS: N-methyl scopolamine. Rm3: Murine muscarinic acetylcholine receptor 3. Materials: All cell cultures, as well as materials for general molecular biology experiments, were provided by Invigtogen (Carlsbad, CA). [3H] N-methyl gasified scopolamine (NMS) (80 Ci/mmol) was obtained from NEN Life Science Products, Inc. (Boston, MA). All chemicals, known to LOPAC, are supplied by Sigma (St. Louis, MO). Oligonucleotides were obtained from Genset (San Diego, CA). MG 132 (benzyl carbonyl-L-bright-L-bright-L-bright) was purchased from Callbiochem (La Jolla, CA). The native marine villus intestinal enzyme is from Biotum (Hayward, CA). The plastid pRLCMV containing the jellyfish luciferase vector was taken from Promega (Madison, WI). The jellyfish luciferase reporter gene detection kit (LucLiteTM) was obtained from Packard (Meriden, CT).

Construction of the Rm3 Receptor Gene Fusion: Both wild-type Rm3-Rlu and Rm3(GURC)-Rlu were made in the manner described in Figure 5. In short, the manufacture of Rm3-Rlu has two steps. First, a full length of the jellyfish luciferase-encoding cDNA (Rlu; 312 amino acids) was obtained by PCR amplification of the plastid pRLCMV containing the aequor luciferase vector, EcoR I and Xba I The EcoR I-Xba I fragment was further sub-transformed into the pCDNA3.1 (+) vector to constitute Riu_pCDNA3.1. In the second step, a full-length cDNA encoding the muscarinic acetylcholine receptor type 3 (Rm3) of mouse is obtained by PCR to amplify the plastid containing Rm3-pCD (National Health 156362.doc - 61 - 201131168 Dr. J. Wess, a research institute, generously provided). The PCR product was digested with Nhe I and EcoR I, and then inserted into Rlu-pCDNA3.1 with Nhe I and EcoR I. The result was that the carbon end of Rm3-Rli^ Rm3 to be constructed was inserted between the carbon end of Rlu and the nitrogen end of Rlu. The three amino acids link to Glu-Asn-Ser, introducing an EcoR I restriction site (fifth panel).

Rm3(GURCl)-Rlu replaces the 250-494 amino acid of Rm3 with GURC (Fig. 3 and Fig. 5), and its construction steps are as follows. First, a PCR fragment encoding GURC1 was inserted, and the Κρη I and EcoR I sites of Rlu-pCNDA3.1 were inserted to become GURC 1-Rlu. Secondly, the PCR product encoding the 1-49 amino acid of Rm3 (from the Met at the nitrogen end to the junction of TMV to IC3) was sub-selected into the upstream Nhe I and Κρη I sites of GURC 1-Rlu. . Finally, a PCR product encoding the amino acid at positions 495-589 of Rm3 was inserted into the EcoR I site by non-directional selection. Each construction step is then confirmed by a DNA sequence analyzer. There are two mutation sites in this construct. The introduction of the Κρη I site will cause the two amino acids to be modified (YW and GT) at the nitrogen end and the introduced GURC 1 clamp, and the added EcoR I site at its carbon end and the introduced GURC 1 clamp The joint is to change the SA of Rm3 to EF. Cell Culture and Transfection: HEK 293 or COS-7 cells were grown in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal calf serum, 2 mM L-glutamate, 100 gg/ml Penicillin also has 100 units/ml of streptomycin and is placed in a CO2 incubator at 37 ° C in a humidity of 5%. At the time of transfection, lxlO6 cells were seeded into l〇〇-mm plates for 24 hours, then transfected with 4 gg of plastid DNA per plate, and liposome reagent was taken and 156362.doc •62· 201131168 Manufacturer Instructions are used. To create a cell line that stably expressed the receptor, the cells were first harvested and diluted 2 days after transfection, and stored in DMEM supplemented with 500 pg/ml sulfated Geneticin. This medium was replaced every 3 days with DMEM supplemented with 500 pg/ml sulfated Geneticin until the cells were completely resistant to sulfated Geneticin. To test how the compound affects the steady-state receptor performance, cells were isolated by transfection with a 96-cell culture plate (Greiner America Inc.) treated with polylysine for 20 hours, depending on the concentration. The test compound was treated for 20 to 24 hours for luciferase activity assay. Luciferase activity assay: Untreated or drug-treated cells were washed with phosphate buffered saline solution (PBS). The cells were placed in a 50 μΐ lysate (0.25% ΝΡ-40 was added to the assay: 100 mM sodium phosphate, pH 7.4, 500 mM sodium chloride) and allowed to stand at room temperature for 30 minutes. Luciferase activity was measured immediately with a Wallace 1450 Microbeta counter (Perkin-Elmer) after adding 100 μΐ of 2 μΜ marine lumen intestinal fluorescer to the assay. Ligand coupling test: COS-7 cells were transiently transfected with various constructs. Two days after transfection, rinse with ice-cold PBS. The cells were precipitated in PBS/0.5 mM EDTA and re-dissolved in ice-cold coupling solution (25 mM sodium phosphate, pH 7.4, 2 mM EDTA and 10 mM MgCl2). The cells were disrupted by homogenization with a 3x1 Os agitation method. The crude homogenate of the cells was subjected to [3H] NMS coupling test. To investigate the effect of different ligands on Rlu-labeled fusion receptors, cells were treated overnight with ΙμΜ atropine 24 hours after transfection (20 hours). Rinse the untreated or treated cells thoroughly with ice-cold PBS to flush out the lighter. Engagement test 156362.doc -63· 201131168 is to use cell homogenate with [3h]nms as radioligand. The sample (15 〇 1) was incubated in a coupling solution at room temperature for 2 hours in a 96-well culture dish. In order to measure the maximum value of the saturation coupling site, 2 nM [3h] nms was used. In the competitive coupling test, the membrane homogenate was first incubated with different concentrations of ligand for 30 minutes at room temperature, followed by [屮]1^]^8 of 11.5 11 cubits. The non-specific coupling test was measured with the addition of 10 μΜ of the atropine. The reaction was stopped by incubation at room temperature for 9 minutes. Samples were collected by rapid filtration using a Whatman GF/c % filter and rinsed three times with ice-cold buffer (Tds_HCl, i EDTA 'pH 7.4) at 5 paws. Once placed in the scintillation buffer, the sample in the filter was detected using a Packard scintillation counter (Hewlett packard, pal〇 AU〇, ca). Reporter gene test: The NFTA reporter gene was used to measure intracellular calcitic changes in HEK293 cells called medium. HEK293 cells were seeded in white plates (10,000 cells per cell). After 24 hours, the cells were co-transfected with the plastids of the receptor and the reporter plastid (pNFAT_luc) using the liposomeTM reagent. The cells were then placed in a medium containing no phenol red or serum, and cultured for 2 to 6 hours with or without a drug. Luciferase activity was then measured by the LucLiteTM Luciferase Gene Assay Kit according to the manufacturer's instructions. Screening for LOPAC compounds: All compounds were first dissolved in 4 μΜ of DMSO and injected into the 2nd to the 丨丨 column of the 96-well plate. The atropine (4 〇〇 μΜ in DMSO) was injected into column 1 and the DMS was injected into column 12. One microliter of normal cells containing 1 μM was cultured by MiniTrak (Packard BioSdence Inc.) and transferred to a culture dish. 156362.doc • 64 · 201131168 HEK293 cells (100 μΐ approximately 50,000 cells) stably loaded into the culture dish with Multidrop (LabSystems) to make a final medium of 200 μΐ and a final DMS0 content of 0.5%. The cells were cultured for another 20 hours and the medium was extracted. All reagents were operated with Multidrop. Luciferase activity was measured in the manner described above. Other useful methods: Protein testing is based on the Bradford method using serum proteins. All data were calculated using the nonlinear least squares optimal curve method and calculated using the GraphPad Prism computer program (San Diego, CA). Example 1

The Rm3(GURC)_Rlu fusion protein exhibits a lower degree of expression than the wild species receptor. The lucerase gene of the sea pansy (Renilla reniform) and the carbon end of the wild species Rm3 are fused in the same reading frame. The fused protein showed similar coupling affinity to [3H]NMS compared to the unlabeled wild species receptor (this data is not presented). However, the saturation coupling test showed that the Rlu-labeled receptor increased the coupling point of [3H]NMS by at least 2 times compared to the unlabeled wild species receptor (Fig. 6). After replacing the 250th to 494 amino acids with GURC1 as described in the Materials and Methods section, the coupling point to [3H]NMS is reduced by more than 2 times compared with Rm3-Rlu (Sixth Figure, Rm3 (GURC)-Rlu). Overall, the steady-state protein expression of Rm3(GURC)-Rlu showed at least a 4-fold reduction. These results show that the substitution of GURC1 at a position in the TM5-IC3-TM6 fragment of the receptor and the addition of Rlu at its carbon end can produce a protein with a lower steady state protein 156362.doc -65- 201131168 Mutation receptor. Example 2

The Rm3(GURC)-RU fusion was compared to the genital agent and the antagonist with the same coupling affinity as the genitor and the antagonist. As shown in the seventh and seventh beta images, the coupling affinity of the antagonist atropine and nms to Rm3(GURC)-Rlu is comparable to that of the wild species receptor (the competing curve shifts to the left), Shows a slight increase (up to 2 times). However, the coupling affinity of 4 DAMP to Rm3(GURC)-Rlu has a 1-fold increase compared to wild species receptors (Section 7C). The antagonist carbachol showed similar affinity to the wild type receptor and to the fused mutant receptor (seventh D-drawing). Thus, the 'Rm3(GURC)-Rlu fusion receptor retains the ligand (adjuvant and antagonist) coupling properties of the wild species receptor. Example 3 配ιη3(〇υΐΚ:)·ΙΠιι's ligand specialization leads to upregulation. Adding Rlu to the receptor directly measures luciferase activity and helps determine the performance of the fusion receptor. As shown in Figure 8A, 'all antagonists against muscarinic acetylcholine receptors (NMS, atropine, 4-DAMP 'himbacine and QNB '1〇μΜ), Rm3(GURC)-Rlu Photozyme activity can result in a 4 to 5 fold increase compared to untreated cells. The agonist carbon sputum test (10 μΜ) showed a 2-fold increase in luciferase activity at Rm3(GURC)-Rlu (Figure 8A). In contrast, the ligands specific to adrenergic receptors (isoproterenol, yohimbine, ICI-1 18551) and other non-ligand compounds (S103003, S104431 and S105201) are in the same situation. Fusion 156362.doc 66· 201131168 The receptor has no significant effect on luciferase activity. Moreover, compounds that are not known to interact with muscarinic receptors are known as serotonin receptor-specific ligands (serotonin, dimethophyllin), as well as the exclusive ligand for histamine receptors (imetit, Reni It does not change its luciferase activity (data not shown). These results indicate that the upregulation of the enzyme activity of this fusion receptor is particularly relevant to ligands. To rule out the possibility that these ligands directly affect luciferase activity, cells are transfected with the wild-type Rlu gene prior to exposure to the test compound, while in another experiment the ligand is directly administered for luciferase activity assay. . The ligands had no effect on the activity of the luciferase enzyme in both experiments (data not shown). Example 4 Determination of Receptor Upregulation by Ligand by Radioligand Coupling Test To confirm that the increase in luciferase activity caused by a ligand is due to an increase in the number of receptors, we measure by coupling test. [3H] The number of couplings S of NMs to determine receptor expression in untreated and ligand-treated cells. Use cells that exhibit Rm3(GURC)_Rlu! The [3H]NMS coupling site was increased by 4 times in 20 hours after the treatment with μΜ atropine (Fig. 8). The wild species Rm3_Rlu treated with atropine did not significantly affect the [3H] NMS coupling site. These results are consistent with our results from the luciferase assay (Figure 8A), confirming that the increase in luciferase activity is due to a substantial increase in the number of receptors. Example 5 Ligand-induced Rm3(GURC)-Rlu upregulation time and dose-dependent Ligand-induced Rm3(GURC)-Rlu upregulation with time dependence 156362.doc -67- 201131168 dose dependence. A significant up-regulation can be detected in cultures of 37t for at least 6 hours. If treated with 10 μL of atropine, the activity of luciferase will gradually increase over time from 6 hours to 96 hours (not shown in the data). In general, considering the long-term culture at 37 °C, there are concerns about the stability of the compound, and the experiment will use a culture time of 20 to 24 hours. As shown in Figure 9, atropine and 4-DAMP enhance the luciferase activity of Rm3(GURC)-Rlu in a concentration-dependent manner. All other tested antagonists also exhibited dose-dependent upregulation of this mutant fusion receptor (not shown in the data). In any case, the pharmacodynamics (ECw values) of these ligands differ greatly from each other if compared to the Ki values measured by the other test methods, which are coupled to the test s (the seventh figure). The ECso value (about 6 nM) of the atropine in the fluorescence activity test was only about 5 times greater than the result of the coupled competition test (about 1.3 nM). In this test, the Ec5〇 values were also only slightly shifted to the right of the antagonist/reverse agonist as well as MNS, QNB and Himbacine (data not shown). However, the fluorescence activity test ECS() value of 4-DAMP (approximately 900 nM) was approximately 1 〇〇〇 more than the result of the coupled competition test (approximately 1 nM) (Fig. 7). The agonist carbon brewing test is also a dose-induced receptor upregulation (p. IX), but its EC5〇 (greater than 100 μΜ) is at least 1x right offset from the coupled competition test. (Sixth map). The EC5〇 value of Oxotremorine® is 1〇〇〇 higher than its Ki value (Figure 9). The right shift in ECm values in luciferase activity assays may be due to ligand instability and ligand-induced downregulation (at 37 ° C, over 20 hours of treatment) (see Related discussion). In fact, we found that 4-DAMP is less stable than atropine under this condition, which can be 156362.doc • 68 - 201131168 It was found in the experiment that the coupling of the ligand can be measured after treatment in the same situation as the uplift test. Affinity is reduced (data not shown). Example 6 developed a screening platform based on the ligand-associated upregulation of Rm3(GURC)-Rlu. We designed a library of known compounds in the format of 96 cells and 384 cells to screen LOPAC known compounds (about 7 〇〇 compounds), including ligands with many virulence receptors. DMS 〇 up to 1% concentration does not affect this test. All compounds were tested at a concentration of 2 μΜ and in a % or 384 grid format with a single point of 〇 5% £)^13〇 final concentration. Atropine was used as a positive control and set to a 2%% reaction, while pure DMSO was used as a negative control (set to 1% reaction). Both formats are approximating the standard deviation (less than... 乂彡 and /, values (〇5 to 〇7) (data not shown). One of the results of the screening test results can be seen in the tenth figure (96 cells). Compounds with a reaction amount greater than 130 Å/〇 were selected for re-confirmation and repeated three times. The ratio of re-confirmation was generally greater than 75%. The known Rm3 ligands were recognized very accurately (Tenth Figure. A lower affinity ligand (Ki > 2 μΜ) (like carbachol) and/or unstable (like acetylcholine) cannot be identified using a 13% screening standard. Many compounds show high activity (greater than 150%) but are not ligands for Rm3. These compounds include octocl〇thepin maleate (D2 dopamine receptor antagonist), thi〇peramide ( H3 histamine receptor antagonist), As_ 1397 (selective inhibitor of cholinesterase), thioridazine (dopamine receptor antagonist), quinidine sulfate (sodium ion channel blocker), and Nika Diping 156362.doc •69- 201131168 (nano-ion channel antagonists)^ these Compounds are then tested for ligand-coupled competition and functional nfat reporters for wild-type Rm3 to directly test their interaction with receptors. Indeed, these compounds inhibit [H]NMS coupling and carbon The gene expression of choline-activated NFAT (Fig. 11) 'The effect depends on the dose. The EC value of these compounds in the up-regulation test is usually the result of the coupled competitive test or the NFAT-reported gene test. Consistently. We also screened 4〇, other species, and found several targets, and confirmed by [3H]NMS coupled competitive detection method and hydrolysis detection method (data not shown)^ These results show This test can accurately identify the target and can be repeated in a large number of formats of 96 and 3 84. Example 7 Other ligands of the invention upregulate G-protein coupled receptors in order to examine the proposed construction of the present invention The method of ligand-upregulation of GPCRs is used to develop the general versatility of the assay, and the design of Rni3(GURC)-Rlu is also extended to other gpcRs in Figure 2. All of these substituted GPCRs are The detection also showed a ligand-dependent upregulation. The data of eight substituted GpCs can be seen in Figures 12 and 13, showing that the substituted GPCRs can be up-regulated by ligands, agonists or There are antagonists. The histamine H3 receptor ligand is specifically regulated. The method of recognizing 殓甩, the amibamine receptor Η3 can be changed to ligand upregulation and with different agonists and antagonists. And non-ligands are tested. Figure 14 shows this result. Imitet and methyl-histamine are agonists. Clobenpropit, thioperamide and 156362.doc • 70· 201131168 iodophenopropin is an antagonist. Other compounds are not ligands for the histamine receptor H3. This result indicates that the histamine receptor H3 exhibits a ligand-specific upregulation. The methyl histamine used in these experiments is quite potent. The specific upregulation of the ligand of the alkali acid receptor. Similar to the method of sputum, both nicotinic acid receptors and β2 adrenergic receptors are constructed to be upregulated by ligands and tested by a series of agonists, antagonists and non-ligands. Figure 15 shows the results of these tests. Methyl nicotine, niacin, and NAADP are agonists of this nicotinic acid receptor. Atropine and acesulfonide are not ligands for this nicotinic acid receptor. None of the compounds presented herein are ligands for the β2-adrenergic receptor. This result indicates that the nicotinic acid receptor is upregulated by the ligand, and the ligand of the modified nicotinic acid receptor cannot up-regulate the β2-adrenergic receptor which is modified as such. The ligand upregulation of the terminal alkali acid receptor relies on the substituted GURC. Dispersion The method proposed by the invention can produce two improved species of nicotinic acid receptors: one of which has GURC and the other does not. As shown in Figure 16, only the nicotinic acid receptor containing GURC can be up-regulated by the ligand, indicating that this GURC allows the GPCR to be up-regulated by the ligand. These results demonstrate a general strategy for identifying a GPCR ligand, and the GPCR regulator is also included in the identified ligand, eliminating the need for traditional secondary subtests. EXAMPLES Chemical Synthesis of Ligand-Chip-Posted Nucleic Acids The ligand-enhanced GPCR-encoding nucleic acid produced by the method of the present invention can be chemically synthesized. The so-called chemical synthesis method is familiar to those skilled in the art 156362.doc -71 - 201131168. An example method can be found in the paper by Cello et al. (Science (2002) 297:1016-8; referenced herein is based on its entirety). A ligand-upregulated GPCR-encoding nucleic acid can be used as a design reference based on its parental GPCR and its GPCR upregulation (GURC)-based gene code and amino acid sequence. The above nucleic acids can be synthesized chemically by recombining a large number of repetitive nucleic acid fragments, which are obtained from 400 to 600 basic paired interlaced fragments. These fragments are synthesized from a pure oligopeptide group. In some embodiments the parental GPCR is a native GPCR or an altered native GPCR. In certain embodiments, the parental GPCR is a known GPCR or a modified known GPCR. In certain embodiments, the parental GPCR is an orphan ligand GPCR or a modified orphan ligand GPCR. In certain embodiments, the parental GPCR is an orphan ligand GPCR to which a ligand has been found or a modified orphan ligand GPCR to which a ligand has been found. In certain embodiments, a ligand-upregulated GPCR comprises a reporter multi-peptide that is fused to a parental GPCR substituted with GURC in the region of action. In certain embodiments, the reporter protein is a luciferase, such as a wild or modified species of jellyfish luciferase. Example 9 GPCR Activation Assay 4 Induction: Mammalian cells or melanocytes are used to express GPCRs. The GPCR of this combined expression can be a native GPCR or a native GPCR mutant that is activated by 156362.doc-72-201131168. In certain embodiments, the mammalian cell is a HEK-293 cell'-HEK-293T, a CHO cell, or a C O S-7 cell. f Ting #attack: On the first day, take the HEK293 cells of the 6<106/10 cm culture book. The next day, two reaction tubes were prepared (the ratios below are in units of each dish): tube A was placed with 4 pg of DNA (eg, pCMV vector; pCMV vector carrying the acceptor DNA, etc.) 0.5 ml of serum-free DMEM (Gibco BRL) was mixed; in vitro, 24 μL of liposomes (Gibco BRL) were mixed in 0.5 ml of serum-free DMEM (Gibco BRL). Tubes A and B were each turned upside down and mixed thoroughly, and cultured at room temperature for 30 to 45 minutes. The mixture is called a "transfection mixture". The dialed HEK293 cells were washed with 1X PBS, and 5 ml of serum-free DMEM was added. 1 ml of the transfection mixture was added to the cells, and cultured at 37 ° C / 5% C 2 for 4 hours. The transfection mixture was aspirated and an additional 10 ml of DMEM and 10% fetal calf serum were added. The cells were cultured at 37 ° C / 5% CO 2 . After 48 hours of incubation, cells were harvested for analysis. Approximately 12χ106 of HEK293 cells were placed in a 15 cm tissue culture dish. It was grown in DME high sugar medium containing 10% fetal calf serum, 1% sodium pyruvate, levoglutamine and antibiotic. HEK293 cells were transfected in dishes for 24 hours (or approximately 80% pooling) and transfected with 12 pg of DNA. This 12 pg of DNA was mixed with 60 μL of liposome and 20 mL of serum-free DME high glucose medium. The medium is aspirated from the dish and rinsed once again with serum-free medium. A mixture of DNA, liposome and medium was added to the culture dish along with 10 mL of serum-free medium. After incubation at 37 °C for 24 156362.doc -73-201131168 hours, the medium was aspirated and then added to the serum-containing medium 25 ml ^ for 48 hours. 'The medium was aspirated and added to the final concentration of 5 〇 0 pg / mL. Geneticin (G418 Pharmacy). The transfected cells are selected to obtain cells that have been resistant to G418 and have been transfected. The medium is replaced every 4 to 5 days during this selection. During the selection process, the cells will grow into a stable colony or may be precipitated for stable pure selection. Should be rotted into a combined test. 57 G T" Py iS Weng "• test: If the G protein-contracting receptor is in the activated state, whether due to ligand coupling or from the essential activation, coupled with a G protein The experience stimulates the release of GDP and thus leads to the binding of GTP to the G protein. The alpha subunit of the G protein receptor is a gtp hydrolase that slowly hydrolyzes GTP to GDP, at which point the receptor is usually in a deactivated state. Activated receptors continue to convert GDP to GTP. The non-hydrolyzable GTP analog [S]GTPyS can be used to exhibit an increase in the degree of surface area, and the coupling of [358] 〇τργ8 to the membrane represents the activated receptor. The use of [35s; jGTPyS for the degree of activation has several advantages: (a) it can generally be used for all G-protein coupled receptors; (b) it is closer to the surface of the cell membrane. Some molecules that affect the intracellular reaction process will be hooked up. This assay utilizes a G-protein coupled receptor to stimulate the ability of a receptor to be expressed on a membrane. Therefore, this assay can be used in direct identification to screen for candidate compounds for native or non-native GPCRs. This assay is widely available and has been applied to drug development for all G-protein coupled receptors. [35S] GTPyS test is in 1〇]^8 of 2〇„1河 and 丨(9)mM MgCh (this amount can be adjusted for best effect, but usually 2〇Claw is suitable) Ph 7.4 [35s]GTPyS containing 〇3 to 1.2 nM is the coupling solution (this 156362.doc •74· 201131168 can be adjusted for best results 'but usually 1.2) and 12.5 to 75 pg Membrane proteins (such as hEK293 cells with & fusion protein; this amount can be adjusted for optimal results) and 10 μΜ of GDP (this amount can be adjusted for best results) for a total of i hours. A few drops of malt lectin (25 μl; Amersham) were incubated for 3 min at room temperature. The tubes were then centrifuged at 150 〇 xg for 5 minutes at room temperature and measured by a scintillation counter. A Flash PlateTM adenylate cyclase kit (New England Nuclear; Cat N〇SMp〇〇4A) was designed for cell-based assays that can be modified for use in unprocessed plastid membranes. Plates can include a flash coating and a cAMP recognized by an antibody. The cAMp produced in the cell can be straight. The following briefly describes how to measure changes in the amount of cAMp in whole cells that exhibit receptors. Transfected cells are harvested after approximately 24 hours of transient transfection. Carefully pipette the medium to discard it. The cells of the dish are then gently added to the PBS of 1 〇 and carefully pipetted. Add the ml of cell lysate and 3 w of pBs to each dish. Pipette the cells out of the dish and place the cells Place the suspension in a 50 ml conical centrifuge tube. Centrifuge the cells at room temperature for five minutes at rpm, then carefully disperse the cell pellet in a suitable volume of PBS (approximately each dish). 3 mi) Use a hemocytometer to estimate the number of cells and add additional PBS to get the appropriate number of cells (finally about 50 μM per cell). CAMP Standard and Measurei〇n Buffer [1251] "Elbow 1" containing i ^ 1 &; Take 50 μl of the measurement reference liquid added to U as follows. Prepared according to the instructions of the instrument supplier 156362.doc • 75· 201131168. The Assay Buffer is immediately dispensed with '50 μΐ of the stimulation buffer' 3 μΐ of the test compound (12 μΜ final concentration) and 50 μΐ cells. The liquid is stored on ice before use. The test is to first add 5 〇 μ1 of the cAMP standard to the appropriate compartment and then add 50 μl of PBSA to the cells numbered Η-11 and Η-12. Add 50 μΐ of stimulation buffer to all cells. DMSO (or other selected compound) is added to the appropriate compartment using a pointed tool that dispenses 3 μl of liquid. The final concentration of the test compound is 12 μΜ and the total test volume is 1 μ μ〇〇. Inject the cells into appropriate cells and incubate for 60 minutes at room temperature. The 100 μΐ measurement mixture (Detection Mix) contains c AMP and is added to the cell. The plates were incubated for an additional 2 hours and counted in a Wallace MicroBeta scintillation counter. Next, the cAMP data curve of each plate was used to infer the number of cAMp in each cell. & for the combined GPChe tour 4: TSHR is a Gs-coupled GPCR, which can lead to the accumulation of cAMP in its activated state. "We can change the amino acid of position 623 of TSHR to make it essentially activated. State (that is, to change mono-alanine to iso-leucine). One (1) coupled receptor is sufficiency to inhibit adenylate cyclase, thus reducing the production of CAMp, which also makes the detection of cAMP content even more challenging. To measure the decrease in the amount of cAMP produced as a measure of the degree of activation of a Gi-coupled receptor, the TSHR can be co-transfected as a "signal amplifier" with a GPCR linked to Gi, this TSHr Preferably, it is non-endogenous, essentially activated (TSHR-A6231) (or an endogenous, essentially activated Gs-coupled receptor), thus establishing the underlying background value of cAMP. To make a non-native G-coupled receptor, the non-native target GPCR is co-transfected with the signal amplifier, so that it is the material used in the screening test. If we perform the cAMP test, we can use this procedure to generate signals very efficiently; this procedure is especially suitable for directly identifying the test compound on the Gi-coupled receptor pair. Note that in the case of Gi-coupled GPCRs, the reverse agonist of the target GPCR will increase the cAMP signal and the agonist will reduce the cAMP signal if the method proposed here is adopted. On the first day, 6x104/cell HEK293 was placed in the plate. The next day, prepare two reaction tubes (the ratio is the unit for each dish): Tube A is 2 pg of DNA for each receptor to be transfected into mammalian cells, up to 4 gg of DNA. (eg, pCMV vector; pCMV vector carrying mutant THSR (TSJHR-A6231), etc.) in 1.2 ml serum-free DMEM (Irvine Scientific, Irvine, CA); tube B is 120 μΐ Liposomes (Gibco BRL) were mixed in 1.2 ml serum-free DMEM. The test tubes A and B were each inverted upside down (several times) and cultured at room temperature for 30 to 45 minutes. The mixture is referred to as the "transfection mixture". The fractionated HEK293 cells were washed with 1X PBS, and 10 ml of serum-free DMEM was added. 2.4 ml of the transfection mixture was added to the cell culture dish, and cultured at 37 ° C / 5% CO 2 for 4 hours. The transfection mixture was aspirated and an additional 25 ml of DMEM and 10% fetal calf serum were added. The cells were cultured at 37 ° C / 5% CO 2 . After 24 hours of incubation, cells can be collected for analysis. A Flash PlateTM adenosine cyclase test kit (New England Nuclear; Cat. No. SMP004A) is designed to be cell-based 156362.doc -77- 201131168's test' but we can also improve it to It can be applied to unprocessed plastid membranes, depending on the actual needs of those skilled in the art. This Flash Plate has a scintillator coating in a small compartment that contains a special antibody that recognizes cAMP. The c amp produced in the culture disc sub-segment can be directly competed with the radioactive cAMP tracer antibody against CAMP to obtain quantitative analysis data. The simple procedure presented below can be used to measure changes in all intracellular cAMP levels that exhibit this receptor. Transfected cells were harvested approximately 24 hours after transient transfection. Carefully remove the medium and discard it. The cells of each dish were gently added to 丨〇 ml of PBS and carefully pipetted. Add 1 ml of Sigma cell lysate and 3 ml of PBS to each culture JDL. The cells were removed from the Petri dish by pipette and the cell suspension was placed in a 50 ml conical centrifuge tube. The cells were centrifuged at 1,100 rpm for 5 minutes at room temperature. The cells were then carefully dispersed in a suitable volume of PBS (approximately 3 ml per dish). The number of cells was estimated using a hemocytometer and additional PBS was added to obtain the appropriate number of cells (final approximately 50 μM per cell). cAMP standard solution and measurement buffer (detection buffer) (containing 1# (^ tracer [1251] into ^4? 5〇41 added to 111111 measurement reference solution) are prepared according to the instrument supplier's instructions The Assay Buffer was prepared immediately prior to the screening test, using 50 μΐ of Stimulation Buffer, 3 μΐ of the test compound (12 μΜ final concentration) and 50 μΐ cells. The solution is stored on ice before use. The test is to add 50 μΐ of cAMP standard solution to the appropriate compartment, then add 156362.doc •78· 201131168 50 μΐ of PBSA to the numbers H-ll and H. In a small cell of -12, 50 μΐ of the stimulation buffer is added to all the cells. The selected compound (for example, TSH) is added to the appropriate cell using a pointed tool that can dispense 3 μΐ of liquid, and the final test is performed. The compound concentration was 12 μΜ and the total test volume was 100 μΐ. The cells were injected into a small compartment and incubated at room temperature for 60 minutes. Then 100 μΐ of the detection mixture containing the tracer cAMP was added to the cell. Medium. The plate is cultured for 2 hours and then used as Wall. The ace MicroBeta scintillation counter is calculated. Next, the cAMP data curve of each culture plate is used to infer the number of cAMP in each cell. From the interception is the test method of the ritual: Gre-Luc report test (Gs related HEK293 and HEK293T cells were placed in 96-well plates at a concentration of 2 x 104 cells per cell and transfected with liposome Reagent (BRL) according to the manufacturer's instructions. Transfection per six-cell unit The desired DNA/lipid mixture was prepared as follows: 100 μL of DMEM containing 260 ng of plastid DNA was slowly mixed with 100 μM of DMEM containing 2 μL of lipid (260 ng of plastid DNA containing 200 ng of 8xCRE) -Luc reported plastids, 50 ng of pCMV containing native or non-native receptors or only pCMV, 10 ng of GPRS-expressing plastids (GPRS placed in pcDNA3 (Invitrogen)). 8xCRE-Luc reported plastids were prepared as follows The vector SRIF-β-gal was obtained by culturing the rat system promoter (-71/+51) at the BglV-Hindlll site with ppgal-Basic Vector (Clontech). The PCR method can be obtained from the adenovirus template AdpCFl26CCRE8. Eight (8) cAMP response elements (see 7 Human Gene Therapy 1883) (1996))' and selected the Kpn-BglV site of the SRIF-p-gal vector to become the 8xCRE-P-gal reporter 156362.doc-79-201131168. The 8xCRE-Luc reporter plastid was produced by replacing the galactosidase gene on the SRIF-p-gal reporter vector with the luciferase gene obtained from the pGL3-basic vector (Promega) at the Hindlll-BamHI site. After 30 minutes of incubation, the DNA/lipid mixture was diluted with 400 μM DMEM and 100 μM of the diluted mixture was added to each cell. After 4 hours of incubation in a cell culture incubator, DMEM spiked with 10% FCS was added to each cell for 100 μM. The transfected cells were then supplemented with 200 μM per cell in DMEM containing 10% FCS. Eight (8) hours later, the cells were washed with PBS and replaced with 100 μL of phenol red-free DMEM. The LuciferTMTM Reported Gene Assay Kit (Packard) was measured every other day for its fluorescence activity using a 1450 MicroBetaTM scintillation and fluorescence counter (Wallace). API Reporting Test (Recipients Associated with Gq)' The stimulation amount of Gq, which is based on the known Gq-associated phosphatase C, allows the gene with the API element in the promoter to be activated. The -PathdetectTMAP-lcis-Reporting System (Stratagene, Catalogue #219073) can be used in the CREB reporter test procedure presented above, but the elements used in the calcium phosphate coprecipitation technique are: 410 ng pAPl-Luc, 80 ng pCMV Reported plastids and 20 ng of CMV-SEAP. One of the methods of SRF-UJC reporter assay (receptor associated with Gq), using Gq-associated phosphatase C, allows the gene with a serum response factor in the promoter to be activated. A PathdetectTM AP-1 cis-Reporting System (Stratagene) can be used to measure Gq coupling reactions on cells such as COS7. Cells are transfected with the plastid elements of this system, as well as those expressing an endogenous or non-endogenous GPCR, to Mammalian 156362.doc -80· 201131168

The TransfectionTM Kit (Stratagene, Catalogue #200285) is used according to the manufacturer's instructions. Simply put, '410 ng of pAP-Luc, 80 ng of pCMV reported plastids, and 20 ng of CMV-SEAP (secreted alkaline phosphatase-expressing plastids; measuring alkaline phosphatase activity of transfected cells in culture medium is To control the transfection effect between different samples), the calcium phosphate coprecipitation test was completed by mixing according to the manufacturer's instructions. Half of the precipitate was evenly distributed in 3 cells in a 96-well plate, and the cells were kept in a serum-free medium for 24 hours. The cells were then incubated with the selected compounds for the last 5 hours. The cells were then lysed and their luciferase activity was measured using a LuciteTM Kit (Packard, Cat. #6016911) and a "Trilux 1450 Microbeta" liquid scintillation and fluorescence counter (Wallace) according to the manufacturer's instructions. The resulting data can be analyzed using the GraphPad PrismTM 2.0a (GraphPad Software Inc.) suite of programs. Intracellular 1P3 accumulation assay (receptors associated with Gq) %—large, silent-receptor (native and/or non-native) cells are dispensed into 24 culture dishes, usually 1 X 1 per cell 5 cells (although this number can be adjusted according to the situation). On the next day, these cells were first transfected with 50 μL per serum in serum-free DMEM mixed with 〇25pg DNA, and 50 μΐ per serum in serum-free DMEM containing 2pg of liposomes. This solution was slowly mixed and incubated at room temperature for 15 to 30 minutes. Rinse with 0.5 ml of PBS and mix 400 μl of serum-free medium with the transfected medium and add to the cells. The cells were further cultured for 3 to 4 hours at 37 ° C / 5% CO 2 and then the transfection medium was taken out and replaced with 1 ml of a general growth medium per cell. On the third day, cells were labeled with 3H-myo-inositol. Briefly, the medium was removed and rinsed with 0.5 ml of PBS. Then take no inositol/blood 156362.doc -81· 201131168 Clear medium (GIBCO BRL), add 5 ml per cell, and also 3 p-myo-inositol per cell, 25 pCi, and then 16 to 18 hours. Culture of °C/5% C02. On the fourth day, the cells were washed with 0.5 ml of PBS and then added to 0 45 ml. The test medium contained inositol but no serum containing 1 μM of sputum and 1 mM of vaporized lithium, or 〇.4 ml test. The medium was adjusted to a final concentration of 1 〇 ml of 50 μL of ketone ketone (ket). The cells are again at 37. (: Incubate for 30 minutes. The cells were then added with 0.5 ml of PBS and 200 μM of freshly prepared ice-cold stop solution (1 Μ potassium hydroxide; i8 mM sodium borate; 3.8 mM EDTA) per square. This solution was placed on ice. After 5 to 1 minute, the cells were dissolved and then neutralized with 2 μm of freshly prepared ice-cold neutralized solution (7.5% hydrochloric acid). The lysate was transferred to a 5 ml microcentrifuge tube. Ml chloroform / sterol mixture (1:2) This solution was spun for 15 seconds and the supernatant was treated with Biorad AGI-X8TM anion exchange resin (100_2〇0 mesh). First, the resin was j:! 25 W/ v Water wash and then add 0.9 ml of the supernatant to the tube. The tube is then rinsed with 丨〇mi 5 mM inositol and 10 ml of 5 mM sodium borate/60 mM sodium formate. Into the scintillation counter, the scintillator containing 丨〇m丨 and 0.1 ml of 0_1 citric acid/1 Μ曱 acid. The anion exchange tube was then rinsed with 10 ml of 0.1 Μ formic acid/1 Μ ammonium formate'. Rinse twice with dd Η20 and store in 4°C water. Use fluorometric imaging piate reader (FL1PR) as the fluorometric imaging piate reader (FL1PR) The intracellular concentration measurement method '. 芑 免 散 散 windings' and pCMV (reverse control group) stably transfected cells were taken from their cell lines and firstly treated with poly-D-lysine treated 96-cell culture. In the plate (7) "(7)!!-

Dickinson, #356640), 5.5x104 cells per cell and complete culture 156362.doc •82· 201131168 Nutrients (10% FBS in EMEM, 2 mM glutamine, 1 mM sodium pyruvate) Test every other day. Prepare a culture buffer of Fluo4-AM (Molecular Probe, #F14202), and dissolve 1 mg of Fluo4-AM in 467 μΐ of DMSO and 467 μM of Pluoronic acid (Molecular Probe, #Ρ3000) to obtain 1 mM stock solution. Store at -20 ° C for one month. Fluo4-AM is a fluorescent cesium atomic reporter. The test compound was prepared as a wash solution (1x HBSS/2.5 mM Probenicid/20 mM HEPES, pH 7.4). At the time of the assay, the medium was removed from the cells and the cells were placed in 100 μΐ of 4 μΜ Fluo4-AM/2.5 mM Probenicid (Sigma, #P8761)/20 mM HEPES/all medium pH 7.4. Place at 37 ° C / 5% C02 for 60 minutes. After 1 hour of incubation, the Fluo4-AM medium was taken out and washed twice with a 100 μM wash. Leave 100 μΐ of rinse solution in each cell. The plate was returned to 37 ° C / 5% C02 for 60 minutes. The procedure of FLIPR (Molecular Device) was to add 50 μΐ of the compound to be tested at the 30th second and then record the change in intracellular calcium concentration ([Ca2+)) caused by the analyte for 150 seconds. All fluorescence change readings used to determine agonist activity were calculated using the FLIPR program. The software program attached to the instrument will normalize the fluorescence reading to the starting value corresponding to zero. In certain embodiments, the cell containing the target receptor further comprises 15/16 of an unspecified G protein alpha or a transformed Gq/Gi alpha unit. Although FLIPR assays using stable transfected cells to measure promoter activity have been proposed above, those skilled in the art should be able to adjust this method 156362.doc -83 - 201131168 to determine the activity of the antagonist. Moreover, those who claim to have basic skills in the industry should be able to think of the possibility of using transiently transfected cells. From the results and discussion above, it will be apparent that the present invention proposes an important new method for making a ligand-upregulated GPCR. In particular, the present invention provides a system for screening a library of compounds for finding GPCR modulators. Thus, the methods and systems of the present invention can be used in a variety of applications, such as research, medicine, therapy, or other applications. Therefore, the present invention is an important contribution in the industry. Applicants reserve the right to exclude any one or more of the native GPCRs, parental GPCRs, or ligand-upregulated GPCRs from any of the embodiments of the invention. The Applicant also reserves the right to exclude any polynucleotide or polypeptide from any of the embodiments of the present invention. [Simplified Schematic] The first figure is a schematic diagram of the structure of G-protein coupled receptors, inter-membrane regions (TM1, TM2, TM3, TM4, TM5, TM6, and TM7) and intracellular portions (IC1, IC2, IC3). The monthly package ECp points (EC1, EC2 mouth EC3) are each numbered. The second panel is a table showing 28 exemplary G protein-coupled receptors suitable for use in the methods of the present invention. There are three columns in the table: GPCR species (N1 to N28) which is the code for this GPCR, followed by the name of the GPCR and its number registered in GenBank. These registration numbers provide access to their unique GenBank entries, including polynucleotides, peptides, and other noted features. The third figure is a 156362.doc-84·201131168 nucleotide and multi-peptide sequence sequence of the "GPCR Uplifting Block (GURC)" used in the present invention. SEQ ID NOS: 2-8 are representative GURC amino acid sequences, and SEQ ID NO: 9 is a representative GURC encoding nucleic acid sequence. The bottom lined unit indicates the relative mutation compared to the GURC1 amino acid sequence. SEQ ID NOS: 2-8 can be flanked by "GT" and "EF" as shown in the multi-peptide of Figure 4. In many embodiments, the GURC of the present invention carries the sequence "SREKKAAK (SEQ ID NChl)" at its near carbon terminus. Figures 4-1 to 4-10 show 28 multi-peptide sequences (SEQ ID NOS 10-37) that have been modified to be examples of GPCRs that can be promoted by ligands. For GPCRs N1 to N28, the parental GPCR is a native GPCR as shown in Figure 2. In each sequence, the position of the GPCR modification is indicated by the bottom line, and the amino acid remaining in the TM5 and TM6 intermembrane regions of the parental GPCR and flanked by the substituted GURC is indicated in bold. Each of the multi-peptides is a jellyfish luciferase fusion protein, and between the carbon-terminal amino acid of the GPCR modified GPCR and the nitrogen-terminal thiol acid of the luciferase, there are two amino acids. [Glu-Asn-SeΓ (ENS)] is a spacer. In each of the sequences, the GURC used in the ligand-up GPCR was clamped with "GT" and "EF" amino acids. Each modified GPCR can be up-regulated by a ligand, and representative data of many GPCRs will be presented later. The fifth panel is a graphical representation of the native species Rm3 and the modified species Rm3 receptor. The above figure shows that the carbon end of the whole Rm3 and the nitrogen end Met of the jellyfish luciferase (Rlu) are fused by a spacer composed of three amino acids Glu-Asn-Ser. The following figure shows that the 250-494 amino acid fragment of wild species Rm3 was replaced by GURC1 and was ligated via ΚρηΙ and EcoRI to form the modified GURC1 at the nitrogen end 156362.doc -85 - 201131168 (Tyr-Trp to Gly-Thr Or a mutant at the carbon end (Ser-Ala to Glu-Phe). This modified Rm3 (GURCl) is then interfaced with the nitrogen terminal Met of the jellyfish luciferase via a spacer composed of three amino acids Glu-Asn-Ser. The sixth panel is a bar graph showing the comparison of native species Rm3, Rm3-Rlu, and Rm3(GURC)-Rlu in COS-7 cells. COS-7 cells were transiently transfected with plastids of native species Rm3, Rm3-Rlu, Rm.3 (GURC)-Rlu, etc., to be harvested in 48 hours. The homogenate of the cells (20 pg/well) was first incubated at 2 nM [3H]NMS for 2 hours at room temperature, without the addition of atropine (fully coupled) or the addition of 10 μM of atropine (non-specific coupling). The data in the table is represented by three standard procedures, three times each time, and the unit is CPM per well. Figures 7 through 7D show the binding of [3H]NMS to the native species Rm3, Rm3 (GURC)-Rlu under the action of agonists and antagonists. Temporally expressed native species Rm3 (solid square) and Rm3 (GURC)-Rlu (open triangle) COS-7 cell homogenate (20 pg/well) first at room temperature with buffer plus different concentrations of the above Body, culture for 30 minutes. [3H] NMS was added to the mixture at room temperature for 1 hour at a concentration of 500 pM. The data in the table is represented by the standard experimental procedure, which is repeated three times each time. The unit is the percentage of the control group (100% with no ligand), and the bar graphs of the eighth and eighth B are shown in The ligand upregulation of Rm3(GURC)-Rlu was transiently expressed in HEK293 cells. (A) HEK293 cells were first transfected with Rm3 (GURC)-Rlu. After 24 hours, the cells (10 cm dish) were subdivided into 96 cell culture dishes (50,000 cells per cell) and 1 〇 μΜ of each compound was added to the culture solution. The fluorescence activity test is carried out after 20 to 24 hours of 156362.doc • 86 - 201131168, and the method is described in detail later. The data in the table is represented by six independent procedures, each of which is repeated twice, and the unit is the average of the fluorescent activity (RLU). (B) The [3H] NMS coupling test was used to verify the upregulation of the receptor due to the ligand. HEK293 cells stably expressing Rm3(GURC)-Rlu were first placed in the culture medium for 20 hours, respectively, without atropine (open) or with ΙμΜ atropine (solid). The cells were harvested and rinsed thoroughly with cold PBS to remove the coupled ligand. The coupling test is to effect the homogenate of the cells with [3H]NMS (2 ηΜ) as described later. The data in the table are based on untreated cells (100%) and repeated twice in each of three independent experiments to obtain the mean ± standard deviation. The ninth figure shows that the phenomenon that Rm3(GURC)-Rlu is up-regulated by the ligand is related to the dose. HEK293 cells stably expressing Rm3(GURC)-Rlu were divided into 96-cell cell culture plates, and different concentrations of atropine (closed squares), 4-DAMP (solid triangles), carbachol (open squares), and oxotremorine were added. Μ (open triangle), after 20 to 24 hours of incubation, fluorescence activity test, the method is detailed later. The data in the table is based on the fluorescence activity of the un-ligand control group (100%), and is represented by three independent experiments, three times each time. The tenth figure is a bar graph showing an example of a LOPAC compound in a 96-well culture dish. A total of 9 dishes of LOPAC were screened at 2 μΜ. This picture is an example of this. The positive control group (2 μΜ atropine) was 200% and the negative control group (0.5% DMSO) was 100%. Compounds which can cause more than 140% of the effects are as follows: 1, atopine sulfate; 2, aminobenzyltropine; 3, benzyltropine; 4, As-1397; 5,4-DAMP; 6, hexahydro-sila-difenidol; 7, 156362.doc -87- 201131168 hexahydro-sila-difenidol, p-fluoro; 8, himbacine; 9, desertified ipratropium; 10, pilocarpine; ll, pirenzepine; 12, quinidine sulfate; -) East K-base; 14, (-) n-butyl-E-Kinine. Figure 11 is a table showing the repeated manipulation of the NFAT-reporter gene assay. Receptor up-regulation is performed using the aforementioned HEK293 cells which stably express Rm3(GURC)-Rlu. Ligand-coupled competition assays were performed on the membrane of C〇S-7 cells transfected as native species as shown in the third panel. The NFAT-reporter gene assay is a HEK293 cell transfected with the native species Rm3 as described previously. The data in the table is representative of the repeated three-time experiment. Figures 12 through 12D show the effect of the GPCR modified by the proposed method in the presence of the desired ligand. The number of 'GPCRs in each figure is measured by luciferase assay and plotted in rLU (relative brightness). The ligand and the non-ligand are each applied at different molar concentrations. In the figure, one axis is in "concentration (l 〇 gM)". Figure a is the upregulation of serotonin (serotonin) receptor 2B. Serotonin is an agonist, while Cl〇Zapine and ergobenzyl ester are antagonists. Panel B is the upregulation of serotonin (serotonin) stylistic 2C. Serotonin and quipazine maleate are agonists, while SB-206553, LY-53 85 8 maleate and methalin are antagonists; atropine is not a ligand. Figure c is the upregulation of neurotensin receptor 1. Neurotensin is an agonist, while 272349 is not a ligand with haishalin. The map is the upregulation of hormone receptors in the stomach. The compounds used in Figure A are all agonists. The 12th to thirteenth D-D diagrams show that the GPCRs according to the method of the present invention are modified by 156362.doc •88·201131168. In each of the figures, the number of GPCRs was measured using the luciferase assay and plotted in terms of RLU (relative brightness). The ligand and the non-ligand are each applied at different molar concentrations, and one axis in the figure is in "concentration (logM)". Panel A is the upregulation of dopamine receptor D1. Dopamine is an agonist and the rest of the compounds are antagonists. Panel B is the upregulation of dopamine receptor D4. All compounds in the figure are antagonists. Figure C shows the upregulation of murine acetylcholine and muscarinic 3 receptors. Carbon guanidine and oxotremoriline are agonists; 4-DAMP and atropine are antagonists. The D picture is the ascending hormone receptor and the upregulation of alpha-2A. UK14304 and clopidogrel are agonists; yohimbine and resveratrol are antagonists. Figures 14A and 14B show different effects of anti-histamine H3 receptor-dependent ligands. A is a bar graph showing the effect of modifying the antihistamine H3 receptor in accordance with the method of the present invention. Imetit, methyl anti-histamine is an agonist; clobenpropit, thioperamide and iodophenopropit are antagonists; the rest of the compounds are ligands. All compounds were applied at a concentration of 2 μΜ. The asterisk (*) indicates the ligand. Panel B shows the reaction of the antihistamine receptor Η3 with the concentrations of the four compounds. The fifteenth and fifteenth panels show the different effects of niacin receptors on ligands. Panel A shows the dose and response of nicotinic acid to chemicals such as guanidine nicotine, nicotinic acid, NAADP-β, atropine, and ATII peptide. The first three are agonists of nicotinic acid receptors; or the two are not ligands for nicotinic acid receptors. Panel B shows the response of β2 adrenergic receptors, which are not dose-dependent, to sputum nicotine, DAD, and NAADP-β. None of these compounds are ligands for the β2 adrenergic receptor. 156362.doc -89- 201131168 Figures 16A and 16B show the correlation between the upregulated condition of the modified nicotinic acid receptor and the GPCR uplift. The compounds are directed against RLU-modified nicotinic acid receptors: one with GURC (Figure 16A) and the other without GURC (Figure 16B). This nicotinic acid receptor containing GURC can be raised only in the presence of a ligand. [Key element symbol description] C TERM carbon end EC1 extracellular part 1 EC2 extracellular part 2 EC3 extracellular part 3 EXTRACELLULAR extracellular IC1 intracellular part 1 IC2 intracellular part 2 IC3 intracellular part 3 INTRACELLULAR intracellular N TERM nitrogen end TM1 Intermembrane region 1 TM2 Intermembrane region 2 TM3 Intermembrane region 3 TM4 Intermembrane region 4 TM5 Intermembrane region 5 TM6 Intermembrane region 6 TM7 Intermembrane region 7 156362.doc -90- 201131168 Sequence Listing <110> Ainina Pharmaceuticals <120> Method and synthesis for distinguishing G protein-coupled receptor regulators

<130> 61.TW1.REG <140> 093112289 <141> 2004-04-30 <150> 60/467,489 <151> 2003-04-30 <150> 60/516,168 <m> 2003-10-31 <160> 65 <170> Patentln version 3.2 <210> 1 <211> 8 <212> PRT <213>manual<220><223> GURC 匣<400&gt ; 1

Ser Arg Glu Lys Lys Ala Ala Lys 1 5 <210〉 2 <211> 55 <212> PRT <213>manual <220><223>GURC 匣 <400〉 2

Arg Val Phe Gin Glu Ala Lys Arg Gin Leu Gin Lys lie Asp Lys Ser 15 10 15

Glu Gly Arg Phe His Val Gin Asn Leu Ser Gin Val Glu Gin Asp Gly 20 25 30

Arg Thr Gly His Gly Leu Arg Arg Ser Ser Lys Phe Cys Ser Arg Glu 35 40 45

Lys Lys Ala Ala Lys Thr Leu 50 55 156362. Sequence Listing.doc 201131168 <210> 3 <211> 55 <212> PRT <213>Manual<220><223> GURC 匣<400> 3

Arg Val Phe Gin Val Ala Gin Arg Gin Leu Gin Lys lie Asp Lys Ser 15 10 15

Glu Gly Arg Phe His Ala Gin Asn Leu Ser Gin Val GIu Gin Asp Gly 20 25 30

Arg Ser Gly His Gly His Arg Arg Ala Ser Lys Phe Cys Ser Arg Glu 35 40 45

Lys Lys Ala Ala Lys Thr Leu 50 55 <210> 4 <211> 55 <212> PRT <213>manual <220><223> GURC 匣 <400> 4

Arg Val Phe Gin Val Ala Gin Arg Gin Leu Gin Lys lie Asp Arg Ser 15 10 15

Glu Gly Arg Phe His Ala Gin Asn Leu Ser Gin Val Glu Gin Asp Gly 20 25 30

Arg Ser Gly His Gly His Arg Arg Ser Ser Lys Phe Cys Ser Arg Glu 35 40 45

Lys Lys Ala Ala Lys Thr Leu 50 55 <210> 5 <211> 55 <212> PRT < 213 > Labor <220><223> GURC 匣156362 - Sequence Listing.doc 201131168 <400> 5

Arg Val Phe Gin Val Ala Lys Lys Gin Leu Gin Lys lie Asp Arg Ser 15 10 15

Glu Gly Arg Phe His Thr Gin Asn Leu Ser Gin Val Glu Gin Asp Gly 20 25 30

Arg Ser Gly His Gly Leu Arg Arg Ser Ser Lys Phe Tyr Ser Arg Glu 35 40 45

Lys Lys Ala Ala Lys Thr Leu 50 55 <210> 6 <211> 55 <212> PRT <213>manual <220><223>GURC 匣 <400> 6

Arg Val Phe Gin Val Ala Lys Arg Gin Leu Gin Lys lie Asp Lys Ser 1 5 10 15

Glu Gly Arg Phe His Ala Gin Asn Leu Ser Gin Val Glu Gin Asp Gly 20 25 30

Arg Thr Gly His Gly Leu Arg Arg Ser Ser Lys Phe Cys Ser Arg Glu 35 40 45

Lys Lys Ala Ala Lys Thr Leu 50 55 <210> 7 <211> 55 <212> PRT <213>manual <220><223> GURC 匣 <400>

Arg Val Phe Gin Val Ala Lys Arg Gin Leu Gin Lys lie Asp Lys Ser 15 10 15

Glu Gly Arg Phe His Ala Gin Asn Leu Ser Gin Val Glu Gin Asp Gly 20 25 30 156362 - Sequence Listing.doc 201131168

Arg Ser Gly His Gly Leu Arg Ser Ser Ser Lys Phe Cys Ser Arg Glu 35 40 45

Lys Lys Ala Ala Lys Thr Leu 50 55 <210> 8 <211> 55 <212> PRT <213>manual <220><223> GURC 匣 <400〉 8

Arg Val Phe Gin Glu Ala Lys Arg Gin Leu Gin Lys lie Asp Lys Ser 15 10 15

Glu Gly Arg Phe His Ala Gin Asn Leu Ser Gin Val Glu Gin Asp Gly 20 25 30

Arg Thr Gly His Gly Leu Arg Arg Ser Ser Lys Phe Cys Ser Arg Glu 35 40 45

Lys Lys Ala Ala Lys Thr Leu 50 55 <210> 9 <211> 177 <212> DNA <213>Manual<220><223> GURC 匣<400> 9 ggtaccaggg tctttcagga ggccaaaagg cagctccaga agattgacaa atctgagggc Cgcttccaig tccagaacci tagccaggtg gagcaggatg ggcggacggg gcatggactc cgcagatctt ccaagttctg ctccagggaa aagaaagcag ccaaaacctt ggaattc <210> 10 <211> 717 <212> PRT < 213 > Artificial <220><223> Derived from rat cholinergic receptor, Physostine 3 156362 - Sequence Listing.doc 201131168 <400> 10

Met Thr Leu His Ser Asn Ser Thr Thr Ser Pro Leu Phe Pro Asn He 15 10 15

Ser Ser Ser Trp Val His Ser Pro Ser Glu Ala Gly Leu Pro Leu Gly 20 25 30

Thr Val Thr Gin Leu Gly Ser Tyr Asn lie Ser Gin Glu Thr Gly Asn 35 40 45

Phe Ser Ser Asn Asp Thr Ser Ser Asp Pro Leu Gly Gly His Thr lie 50 55 60

Trp Gin Val Val Phe lie Ala Phe Leu Thr Gly Phe Leu Ala Leu Val 65 70 75 80

Thr lie lie Gly Asn lie Leu Val I]e Val Ala Phe Lys Val Asn Lys 85 90 95

Gin Leu Lys Thr Val Asn Asn Tyr Phe Leu Leu Ser Leu Ala Cys Ala 100 105 110

Asp Leu lie lie Gly Val lie Ser Met Asn Leu Phe Thr Thr Tyr lie 115 120 125 lie Met Asn Arg Trp Ala Leu Gly Asn Leu Ala Cys Asp Leu Trp Leu 130 135 140

Ser lie Asp Tyr Val Ala Ser Asn Ala Ser Val Met Asn Leu Leu Val 145 150 155 160 lie Ser Phe Asp Arg Tyr Phe Ser lie Thr Arg Pro Leu Thr Tyr Arg 165 170 175

Aia Lys Arg Thr Thr Lys Arg Arg Gly Val Met lie Gly Leu Ala Trp 180 185 190

Val lie Ser Phe Val Leu Trp Ala Pro Ala lie Leu Phe Trp Gin Tyr 195 200 205

Phe Val Gly Lys Arg Thr Val Pro Pro Gly Glu Cys Phe He Gin Phe 210 215 220

Leu Ser Glu Pro Thr lie Thr Phe Gly Thr Ala lie Ala Ala Phe Tyr 225 230 235 240 156362 - Sequence Listing.doc 201131168

Met Pro Val Thr lie Met Thr lie Leu Gly Thr Arg Val Phe Gin Glu 245 250 255

Ala Lys Arg Gin Leu Gin Lys lie Asp Lys Ser Glu Gly Arg Phe His 260 265 270

Val Gin Asn Leu Ser Gin Val Glu Gin Asp Gly Arg Thr Gly His Gly 275 280 285

Leu Arg Arg Ser Ser Lys Phe Cys Ser Arg Glu Lys Lys Ala Ala Lys 290 295 300

Thr Leu Glu Phe lie Leu Leu Ala Phe lie lie Thr Trp Thr Pro Tyr 305 310 315 320

Asn lie Met Val Leu Val Asn Thr Phe Cys Asp Ser Cys lie Pro Lys 325 330 335

Thr Tyr Trp Asn Leu Gly Tyr Trp Leu Cys Tyr lie Asn Ser Thr Val 340 345 350

Asn Pro Val Cys Tyr Ala Leu Cys Asn Lys Thr Phe Arg Thr Thr Phe 355 360 365

Lys Thr Leu Leu Leu Cys Gin Cys Asp Lys Arg Lys Arg Arg Lys Gin 370 375 380

Gin Tyr Gin Gin Arg Gin Ser Val lie Phe His Lys Arg Val Pro Glu 385 390 395 400

Gin Ala Leu Glu Asn Ser Met Thr Ser Lys Val Tyr Asp Pro Glu Gin 405 410 415

Arg Lys Arg Met lie Thr Gly Pro Gin Trp Trp Ala Arg Cys Lys Gin 420 425 430

Met Asn Val Leu Asp Ser Phe lie Asn Tyr Tyr Asp Ser Glu Lys His 435 440 445

Ala Glu Asn Ala Val lie Phe Leu His Gly Asn Ala Ala Ser Ser Tyr 450 455 460

Leu Trp Arg His Val Val Pro His lie Glu Pro Val Ala Arg Cys lie 465 470 475 480 156362 - Sequence Listing.doc 201131168 lie Pro Asp Leu He Gly Met Gly Lys Ser Gly Lys Ser Gly Asn Gly 485 490 495

Ser Tyr Arg Leu Leu Asp His Tyr Lys Tyr Leu Thr Ala Trp Phe Glu 500 505 510

Leu Leu Asn Leu Pro Lys Lys lie Asn Phe Val Gly His Asp Trp Gly 515 520 525

Ala Cys Leu Ala Phe His Tyr Ser Tyr Glu His Gin Asp Lys He Lys 530 535 540

Ala lie Val His Ala Glu Ser Val Val Asp Val lie Glu Ser Trp Asp 545 550 555 560

Glu Trp Pro Asp lie Glu Glu Asp lie Ala Leu lie Lys Ser Glu Glu 565 570 575

Gly Glu Lys Met Val Leu Glu Asn Asn Phe Phe Val Glu Thr Met Leu 580 585 590

Pro Ser Lys lie Met Arg Lys Leu Glu Pro Glu Glu Phe Ala Ala Tyr 595 600 605

Leu Glu Pro Phe Lys Glu Lys Gly Glu Val Arg Arg Pro Thr Leu Ser 610 615 620

Trp Pro Arg Glu lie Pro Leu Val Lys Gly Gly Lys Pro Asp Val Val 625 630 635 640

Gin lie Val Arg Asn Tyr Asn Ala Tyr Leu Arg Ala Ser Asp Asp Leu 645 650 655

Pro Lys Met Phe lie Glu Ser Asp Pro Gly Phe Phe Ser Asn Ala lie 660 665 670

Val Glu Gly Ala Lys Lys Phe Pro Asn Thr Glu Phe Val Lys Val Lys 675 680 685

Gly Leu His Phe Ser Gin Glu Asp Ala Pro Asp Glu Met Gly Lys Tyr 690 695 700 lie Lys Ser Phe Val Glu Arg Val Leu Lys Asn Glu Gin 705 710 715 156362 - Sequence Listing.doc 201131168 <220> 11 <21 676 <212> PRT < 213 > Artificial <220><223> Derived from Human Melanin Agglutination Receptor 2 <400> 11

Met Asn Pro Phe His Ala Ser Cys Trp Asn Thr Ser Ala Glu Leu Leu 15 10 15

Asn Lys Ser Trp Asn Lys Glu Phe Ala Tyr Gin Thr Ala Ser Val Val 20 25 30

Asp Thr Val lie Leu Pro Ser Met lie Gly lie lie Cys Ser Thr Gly 35 40 45

Leu Val Gly Asn lie Leu lie Val Phe Thr lie lie Arg Ser Arg Lys 50 55 60

Lys Thr Val Pro Asp lie Tyr lie Cys Asn Leu Ala Val Ala Asp Leu 65 70 75 80

Val His lie Val Gly Met Pro Phe Leu lie His Gin Trp Ala Arg Gly 85 90 95

Gly Glu Trp Val Phe Gly Gly Pro Leu Cys Thr lie lie Thr Ser Leu 100 105 110

Asp Thr Cys Asn Gin Phe Ala Cys Ser Ala lie Met Thr Val Met Ser 115 120 125

Val Asp Arg Tyr Phe Ala Leu Val Gin Pro Phe Arg Leu Thr Arg Trp 130 135 140

Arg Thr Arg Tyr Lys Thr lie Arg lie Asn Leu Gly Leu Trp Ala Ala 145 150 155 160

Ser Phe lie Leu Ala Leu Pro Val Trp Val Tyr Ser Lys Val lie Lys 165 170 175

Phe Lys Asp Gly Val Glu Ser Cys Ala Phe Asp Leu Thr Ser Pro Asp 180 185 190

Asp Val Leu Trp Tyr Thr Leu Tyr Leu Thr lie Thr Thr Phe Phe Phe 195 200 205 156362 · Sequence Listing.doc 201131168

Pro Leu Pro Leu lie Leu Val Cys Tyr lie Gly Thr Arg Val Phe Gin 210 215 220

Glu Ala Lys Arg Gin Leu Gin Lys lie Asp Lys Ser Glu Gly Arg Phe 225 230 235 240

His Val Gin Asn Leu Ser Gin Val Glu Gin Asp Gly Arg Thr Gly His 245 250 255

Gly Leu Arg Arg Ser Ser Lys Phe Cys Ser Arg Glu Lys Lys Ala Ala 260 265 270

Lys Thr Leu Glu Phe Leu Val Val Val Phe lie Leu Ser Ala Ala Pro 275 280 285

Tyr His Val lie Gin Leu Val Asn Leu Gin Met Glu Gin Pro Thr Leu 290 295 300

Ala Phe Tyr Val Gly Tyr Tyr Leu Ser lie Cys Leu Ser Tyr Ala Ser 305 310 315 320

Ser Ser lie Asn Pro Phe Leu Tyr lie Leu Leu Ser Gly Asn Phe Gin 325 330 335

Lys Arg Leu Pro Gin lie Gin Arg Arg Ala Thr Glu Lys Glu lie Asn 340 345 350

Asn Met Gly Asn Thr Leu Lys Ser His Phe Glu Asn Ser Met Thr Ser 355 360 365

Lys Val Tyr Asp Pro Glu Gin Arg Lys Arg Met lie Thr Gly Pro Gin 370 375 380

Trp Trp Ala Arg Cys Lys Gin Met Asn Val Leu Asp Ser Phe lie Asn 385 390 395 400

Tyr Tyr Asp Ser Glu Lys His Ala Glu Asn Ala Val lie Phe Leu His 405 410 415

Gly Asn Ala Ala Ser Ser Tyr Leu Trp Arg His Val Val Pro His lie 420 425 430

Glu Pro Val Ala Arg Cys lie lie Pro Asp Leu lie Gly Met Gly Lys 435 440 445 9- 156362 - Sequence Listing, doc 201131168

Ser Gly Lys Ser Gly Asn Gly Ser Tyr Arg Leu Leu Asp His Tyr Lys 450 455 460

Tyr Leu Thr Ala Trp Phe Glu Leu Leu Asn Leu Pro Lys Lys lie Asn 465 470 475 480

Phe Val Gly His Asp Trp Gly Ala Cys Leu Ala Phe His Tyr Ser Tyr 485 490 495

Glu His Gin Asp Lys lie Lys Ala He Val His Ala Glu Ser Val Val 500 505 510

Asp Val lie Glu Ser Trp Asp Glu Trp Pro Asp lie Glu Glu Asp lie 515 520 525

Ala Leu lie Lys Ser Glu Glu Gly Glu Lys Met Val Leu Glu Asn Asn 530 535 540

Phe Phe Val Glu Thr Met Leu Pro Ser Lys lie Met Arg Lys Leu Glu 545 550 555 560

Pro Glu Glu Phe Ala Ala Tyr Leu Glu Pro Phe Lys Glu Lys Gly Glu 565 570 575

Val Arg Arg Pro Thr Leu Ser Trp Pro Arg Glu lie Pro Leu Val Lys 580 585 590

Gly Gly Lys Pro Asp Val Val Gin lie Val Arg Asn Tyr Asn Ala Tyr 595 600 605

Leu Arg Ala Ser Asp Asp Leu Pro Lys Met Phe lie Glu Ser Asp Pro 610 615 620

Gly Phe Phe Ser Asn Ala lie Val Glu Gly Ala Lys Lys Phe Pro Asn 625 630 635 640

Thr Glu Phe Val Lys Val Lys Gly Leu His Phe Ser Gin Glu Asp Ala 645 650 655

Pro Asp Glu Met Gly Lys Tyr lie Lys Ser Phe Val Glu Arg Val Leu 660 665 670

Lys Asn Glu Gin 675 <210> 12 -10- 156362 · Sequence Listing.doc 201131168 <211> 662 <212> PRT <213>Manual<220><223> Derived from Human Gallbladder #g Receptor, muscarinic 4 <400> 12

Met Ala Asn Phe Thr Pro Val Asn Gly Ser Ser Gly Asn Gin Ser Val 15 10 15

Arg Leu Val Thr Ser Ser Ser As As Arg Tyr Glu Thr Val Glu Met 20 25 30

Val Phe lie Ala Thr Val Thr Gly Ser Leu Ser Leu Val Thr Val Val 35 40 45

Gly Asn lie Leu Val Met Leu Ser lie Lys Val Asn Arg Gin Leu Gin 50 55 60

Thr Val Asn Asn Tyr Phe Leu Phe Ser Leu Ala Cys Ala Asp Leu lie 65 70 75 80 lie Gly Ala Phe Ser Met Asn Leu Tyr Thr Val Tyr lie lie Lys Gly 85 90 95

Tyr Trp Pro Leu Gly Ala Val Val Cys Asp Leu Trp Leu Ala Leu Asp 100 105 110

Tyr Val Val Ser Asn Ala Ser Val Met Asn Leu Leu lie lie Ser Phe 115 120 125

Asp Arg Tyr Phe Cys Val Thr Lys Pro Leu Thr Tyr Pro Ala Arg Arg 130 135 140

Thr Thr Lys Met Ala Gly Leu Met lie Ala Ala Ala Trp Val Leu Ser 145 150 155 160

Phe Val Leu Trp Ala Pro Ala lie Leu Phe Trp Gin Phe Val Val Gly 165 170 175

Lys Arg Thr Val Pro Asp Asn Gin Cys Phe lie Gin Phe Leu Ser Asn 180 185 190

Pro Ala Val Thr Phe Gly Thr Ala lie Ala Ala Phe Tyr Leu Pro Val 195 200 205 -11 - 156362 - Sequence Listing.doc 201131168

Val lie Met Thr Val Leu Gly Thr Arg Val Phe Gin Glu Ala Lys Arg 210 215 220

Gin Leu Gin Lys lie Asp Lys Ser Glu Gly Arg Phe His Val Gin Asn 225 230 235 240

Leu Ser Gin Val Glu Gin Asp Gly Arg Thr Gly His Gly Leu Arg Arg 245 250 255

Ser Ser Lys Phe Cys Ser Arg Glu Lys Lys Ala Ala Lys Thr Leu Glu 260 265 270

Phe lie Leu Leu Ala Phe lie Leu Thr Trp Thr Pro Tyr Asn Val Met 275 280 285

Val Leu Val Asn Thr Phe Cys Gin Ser Cys lie Pro Asp Thr Val Trp 290 295 300

Ser lie Gly Tyr Trp Leu Cys Tyr Val Asn Ser Thr lie Asn Pro Ala 305 310 315 320

Cys Tyr Ala Leu Cys Asn Ala Thr Phe Lys Lys Thr Phe Arg His Leu 325 330 335

Leu Leu Cys Gin Tyr Arg Asn lie Gly Thr Ala Arg Glu Asn Ser Met 340 345 350

Thr Ser Lys Val Tyr Asp Pro Glu Gin Arg Lys Arg Met lie Thr Gly 355 360 365

Pro Gin Trp Trp Ala Arg Cys Lys Gin Met Asn Val Leu Asp Ser Phe 370 375 380 lie Asn Tyr Tyr Asp Ser Glu Lys His Ala Glu Asn Ala Val lie Phe 385 390 395 400

Leu His Gly Asn Ala Ala Ser Ser Tyr Leu Trp Arg His Val Val Pro 405 410 415

His lie Glu Pro Val Ala Arg Cys lie lie Pro Asp Leu lie Gly Met 420 425 430

Gly Lys Ser Gly Lys Ser Gly Asn Gly Ser Tyr Arg Leu Leu Asp His 435 440 445

Tyr Lys Tyr Leu Thr Ala Trp Phe Glu Leu Leu Asn Leu Pro Lys Lys 450 455 460 -12- 156362 - Sequence Listing.doc 201131168 lie Asn Phe Val Gly His Asp Trp Gly Ala Cys Leu Ala Phe His Tyr 465 470 475 480

Ser Tyr Glu His Gin Asp Lys lie Lys Ala lie Val His Ala Glu Ser 485 490 495

Val Val Asp Val lie Glu Ser Trp Asp Glu Trp Pro Asp lie Glu Glu 500 505 510

Asp He Ala Leu lie Lys Ser Glu Glu Gly Glu Lys Met Val Leu Glu 515 520 525

Asn Asn Phe Phe Val Glu Thr Met Leu Pro Ser Lys lie Met Arg Lys 530 535 540

Leu Glu Pro Glu Glu Phe Ala Ala Tyr Leu Glu Pro Phe Lys Glu Lys 545 550 555 560

Gly Glu Val Arg Arg Pro Thr Leu Ser Trp Pro Arg Glu lie Pro Leu 565 570 575

Val Lys Gly Gly Lys Pro Asp Val Val Gin lie Val Arg Asn Tyr Asn 580 585 590

Ala Tyr Leu Arg Ala Ser Asp Asp Leu Pro Lys Met Phe lie Glu Ser 595 600 605

Asp Pro Gly Phe Phe Ser Asn Ala lie Val Glu Gly Ala Lys Lys Phe 610 615 620

Pro Asn Thr Glu Phe Val Lys Val Lys Gly Leu His Phe Ser Gin Glu 625 630 635 640

Asp Ala Pro Asp Glu Met Gly Lys Tyr lie Lys Ser Phe Val Glu Arg 645 650 655

Val Leu Lys Asn Glu Gin 660 <210> 13 <211> 708 <212> PRT < 213 > Labor <220><223> Derived from Human Toxic Acid Receptor-13 - 156362 - Sequence Listing .doc 201131168 <400> 13

Met Asn Arg His His Leu Gin Asp His Phe Leu Glu lie Asp Lys Lys 15 10 15

Asn Cys Cys Val Phe Arg Asp Asp Phe lie Val Lys Val Leu Pro Pro 20 25 30

Val Leu Gly Leu Glu Phe lie Phe Gly Leu Leu Gly Asn Gly Leu Ala 35 40 45

Leu Trp lie Phe Cys Phe His Leu Lys Ser Trp Lys Ser Ser Arg lie 50 55 60

Phe Leu Phe Asn Leu Ala Val Ala Asp Phe Leu Leu lie lie Cys Leu 65 70 75 80

Pro Phe Leu Met Asp Asn Tyr Val Arg Arg Trp Asp Trp Lys Phe Gly 85 90 95

Asp lie Pro Cys Arg Leu Met Leu Phe Met Leu Ala Met Asn Arg Gin 100 105 110

Gly Ser He lie Phe Leu Thr Val Val Ala Val Asp Arg Tyr Phe Arg 115 120 125

Val Val His Pro His His Ala Leu Asn Lys lie Ser Asn Arg Thr Ala 130 135 140

Ala lie lie Ser Cys Leu Leu Trp Gly lie Thr lie Gly Leu Thr Val 145 150 155 160

His Leu Leu Lys Lys Lys Met Pro lie Gin Asn Gly Gly Ala Asn Leu 165 170 175

Cys Ser Ser Phe Ser lie Cys His Thr Phe Gin Trp His Glu Ala Met 180 185 190

Phe Leu Leu Glu Phe Phe Leu Pro Leu Gly lie lie Leu Phe Cys Gly 195 200 205

Thr Arg Val Phe Gin Glu Ala Lys Arg Gin Leu Gin Lys lie Asp Lys 210 215 220

Ser Glu Gly Arg Phe His Val Gin Asn Leu Ser Gin Val Glu Gin Asp 225 230 235 240 • 14· 156362 · Sequence Listing.doc 201131168

Gly Arg Thr Gly His Gly Leu Arg Arg Ser Ser Lys Phe Cys Ser Arg 245 250 255

Glu Lys Lys Ala Ala Lys Thr Leu Glu Phe Val Ala lie Val Phe Val 260 265 270

He Cys Phe Leu Pro Ser Val Val Val Arg lie Arg lie Phe Trp Leu 275 280 285

Leu His Thr Ser Gly Thr Gin Asn Cys Glu Val Tyr Arg Ser Val Asp 290 295 300

Leu Ala Phe Phe He Thr Leu Ser Phe Thr Tyr Met Asn Ser Met Leu 305 310 315 320

Asp Pro Val Val Tyr Tyr Phe Ser Ser Pro Ser Phe Pro Asn Phe Phe 325 330 335

Ser Thr Leu lie Asn Arg Cys Leu Gin Arg Lys Met Thr Gly Glu Pro 340 345 350

Asp Asn Asn Arg Ser Thr Ser Val Glu Leu Thr Gly Asp Pro Asn Lys 355 360 365

Thr Arg Gly Ala Pro Glu Ala Leu Met Ala Asn Ser Gly Glu Pro Trp 370 375 380

Ser Pro Ser Tyr Leu Gly Pro Thr Ser Pro Glu Asn Ser Met Thr Ser 385 390 395 400

Lys Val Tyr Asp Pro Glu Gin Arg Lys Arg Met lie Thr Gly Pro Gin 405 410 415

Trp Trp Ala Arg Cys Lys Gin Met Asn Val Leu Asp Ser Phe lie Asn 420 425 430

Tyr Tyr Asp Ser Glu Lys His Ala Glu Asn Ala Val lie Phe Leu His 435 440 445

Gly Asn Ala Ala Ser Ser Tyr Leu Trp Arg His Val Val Pro His lie 450 455 460

Glu Pro Val Ala Arg Cys He lie Pro Asp Leu lie Gly Met Gly Lys 465 470 475 480 -15· 156362 - Sequence Listing.doc 201131168

Ser Gly Lys Ser Gly Asn Gly Ser Tyr Arg Leu Leu Asp His Tyr Lys 485 490 495

Tyr Leu Thr Ala Trp Phe Glu Leu Leu Asn Leu Pro Lys Lys lie Asn 500 505 510

Phe Val Gly His Asp Trp Gly Ala Cys Leu Ala Phe His Tyr Ser Tyr 515 520 525

Glu His Gin Asp Lys lie Lys Ala lie Val His Ala Glu Ser Val Val 530 535 540

Asp Val lie Glu Ser Trp Asp Glu Trp Pro Asp lie Glu Glu Asp lie 545 550 555 560

Ala Leu lie Lys Ser Glu Glu Gly Glu Lys Met Val Leu Glu Asn Asn 565 570 575

Phe Phe Val Glu Thr Met Leu Pro Ser Lys He Met Arg Lys Leu Giu 580 585 590

Pro Glu Glu Phe Ala Ala Tyr Leu Glu Pro Phe Lys Glu Lys Gly Glu 595 600 605

Val Arg Arg Pro Thr Leu Ser Trp Pro Arg Glu lie Pro Leu Val Lys 610 615 620

Gly Gly Lys Pro Asp Val Val Gin lie Val Arg Asn Tyr Asn Ala Tyr 625 630 635 640

Leu Arg Ala Ser Asp Asp Leu Pro Lys Met Phe lie Glu Ser Asp Pro 645 650 655

Gly Phe Phe Ser Asn Ala He Val Glu Gly Ala Lys Lys Phe Pro Asn 660 665 670

Thr Glu Phe Val Lys Val Lys Gly Leu His Phe Ser Gin Glu Asp Ala 675 680 685

Pro Asp Glu Met Gly Lys Tyr lie Lys Ser Phe Val Glu Arg Val Leu 690 695 700

Lys Asn Glu Gin 705 16- 156362 - Sequence Listing.doc 201131168 <210> 14 <211> 649 <212> PRT <213>Manual<220><223> Derived from human histamine 4 receptor <400> 14

Mel: Pro Asp Thr Asn Ser Thr lie Asn Leu Ser Leu Ser Thr Arg Val 15 10 15

Thr Leu Ala Phe Phe Met Ser Leu Val Ala Phe Ala lie Met Leu Gly 20 25 30

Asn Ala Leu Val lie Leu Ala Phe Val Val Asp Lys Asn Leu Arg His 35 40 45

Arg Ser Ser Tyr Phe Phe Leu Asn Leu Ala lie Ser Asp Phe Phe Val 50 55 60

Gly Val lie Ser He Pro Leu Tyr lie Pro His Thr Leu Phe Glu Trp 65 70 75 80

Asp Phe Gly Lys Glu lie Cys Val Phe Trp Leu Thr Thr Asp Tyr Leu 85 90 95

Leu Cys Thr Ala Ser Val Tyr Asn lie Val Leu lie Ser Tyr Asp Arg 100 105 110

Tyr Leu Ser Val Ser Asn Ala Val Ser Tyr Arg Thr Gin His Thr Gly 115 120 125

Val Leu Lys lie Val Thr Leu Met Val Ala Val Trp Val Leu Ala Phe 130 135 140

Leu Val Asn Gly Pro Met lie Leu Val Ser Glu Ser Trp Lys Asp Glu 145 150 155 160

Gly Ser Glu Cys Glu Pro Gly Phe Phe Ser Glu Trp Tyr He Leu Ala 165 170 175 lie Thr Ser Phe Leu Glu Phe Val lie Pro Val lie Leu Val Ala Tyr 180 185 190

Phe Gly Thr Arg Val Phe Gin Glu Ala Lys Arg Gin Leu Gin Lys lie 195 200 205 17· 156362 - Sequence Listing.doc 201131168

Asp Lys Ser Glu Gly Arg Phe His Val Gin Asn Leu Ser Gin Val Glu 210 215 220

Gin Asp Gly Arg Thr Gly His Gly Leu Arg Arg Ser Ser Lys Phe Cys 225 230 235 240

Ser Arg Glu Lys Lys Ala Ala Lys Thr Leu Glu Phe Leu Leu Gly Val 245 250 255

Phe Ala Val Cys Trp Ala Pro Tyr Ser Leu Phe Thr lie Val Leu Ser 260 265 270

Phe Tyr Ser Ser Ala Thr Gly Pro Lys Ser Val Trp Tyr Arg He Ala 275 280 285

Phe Trp Leu Gin Trp Phe Asn Ser Phe Val Asn Pro Leu Leu Tyr Pro 290 295 300

Leu Cys His Lys Arg Phe Gin Lys Ala Phe Leu Lys lie Phe Cys He 305 310 315 320

Lys Lys Gin Pro Leu Pro Ser Gin His Ser Arg Ser Val Ser Ser Glu 325 330 335

Asn Ser Met Thr Ser Lys Val Tyr Asp Pro Glu Gin Arg Lys Arg Met 340 345 350 lie Thr Gly Pro Gin Trp Trp Ala Arg Cys Lys Gin Met Asn Val Leu 355 360 365

Asp Ser Phe lie Asn Tyr Tyr Asp Ser Glu Lys His Ala Glu Asn Ala 370 375 380

Val lie Phe Leu His Gly Asn Ala Ala Ser Ser Tyr Leu Trp Arg His 385 390 395 400

Val Val Pro His lie Glu Pro Val Ala Arg Cys lie He Pro Asp Leu 405 410 415 lie Gly Met Gly Lys Ser Gly Lys Ser Gly Asn Gly Ser Tyr Arg Leu 420 425 430

Leu Asp His Tyr Lys Tyr Leu Thr Ala Trp Phe Glu Leu Leu Asn Leu 435 440 445 18· 156362-Sequence List.doc 201131168

Pro Lys Lys lie Asn Phe Val Gly His Asp Trp Gly Ala Cys Leu Ala 450 455 460

Phe His Tyr Ser Tyr Glu His Gin Asp Lys lie Lys Ala lie Val His 465 470 475 480

Ala Glu Ser Val Val Asp Val lie Glu Ser Trp Asp Glu Trp Pro Asp 485 490 495 lie Glu Glu Asp lie Ala Leu lie Lys Ser Glu Glu Gly Glu Lys Met 500 505 510

Val Leu Glu Asn Asn Phe Phe Val Glu Thr Met Leu Pro Ser Lys lie 515 520 525

Met Arg Lys Leu Glu Pro Glu Glu Phe Ala Ala Tyr Leu Glu Pro Phe 530 535 540

Lys Glu Lys Gly Glu Val Arg Arg Pro Thr Leu Ser Trp Pro Arg Glu 545 550 555 560 lie Pro Leu Val Lys Gly Gly Lys Pro Asp Val Val Gin lie Val Arg 565 570 575

Asn Tyr Asn Ala Tyr Leu Arg Ala Ser Asp Asp Leu Pro Lys Met Phe 580 585 590 lie Glu Ser Asp Pro Gly Phe Phe Ser Asn Ala lie Val Glu Gly Ala 595 600 605

Lys Lys Phe Pro Asn Thr Glu Phe Val Lys Val Lys Gly Leu His Phe 610 615 620

Ser Gin Glu Asp Ala Pro Asp Glu Met Gly Lys Tyr He Lys Ser Phe 625 630 635 640

Val Glu Arg Val Leu Lys Asn Glu Gin 645 <210> 15 <211> 705 <212> PRT < 213 > 213 <220><223> Derived from human polypeptide ghrelin receptor <400> 15 19· 156362 - Sequence Listing.doc 201131168

Met Trp Asn Ala Thr Pro Ser Glu Glu Pro Gly Phe Asn Leu Thr Leu 15 10 15

Ala Asp Leu Asp Trp Asp Ala Ser Pro Gly Asn Asp Ser Leu Gly Asp 20 25 30

Glu Leu Leu Gin Leu Phe Pro Ala Pro Leu Leu Ala Gly Val Thr Ala 35 40 45

Thr Cys Val Ala Leu Phe Val Val Gly lie Ala Gly Asn Leu Leu Thr 50 55 60

Met Leu Val Val Ser Arg Phe Arg Glu Leu Arg Thr Thr Thr Asn Leu 65 70 75 80

Tyr Leu Ser Ser Met Ala Phe Ser Asp Leu Leu lie Phe Leu Cys Met 85 90 95

Pro Leu Asp Leu Val Arg Leu Trp Gin Tyr Arg Pro Trp Asn Phe Gly 100 105 110

Asp Leu Leu Cys Lys Leu Phe Gin Phe Val Ser Glu Ser Cys Thr Tyr 115 120 125

Ala Thr Val Leu Thr lie Thr Ala Leu Ser Val Glu Arg Tyr Phe Ala 130 135 140 lie Cys Phe Pro Leu Arg Ala Lys Val Val Val Thr Lys Gly Arg Val 145 150 155 160

Lys Leu Val lie Phe Val lie Trp Ala Val Ala Phe Cys Ser Ala Gly 165 170 175

Pro He Phe Val Leu Val Gly Val Glu His Glu Asn Gly Thr Asp Pro 180 185 190

Trp Asp Thr Asn Glu Cys Arg Pro Thr Glu Phe Ala Val Arg Ser Gly 195 200 205

Leu Leu Thr Val Met Val Trp Val Ser Ser lie Phe Phe Phe Leu Pro 210 215 220

Val Phe Cys Leu Thr Val Leu Gly Thr Arg Val Phe Gin Glu Ala Lys 225 230 235 240 •20· 156362·Sequence List.doc 201131168

Arg Gin Leu Gin Lys lie Asp Lys Ser Glu Gly Arg Phe His Val Gin 245 250 255

Asn Leu Ser Gin Val Glu Gin Asp Gly Arg Thr Gly His Gly Leu Arg 260 265 270

Arg Ser Ser Lys Phe Cys Ser Arg Glu Lys Lys Ala Ala Lys Thr Leu 275 280 285

Glu Phe Ala Val Val Val Phe Ala Phe lie Leu Cys Trp Leu Pro Phe 290 295 300

His Val Gly Arg Tyr Leu Phe Ser Lys Ser Phe Glu Pro Gly Ser Leu 305 310 315 320

Glu lie Ala Gin lie Ser Gin Tyr Cys Asn Leu Val Ser Phe Val Leu 325 330 335

Phe Tyr Leu Ser Ala Ala lie Asn Pro lie Leu Tyr Asn lie Met Ser 340 345 350

Lys Lys Tyr Arg Val Ala Val Phe Arg Leu Leu Gly Phe Glu Pro Phe 355 360 365

Ser Gin Arg Lys Leu Ser Thr Leu Lys Asp Glu Ser Ser Arg Ala Trp 370 375 380

Thr Giu Ser Ser lie Asn Thr Glu Asn Ser Met Thr Ser Lys Val Tyr 385 390 395 400

Asp Pro Glu Gin Arg Lys Arg Met lie Thr Gly Pro Gin Trp Trp Ala 405 410 415

Arg Cys Lys Gin Met Asn Val Leu Asp Ser Phe lie Asn Tyr Tyr Asp 420 425 430

Ser Glu Lys His Ala Glu Asn Ala Val lie Phe Leu His Gly Asn Ala 435 440 445

Ala Ser Ser Tyr Leu Trp Arg His Val Val Pro His lie Glu Pro Val 450 455 460

Ala Arg Cys lie lie Pro Asp Leu He Gly Met Gly Lys Ser Gly Lys 465 470 475 480

Ser Gly Asn Gly Ser Tyr Arg Leu Leu Asp His Tyr Lys Tyr Leu Thr 485 490 495 -21 - 156362 - Sequence Listing.doc 201131168

Ala Trp Phe Glu Leu Leu Asn Leu Pro Lys Lys lie Asn Phe Val Gly 500 505 510

His Asp Trp Gly Ala Cys Leu Ala Phe His Tyr Ser Tyr Glu His Gin 515 520 525

Asp Lys lie Lys Ala lie Val His Ala Glu Ser Val Val Asp Val He 530 535 540

Glu Ser Trp Asp Glu Trp Pro Asp lie Glu Glu Asp lie Ala Leu lie 545 550 555 560

Lys Ser Glu Glu Gly Glu Lys Met Val Leu Glu Asn Asn Phe Phe Val 565 570 575

Glu Thr Met Leu Pro Ser Lys lie Met Arg Lys Leu Glu Pro Glu Glu 580 585 590

Phe Ala Ala Tyr Leu Glu Pro Phe Lys Glu Lys Gly Glu Val Arg Arg 595 600 605

Pro Thr Leu Ser Trp Pro Arg Glu lie Pro Leu Val Lys Gly Gly Lys 610 615 620

Pro Asp Val Val Gin lie Val Arg Asn Tyr Asn Ala Tyr Leu Arg Ala 625 630 635 640

Ser Asp Asp Leu Pro Lys Met Phe lie Glu Ser Asp Pro Gly Phe Phe 645 650 655

Ser Asn Ala lie Val Glu Gly Ala Lys Lys Phe Pro Asn Thr Glu Phe 660 665 670

Val Lys Val Lys Gly Leu His Phe Ser Gin Glu Asp Ala Pro Asp Glu 675 680 685

Met Gly Lys Tyr lie Lys Ser Phe Val Glu Arg Val Leu Lys Asn Glu 690 695 700

Gin

705 <210> 16 <211> 716 <212> PRT 22· 156362 - Sequence Listing.doc 201131168 <213>Manual<220><223> Derived from Human CXCR3 Chemokine Receptor <400>; 16

Met Val Leu Glu Val Ser Asp His Gin Val Leu Asn Asp Ala Glu Val 15 10 15

Ala Ala Leu Leu Glu Asn Phe Ser Ser Ser Tyr Asp Tyr Gly Glu Asn 20 25 30

Glu Ser Asp Ser Cys Cys Thr Ser Pro Pro Cys Pro Gin Asp Phe Ser 35 40 45

Leu Asn Phe Asp Arg Ala Phe Leu Pro Ala Leu Tyr Ser Leu Leu Phe 50 55 60

Leu Leu Gly Leu Leu Gly Asn Gly Ala Val Ala Ala Val Leu Leu Ser 65 70 75 80

Arg Arg Thr Ala Leu Ser Ser Thr Asp Thr Phe Leu Leu His Leu Ala 85 90 95

Val Ala Asp Thr Leu Leu Val Leu Thr Leu Pro Leu Trp Ala Val Asp 100 105 110

Ala Ala Val Gin Trp Val Phe Gly Ser Gly Leu Cys Lys Val Ala Gly 115 120 125

Ala Leu Phe Asn lie Asn Phe Tyr Ala Gly Ala Leu Leu Leu Ala Cys 130 135 140 lie Ser Phe Asp Arg Tyr Leu Asn He Val His Ala Thr Gin Leu Tyr 145 150 155 160

Arg Arg Gly Pro Pro Ala Arg Val Thr Leu Thr Cys Leu Ala Val Trp 165 170 175

Gly Leu Cys Leu Leu Phe Ala Leu Pro Asp Phe lie Phe Leu Ser Ala 180 185 190

His His Asp Glu Arg Leu Asn Ala Thr His Cys Gin Tyr Asn Phe Pro 195 200 205

Gin Val Gly Arg Thr Ala Leu Arg Val Leu Gin Leu Val Ala Gly Phe 210 215 220 -23- 156362 · Sequence Listing.doc 201131168

Leu Leu Pro Leu Leu Val Met Ala Tyr Cys Gly Thr Arg Val Phe Gin 225 230 235 240

Glu Ala Lys Arg Gin Leu Gin Lys lie Asp Lys Ser Glu Gly Arg Phe 245 250 255

His Val Gin Asn Leu Ser Gin Val Glu Gin Asp Gly Arg Thr Gly His 260 265 270

Gly Leu Arg Arg Ser Ser Lys Phe Cys Ser Arg Glu Lys Lys Ala Ala 275 280 285

Lys Thr Leu Glu Phe Val Val Val Ala Phe Ala Leu Cys Trp Thr Pro 290 295 300

Tyr His Leu Val Val Leu Val Asp lie Leu Met Asp Leu Gly Ala Leu 305 310 315 320

Ala Arg Asn Cys Gly Arg Glu Ser Arg Val Asp Val Ala Lys Ser Val 325 330 335

Thr Ser Gly Leu Gly Tyr Met His Cys Cys Leu Asn Pro Leu Leu Tyr 340 345 350

Ala Phe Val Gly Val Lys Phe Arg Glu Arg Met Trp Met Leu Leu Leu 355 360 365

Arg Leu Gly Cys Pro Asn Gin Arg Gly Leu Gin Arg Gin Pro Ser Ser 370 375 380

Ser Arg Arg Asp Ser Ser Trp Ser Glu Thr Ser Glu Ala Ser Tyr Ser 385 390 395 400

Gly Leu Glu Asn Ser Met Thr Ser Lys Val Tyr Asp Pro Glu Gin Arg 405 410 415

Lys Arg Met lie Thr Gly Pro Gin Trp Trp Ala Arg Cys Lys Gin Met 420 425 430

Asn Val Leu Asp Ser Phe lie Asn Tyr Tyr Asp Ser Glu Lys His Ala 435 440 445

Glu Asn Ala Val lie Phe Leu His Gly Asn Ala Ala Ser Ser Tyr Leu 450 455 460 • 24· 156362 - Sequence Listing.doc 201131168

Trp Arg His Val Val Pro His He Glu Pro Val Ala Arg Cys lie lie 465 470 475 480

Pro Asp Leu He Gly Met Gly Lys Ser Gly Lys Ser Gly Asn Gly Ser 485 490 495

Tyr Arg Leu Leu Asp His Tyr Lys Tyr Leu Thr Ala Trp Phe Glu Leu 500 505 510

Leu Asn Leu Pro Lys Lys lie Asn Phe Val Gly His Asp Trp Gly Ala 515 520 525

Cys Leu Ala Phe His Tyr Ser Tyr Glu His Gin Asp Lys lie Lys Ala 530 535 540 lie Val His Ala Glu Ser Val Val Asp Val lie Glu Ser Trp Asp Glu 545 550 555 560

Trp Pro Asp lie Glu Glu Asp lie Ala Leu lie Lys Ser Glu Glu Gly 565 570 575

Glu Lys Met Val Leu Glu Asn Asn Phe Phe Val Glu Thr Met Leu Pro 580 585 590

Ser Lys lie Met Arg Lys Leu Glu Pro Glu Glu Phe Ala Ala Tyr Leu 595 600 605

Glu Pro Phe Lys Glu Lys Gly Glu Val Arg Arg Pro Thr Leu Ser Trp 610 615 620

Pro Arg Glu He Pro Leu Val Lys Gly Gly Lys Pro Asp Val Val Gin 625 630 635 640 lie Val Arg Asn Tyr Asn Ala Tyr Leu Arg Ala Ser Asp Asp Leu Pro 645 650 655

Lys Met Phe lie Glu Ser Asp Pro Gly Phe Phe Ser Asn Ala He Val 660 665 670

Glu Gly Ala Lys Lys Phe Pro Asn Thr Glu Phe Val Lys Val Lys Gly 675 680 685

Leu His Phe Ser Gin Glu Asp Ala Pro Asp Glu Met Gly Lys Tyr lie 690 695 700 -25- 156362 - Sequence Listing.doc 201131168

Lys Ser Phe Val Glu Arg Val Leu Lys Asn Glu Gin 705 710 715 <210> 17 <211> 749 <212> PRT <213>Manual<220><223> Derived from human enterokin Body <400〉 17

Met Gly Ser Pro Trp Asn Gly Ser Asp Gly Pro Glu Gly Ala Arg Glu 15 10 15

Pro Pro Trp Pro Ala Leu Pro Pro Cys Asp Glu Arg Arg Cys Ser Pro 20 25 30

Phe Pro Leu Gly Ala Leu Val Pro Val Thr Ala Val Cys Leu Cys Leu 35 40 45

Phe Val Val Gly Val Ser Gly Asn Val Val Thr Val Met Leu lie Gly 50 55 60

Arg Tyr Arg Asp Met Arg Thr Thr Thr Asn Leu Tyr Leu Gly Ser Met 65 70 75 80

Ala Val Ser Asp Leu Leu lie Leu Leu Gly Leu Pro Phe Asp Leu Tyr 85 90 95

Arg Leu Trp Arg Ser Arg Pro Trp Val Phe Gly Pro Leu Leu Cys Arg 100 105 110

Leu Ser Leu Tyr Val Gly Glu Gly Cys Thr Tyr Ala Thr Leu Leu His 115 120 125

Met Thr Ala Leu Ser Val Glu Arg Tyr Leu Ala lie Cys Arg Pro Leu 130 135 140

Arg Ala Arg Val Leu Val Thr Arg Arg Arg Val Arg Ala Leu lie Ala 145 150 155 160

Val Leu Trp Ala Val Ala Leu Leu Ser Ala Gly Pro Phe Leu Phe Leu 165 170 175

Val Gly Val Glu Gin Asp Pro Gly lie Ser Val Val Pro Gly Leu Asn 180 185 190 -26- 156362 - Sequence Listing.doc 201131168

Gly Thr Ala Arg lie Ala Ser Ser Pro Leu Ala Ser Ser Pro Pro Leu 195 200 205

Trp Leu Ser Arg Ala Pro Pro Pro Ser Pro Pro Ser Gly Pro Glu Thr 210 215 220

Ala Glu Ala Ala Ala Leu Phe Ser Arg Glu Cys Arg Pro Ser Pro Ala 225 230 235 240

Gin Leu Gly Ala Leu Arg Val Met Leu Trp Val Thr Thr Ala Tyr Phe 245 250 255

Phe Leu Pro Phe Leu Cys Leu Ser lie Leu Gly Thr Arg Val Phe Gin 260 265 270

Glu Ala Lys Arg Gin Leu Gin Lys lie Asp Lys Ser Glu Gly Arg Phe 275 280 285

His Val Gin Asn Leu Ser Gin Val Glu Gin Asp Gly Arg Thr Gly His 290 295 300

Gly Leu Arg Arg Ser Ser Lys Phe Cys Ser Arg Glu Lys Lys Ala Ala 305 310 315 320

Lys Thr Leu Glu Phe Val Val Leu Ala Phe lie lie Cys Trp Leu Pro 325 330 335

Phe His Val Gly Arg lie lie Tyr lie Asn Thr Glu Asp Ser Arg Met 340 345 350

Met Tyr Phe Ser Gin Tyr Phe Asn lie Val Ala Leu Gin Leu Phe Tyr 355 360 365

Leu Ser Ala Ser lie Asn Pro lie Leu Tyr Asn Leu lie Ser Lys Lys 370 375 380

Tyr Arg Ala Ala Ala Phe Lys Leu Leu Leu Ala Arg Lys Ser Arg Pro 385 390 395 400

Arg Gly Phe His Arg Ser Arg Asp Thr Ala Gly Glu Val Ala Gly Asp 405 410 415

Thr Gly Gly Asp Thr Val Gly Tyr Thr Glu Thr Ser Ala Asn Val Lys 420 425 430 27- 156362 - Sequence Listing.doc 201131168

Thr Met Gly Glu Asn Ser Met Thr Ser Lys Val Tyr Asp Pro Glu Gin 435 440 445

Arg Lys Arg Met lie Thr Gly Pro Gin Trp Trp Ala Arg Cys Lys Gin 450 455 460

Met Asn Val Leu Asp Ser Phe lie Asn Tyr Tyr Asp Ser Glu Lys His 465 470 475 480

Ala Glu Asn Ala Val lie Phe Leu His Gly Asn Ala Ala Ser Ser Tyr 485 490 495

Leu Trp Arg His Val Val Pro His lie Glu Pro Val Ala Arg Cys lie 500 505 510 lie Pro Asp Leu lie Gly Met Gly Lys Ser Gly Lys Ser Gly Asn Gly 515 520 525

Ser Tyr Arg Leu Leu Asp His Tyr Lys Tyr Leu Thr Ala Trp Phe Glu 530 535 540

Leu Leu Asn Leu Pro Lys Lys lie Asn Phe Val Gly His Asp Trp Gly 545 550 555 560

Ala Cys Leu Ala Phe His Tyr Ser Tyr Glu His Gin Asp Lys He Lys 565 570 575

Ala lie Val His Ala Glu Ser Val Val Asp Val lie Glu Ser Trp Asp 580 585 590

Glu Trp Pro Asp lie Glu Glu Asp lie Ala Leu lie Lys Ser Glu Glu 595 600 605

Gly Glu Lys Met Val Leu Glu Asn Asn Phe Phe Val Glu Thr Met Leu 610 615 620

Pro Ser Lys lie Met Arg Lys Leu Glu Pro Glu Glu Phe Ala Ala Tyr 625 630 635 640

Leu Glu Pro Phe Lys Glu Lys Gly Glu Val Arg Arg Pro Thr Leu Ser 645 650 655

Trp Pro Arg Glu lie Pro Leu Val Lys Gly Gly Lys Pro Asp Val Val 660 665 670

Gin lie Val Arg Asn Tyr Asn Ala Tyr Leu Arg Ala Ser Asp Asp Leu 675 680 685 -28· 156362 · Sequence Listing.doc 201131168

Pro Lys Met Phe lie Glu Ser Asp Pro Gly Phe Phe Ser Asn Ala lie 690 695 700

Val Glu Gly Ala Lys Lys Phe Pro Asn Thr Glu Phe Val Lys Val Lys 705 710 715 720

Gly Leu His Phe Ser Gin Glu Asp Ala Pro Asp Glu Met Gly Lys Tyr 725 730 735 lie Lys Ser Phe Val Glu Arg Val Leu Lys Asn Glu Gin 740 745 <210> 18 <211> 770 <212> PRT <;213>Manual<220><223> Derived from human 5-hydroxytryptamine (serotonin) receptor 2A <400>

Met Asp lie Leu Cys Glu Glu Asn Thr Ser Leu Ser Ser Thr Thr Asn 15 10 15

Ser Leu Met Gin Leu Asn Asp Asp Thr Arg Leu Tyr Ser Asn Asp Phe 20 25 30

Asn Ser Gly Glu Ala Asn Thr Ser Asp Ala Phe Asn Trp Thr Val Asp 35 40 45

Ser Glu Asn Arg Thr Asn Leu Ser Cys Glu Gly Cys Leu Ser Pro Ser 50 55 60

Cys Leu Ser Leu Leu His Leu Gin Glu Lys Asn Trp Ser Ala Leu Leu 65 70 75 80

Thr Ala Val Val lie lie Leu Thr lie Ala Gly Asn lie Leu Val lie 85 90 95

Met Ala Val Ser Leu Glu Lys Lys Leu Gin Asn Ala Thr Asn Tyr Phe 100 105 110

Leu Met Ser Leu Ala lie Ala Asp Met Leu Leu Gly Phe Leu Val Met 115 120 125

Pro Val Ser Met Leu Thr lie Leu Tyr Gly Tyr Arg Trp Pro Leu Pro 130 135 140 -29- 156362 - Sequence Listing.doc 201131168

Ser Lys Leu Cys Ala Val Trp lie Tyr Leu Asp Val Leu Phe Ser Thr 145 150 155 160

Ala Ser lie Met His Leu Cys Ala lie Ser Leu Asp Arg Tyr Val Ala 165 170 175 lie Gin Asn Pro lie His His Ser Arg Phe Asn Ser Arg Thr Lys Ala 180 185 190

Phe Leu Lys lie lie Ala Val Trp Thr lie Ser Val Gly lie Ser Met 195 200 205

Pro lie Pro Val Phe Gly Leu Gin Asp Asp Ser Lys Val Phe Lys Glu 210 215 220

Gly Ser Cys Leu Leu Ala Asp Asp Asn Phe Val Leu lie Gly Ser Phe 225 230 235 240

Val Ser Phe Phe lie Pro Leu Thr lie Met Val lie Thr Gly Thr Arg 245 250 255

Val Phe Gin Glu Ala Lys Arg Gin Leu Gin Lys lie Asp Lys Ser Glu 260 265 270

Gly Arg Phe His Val Gin Asn Leu Ser Gin Val Glu Gin Asp Gly Arg 275 280 285

Thr Gly His Gly Leu Arg Arg Ser Ser Lys Phe Cys Ser Arg Glu Lys 290 295 300

Lys Ala Ala Lys Thr Leu Glu Phe Val Phe Phe Leu Phe Val Val Met 305 310 315 320

Trp Cys Pro Phe Phe lie Thr Asn lie Met Ala Val lie Cys Lys Glu 325 330 335

Ser Cys Asn Glu Asp Val lie Gly Ala Leu Leu Asn Val Phe Val Trp 340 345 350 lie Gly Tyr Leu Ser Ser Ala Val Asn Pro Leu Val Tyr Thr Leu Phe 355 360 365

Asn Lys Thr Tyr Arg Ser Ala Phe Ser Arg Tyr lie Gin Cys Gin Tyr 370 375 380 • 30· 156362 - Sequence Listing.doc 201131168

Lys Glu Asn Lys Lys Pro Leu Gin Leu lie Leu Val Asn Thr He Pro 385 390 395 400

Ala Leu Ala Tyr Lys Ser Ser Gin Leu Gin Met Gly Gin Lys Lys Asn 405 410 415

Ser Lys Gin Asp Ala Lys Thr Thr Asp Asn Asp Cys Ser Met Val Ala 420 425 430

Leu Gly Lys Gin His Ser Glu Glu Ala Ser Lys Asp Asn Ser Asp Gly 435 440 445

Val Asn Glu Lys Val Ser Cys Val Glu Asn Ser Met Thr Ser Lys Val 450 455 460

Tyr Asp Pro Glu Gin Arg Lys Arg Met lie Thr Gly Pro Gin Trp Trp 465 470 475 480

Ala Arg Cys Lys Gin Met Asn Val Leu Asp Ser Phe lie Asn Tyr Tyr 485 490 495

Asp Ser Glu Lys His Ala Glu Asn Ala Val lie Phe Leu His Gly Asn 500 505 510

Ala Ala Ser Ser Tyr Leu Trp Arg His Val Val Pro His lie Glu Pro 515 520 525

Val Ala Arg Cys lie lie Pro Asp Leu lie Gly Met Gly Lys Ser Gly 530 535 540

Lys Ser Gly Asn Gly Ser Tyr Arg Leu Leu Asp His Tyr Lys Tyr Leu 545 550 555 560

Thr Ala Trp Phe Glu Leu Leu Asn Leu Pro Lys Lys lie Asn Phe Val 565 570 575

Gly His Asp Trp Gly Ala Cys Leu Ala Phe His Tyr Ser Tyr Glu His 580 585 590

Gin Asp Lys lie Lys Ala lie Val His Ala Glu Ser Val Val Asp Val 595 600 605 lie Glu Ser Trp Asp Glu Trp Pro Asp He Glu Glu Asp lie Ala Leu 610 615 620 -31 · 156362 · Sequence Listing.doc 201131168 lie Lys Ser Glu Glu Gly Glu Lys Met Val Leu Glu Asn Asn Phe Phe 625 630 635 640

Val Glu Thr Met Leu Pro Ser Lys lie Met Arg Lys Leu Glu Pro Glu 645 650 655

Glu Phe Ala Ala Tyr Leu Glu Pro Phe Lys Glu Lys Gly Glu Val Arg 660 665 670

Arg Pro Thr Leu Ser Trp Pro Arg Glu lie Pro Leu Val Lys Gly Gly 675 680 685

Lys Pro Asp Val Val Gin lie Val Arg Asn Tyr Asn Ala Tyr Leu Arg 690 695 700

Ala Ser Asp Asp Leu Pro Lys Met Phe He Glu Ser Asp Pro Gly Phe 705 710 715 720

Phe Ser Asn Ala lie Val Glu Gly Ala Lys Lys Phe Pro Asn Thr Glu 725 730 735

Phe Val Lys Val Lys Gly Leu His Phe Ser Gin Glu Asp Ala Pro Asp 740 745 750

Glu Met Gly Lys Tyr lie Lys Ser Phe Val Glu Arg Val Leu Lys Asn 755 760 765

Glu Gin 770 <230> 19 <211> 762 <212> PRT < 213 > Labor <220><223> Derived from human 5-hydroxytryptamine (serotonin) receptor 2B <400> 19

Met Ala Leu Ser Tyr Arg Val Ser Glu Leu Gin Ser Thr lie Pro Glu 15 10 15

His lie Leu Gin Ser Thr Phe Val His Val lie Ser Ser Asn Trp Ser 20 25 30

Gly Leu Gin Thr Glu Ser lie Pro Glu Glu Met Lys Gin lie Val Glu 35 40 45 32· 156362 - Sequence Listing.doc 201131168

Glu Gin Gly Asn Lys Leu His Trp Ala Ala Leu Leu lie Leu Met Val 50 55 60 lie lie Pro Thr lie Gly Gly Asn Thr Leu Val lie Leu Ala Val Ser 65 70 75 80

Leu Glu Lys Lys Leu Gin Tyr Ala Thr Asn Tyr Phe Leu Met Ser Leu 85 90 95

Ala Val Ala Asp Leu Leu Val Gly Leu Phe Val Met Pro lie Ala Leu 100 105 110

Leu Thr lie Met Phe Glu Ala Met Trp Pro Leu Pro Leu Val Leu Cys 115 120 125

Pro Ala Trp Leu Phe Leu Asp Val Leu Phe Ser Thr Ala Ser lie Met 130 135 140

His Leu Cys Ala lie Ser Val Asp Arg Tyr lie Ala lie Lys Lys Pro 145 150 155 160 lie Gin Ala Asn Gin Tyr Asn Ser Arg Ala Thr Ala Phe lie Lys lie 165 170 175

Thr Val Val Trp Leu lie Ser He Gly lie Ala lie Pro Val Pro lie 180 185 190

Lys Gly lie Glu Thr Asp Val Asp Asn Pro Asn Asn lie Thr Cys Val 195 200 205

Leu Thr Lys Glu Arg Phe Gly Asp Phe Met Leu Phe Gly Ser Leu Ala 210 215 220

Ala Phe Phe Thr Pro Leu Ala lie Met lie Val Thr Gly Thr Arg Val 225 230 235 240

Phe Gin Glu Ala Lys Arg Gin Leu Gin Lys lie Asp Lys Ser Glu Gly 245 250 255

Arg Phe His Val Gin Asn Leu Ser Gin Val Glu Gin Asp Gly Arg Thr 260 265 270

Gly His Gly Leu Arg Arg Ser Ser Lys Phe Cys Ser Arg Glu Lys Lys 275 280 285 33- 156362 - Sequence Listing.doc 201131168

Ala Ala Lys Thr Leu Glu Phe Val Phe Phe Leu Phe Leu Leu Met Trp 290 295 300

Cys Pro Phe Phe lie Thr Asn lie Thr Leu Val Leu Cys Asp Ser Cys 305 310 315 320

Asn Gin Thr Thr Leu Gin Met Leu Leu Glu lie Phe Val Trp He Gly 325 330 335

Tyr Val Ser Ser Gly Val Asn Pro Leu Val Tyr Thr Leu Phe Asn Lys 340 345 350

Thr Phe Arg Asp Ala Phe Gly Arg Tyr lie Thr Cys Asn Tyr Arg Ala 355 360 365

Thr Lys Ser Val Lys Thr Leu Arg Lys Arg Ser Ser Lys lie Tyr Phe 370 375 380

Arg Asn Pro Met Ala Glu Asn Ser Lys Phe Phe Lys Lys His Gly lie 385 390 395 400

Arg Asn Gly lie Asn Pro Ala Met Tyr Gin Ser Pro Met Arg Leu Arg 405 410 415

Ser Ser Thr lie Gin Ser Ser Ser lie Leu Leu Asp Thr Leu Leu 420 425 430

Leu Thr Glu Asn Glu Gly Asp Lys Thr Glu Glu Gin Val Ser Tyr Val 435 440 445

Glu Asn Ser Met Thr Ser Lys Val Tyr Asp Pro Glu Gin Arg Lys Arg 450 455 460

Met lie Thr Gly Pro Gin Trp Trp Ala Arg Cys Lys Gin Met Asn Val 465 470 475 480

Leu Asp Ser Phe lie Asn Tyr Tyr Asp Ser Glu Lys His Ala Glu Asn 485 490 495

Ala Val lie Phe Leu His Gly Asn Ala Ala Ser Ser Tyr Leu Trp Arg 500 505 510

His Val Val Pro His lie Glu Pro Val Ala Arg Cys lie lie Pro Asp 515 520 525

Leu lie Gly Met Gly Lys Ser Gly Lys Ser Gly Asn Gly Ser Tyr Arg 530 535 540 -34- 156362 - Sequence Listing.doc 201131168

Leu Leu Asp His Tyr Lys Tyr Leu Thr Ala Trp Phe Glu Leu Leu Asn 545 550 555 560

Leu Pro Lys Lys lie Asn Phe Val Gly His Asp Trp Gly Ala Cys Leu 565 570 575

Ala Phe His Tyr Ser Tyr Glu His Gin Asp Lys lie Lys Ala lie Val 580 585 590

His Ala Glu Ser Val Val Asp Val lie Glu Ser Trp Asp Glu Trp Pro 595 600 605

Asp lie Glu Glu Asp He Ala Leu lie Lys Ser Glu Glu Gly Glu Lys 610 615 620

Met Val Leu Glu Asn Asn Phe Phe Val Glu Thr Met Leu Pro Ser Lys 625 630 635 640 lie Met Arg Lys Leu Glu Pro Glu Glu Phe Ala Ala Tyr Leu Glu Pro 645 650 655

Phe Lys Glu Lys Gly Glu Val Arg Arg Pro Thr Leu Ser Trp Pro Arg 660 665 670

Glu lie Pro Leu Val Lys Gly Gly Lys Pro Asp Val Val Gin lie Val 675 680 685

Arg Asn Tyr Asn Ala Tyr Leu Arg Ala Ser Asp Asp Leu Pro Lys Met 690 695 700

Phe lie Glu Ser Asp Pro Gly Phe Phe Ser Asn Ala lie Val Glu Gly 705 710 715 720

Ala Lys Lys Phe Pro Asn Thr Glu Phe Val Lys Val Lys Gly Leu His ΊΤ5 730 735

Phe Ser Gin Glu Asp Ala Pro Asp Glu Met Gly Lys Tyr lie Lys Ser 740 745 750

Phe Val Glu Arg Val Leu Lys Asn Glu Gin 755 760

<210> 20 <211> 749 <212> PRT -35-156362·SEQ ID NO.doc 201131168 <213>Manual<220><223> Derived from human 5-hydroxytryptamine (serotonin) Receptor 2C <400〉 20

Met Val Asn Leu Arg Asn Ala Val His Ser Phe Leu Val His Leu lie 15 10 15

Gly Leu Leu Val Trp Gin Cys Asp lie Ser Val Ser Pro Val Ala Ala 20 25 30 lie Val Thr Asp lie Phe Asn Thr Ser Asp Gly Gly Arg Phe Lys Phe 35 40 45

Pro Asp Gly Val Gin Asn Trp Pro Ala Leu Ser lie Val lie lie lie 50 55 60

He Met Thr lie Gly Gly Asn lie Leu Val lie Met Ala Val Ser Met 65 70 75 80

Glu Lys Lys Leu His Asn Ala Thr Asn Tyr Phe Leu Met Ser Leu Ala 85 90 95 lie Ala Asp Met Leu Val Gly Leu Leu Val Met Pro Leu Ser Leu Leu 100 105 110

Ala lie Leu Tyr Asp Tyr Val Trp Pro Leu Pro Arg Tyr Leu Cys Pro 115 120 125

Val Trp lie Ser Leu Asp Val Leu Phe Ser Thr Ala Ser lie Met His 130 135 140

Leu Cys Ala lie Ser Leu Asp Arg Tyr Val Ala lie Arg Asn Pro lie 145 150 155 160

Glu His Ser Arg Phe Asn Ser Arg Thr Lys Ala lie Met Lys lie Ala 165 170 175 lie Val Trp Ala lie Ser lie Gly Val Ser Val Pro lie Pro Val lie 180 185 190

Gly Leu Arg Asp Glu Glu Lys Val Phe Val Asn Asn Thr Thr Cys Val 195 200 205

Leu Asn Asp Pro Asn Phe Val Leu lie Gly Ser Phe Val Ala Phe Phe 210 215 220 -36· 156362 - Sequence Listing.doc 201131168 lie Pro Leu Thr lie Met Val lie Thr Gly Thr Arg Val Phe Gin Glu 225 230 235 240

Ala Lys Arg Gin Leu Gin Lys lie Asp Lys Ser Glu Gly Arg Phe His 245 250 255

Val Gin Asn Leu Ser Gin Val Glu Gin Asp Gly Arg Thr Gly His Gly 260 265 270

Leu Arg Arg Ser Ser Lys Phe Cys Ser Arg Glu Lys Lys Ala Ala Lys 275 280 2S5

Thr Leu Glu Phe Val Phe Phe Val Phe Leu lie Met Trp Cys Pro Phe 290 295 300

Phe lie Thr Asn lie Leu Ser Val Leu Cys Glu Lys Ser Cys Asn Gin 305 310 315 320

Lys Leu Met Glu Lys Leu Leu Asn Val Phe Val Trp lie Gly Tyr Val 325 330 335

Cys Ser Gly lie Asn Pro Leu Val Tyr Thr Leu Phe Asn Lys lie Tyr 340 345 350

Arg Arg Ala Phe Ser Asn Tyr Leu Arg Cys Asn Tyr Lys Val Glu Lys 355 360 365

Lys Pro Pro Val Arg Gin lie Pro Arg Val Ala Ala Thr Ala Leu Ser 370 375 380

Gly Arg Glu Leu Asn Val Asn lie Tyr Arg His Thr Asn Glu Pro Val 385 390 395 400 lie Glu Lys Ala Ser Asp Asn Glu Pro Gly lie Glu Met Gin Val Glu 405 410 415

Asn Leu Glu Leu Pro Val Asn Pro Ser Ser Val Val Ser Glu Arg lie 420 425 430

Ser Ser Val Glu Asn Ser Met Thr Ser Lys Val Tyr Asp Pro Glu Gin 435 440 445

Arg Lys Arg Met He Thr Gly Pro Gin Trp Trp Ala Arg Cys Lys Gin 450 455 460 •37· 156362-Sequence List.doc 201131168

Met Asn Val Leu Asp Ser Phe lie Asn Tyr Tyr Asp Ser Glu Lys His 465 470 475 480

Ala Glu Asn Ala Val lie Phe Leu His Gly Asn Ala Ala Ser Ser Tyr 485 490 495

Leu Trp Arg His Val Val Pro His lie Glu Pro Val Ala Arg Cys lie 500 505 510 lie Pro Asp Leu lie Gly Met Gly Lys Ser Gly y ys Ser Gly Asn Gly 515 520 525

Ser Tyr Arg Leu Leu Asp His Tyr Lys Tyr Leu Thr Ala Trp Phe Glu 530 535 540

Leu Leu Asn Leu Pro Lys Lys lie Asn Phe Val Gly His Asp Trp Gly 545 550 555 560

Ala Cys Leu Ala Phe His Tyr Ser Tyr Glu His Gin Asp Lys lie Lys 565 570 575

Ala lie Val His Ala Glu Ser Val Val Asp Val lie Glu Ser Trp Asp 580 585 590

Glu Trp Pro Asp lie Glu Glu Asp lie Ala Leu lie Lys Ser Glu Glu 595 600 605

Gly Glu Lys Met Val Leu Glu Asn Asn Phe Phe Val Glu Thr Met Leu 610 615 620

Pro Ser Lys lie Met Arg Lys Leu Glu Pro Glu Glu Phe Ala Ala Tyr 625 630 635 640

Leu Glu Pro Phe Lys Glu Lys Gly Glu Val Arg Arg Pro Thr Leu Ser 645 650 655

Trp Pro Arg Glu lie Pro Leu Val Lys Gly Gly Lys Pro Asp Val Val 660 665 670

Gin lie Val Arg Asn Tyr Asn Ala Tyr Leu Arg Ala Ser Asp Asp Leu 675 680 685

Pro Lys Met Phe lie Glu Ser Asp Pro Gly Phe Phe Ser Asn Ala lie 690 695 700 -38- 156362 · Sequence Listing.doc 201131168

Val Glu Gly Ala Lys Lys Phe Pro Asn Thr Glu Phe Val Lys Val Lys 705 710 715 720

Gly Leu His Phe Ser Gin Glu Asp Ala Pro Asp Glu Met Gly Lys Tyr 725 730 735 lie Lys Ser Phe Val Glu Arg Val Leu Lys Asn Glu Gin 740 745 <210> 21 <211> 647 <212> PRT <;213>Manual<220><223> derived from human dopamine receptor D3 <400> 21

Met Ala Ser Leu Ser Gin Leu Ser Ser His Leu Asn Tyr Thr Cys Gly 15 10 15

Ala Glu Asn Ser Thr Gly Ala Ser Gin Ala Arg Pro His Ala Tyr Tyr 20 25 30

Ala Leu Ser Tyr Cys Ala Leu lie Leu Ala lie Val Phe Gly Asn Gly 35 40 45

Leu Val Cys Met Ala Val Leu Lys Glu Arg Ala Leu Gin Thr Thr Thr 50 55 60

Asn Tyr Leu Val Val Ser Leu Ala Val Ala Asp Leu Leu Val Ala Thr 65 70 75 80

Leu Val Met Pro Trp Val Val Tyr Leu Glu Val Thr Gly Gly Val Trp 85 90 95

Asn Phe Ser Arg He Cys Cys Asp Val Phe Val Thr Leu Asp Val Met 100 105 110

Met Cys Thr Ala Ser lie Leu Asn Leu Cys Ala lie Ser lie Asp Arg 115 120 125

Tyr Thr Ala Val Val Met Pro Val His Tyr Gin His Gly Thr Gly Gin 130 135 140

Ser Ser Cys Arg Arg Val Ala Leu Met lie Thr Ala Val Trp Val Leu 145 150 155 160 -39- 156362 - Sequence Listing.doc 201131168

Ala Phe Ala Val Ser Cys Pro Leu Leu Phe Gly Phe Asn Thr Thr Gly 165 170 175

Asp Pro Thr Val Cys Ser lie Ser Asn Pro Asp Phe Val lie Tyr Ser 180 185 190

Ser Val Val Ser Phe Tyr Leu Pro Phe Gly Val Thr Val Leu Val Gly 195 200 205

Thr Arg Val Phe Gin Glu Ala Lys Arg Gin Leu Gin Lys lie Asp Lys 210 215 220

Ser Glu Gly Arg Phe His Val Gin Asn Leu Ser Gin Val Glu Gin Asp 225 230 235 240

Gly Arg Thr Gly His Gly Leu Arg Arg Ser Ser Lys Phe Cys Ser Arg 245 250 255

Glu Lys Lys Ala Ala Lys Thr Leu Glu Phe Val Leu Gly Ala Phe lie 260 265 270

Val Cys Trp Leu Pro Phe Phe Leu Thr His Val Leu Asn Thr His Cys 275 280 285

Gin Thr Cys His Val Ser Pro Glu Leu Tyr Ser Ala Thr Thr Trp Leu 290 295 300

Gly Tyr Val Asn Ser Ala Leu Asn Pro Val lie Tyr Thr Thr Phe Asn 305 310 315 320 lie Glu Phe Arg Lys Ala Phe Leu Lys lie Leu Ser Cys Glu Asn Ser 325 330 335

Met Thr Ser Lys Val Tyr Asp Pro Glu Gin Arg Lys Arg Met lie Thr 340 345 350

Gly Pro Gin Trp Trp Ala Arg Cys Lys Gin Met Asn Val Leu Asp Ser 355 360 365

Phe lie Asn Tyr Tyr Asp Ser Glu Lys His Ala Glu Asn Ala Val lie 370 375 380

Phe Leu His Gly Asn Ala Ala Ser Ser Tyr Leu Trp Arg His Val Val 385 390 395 400 -40- 156362 · Sequence Listing.doc 201131168

Pro His lie Glu Pro Val Ala Arg Cys lie lie Pro Asp Leu lie Gly 405 410 415

Met Gly Lys Ser Gly Lys Ser Gly Asn Gly Ser Tyr Arg Leu Leu Asp 420 425 430

His Tyr Lys Tyr Leu Thr Ala Trp Phe Glu Leu Leu Asn Leu Pro Lys 435 440 445

Lys lie Asn Phe Val Gly His Asp Trp Gly Ala Cys Leu Ala Phe His 450 ( 455 460

Tyr Ser Tyr Glu His Gin Asp Lys lie Lys Ala lie Val His Ala Glu 465 470 475 480

Ser Val Val Asp Val lie Glu Ser Trp Asp Glu Trp Pro Asp He Glu 485 490 495

Glu Asp He Ala Leu lie Lys Ser Glu Glu Gly Glu Lys Met Val Leu 500 505 510

Glu Asn Asn Phe Phe Val Glu Thr Met Leu Pro Ser Lys lie Met Arg 515 520 525

Lys Leu Glu Pro Glu Glu Phe Ala Ala Tyr Leu Glu Pro Phe Lys Glu 530 535 540

Lys Gly Glu Val Arg Arg Pro Thr Leu Ser Trp Pro Arg Glu lie Pro 545 550 555 560

Leu Val Lys Gly Gly Lys Pro Asp Val Val Gin lie Val Arg Asn Tyr 565 570 575

Asn Ala Tyr Leu Arg Ala Ser Asp Asp Leu Pro Lys Met Phe lie Glu 580 585 590

Ser Asp Pro Gly Phe Phe Ser Asn Ala lie Val Glu Gly Ala Lys Lys 595 600 605

Phe Pro Asn Thr Glu Phe Val Lys Val Lys Gly Leu His Phe Ser Gin 610 615 620

Glu Asp Ala Pro Asp Glu Met Gly Lys Tyr lie Lys Ser Phe Val Glu 625 630 635 640

Arg Val Leu Lys Asn Glu Gin 645 •41 · 156362 - Sequence Listing.doc 201131168 <210> 22 <211> 655 <212> PRT <213>Manual<220><223> Derived from Human Dopamine Receptor D4 <400> 22

Met Gly Asn Arg Ser Thr Ala Asp Ala Asp Gly Leu Leu Ala Gly Arg 15 10 15

Gly Pro Ala Ala Gly Ala Ser Ala Gly Ala Ser Ala Gly Leu Ala Gly 20 25 30

Gin Gly Ala Ala Ala Leu Val Gly Gly Val Leu Leu lie Gly Ala Val 35 40 45

Leu Ala Gly Asn Ser Leu Val Cys Val Ser Val Ala Thr Glu Arg Ala 50 55 60

Leu Gin Thr Pro Thr Asn Ser Phe lie Val Ser Leu Ala Ala Ala Asp 65 70 75 80

Leu Leu Leu Ala Leu Leu Val Leu Pro Leu Phe Val Tyr Ser Glu Val 85 90 95

Gin Gly Gly Ala Trp Leu Leu Ser Pro Arg Leu Cys Asp Ala Leu Met 100 105 110

Ala Met Asp Val Met Leu Cys Thr Ala Ser lie Phe Asn Leu Cys Ala 115 120 125 lie Ser Val Asp Arg Phe Val Ala Val Ala Val Pro Leu Arg Tyr Asn 130 135 140

Arg Gin Gly Gly Ser Arg Arg Gin Leu Leu Leu lie Gly Ala Thr Trp 145 150 155 160

Leu Leu Ser Ala Ala Val Ala Ala Pro Val Leu Cys Gly Leu Asn Asp 165 170 175

Val Arg Gly Arg Asp Pro Ala Val Cys Arg Leu Glu Asp Arg Asp Tyr 180 185 190

Val Val Tyr Ser Ser Val Cys Ser Phe Phe Leu Pro Cys Pro Leu Met 195 200 205 • 42· 156362 · Sequence Listing.doc 201131168

Leu Leu Leu Tyr Trp Gly Thr Arg Val Phe Gin Glu Ala Lys Arg Gin 210 215 220

Leu Gin Lys lie Asp Lys Ser Glu Gly Arg Phe His Val Gin Asn Leu 225 230 235 240

Ser Gin Val Glu Gin Asp Gly Arg Thr Gly His Gly Leu Arg Arg Ser 245 250 255

Ser Lys Phe Cys Ser Arg Glu Lys Lys Ala Ala Lys Thr Leu Glu Phe 260 265 270

Val Val Gly Ala Phe Leu Leu Cys Trp Thr Pro Phe Phe Val Val His 275 280 285 lie Thr Gin Ala Leu Cys Pro Ala Cys Ser Val Pro Pro Arg Leu Val 290 295 300

Ser Ala Val Thr Trp Leu Gly Tyr Val Asn Ser Aia Leu Asn Pro Val 305 310 315 320 lie Tyr Thr Val Phe Asn Ala Glu Phe Arg Asn Val Phe Arg Lys Ala 325 330 335

Leu Arg Ala Cys Cys Glu Asn Ser Met Thr Ser Lys Val Tyr Asp Pro 340 345 350

Glu Gin Arg Lys Arg Met lie Thr Gly Pro Gin Trp Trp Ala Arg Cys 355 360 365

Lys Gin Met Asn Val Leu Asp Ser Phe lie Asn Tyr Tyr Asp Ser Glu 370 375 380

Lys His Ala Glu Asn Ala Val lie Phe Leu His Gly Asn Ala Ala Ser 385 390 395 400

Ser Tyr Leu Trp Arg His Val Val Pro His lie Glu Pro Val Ala Arg 405 410 415

Cys lie lie Pro Asp Leu lie Gly Met Gly Lys Ser Gly Lys Ser Gly 420 425 430

Asn Gly Ser Tyr Arg Leu Leu Asp His Tyr Lys Tyr Leu Thr Ala Trp 435 440 445 156362 · Sequence Listing. doc -43- 201131168

Phe Glu Leu Leu Asn Leu Pro Lys Lys lie Asn Phe Val Gly His Asp 450 455 460

Trp Gly Ala Cys Leu Ala Phe His Tyr Ser Tyr Glu His Gin Asp Lys 465 470 475 480 lie Lys Ala lie Val His Ala Glu Ser Val Val Asp Val lie Glu Ser 485 490 495

Trp Asp Glu Trp Pro Asp lie Glu Glu Asp lie Ala Leu lie Lys Ser 500 505 510

Glu Glu Gly Glu Lys Met Val Leu Glu Asn Asn Phe Phe Val Glu Thr 515 520 525

Met Leu Pro Ser Lys lie Met Arg Lys Leu Glu Pro Glu Glu Phe Ala 530 535 540

Ala Tyr Leu Glu Pro Phe Lys Glu Lys Gly Glu Val Arg Arg Pro Thr 545 550 555 560

Leu Ser Trp Pro Arg Glu lie Pro Leu Val Lys Gly Gly Lys Pro Asp 565 570 575

Val Val Gin lie Val Arg Asn Tyr Asn Ala Tyr Leu Arg Ala Ser Asp 580 585 590

Asp Leu Pro Lys Met Phe lie Glu Ser Asp Pro Gly Phe Phe Ser Asn 595 600 605

Ala lie Val Glu Gly Ala Lys Lys Phe Pro Asn Thr Glu Phe Val Lys 610 615 620

Val Lys Gly Leu His Phe Ser Gin Glu Asp Ala Pro Asp Glu Met Gly 625 630 635 640

Lys Tyr lie Lys Ser Phe Val Glu Arg Val Leu Lys Asn Glu Gin 645 650 655 <210> 23 <211> 756 <212> PRT <213>Manual<220><223> Derived from human dopamine Receptor D1 • 44- 156362 - Sequence Listing.doc 201131168 <400> 23

Met Arg Thr Leu Asn Thr Ser Ala Met Asp Gly Thr Gly Leu Val Val 15 10 15

Glu Arg Asp Phe Ser Val Arg lie Leu Thr Ala Cys Phe Leu Ser Leu 20 25 30

Leu lie Leu Ser Thr Leu Leu Gly Asn Thr Leu Val Cys Ala Ala Val 35 40 45

He Arg Phe Arg His Leu Arg Ser Lys Val Thr Asn Phe Phe Val lie 50 55 60

Ser Leu Ala Val Ser Asp Leu Leu Val Ala Val Leu Val Met Pro Trp 65 70 75 80

Lys Ala Val Ala Glu lie Ala Gly Phe Trp Pro Phe Gly Ser Phe Cys 85 90 95

Asn He Trp Val Ala Phe Asp lie Met Cys Ser Thr Ala Ser lie Leu 100 105 110

Asn Leu Cys Val lie Ser Val Asp Arg Tyr Trp Ala lie Ser Ser Pro 115 120 125

Phe Arg Tyr Glu Arg Lys Met Thr Pro Lys Ala Ala Phe lie Leu lie 130 135 140

Ser Val Ala Trp Thr Leu Ser Val Leu lie Ser Phe lie Pro Val Gin 145 150 155 160

Leu Ser Trp His Lys Ala Lys Pro Thr Ser Pro Ser Asp Gly Asn Ala 165 170 175

Thr Ser Leu Ala Glu Thr lie Asp Asn Cys Asp Ser Ser Leu Ser Arg 180 185 190

Thr Tyr Ala lie Ser Ser Ser Val lie Ser Phe Tyr lie Pro Val Ala 195 200 205 lie Met lie Val Thr Gly Thr Arg Val Phe Gin Glu Ala Lys Arg Gin 210 215 220

Leu Gin Lys lie Asp Lys Ser Glu Gly Arg Phe His Val Gin Asn Leu 225 230 235 240 •45· 156362-Sequence List.doc 201131168

Ser Gin Val Glu Gin Asp Gly Arg Thr Gly His Gly Leu Arg Arg Ser 245 250 255

Ser Lys Phe Cys Ser Arg Glu Lys Lys Ala Ala Lys Thr Leu Glu Phe 260 265 270 lie Met Gly Val Phe Val Cys Cys Trp Leu Pro Phe Phe lie Leu Asn 275 280 285

Cys lie Leu Pro Phe Cys Gly Ser Gly Glu Thr Gin Pro Phe Cys lie 290 295 300

Asp Ser Asn Thr Phe Asp Val Phe Val Trp Phe Gly Trp Ala Asn Ser 305 310 315 320

Ser Leu Asn Pro He He Tyr Ala Phe Asn Ala Asp Phe Arg Lys Ala 325 330 335

Phe Ser Thr Leu Leu Gly Cys Tyr Arg Leu Cys Pro Ala Thr Asn Asn 340 345 350

Ala lie Glu Thr Val Ser lie Asn Asn Asn Gly Ala Ala Met Phe Ser 355 360 365

Ser His His Glu Pro Arg Gly Ser lie Ser Lys Glu Cys Asn Leu Val 370 375 380

Tyr Leu lie Pro His Ala Val Gly Ser Ser Glu Asp Leu Lys Lys Glu 385 390 395 400

Glu Ala Ala Gly lie Ala Arg Pro Leu Glu Lys Leu Ser Pro Ala Leu 405 410 415

Ser Val lie Leu Asp Tyr Asp Thr Asp Val Ser Leu Glu Lys He Gin 420 425 430

Pro Met Thr Gin Asn Gly Gin His Pro Thr Glu Asn Ser Met Thr Ser 435 440 445

Lys Val Tyr Asp Pro Glu Gin Arg Lys Arg Met lie Thr Gly Pro Gin 450 455 460

Trp Trp Ala Arg Cys Lys Gin Met Asn Val Leu Asp Ser Phe lie Asn 465 470 475 480 • 46· 156362 - Sequence Listing.doc 201131168

Tyr Tyr Asp Ser Glu Lys His Ala Glu Asn Ala Val lie Phe Leu His 485 490 495

Gly Asn Ala Ala Ser Ser Tyr Leu Trp Arg His Val Val Pro His lie 500 505 510

Glu Pro Val Ala Arg Cys lie lie Pro Asp Leu lie Gly Met Gly Lys 515 520 525

Ser Gly Lys Ser Gly Asn Gly Ser Tyr Arg Leu Leu Asp His Tyr Lys 530 535 540

Tyr Leu Thr Ala Trp Phe Glu Leu Leu Asn Leu Pro Lys Lys lie Asn 545 550 555 560

Phe Val Gly His Asp Trp Gly Ala Cys Leu Ala Phe His Tyr Ser Tyr 565 570 575

Glu His Gin Asp Lys lie Lys Ala lie Val His Ala Glu Ser Val Val 580 585 590

Asp Val lie Glu Ser Trp Asp Glu Trp Pro Asp lie Glu Glu Asp lie 595 600 605

Ala Leu lie Lys Ser Glu Glu Gly Glu Lys Met Val Leu Glu Asn Asn 610 615 620

Phe Phe Val Glu Thr Met Leu Pro Ser Lys lie Met Arg Lys Leu Glu 625 630 635 640

Pro Glu Glu Phe Ala Ala Tyr Leu Glu Pro Phe Lys Glu Lys Gly Glu 645 650 655

Val Arg Arg Pro Thr Leu Ser Trp Pro Arg Glu lie Pro Leu Val Lys 660 665 670

Gly Gly Lys Pro Asp Val Val Gin lie Val Arg Asn Tyr Asn Ala Tyr 675 680 685

Leu Arg Ala Ser Asp Asp Leu Pro Lys Met Phe lie Glu Ser Asp Pro 690 695 700

Gly Phe Phe Ser Asn Ala lie Val Glu Gly Ala Lys Lys Phe Pro Asn 705 710 715 720

Thr Glu Phe Val Lys Val Lys Gly Leu His Phe Ser Gin Glu Asp Ala 725 730 735 -47- 156362 · Sequence Listing.doc 201131168

Pro Asp Glu Met Gly Lys Tyr lie Lys Ser Phe Val Glu Arg Val Leu 740 745 750

Lys Asn Glu Gin 755 <210> 24 <211> 695 <212> PRT <213> Labor <220><223> Derived from human histamine receptor H2 <400>

Met Ala Pro Asn Gly Thr Ala Ser Ser Phe Cys Leu Asp Ser Thr Ala 1 5 10 15

Cys Lys lie Thr lie Thr Val Val Leu Ala Val Leu lie Leu lie Thr 20 25 30

Val Ala Gly Asn Val Val Val Cys Leu Ala Val Gly Leu Asn Arg Arg 35 40 45

Leu Arg Asn Leu Thr Asn Cys Phe lie Val Ser Leu Ala lie Thr Asp 50 55 60

Leu Leu Leu Leu Gly Leu Leu Val Leu Pro Phe Ser Ala lie Tyr Gin Leu 65 70 75 80

Ser Cys Lys Trp Ser Phe Gly Lys Val Phe Cys Asn lie Tyr Thr Ser 85 90 95

Leu Asp Val Met Leu Cys Thr Ala Ser lie Leu Asn Leu Phe Met He 100 105 110

Ser Leu Asp Arg Tyr Cys Ala Val Met Asp Pro Leu Arg Tyr Pro Val 115 120 125

Leu Val Thr Pro Val Arg Val Ala lie Ser Leu Val Leu lie Trp Val 130 135 140

He Ser lie Thr Leu Ser Phe Leu Ser lie His Leu Gly Trp Asn Ser 145 150 155 160

Arg Asn Glu Thr Ser Lys Gly Asn His Thr Thr Ser Lys Cys Lys Val 165 170 175 •48- 156362 · Sequence Listing.doc 201131168

Gin Val Asn Glu Val Tyr Gly Leu Val Asp Gly Leu Val Thr Phe Tyr 180 185 190

Leu Pro Leu Leu lie Met Cys lie Thr Gly Thr Arg Val Phe Gin Glu 195 200 205

Ala Lys Arg Gin Leu Gin Lys lie Asp Lys Ser Glu Gly Arg Phe His 210 215 220

Val Gin Asn Leu Ser Gin Val Glu Gin Asp Gly Arg Thr Gly His Gly 225 230 235 240

Leu Arg Arg Ser Ser Lys Phe Cys Ser Arg Glu Lys Lys Ala Ala Lys 245 250 255

Thr Leu Glu Phe Val Met Gly Ala Phe lie lie Cys Trp Phe Pro Tyr 260 265 270

Phe Thr Ala Phe Val Tyr Arg Gly Leu Arg Gly Asp Asp Ala lie Asn 275 280 285

Glu Val Leu Glu Ala lie Val Leu Trp Leu Gly Tyr Ala Asn Ser Ala 290 295 300

Leu Asn Pro lie Leu Tyr Ala Ala Leu Asn Arg Asp Phe Arg Thr Gly 305 310 315 320

Tyr Gin Gin Leu Phe Cys Cys Arg Leu Ala Asn Arg Asn Ser His Lys 325 330 335

Thr Ser Leu Arg Ser Asn Ala Ser Gin Leu Ser Arg Thr Gin Ser Arg 340 345 350

Glu Pro Arg Gin Gin Glu Glu Lys Pro Leu Lys Leu Gin Val Trp Ser 355 360 365

Gly Thr Glu Val Thr Ala Pro Gin Gly Ala Thr Asp Arg Glu Asn Ser 370 375 380

Met Thr Ser Lys Val Tyr Asp Pro Glu Gin Arg Lys Arg Met lie Thr 385 390 395 400

Gly Pro Gin Trp Trp Ala Arg Cys Lys Gin Met Asn Val Leu Asp Ser 405 410 415 •49· 156362·Sequence List.doc 201131168

Phe lie Asn Tyr Tyr Asp Ser Glu Lys His Ala Glu Asn Ala Val lie 420 425 430

Phe Leu His Gly Asn Ala Ala Ser Ser Tyr Leu Trp Arg His Val Val 435 440 445

Pro His lie Glu Pro Val Ala Arg Cys lie lie Pro Asp Leu lie Gly 450 455 460

Met Gly Lys Ser Gly Lys Ser Gly Asn Gly Ser Tyr Arg Leu Leu Asp 465 470 475 480

His Tyr Lys Tyr Leu Thr Ala Trp Phe Glu Leu Leu Asn Leu Pro Lys 485 490 495

Lys lie Asn Phe Val Gly His Asp Trp Gly Ala Cys Leu Ala Phe His 500 505 510

Tyr Ser Tyr Glu His Gin Asp Lys lie Lys Ala lie Val His Ala Glu 515 520 525

Ser Val Val Asp Val lie Glu Ser Trp Asp Glu Trp Pro Asp lie Glu 530 535 540

Glu Asp He Ala Leu lie Lys Ser Glu Glu Gly Glu Lys Met Val Leu 545 550 555 560

Glu Asn Asn Phe Phe Val Glu Thr Met Leu Pro Ser Lys lie Met Arg 565 570 575

Lys Leu Glu Pro Glu Glu Phe Ala Ala Tyr Leu Glu Pro Phe Lys Glu 580 585 590

Lys Gly Glu Val Arg Arg Pro Thr Leu Ser Trp Pro Arg Glu lie Pro 595 600 605

Leu Val Lys Gly Gly Lys Pro Asp Val Val Gin lie Val Arg Asn Tyr 610 615 620

Asn Ala Tyr Leu Arg Ala Ser Asp Asp Leu Pro Lys Met Phe lie Glu 625 630 635 640

Ser Asp Pro Gly Phe Phe Ser Asn Ala lie Val Glu Gly Ala Lys Lys 645 650 655 • 50· 156362 · Sequence Listing.doc 201131168

Phe Pro Asn Thr Glu Phe Val Lys Val Lys Gly Leu His Phe Ser Gin 660 665 670

Glu Asp Ala Pro Asp Glu Met Gly Lys Tyr lie Lys Ser Phe Val Glu 675 680 685

Arg Val Leu Lys Asn Glu Gin 690 695 <210> 25 <211> 673 <212> PRT <213> Labor <220><223> Derived from human histamine receptor H3 <400>

Met Glu Arg Ala Pro Pro Asp Gly Pro Leu Asn Ala Ser Gly Ala Leu 1 5 10 15

Ala Gly Asp Ala Ala Ala Ala Gly Gly Ala Arg Gly Phe Ser Ala Ala 20 25 30

Trp Thr Ala Val Leu Ala Ala Leu Met Ala Leu Leu lie Val Ala Thr 35 40 45

Val Leu Gly Asn Ala Leu Val Met Leu Ala Phe Val Ala Asp Ser Ser 50 55 60

Leu Arg Thr Gin Asn Asn Phe Phe Leu Leu Asn Leu Ala lie Ser Asp 65 70 75 80

Phe Leu Val Gly Ala Phe Cys lie Pro Leu Tyr Val Pro Tyr Val Leu 85 90 95

Thr Gly Arg Trp Thr Phe Gly Arg Gly Leu Cys Lys Leu Trp Leu Val 100 105 110

Val Asp Tyr Leu Leu Cys Thr Ser Ser Ala Phe Asn lie Val Leu lie 115 120 125

Ser Tyr Asp Arg Phe Leu Ser Val Thr Arg Ala Val Ser Tyr Arg Ala 130 135 140

Gin Gin Gly Asp Thr Arg Arg Ala Val Arg Lys Met Leu Leu Val Trp 145 150 155 160 -51- 156362 · Sequence Listing.doc 201131168

Val Leu Ala Phe Leu Leu Tyr Gly Pro Ala lie Leu Ser Trp Glu Tyr 165 170 175

Leu Ser Gly Gly Ser Ser lie Pro Glu Gly His Cys Tyr Ala Glu Phe 180 185 190

Phe Tyr Asn Trp Tyr Phe Leu lie Thr Ala Ser Thr Leu Glu Phe Phe 195 200 205

Thr Pro Phe Leu Ser Val Thr Phe Phe Gly Thr Arg Val Phe Gin Glu 210 215 220

Ala Lys Arg Gin Leu Gin Lys lie Asp Lys Ser Glu Gly Arg Phe His 225 230 235 240

Val Gin Asn Leu Ser Gin Val Glu Gin Asp Gly Arg Thr Gly His Gly 245 250 255

Leu Arg Arg Ser Ser Lys Phe Cys Ser Arg Glu Lys Lys Ala Ala Lys 260 265 270

Thr Leu Glu Phe He Val Ser lie Phe Gly Leu Cys Trp Ala Pro Tyr 275 280 285

Thr Leu Leu Met lie lie Arg Ala Ala Cys His Gly His Cys Val Pro 290 295 300

Asp Tyr Trp Tyr Glu Thr Ser Phe Trp Leu Leu Trp Ala Asn Ser Ala 305 310 315 320

Val Asn Pro Val Leu Tyr Pro Leu Cys His His Ser Phe Arg Arg Ala 325 330 335

Phe Thr Lys Leu Leu Cys Pro Gin Lys Leu Lys lie Gin Pro His Ser 340 345 350

Ser Leu Glu His Cys Trp Lys Glu Asn Ser Met Thr Ser Lys Val Tyr 355 360 365

Asp Pro Glu Gin Arg Lys Arg Met lie Thr Gly Pro Gin Trp Trp Ala 370 375 380

Arg Cys Lys Gin Met Asn Val Leu Asp Ser Phe lie Asn Tyr Tyr Asp 385 390 395 400 -52- 156362 - Sequence Listing.doc 201131168

Ser Glu Lys His Ala Glu Asn Ala Val lie Phe Leu His Gly Asn Ala 405 410 415

Ala Ser Ser Tyr Leu Trp Arg His Val Val Pro His lie Glu Pro Val 420 425 430

Ala Arg Cys lie lie Pro Asp Leu lie Gly Met Gly Lys Ser Gly Lys 435 440 445

Ser Gly Asn Gly Ser Tyr Arg Leu Leu Asp His Tyr Lys Tyr Leu Thr 450 455 460

Ala Trp Phe Glu Leu Leu Asn Leu Pro Lys Lys He Asn Phe Val Gly 465 470 475 480

His Asp Trp Gly Ala Cys Leu Ala Phe His Tyr Ser Tyr Glu His Gin 485 490 495

Asp Lys lie Lys Ala lie Val His Ala Glu Ser Val Val Asp Val lie 500 505 510

Glu Ser Trp Asp Glu Trp Pro Asp lie Glu Glu Asp lie Ala Leu lie 515 520 525

Lys Ser Glu Glu Gly Glu Lys Met Val Leu Glu Asn Asn Phe Phe Val 530 535 540

Glu Thr Met Leu Pro Ser Lys lie Met Arg Lys Leu Glu Pro Glu Glu 545 550 555 560

Phe Ala Ala Tyr Leu Glu Pro Phe Lys Glu Lys Gly Glu Val Arg Arg 565 570 575

Pro Thr Leu Ser Trp Pro Arg Glu lie Pro Leu Val Lys Gly Gly Lys 580 585 590

Pro Asp Val Val Gin lie Val Arg Asn Tyr Asn Ala Tyr Leu Arg Ala 595 600 605

Ser Asp Asp Leu Pro Lys Met Phe lie Glu Ser Asp Pro Gly Phe Phe 610 615 620

Ser Asn Ala lie Val Glu Gly Ala Lys Lys Phe Pro Asn Thr Glu Phe 625 630 635 640

Val Lys Val Lys Gly Leu His Phe Ser Gin Glu Asp Ala Pro Asp Glu 645 650 655 •53· 156362-Sequence List.doc 201131168

Met Gly Lys Tyr lie Lys Ser Phe Val Glu Arg Val Leu Lys Asn Glu 660 665 670

Gin <210> 26 <211> 660 <212> PRT <213>manual <220><223> derived from porcine adrenergic receptor, alpha-2A <400>

Met Gly Ser Leu Gin Pro Glu Ala Gly Asn Ala Ser Trp Asn Gly Thr 15 10 15

Glu Ala Pro Gly Gly Gly Ala Arg Ala Thr Pro Tyr Ser Leu Gin Val 20 25 30

Thr Leu Thr Leu Val Cys Leu Ala Gly Leu Leu Met Leu Phe Thr Val 35 40 45

Phe Gly Asn Val Leu Val lie lie Ala Val Phe Thr Ser Arg Ala Leu 50 55 60

Lys Ala Pro Gin Asn Leu Phe Leu Val Ser Leu Ala Ser Ala Asp lie 65 70 75 80

Leu Val Ala Thr Leu Val lie Pro Phe Ser Leu Ala Asn Glu Val Met 85 90 95

Gly Tyr Trp Tyr Phe Gly Lys Ala Trp Cys Glu lie Tyr Leu Ala Leu 100 105 110

Asp Val Leu Phe Cys Thr Ser Ser lie Val His Leu Cys Ala lie Ser 115 120 125

Leu Asp Arg Tyr Trp Ser He Thr Gin Ala lie Glu Tyr Asn Leu Lys 130 135 140

Arg Thr Pro Arg Arg lie Lys Ala lie lie Val Thr Val Trp Val lie 145 150 155 160

Ser Ala Val He Ser Phe Pro Pro Leu lie Ser lie Glu Lys Lys Ala 165 170 175 -54- 156362 - Sequence Listing.doc 201131168

Gly Gly Gly Gly Gin Gin Pro Ala Glu Pro Arg Cys Glu lie Asn Asp 180 185 190

Gin Lys Trp Tyr Val lie Ser Ser Cys lie Gly Ser Phe Phe Ala Pro 195 200 205

Cys Leu lie Met lie Leu Val Gly Thr Arg Val Phe Gin Glu Ala Lys 210 215 220

Arg Gin Leu Gin Lys lie Asp Lys Ser Glu Gly Arg Phe His Val Gin 225 230 235 240

Asn Leu Ser Gin Val Glu Gin Asp Gly Arg Thr Gly His Gly Leu Arg 245 250 255

Arg Ser Ser Lys Phe Cys Ser Arg Glu Lys Lys Ala Ala Lys Thr Leu 260 265 270

Glu Phe Val He Gly Val Phe Val Val Cys Trp Phe Pro Phe Phe Phe 275 280 285

Thr Tyr Thr Leu Thr Ala Val Gly Cys Ser Val Pro Pro Thr Leu Phe 290 295 300

Lys Phe Phe Phe Trp Phe Gly Tyr Cys Asn Ser Ser Leu Asn Pro Val 305 310 315 320 lie Tyr Thr lie Phe Asn His Asp Phe Arg Arg Ala Phe Lys Lys lie 325 330 335

Leu Cys Arg Gly Asp Arg Lys Arg lie Val Glu Asn Ser Met Thr Ser 340 345 350

Lys Val Tyr Asp Pro Glu Gin Arg Lys Arg Met lie Thr Gly Pro Gin 355 360 365

Trp Trp Ala Arg Cys Lys Gin Met Asn Val Leu Asp Ser Phe lie Asn 370 375 380

Tyr Tyr Asp Ser Glu Lys His Ala Glu Asn Ala Val lie Phe Leu His 385 390 395 400

Gly Asn Ala Ala Ser Ser Tyr Leu Trp Arg His Val Val Pro His lie 405 410 415 •55· 156362-Sequence List.doc 201131168

Glu Pro Val Ala Arg Cys lie lie Pro Asp Leu lie Gly Met Gly Lys 420 425 430

Ser Gly Lys Ser Gly Asn Gly Ser Tyr Arg Leu Leu Asp His Tyr Lys 435 440 445

Tyr Leu Thr Ala Trp Phe Glu Leu Leu Asn Leu Pro Lys Lys He Asn 450 455 460

Phe Val Gly His Asp Trp Gly Ala Cys Leu Ala Phe His Tyr Ser Tyr 465 470 475 480

Glu His Gin Asp Lys lie Lys Ala lie Val His Ala Glu Ser Val Val 485 490 495

Asp Val lie Glu Ser Trp Asp Glu Trp Pro Asp lie Glu Glu Asp lie 500 505 510

Ala Leu He Lys Ser Glu Glu GJy Glu Lys Met Val Leu Glu Asn Asn 515 520 525

Phe Phe Val Glu Thr Met Leu Pro Ser Lys lie Met Arg Lys Leu Glu 530 535 540

Pro Glu Glu Phe Ala Ala Tyr Leu Glu Pro Phe Lys Glu Lys Gly Glu 545 550 555 560

Val Arg Arg Pro Thr Leu Ser Trp Pro Arg Giu lie Pro Leu Val Lys 565 570 575

Gly Gly Lys Pro Asp Val Val Gin lie Val Arg Asn Tyr Asn Ala Tyr 580 585 590

Leu Arg Ala Ser Asp Asp Leu Pro Lys Met Phe lie Glu Ser Asp Pro 595 600 605

Gly Phe Phe Ser Asn Ala lie Val Glu Gly Ala Lys Lys Phe Pro Asn 610 615 620

Thr Glu Phe Val Lys Val Lys Gly Leu His Phe Ser Gin Glu Asp Ala 625 630 635 640

Pro Asp Glu Met Gly Lys Tyr lie Lys Ser Phe Val Glu Arg Val Leu 645 650 655 -56- 156362 - Sequence Listing.doc 201131168

Lys Asn Glu Gin 660 <210> 27 <211> 690 <212> PRT <213> Labor <220><223> Derived from human galansin receptor 1 <400>

Met Glu Leu Ala Val Gly Asn Leu Ser Glu Gly Asn Ala Ser Cys Pro 15 10 15

Glu Pro Pro Ala Pro Glu Pro Gly Pro Leu Phe Gly lie Gly Val Glu 20 25 30

Asn Phe Val Thr Leu Val Val Phe Gly Leu lie Phe Ala Leu Gly Val 35 40 45

Leu Gly Asn Ser Leu Val lie Thr Val Leu Ala Arg Ser Lys Pro Gly 50 55 60

Lys Pro Arg Ser Thr Thr Asn Leu Phe lie Leu Asn Leu Ser lie Ala 65 70 75 80

Asp Leu Ala Tyr Leu Leu Phe Cys lie Pro Phe Gin Ala Thr Val Tyr 85 90 95

Ala Leu Pro Thr Trp Val Leu Gly Ala Phe lie Cys Lys Phe He His 100 105 110

Tyr Phe Phe Thr Val Ser Met Leu Val Ser lie Phe Thr Leu Ala Ala 115 120 125

Met Ser Val Asp Arg Tyr Val Ala lie Val His Ser Arg Arg Ser Ser 130 135 140

Ser Leu Arg Val Ser Arg Asn Ala Leu Leu Gly Val Gly Cys lie Trp 145 150 155 160

Ala Leu Ser lie Ala Met Ala Ser Pro Val Ala Tyr His Gin Gly Leu 165 170 175

Phe His Pro Arg Ala Ser Asn Gin Thr Phe Cys Trp Glu Gin Trp Pro 180 185 190 -57· 156362-Sequence List.doc 201131168

Asp Pro Arg His Lys Lys Ala Tyr Val Val Cys Thr Phe Val Phe Gly 195 200 205

Tyr Leu Leu Leu Leu Leu Leu lie Cys Phe Cys Gly Thr Arg Val Phe 210 215 220

Gin Glu Ala Lys Arg Gin Leu Gin Lys lie Asp Lys Ser Glu Gly Arg 225 230 235 240

Phe His Val Gin Asn Leu Ser Gin Val Glu Gin Asp Gly Arg Thr Gly 245 250 255

His Gly Leu Arg Arg Ser Ser Lys Phe Cys Ser Arg Glu Lys Lys Ala 260 265 270

Ala Lys Thr Leu Glu Phe Val Val Val Val Phe Gly He Ser Trp Leu 275 280 285

Pro His His lie lie His Leu Trp Ala Glu Phe Gly Val Phe Pro Leu 290 295 300

Thr Pro Ala Ser Phe Leu Phe Arg lie Thr Ala His Cys Leu Ala Tyr 305 310 315 320

Ser Asn Ser Ser Val Asn Pro lie lie Tyr Ala Phe Leu Ser Glu Asn 325 330 335

Phe Arg Lys Ala Tyr Lys Gin Val Phe Lys Cys His lie Arg Lys Asp 340 345 350

Ser His Leu Ser Asp Thr Lys Glu Asn Lys Ser Arg lie Asp Thr Pro 355 360 365

Pro Ser Thr Asn Cys Thr His Val Glu Asn Ser Met Thr Ser Lys Val 370 375 380

Tyr Asp Pro Glu Gin Arg Lys Arg Met lie Thr Gly Pro Gin Trp Trp 385 390 395 400

Ala Arg Cys Lys Gin Met Asn Val Leu Asp Ser Phe lie Asn Tyr Tyr 405 410 415

Asp Ser Glu Lys His Ala Glu Asn Ala Val lie Phe Leu His Gly Asn 420 425 430 •58· 156362-Sequence List.doc 201131168

Ala Ala Ser Ser Tyx Leu Trp Arg His Val Val Pro His lie Glu Pro 435 440 445

Val Ala Arg Cys lie lie Pro Asp Leu lie Gly Met Gly Lys Ser Gly 450 455 460

Lys Ser Gly Asn Gly Ser Tyr Arg Leu Leu Asp His Tyr Lys Tyr Leu 465 470 475 480

Thr Ala Trp Phe Glu Leu Leu Asn Leu Pro Lys Lys lie Asn Phe Val 485 490 495

Gly His Asp Trp Gly Ala Cys Leu Ala Phe His Tyr Ser Tyr Glu His 500 505 510

Gin Asp Lys lie Lys Ala lie Val His Ala Glu Ser Val Val Asp Val 515 520 525 lie Glu Ser Trp Asp Glu Trp Pro Asp lie Glu Glu Asp lie Ala Leu 530 535 540 lie Lys Ser Glu Glu Gly Glu Lys Met Val Leu Glu Asn Asn Phe Phe 545 550 555 560

Val Glu Thr Met Leu Pro Ser Lys lie Met Arg Lys Leu Glu Pro Glu 565 570 575

Glu Phe Ala Ala Tyr Leu Glu Pro Phe Lys Glu Lys Gly Glu Val Arg 580 585 590

Arg Pro Thr Leu Ser Trp Pro Arg Glu lie Pro Leu Val Lys Gly Gly 595 600 605

Lys Pro Asp Val Val Gin lie Val Arg Asn Tyr Asn Ala Tyr Leu Arg 610 615 620

Ala Ser Asp Asp Leu Pro Lys Met Phe lie Glu Ser Asp Pro Gly Phe 625 630 635 640

Phe Ser Asn Ala He Val Glu Gly Ala Lys Lys Phe Pro Asn Thr Glu 645 650 655

Phe Val Lys Val Lys Gly Leu His Phe Ser Gin Glu Asp Ala Pro Asp 660 665 670

Glu Met Gly Lys Tyr lie Lys Ser Phe Val Glu Arg Val Leu Lys Asn 675 680 685 •59- 156362-Sequence List.doc 201131168

Glu Gin 690 <210> 28 <211> 720 <212> PRT < 213 > Labor <220><223> Derived from Human Neuropeptide Y Receptor Y1 <400>

Met Asn Ser Thr Leu Phe Ser Gin Val Glu Asn His Ser Val His Ser 15 10 15

Asn Phe Ser Glu Lys Asn Ala Gin Leu Leu Ala Phe Glu Asn Asp Asp 20 25 30

Cys His Leu Pro Leu Ala Met lie Phe Thr Leu Ala Leu Ala Tyr Gly 35 40 45

Ala Val lie lie Leu Gly Val Ser Gly Asn Leu Ala Leu lie lie lie 50 55 60 lie Leu Lys Gin Lys Glu Met Arg Asn Val Thr Asn lie Leu lie Val 65 70 75 80

Asn Leu Ser Phe Ser Asp Leu Leu Val Ala lie Met Cys Leu Pro Phe 85 90 95

Thr Phe Val Tyr Thr Leu Met Asp His Trp Val Phe Gly Glu Ala Met 100 105 110

Cys Lys Leu Asn Pro Phe Val Gin Cys Val Ser lie Thr Val Ser lie 115 120 125

Phe Ser Leu Val Leu lie Ala Val Glu Arg His Gin Leu lie lie Asn 130 135 140

Pro Arg Gly Trp Arg Pro Asn Asn Arg His Ala Tyr Val Gly lie Ala 145 150 155 360

Val lie Trp Val Leu Ala Val Ala Ser Ser Leu Pro Phe Leu lie Tyr 165 170 175

Gin Val Met Thr Asp Glu Pro Phe Gin Asn Val Thr Leu Asp Ala Tyr 180 185 190 -60- 156362 - Sequence Listing.doc 201131168

Lys Asp Lys Tyr Val Cys Phe Asp Gin Phe Pro Ser Asp Ser His Arg 195 200 205

Leu Ser Tyr Thr Thr Leu Leu Leu Val Leu Gin Tyr Phe Gly Pro Leu 210 215 220

Cys Phe lie Phe lie Cys Gly Thr Arg Val Phe Gin Glu Ala Lys Arg 225 230 235 240

Gin Leu Gin Lys lie Asp Lys Ser Glu Gly Arg Phe His Val Gin Asn 245 250 255

Leu Ser Gin Val Glu Gin Asp Gly Arg Thr Gly His Gly Leu Arg Arg 260 265 270

Ser Ser Lys Phe Cys Ser Arg Glu Lys Lys Ala Ala Lys Thr Leu Glu 275 280 285

Phe lie Val Val Ala Phe Ala Val Cys Trp Leu Pro Leu Thr lie Phe 290 295 300

Asn Thr Val Phe Asp Trp Asn His Gin lie lie Ala Thr Cys Asn His 305 310 315 320

Asn Leu Leu Phe Leu Leu Cys His Leu Thr Ala Met lie Ser Thr Cys 325 330 335

Val Asn Pro lie Phe Tyr Gly Phe Leu Asn Lys Asn Phe Gin Arg Asp 340 345 350

Leu Gin Phe Phe Phe Asn Phe Cys Asp Phe Arg Ser Arg Asp Asp Asp 355 360 365

Tyr Glu Thr lie Ala Met Ser Thr Met His Thr Asp Val Ser Lys Thr 370 375 380

Ser Leu Lys Gin Ala Ser Pro Val Ala Phe Lys Lys lie Asn Asn Asn 385 390 395 400

Asp Asp Asn Glu Lys lie Glu Asn Ser Met Thr Ser Lys Val Tyr Asp 405 410 415

Pro Glu Gin Arg Lys Arg Met lie Thr Gly Pro Gin Trp Trp Ala Arg 420 425 430 156362 - Sequence Listing.doc -61 · 201131168

Cys Lys Gin Met Asn Val Leu Asp Ser Phe lie Asn Tyr Tyr Asp Ser 435 440 445

Glu Lys His Ala Glu Asn Ala Val lie Phe Leu His Gly Asn Ala Ala 450 455 460

Ser Ser Tyr Leu Trp Arg His Val Val Pro His lie Glu Pro Val Ala 465 470 475 480

Arg Cys He lie Pro Asp Leu lie Gly Met Gly Lys Ser Gly Lys Ser 485 490 495

Gly Asn Gly Ser Tyr Arg Leu Leu Asp His Tyr Lys Tyr Leu Thr Ala 500 505 510

Trp Phe Glu Leu Leu Asn Leu Pro Lys Lys lie Asn Phe Val Gly His 515 520 525

Asp Trp Gly Ala Cys Leu Ala Phe His Tyr Ser Tyr Glu His Gin Asp 530 535 540

Lys lie Lys Ala lie Val His Ala Glu Ser Val Val Asp Val lie Glu 545 550 555 560

Ser Trp Asp Glu Trp Pro Asp lie Glu Glu Asp lie Ala Leu lie Lys 565 570 575

Ser Glu Glu Gly Glu Lys Met Val Leu Glu Asn Asn Phe Phe Val Glu 580 585 590

Thr Met Leu Pro Ser Lys He Met Arg Lys Leu Glu Pro Glu Glu Phe 595 600 605

Ala Ala Tyr Leu Glu Pro Phe Lys Glu Lys Gly Glu Val Arg Arg Pro 610 615 620

Thr Leu Ser Trp Pro Arg Glu lie Pro Leu Val Lys Gly Gly Lys Pro 625 630 635 640

Asp Val Val Gin lie Val Arg Asn Tyr Asn Ala Tyr Leu Arg Ala Ser 645 650 655

Asp Asp Leu Pro Lys Met Phe lie Glu Ser Asp Pro Gly Phe Phe Ser 660 665 670 -62- 156362 · Sequence Listing.doc 201131168

Asn Ala lie Val Glu Gly Ala Lys Lys Phe Pro Asn Thr Glu Phe Val 675 680 685

Lys Val Lys Gly Leu His Phe Ser Gin Glu Asp Ala Pro Asp Glu Met 690 695 700

Gly Lys Tyr lie Lys Ser Phe Val Glu Arg Val Leu Lys Asn Glu Gin 705 710 715 720 <210> 29 <211> 702 <212> PRT <213>Manual<220><223> Human angiotensin II receptor 1 <400> 29

Met lie Leu Asn Ser Ser Thr Glu Asp Gly lie Lys Arg lie Gin Asp 15 10 15

Asp Cys Pro Lys Ala Gly Arg His Asn Tyr lie Phe Val Met He Pro 20 25 30

Thr Leu Tyr Ser lie lie Phe Val Val Gly lie Phe Gly Asn Ser Leu 35 40 45

Val Val lie Val lie Tyr Phe Tyr Met Lys Leu Lys Thr Val Ala Ser 50 55 60

Val Phe Leu Leu Asn Leu Ala Leu Ala Asp Leu Cys Phe Leu Leu Thr 65 70 75 80

Leu Pro Leu Trp Ala Val Tyr Thr Ala Met Glu Tyr Arg Trp Pro Phe 85 90 95

Gly Asn Tyr Leu Cys Lys lie Ala Ser Ala Ser Val Ser Phe Asn Leu 100 105 110

Tyr Ala Ser Val Phe Leu Leu Thr Cys Leu Ser lie Asp Arg Tyr Leu 115 120 125

Ala He Val His Pro Met Lys Ser Arg Leu Arg Arg Thr Met Leu Val 130 135 140

Ala Lys Val Thr Cys lie lie lie Trp Leu Leu Ala Gly Leu Ala Ser 145 150 155 160 •63· 156362-Sequence List.doc 201131168

Leu Pro Ala lie lie His Arg Asn Val Phe Phe lie Glu Asn Thr Asn 165 170 175 lie Thr Val Cys Ala Phe His Tyr Glu Ser Gin Asn Ser Thr Leu Pro 180 185 190 lie Gly Leu Gly Leu Thr Lys Asn lie Leu Gly Phe Leu Phe Pro Phe 195 200 205

Leu lie He Leu Thr Ser Gly Thr Arg Val Phe Gin Glu Ala Lys Arg 210 215 220

Gin Leu Gin Lys lie Asp Lys Ser Glu Gly Arg Phe His Val Gin Asn 225 230 235 240

Leu Ser Gin Val Glu Gin Asp Gly Arg Thr Gly His Gly Leu Arg Arg 245 250 255

Ser Ser Lys Phe Cys Ser Arg Glu Lys Lys Ala Ala Lys Thr Leu Glu 260 265 270

Phe He Val Leu Phe Phe Phe Phe Ser Trp lie Pro His Gin lie Phe 275 280 285

Thr Phe Leu Asp Val Leu lie Gin Leu Gly lie lie Arg Asp Cys Arg 290 295 300 lie Ala Asp lie Val Asp Thr Ala Met Pro lie Thr lie Cys lie Ala 305 310 315 320

Tyr Phe Asn Asn Cys Leu Asn Pro Leu Phe Tyr Gly Phe Leu Gly Lys 325 330 335

Lys Phe Lys Arg Tyr Phe Leu Gin Leu Leu Lys Tyr lie Pro Pro Lys 340 345 350

Ala Lys Ser His Ser Asn Leu Ser Thr Lys Met Ser Thr Leu Ser Tyr 355 360 365

Arg Pro Ser Asp Asn Val Ser Ser Ser Thr Lys Lys Pro Ala Pro Cys 370 375 380

Phe Glu Val Glu Glu Asn Ser Met Thr Ser Lys Val Tyr Asp Pro Glu 385 390 395 400 -64- 156362 · Sequence Listing.doc 201131168

Gin Arg Lys Arg Met lie Thr Gly Pro Gin Trp Trp Ala Arg Cys Lys 405 410 415

Gin Met Asn Val Leu Asp Ser Phe He Asn Tyr Tyr Asp Ser Glu Lys 420 425 430

His Ala Glu Asn Ala Val lie Phe Leu His Gly Asn Ala Ala Ser Ser 435 440 445

Tyr Leu Trp Arg His Val Val Pro His lie Glu Pro Val Ala Arg Cys 450 455 460 lie lie Pro Asp Leu lie Gly Met Gly Lys Ser Gly Lys Ser Gly Asn 465 470 475 480

Gly Ser Tyr Arg Leu Leu Asp His Tyr Lys Tyr Leu Thr Ala Trp Phe 485 490 495

Glu Leu Leu Asn Leu Pro Lys Lys lie Asn Phe Val Gly His Asp Trp 500 505 510

Gly Ala Cys Leu Ala Phe His Tyr Ser Tyr Glu His Gin Asp Lys lie 515 520 525

Lys Ala lie Val His Ala Glu Ser Val Val Asp Val lie Glu Ser Trp 530 535 540

Asp Glu Trp Pro Asp lie Glu Glu Asp lie Ala Leu lie Lys Ser Glu 545 550 555 560

Glu Gly Glu Lys Met Val Leu Glu Asn Asn Phe Phe Val Glu Thr Met 565 570 575

Leu Pro Ser Lys lie Met Arg Lys Leu Glu Pro Glu Glu Phe Ala Ala 580 585 590

Tyr Leu Glu Pro Phe Lys Glu Lys Gly Glu Val Arg Arg Pro Thr Leu 595 600 605

Ser Trp Pro Arg Glu lie Pro Leu Val Lys Gly Gly Lys Pro Asp Val 610 615 620

Val Gin lie Val Arg Asn Tyr Asn Ala Tyr Leu Arg Ala Ser Asp Asp 625 630 635 640

Leu Pro Lys Met Phe lie Glu Ser Asp Pro Gly Phe Phe Ser Asn Ala 645 650 655 •65- 156362· Sequence Listing.doc 201131168 lie Val Glu Gly Ala Lys Lys Phe Pro Asn Thr Glu Phe Val Lys Val 660 665 670

Lys Gly Leu His Phe Ser Gin Glu Asp Ala Pro Asp Glu Met Gly Lys 675 680 685

Tyr lie Lys Ser Phe Val Glu Arg Val Leu Lys Asn Glu Gin 690 695 700 <210> 30 <211> 739 <212> PRT <213>manual <220><223> Derived from human neural tone Ardin receptor 1 <400> 30

Met Arg Leu Asn Ser Ser Ala Pro Gly Thr Pro Gly Thr Pro Ala Ala 15 10 15

Asp Pro Phe Gin Arg Ala Gin Ala Gly Leu Glu Glu Ala Leu Leu Ala 20 25 30

Pro Gly Phe Gly Asn Ala Ser Gly Asn Ala Ser Glu Arg Val Leu Ala 35 40 45

Ala Pro Ser Ser Glu Leu Asp Val Asn Thr Asp lie Tyr Ser Lys Val 50 55 60

Leu Val Thr Ala Val Tyr Leu Ala Leu Phe Val Val Gly Thr Val Gly 65 70 75 80

Asn Thr Val Thr Ala Phe Thr Leu Ala Arg Lys Lys Ser Leu Gin Ser 85 90 95

Leu Gin Ser Thr Val His Tyr His Leu Gly Ser Leu Ala Leu Ser Asp 100 105 110

Leu Leu Thr Leu Leu Leu Ala Met Pro Val Glu Leu Tyr Asn Phe lie 115 120 125

Trp Val His His Pro Trp Ala Phe Gly Asp Ala Gly Cys Arg Gly Tyr 130 135 140

Tyr Phe Leu Arg Asp Ala Cys Thr Tyr Ala Thr Ala Leu Asn Val Ala 145 150 155 160 -66- 156362 · Sequence Listing.doc 201131168

Ser Leu Ser Val Glu Arg Tyr Leu Ala lie Cys His Pro Phe Lys Ala 165 170 175

Lys Thr Leu Met Ser Arg Ser Arg Thr Lys Lys Phe lie Ser Ala lie 180 185 190

Trp Leu Ala Ser Ala Leu Leu Thr Val Pro Met Leu Phe Thr Met Gly 195 200 205

Glu Gin Asn Arg Ser Ala Asp Gly Gin His Ala Gly Gly Leu Val Cys 210 215 220

Thr Pro Thr lie His Thr Ala Thr Val Lys Val Val lie Gin Val Asn 225 230 235 240

Thr Phe Met Ser Phe lie Phe Pro Met Val Val lie Ser Val Leu Gly 245 250 255

Thr Arg Val Phe Gin Glu Ala Lys Arg Gin Leu Gin Lys lie Asp Lys 260 265 270

Ser Glu Gly Arg Phe His Val Gin Asn Leu Ser Gin Val Glu Gin Asp 275 2S0 285

Gly Arg Thr Gly His Gly Leu Arg Arg Ser Ser Lys Phe Cys Ser Arg 290 295 300

Glu Lys Lys Ala Ala Lys Thr Leu Glu Phe Val Val lie Ala Phe Val 305 310 315 320

Val Cys Trp Leu Pro Tyr His Val Arg Arg Leu Met Phe Cys Tyr lie 325 330 335

Ser Asp Glu Gin Trp Thr Pro Phe Leu Tyr Asp Phe Tyr His Tyr Phe 340 345 350

Tyr Met Val Thr Asn Ala Leu Phe Tyr Val Ser Ser Thr lie Asn Pro 355 360 365 lie Leu Tyr Asn Leu Val Ser Ala Asn Phe Arg His lie Phe Leu Ala 370 375 380

Thr Leu Ala Cys Leu Cys Pro Val Trp Arg Arg Arg Arg Lys Arg Pro 385 390 395 400 -67- 156362 - Sequence Listing.doc 201131168

Ala Phe Ser Arg Lys Ala Asp Ser Val Ser Ser Asn His Thr Leu Ser 405 410 415

Ser Asn Ala Thr Arg Glu Thr Leu Tyr Glu Asn Ser Met Thr Ser Lys 420 425 430

Val Tyr Asp Pro Glu Gin Arg Lys Arg Met lie Thr Gly Pro Gin Trp 435 440 445 butyl rp Ala Arg Cys Lys G]n Met Asn Va] Leu Asp Ser Phe lie Asn Tyr 450 455 460

Tyr Asp Ser Glu Lys His Ala Glu Asn Ala Val lie Phe Leu His Gly 465 470 475 480

Asn Ala Ala Ser Ser Tyr Leu Trp Arg His Val Val Pro His lie Glu 485 490 495

Pro Val Ala Arg Cys lie Jle Pro Asp Leu I]e G]y Met Gly Lys Ser 500 505 510

Gly Lys Ser Gly Asn Gly Ser Tyr Arg Leu Leu Asp His Tyr Lys Tyr 515 520 525

Leu Thr Ala Trp Phe Glu Leu Leu Asn Leu Pro Lys Lys lie Asn Phe 530 535 540

Val Gly His Asp Trp Gly Ala Cys Leu Ala Phe His Tyr Ser Tyr Glu 545 550 555 560

His Gin Asp Lys lie Lys Ala lie Val His Ala Glu Ser Val Val Asp 565 570 575

Val lie Glu Ser Trp Asp Glu Trp Pro Asp lie Glu Glu Asp lie Ala 580 585 590

Leu lie Lys Ser Glu Glu Gly Glu Lys Met Val Leu Glu Asn Asn Phe 595 600 605

Phe Val Glu Thr Met Leu Pro Ser Lys lie Met Arg Lys Leu Glu Pro 610 615 620

Glu Glu Phe Ala Ala Tyr Leu Glu Pro Phe Lys Glu Lys Gly Glu Val 625 630 635 640 -68 - 156362 - Sequence Listing.doc 201131168

Arg Arg Pro Thr Leu Ser Trp Pro Arg Glu lie Pro Leu Val Lys Gly 645 650 655

Gly Lys Pro Asp Val Val Gin lie Val Arg Asn Tyr Asn Ala Tyr Leu 660 665 670

Arg Ala Ser Asp Asp Leu Pro Lys Met Phe lie Glu Ser Asp Pro Gly 675 680 685

Phe Phe Ser Asn Ala lie Val Glu Gly Ala Lys Lys Phe Pro Asn Thr 690 695 700

Glu Phe Val Lys Val Lys Gly Leu His Phe Ser Gin Glu Asp Ala Pro 705 710 715 720

Asp Glu Met Gly Lys Tyr He Lys Ser Phe Val Glu Arg Val Leu Lys 725 730 735

Asn Glu Gin <210> 31 <211> 673 <212> PRT <213>Manual <220><223> Derived from human melanocortin 4 receptor <400>

Met Val Asn Ser Thr His Arg Gly Met His Thr Ser Leu His Leu Trp 15 10 15

Asn Arg Ser Ser Tyr Arg Leu His Ser Asn Ala Ser Glu Ser Leu Gly 20 25 30

Lys Gly Tyr Ser Asp Gly Gly Cys Tyr Glu Gin Leu Phe Val Ser Pro 35 40 45

Glu Val Phe Val Thr Leu Gly Val lie Ser Leu Leu Glu Asn lie Leu 50 55 60

Val lie Val Ala lie Ala Lys Asn Lys Asn Leu His Ser Pro Met Tyr 65 70 75 80

Phe Phe lie Cys Ser Leu Ala Val Ala Asp Met Leu Val Ser Val Ser 85 90 95 •69· 156362-Sequence List.doc 201131168

Asn Gly Ser Glu Thr lie lie lie Thr Leu Leu Asn Ser Thr Asp Thr 100 105 110

Asp Ala Gin Ser Phe Thr Val Asn He Asp Asn Val, lie Asp Ser Val 115 120 125 lie Cys Ser Ser Leu Leu Ala Ser lie Cys Ser Leu Leu Ser lie Ala 130 135 140

Val Asp Arg Tyr Phe Thr lie Phe Tyr Ala Leu Gin Tyr His Asn lie 145 150 155 160

Met Thr Val Lys Arg Val Gly lie Ser lie Ser Cys lie Trp Ala Ala 165 170 175

Cys Thr Val Ser Gly lie Leu Phe lie lie Tyr Ser Asp Ser Ser Ala 180 185 190

Val lie lie Cys Leu lie Thr Met Phe Phe Thr Met Leu Ala Leu Met 195 200 205

Ala Ser Leu Tyr Val His Met Phe Leu Gly Thr Arg Val Phe Gin Glu 210 215 220

Ala Lys Arg Gin Leu Gin Lys He Asp Lys Ser Glu Gly Arg Phe His 225 230 235 240

Val Gin Asn Leu Ser Gin Val Glu Gin Asp Gly Arg Thr Gly His Gly 245 250 255

Leu Arg Arg Ser Ser Lys Phe Cys Ser Arg Glu Lys Lys Ala Ala Lys 260 265 270

Thr Leu Glu Phe Leu lie Gly Val Phe Val Val Cys Trp Ala Pro Phe 275 280 285

Phe Leu His Leu lie Phe Tyr lie Ser Cys Pro Gin Asn Pro Tyr Cys 290 295 300

Val Cys Phe Met Ser His Phe Asn Leu Tyr Leu lie Leu lie Met Cys 305 310 315 320

Asn Ser lie lie Asp Pro Leu lie Tyr Ala Leu Arg Ser Gin Glu Leu 325 330 335 •70- 156362-Sequence table.doc 201131168

Arg Lys Thr Phe Lys Glu lie lie Cys Cys Tyr Pro Leu Gly Gly Leu 340 345 350

Cys Asp Leu Ser Ser Arg Tyr Glu Asn Ser Met Thr Ser Lys Val Tyr 355 360 365

Asp Pro Glu Gin Arg Lys Arg Met lie Thr Gly Pro Gin Trp Trp Ala 370 375 380

Arg Cys Lys Gin Met Asn Val Leu Asp Ser Phe He Asn Tyr Tyr Asp 385 390 395 400

Ser Glu Lys His Ala Glu Asn Ala Val lie Phe Leu His Gly Asn Ala 405 410 415

Ala Ser Ser Tyr Leu Trp Arg His Val Val Pro His lie Glu Pro Val 420 425 430

Ala Arg Cys lie He Pro Asp Leu He Gly Met Gly Lys Ser Gly Lys 435 440 445

Ser Gly Asn Gly Ser Tyr Arg Leu Leu Asp His Tyr Lys Tyr Leu Thr 450 455 460

Ala Trp Phe Glu Leu Leu Asn Leu Pro Lys Lys lie Asn Phe Val Gly 465 470 475 480

His Asp Trp Gly Ala Cys Leu Ala Phe His Tyr Ser Tyr Glu His Gin 485 490 495

Asp Lys lie Lys Ala lie Val His Ala Glu Ser Val Val Asp Val He 500 505 510

Glu Ser Trp Asp Glu Trp Pro Asp lie Glu Glu Asp lie Ala Leu lie 515 520 525

Lys Ser Glu Glu Gly Glu Lys Met Val Leu Glu Asn. Asn Phe Phe Val 530 535 540’

Glu Thr Met Leu Pro Ser Lys lie Met Arg Lys Leu Glu Pro Glu Glu 545 550 555 560

Phe Ala Ala Tyr Leu Glu Pro Phe Lys Glu Lys Gly Glu Val Arg Arg 565 570 575 -71 - 156362 - Sequence Listing.doc 201131168

Pro Thr Leu Ser Trp Pro Arg Glu lie Pro Leu Val Lys Gly Gly Lys* 580 585 590

Pro Asp Val Val Gin lie Val Arg Asn Tyr Asn Ala Tyr Leu Arg Ala 595 600 605

Ser Asp Asp Leu Pro Lys Met Phe lie Glu Ser Asp Pro Gly Phe Phe 610 615 620

Ser Asn Ala lie Val Glu Gly Ala Lys Lys Phe Pro Asn Thr Glu Phe 625 630 635 640

Val Lys Val Lys Gly Leu His Phe Ser Gin Glu Asp Ala Pro Asp Glu 645 650 655

Met Gly Lys Tyr lie Lys Ser Phe Val Glu Arg Val Leu Lys Asn Glu 660 665 670

Gin <210> 32 <211> 818 <212> PRT <213>Manual <220><223> Derived from human glucagon-like peptide 1 receptor <400>

Met Ala Gly Ala Pro Gly Pro Leu Arg Leu Ala Leu Leu Leu Leu Gly 15 10 15

Met Val Gly Arg Ala Gly Pro Arg Pro Gin Gly Ala Thr Val Ser Leu 20 25 30

Trp Glu Thr Val Gin Lys Trp Arg Glu Tyr Arg Arg Gin Cys Gin Arg 35 40 45

Ser Leu Thr Glu Asp Pro Pro Pro Ala Thr Asp Leu Phe Cys Asn Arg 50 55 60

Thr Phe Asp Glu Tyr Ala Cys Trp Pro Asp Gly Glu Pro Gly Ser Phe 65 70 75 80

Val Asn Val Ser Cys Pro Trp Tyr Leu Pro Trp Ala Ser Ser Val Pro 85 90 95 • 72· 156362 - Sequence Listing.doc 201131168

Gin Gly His Val Tyr Arg Phe Cys Thr Ala GIu Gly Leu Trp Leu Gin 100 105 110

Lys Asp Asn Ser Ser Leu Pro Trp Arg Asp Leu Ser Glu Cys Glu Glu 115 120 125

Ser Lys Arg Gly Glu Arg Ser Ser Pro Glu Glu Gin Leu Leu Phe Leu 130 135 140

Tyr lie lie Tyr Thr Val Gly Tyr Ala Leu Ser Phe Ser Ala Leu Val 145 150 155 160 lie Ala Ser Ala lie Leu Leu Gly Phe Arg His Leu His Cys Thr Arg 165 170 175

Asn Tyr lie His Leu Asn Leu Phe Ala Ser Phe lie Leu Arg Ala Leu 180 185 190

Ser Val Phe lie Lys Asp Ala Ala Leu Lys Trp Met Tyr Ser Thr Ala 195 200 205

Ala Gin Gin His Gin Trp Asp Gly Leu Leu Ser Tyr Gin Asp Ser Leu 210 215 220

Ser Cys Arg Leu Val Phe Leu Leu Met Gin Tyr Cys Val Ala Ala Asn 225 230 235 240

Tyr Tyr Trp Leu Leu Val Glu Gly Val Tyr Leu Tyr Thr Leu Leu Ala 245 250 255

Phe Ser Val Phe Ser Glu Gin Trp lie Phe Arg Leu Tyr Val Ser lie 260 265 270

Gly Trp Gly Val Pro Leu Leu Phe Val Val Pro Trp Gly lie Val Lys 275 280 285

Tyr Leu Tyr Glu Asp Glu Gly Cys Trp Thr Arg Asn Ser Asn Met Asn 290 295 300

Tyr Trp Leu lie lie Arg Leu Pro lie Leu Phe Gly lie Gly Val Asn 305 310 315 320

Phe Leu lie Phe Val Arg Val lie Cys Gly Thr Arg Val Phe Gin Glu 325 330 335 -73· 156362 - Sequence Listing.doc 201131168

Ala Lys Arg Gin Leu Gin Lys lie Asp Lys Ser Glu Gly Arg Phe His 340 345 350

Val Gin Asn Leu Ser Gin Val Glu Gin Asp Gly Arg Thr Gly His Gly 355 360 365

Leu Arg Arg Ser Ser Lys Phe Cys Ser Arg Glu Lys Lys Ala Ala Lys 370 375 380

Thr Leu Glu Phe Arg Leu Ala Lys Ser Thr Leu Thr Leu lie Pro Leu 385 390 395 400

Leu Gly Thr His Glu Val lie Phe Ala Phe Val Met Asp Glu His Ala 405 410 415

Arg Gly Thr Leu Arg Phe lie Lys Leu Phe Thr Glu Leu Ser Phe Thr 420 425 430

Ser Phe Gin Gly Leu Met Val Ala He Leu Tyr Cys Phe Val Asn Asn 435 440 445

Glu Val Gin Leu Glu Phe Arg Lys Ser Trp Glu Arg Trp Arg Leu Glu 450 455 460

His Leu His lie Gin Arg Asp Ser Ser Met Lys Pro Leu Lys Cys Pro 465 470 475 480

Thr Ser Ser Leu Ser Ser Gly Ala Thr Ala Gly Ser Ser Met Tyr Thr 485 490 495

Ala Thr Cys Gin Ala Ser Cys Ser Glu Asn Ser Met Thr Ser Lys Val 500 505 510

Tyr Asp Pro Glu Gin Arg Lys Arg Met lie Thr Gly Pro Gin Trp Trp 515 520 525

Ala Arg Cys Lys Gin Met Asn Val Leu Asp Ser Phe lie Asn Tyr Tyr 530 535 540

Asp Ser Glu Lys His Ala Glu Asn Ala Val lie Phe Leu His Gly Asn 545 550 555 560

Ala Ala Ser Ser Tyr Leu Trp Arg His Val Val Pro His lie Glu Pro 565 570 575

Val Ala Arg Cys lie lie Pro Asp Leu lie Gly Met Gly Lys Ser Gly 580 585 590 -74- 156362 - Sequence Listing.doc 201131168

Lys Ser Gly Asn Gly Ser Tyr Arg Leu Leu Asp His Tyr Lys Tyr Leu 595 600 605

Thr Ala Trp Phe Glu Leu Leu Asn Leu Pro Lys Lys lie Asn Phe Val 610 615 620

Gly His Asp Trp Gly Ala Cys Leu Ala Phe His Tyr Ser Tyr Glu His 625 630 635 640

Gin Asp Lys lie Lys Ala lie Val His Ala Glu Ser Val Val Asp Val 645 650 655 lie Glu Ser Trp Asp Glu Trp Pro Asp lie Glu Glu Asp lie Ala Leu 660 665 670 lie Lys Ser Glu Glu Gly Glu Lys Met Val Leu Glu Asn Asn Phe Phe 675 680 685

Val Glu Thr Met Leu Pro Ser Lys lie Met Arg Lys Leu Glu Pro Glu 690 695 700

Glu Phe Ala Ala Tyr Leu Glu Pro Phe Lys Glu Lys Gly Glu Val Arg 705 710 715 720

Arg Pro Thr Leu Ser Trp Pro Arg Glu lie Pro Leu Val Lys Gly Gly 725 730 735

Lys Pro Asp Val Val Gin lie Val Arg Asn Tyr Asn Ala Tyr Leu Arg 740 745 750

Ala Ser Asp Asp Leu Pro Lys Met Phe lie Glu Ser Asp Pro Gly Phe 755 760 765

Phe Ser Asn Ala lie Val Glu Gly Ala Lys Lys Phe Pro Asn Thr Glu 770 775 780

Phe Val Lys Val Lys Gly Leu His Phe Ser Gin Glu Asp Ala Pro Asp 7B5 790 795 800

Glu Met Gly Lys Tyr lie Lys Ser Phe Val Glu Arg Val Leu Lys Asn 805 810 815

Glu Gin -75-156362 - Sequence Listing.doc 201131168 <210> 33 <211> 660 <212> PRT < 213 > Labor <220><223> Derived from Human Adenosine A1 Receptor <400> 33

Met Pro Pro Ser lie Ser Ala Phe Gin Ala Ala Tyr lie Gly lie Glu 15 10 15

Val Leu lie Ala Leu Val Ser Val Pro Gly Asn Val Leu Val lie Trp 20 25 30

Ala Val Lys Val Asn Gin Ala Leu Arg Asp Ala Thr Phe Cys Phe lie 35 40 45

Val Ser Leu Ala Val Ala Asp Val Ala Val Gly Ala Leu Val lie Pro 50 55 60

Leu Ala lie Leu lie Asn lie Gly Pro Gin Thr Tyr Phe His Thr Cys 65 70 75 80

Leu Met Val Ala Cys Pro Val Leu lie Leu Thr Gin Ser Ser lie Leu 85 90 95

Ala Leu Leu Ala lie Ala Val Asp Arg Tyr Leu Arg Val Lys lie Pro 100 105 110

Leu Arg Tyr Lys Met Val Val Thr Pro Arg Arg Ala Ala Val Ala lie 115 120 125

Ala Gly Cys Trp He Leu Ser Phe Val Val Gly Leu Thr Pro Met Phe 130 135 140

Gly Trp Asn Asn Leu Ser Ala Val Glu Arg Ala Trp Ala Ala Asn Gly 145 150 155 160

Ser Met Gly Glu Pro Val He Lys Cys Glu Phe Glu Lys Val lie Ser 165 170 175

Met Glu Tyr Met Val Tyr Phe Asn Phe Phe Val Trp Val Leu Pro Pro 180 185 190

Leu Leu Leu Met Val Leu lie Gly Thr Arg Val Phe Gin Glu Ala Lys 195 200 205 76- J 56362 - Sequence Listing, doc 201131168

Arg Gin Leu Gin Lys lie Asp Lys Ser Glu Gly Arg Phe His Val Gin 210 215 220

Asn Leu Ser Gin Val Glu Gin Asp Gly Arg Thr Gly His Gly Leu Arg 225 230 235 240

Arg Ser Ser Lys Phe Cys Ser Arg Glu Lys Lys Ala Ala Lys Thr Leu 245 250 255

Glu Phe lie Leu Phe Leu Phe Ala Leu Ser Trp Leu Pro Leu His lie 260 265 270

Leu Asn Cys He Thr Leu Phe Cys Pro Ser Cys His Lys Pro Ser lie 275 280 285

Leu Thr Tyr lie Ala lie Phe Leu Thr His Gly Asn Ser Ala Met Asn 290 295 300

Pro lie Val Tyr Ala Phe Arg lie Gin Lys Phe Arg Val Thr Phe Leu 305 310 315 320

Lys lie Trp Asn Asp His Phe Arg Cys Gin Pro Ala Pro Pro lie Asp 325 330 335

Glu Asp Leu Pro Glu Glu Arg Pro Asp Asp Glu Asn Ser Met Thr Ser 340 345 350

Lys Val Tyr Asp Pro Glu Gin Arg Lys Arg Met lie Thr Gly Pro Gin 355 360 365

Trp Trp Ala Arg Cys Lys Gin Met Asn Val Leu Asp Ser Phe lie Asn 370 375 380

Tyr Tyr Asp Ser Glu Lys His Ala Glu Asn Ala Val lie Phe Leu His 385 390 395 400

Gly Asn Ala Ala Ser Ser Tyr Leu Trp Arg His Val Val Pro His lie 405 410 415

Glu Pro Val Ala Arg Cys lie lie Pro Asp Leu lie Gly Met Gly Lys 420 425 430

Ser Gly Lys Ser Gly Asn Gly Ser Tyr Arg Leu Leu Asp His Tyr Lys 435 440 445 156362 - Sequence Listing.doc •77· 201131168

Tyr Leu Thr Ala Trp Phe Glu Leu Leu Asn Leu Pro Lys Lys lie Asn 450 455 460

Phe Val Gly His Asp Trp Gly Ala Cys Leu Ala Phe His Tyr Ser Tyr 465 470 475 480

Glu His Gin Asp Lys lie Lys Ala lie Val His Ala Glu Ser Val Val 485 490 495

Asp Val lie Glu Ser Trp Asp Glu Trp Pro Asp lie Glu Glu Asp lie 500 505 510

Ala Leu lie Lys Ser Glu Glu Gly Glu Lys Met Val Leu Glu Asn Asn 515 520 525

Phe Phe Val Glu Thr Met Leu Pro Ser Lys lie Met Arg Lys Leu Glu 530 535 540

Pro Glu Glu Phe Ala Ala Tyr Leu Glu Pro Phe Lys Glu Lys Gly Glu 545 550 555 560

Val Arg Arg Pro Thr Leu Ser Trp Pro Arg Glu lie Pro Leu Val Lys 565 570 575

Gly Gly Lys Pro Asp Val Val Gin lie Val Arg Asn Tyr Asn Ala Tyr 580 585 590

Leu Arg Ala Ser Asp Asp Leu Pro Lys Met Phe He Glu Ser Asp Pro 595 600 605

Gly Phe Phe Ser Asn Ala lie Val Glu Gly Ala Lys Lys Phe Pro Asn 610 615 620

Thr Glu Phe Val Lys Val Lys Gly Leu His Phe Ser Gin Glu Asp Ala 625 630 635 640

Pro Asp Glu Met Gly Lys Tyr lie Lys Ser Phe Val Glu Arg Val Leu 645 650 655

Lys Asn Glu Gin 660 <210> 34 <211> 789 <212> PRT < 213 > Artificial 156362 - Sequence Listing. doc -78 - 201131168 <220><223> Derived from human cannabinoid receptor 1 <400〉 34

Met Lys Ser lie Leu Asp Gly Leu Ala Asp Thr Thr Phe Arg Thr lie 15 10 15

Thr Thr Asp Leu Leu Tyr Val Gly Ser Asn Asp lie Gin Tyr Glu Asp 20 25 30 lie Lys Gly Asp Met Ala Ser Lys Leu Gly Tyr Phe Pro Gin Lys Phe 35 40 45

Pro Leu Thr Ser Phe Arg Gly Ser Pro Phe Gin Glu Lys Met Thr Ala 50 55 60

Gly Asp Asn Pro Gin Leu Val Pro Ala Asp Gin Val Asn lie Thr Glu 65 70 75 80

Phe Tyr Asn Lys Ser Leu Ser Ser Phe Lys Glu Asn Glu Glu Asn lie 85 90 95

Gin Cys Gly Glu Asn Phe Met Asp lie Glu Cys Phe Met Val Leu Asn 100 105 110

Pro Ser Gin Gin Leu Ala lie Ala Val Leu Ser Leu Thr Leu Gly Thr 115 120 125

Phe Thr Val Leu Glu Asn Leu Leu Val Leu Cys Val lie Leu His Ser 130 135 140

Arg Ser Leu Arg Cys Arg Pro Ser Tyr His Phe lie Gly Ser Leu Ala 145 150 155 160

Val Ala Asp Leu Leu Gly Ser Val lie Phe Val Tyr Ser Phe lie Asp 165 170 175

Phe His Val Phe His Arg Lys Asp Ser Arg Asn Val Phe Leu Phe Lys ISO 185 190

Leu Gly Gly Val Thr Ala Ser Phe Thr Ala Ser Val Gly Ser Leu Phe 195 200 205

Leu Thr Ala lie Asp Arg Tyr lie Ser lie His Arg Pro Leu Ala Tyr 210 215 220 -79- 156362 - Sequence Listing.doc 201131168

Lys Arg lie Val Thr Arg Pro Lys Ala Val Val Ala Phe Cys Leu Met 225 230 235 240

Trp Thr lie Ala lie Val lie Ala Val Leu Pro Leu Leu Gly Trp Asn 245 250 255

Cys Glu Lys Leu Gin Ser Val Cys Ser Asp lie Phe Pro His lie Asp 260 265 270

Glu Thr Tyr Leu Met Phe Trp lie Gly Val Thr Ser Val Leu Leu Leu 275 280 285

Phe lie Val Gly Thr Arg Val Phe Gin Glu Ala Lys Arg Gin Leu Gin 290 295 300

Lys lie Asp Lys Ser Glu Gly Arg Phe His Val Gin Asn Leu Ser Gin 305 310 315 320

Val Glu Gin Asp Gly Arg Thr Gly His Gly Leu Arg Arg Ser Ser Lys 325 330 335

Phe Cys Ser Arg Glu Lys Lys Ala Ala Lys Thr Leu Glu Phe lie Leu 340 345 350

Val Val Leu lie lie Cys Trp Gly Pro Leu Leu Ala lie Met Val Tyr 355 360 365

Asp Val Phe Gly Lys Met Asn Lys Leu lie Lys Thr Val Phe Ala Phe 370 375 380

Cys Ser Met Leu Cys Leu Leu Asn Ser Thr Val Asn Pro lie lie Tyr 385 390 395 400

Ala Leu Arg Ser Lys Asp Leu Arg His Ala Phe Arg Ser Met Phe Pro 405 410 415

Ser Cys Glu Gly Thr Ala Gin Pro Leu Asp Asn Ser Met Gly Asp Ser 420 425 430

Asp Cys Leu His Lys His Ala Asn Asn Ala Ala Ser Val His Arg Ala 435 440 445

Ala Glu Ser Cys lie Lys Ser Thr Val Lys lie Ala Lys Val Thr Met 450 455 460 -80 - 156362 - Sequence Listing.doc 201131168

Ser Val Ser Thr Asp Thr Ser Ala Glu Ala Leu GIu Asn Ser Met Thr 465 470 475 480

Ser Lys Val Tyr Asp Pro Glu Gin Arg Lys Arg Met lie Thr Gly Pro 485 490 495

Gin Trp Trp Ala Arg Cys Lys Gin Met Asn Val Leu Asp Ser Phe He 500 505 510

Asn Tyr Tyr Asp Ser Glu Lys His Ala Glu Asn Ala Val lie Phe Leu 515 520 525

His Gly Asn Ala Ala Ser Ser Tyr Leu Trp Arg His Val Val Pro His 530 535 540 lie Glu Pro Val Ala Arg Cys lie lie Pro Asp Leu lie Gly Met Gly 545 550 555 560

Lys Ser Gly Lys Ser Gly Asn Gly Ser Tyr Arg Leu Leu Asp His Tyr 565 570 575

Lys Tyr Leu Thr Ala Trp Phe Glu Leu Leu Asn Leu Pro Lys Lys lie 580 585 590

Asn Phe Val Gly His Asp Trp Gly Ala Cys Leu Ala Phe His Tyr Ser 595 600 605

Tyr Glu His Gin Asp Lys lie Lys Ala lie Val His Ala Glu Ser Val 610 615 620

Val Asp Val lie Glu Ser Trp Asp Glu Trp Pro Asp lie Glu Glu Asp 625 630 635 640 lie Ala Leu lie Lys Ser Glu Glu Gly Glu Lys Met Val Leu Glu Asn 645 650 655

Asn Phe Phe Val Glu Thr Met Leu Pro Ser Lys lie Met Arg Lys Leu 660 665 670

Glu Pro Glu Glu Phe Ala Ala Tyr Leu Glu Pro Phe Lys Glu Lys Gly 675 680 685

Glu Val Arg Arg Pro Thr Leu Ser Trp Pro Arg Glu lie Pro Leu Val 690 695 700

Lys Gly Gly Lys Pro Asp Val Val Gin lie Val Arg Asn Tyr Asn Ala 705 710 715 720 -81- 156362 - Sequence Listing.doc 201131168

Tyr Leu Arg Ala Ser Asp Asp Leu Pro Lys Met Phe lie Glu Ser Asp 725 730 735

Pro Gly Phe Phe Ser Asn Ala lie Val Glu Gly Ala Lys Lys Phe Pro 740 745 750

Asn Thr Glu Phe Val Lys Val Lys Gly Leu His Phe Ser Gin Glu Asp 755 760 765

Ala Pro Asp Glu Met Gly Lys Tyr lie Lys Ser Phe Val Glu Arg Val 770 775 780

Lea Lys Asn Glu Gin 785 <210> 35 <211> 693 <212> PRT <213>Manual <220><223> Derived from human melanin agglutinin receptor 1 <400>

Met Asp Leu Glu Ala Ser Leu Leu Pro Thr Gly Pro Asn Ala Ser Asn 15 10 15

Thr Ser Asp Gly Pro Asp Asn Leu Thr Ser Ala Gly Ser Pro Pro Arg 20 25 30

Thr Gly Ser lie Ser Tyr lie Asn lie lie Met Pro Ser Val Phe Gly 35 40 45

Thr lie Cys Leu Leu Gly He lie Gly Asn Ser Thr Val He Phe Ala 50 55 60

Val Val Lys Lys Ser Lys Leu His Trp Cys Asn Asn Val Pro Asp lie 65 70 75 80

Phe lie lie Asn Leu Ser Val Val Asp Leu Leu Phe Leu Leu Gly Met 85 90 95

Pro Phe Met He His Gin Leu Met Gly Asn Gly Val Trp His Phe Gly 100 105 110

Glu Thr Met Cys Thr Leu lie Thr Ala Met Asp Ala Asn Ser Gin Phe 115 120 125 -82 · 156362 - Sequence Listing.doc 201131168

Thr Ser Thr Tyr lie Leu Thr Ala Met Ala lie Asp Arg Tyr Leu Ala 130 135 140

Thr Val His Pro lie Ser Ser Thr Lys Phe Arg Lys Pro Ser Val Ala 145 150 155 160

Thr Leu Val lie Cys Leu Leu Trp Ala Leu Ser Phe lie Ser lie Thr 165 170 175

Pro Val Trp Leu Tyr Ala Arg Leu lie Pro Phe Pro Gly Gly Ala Val 180 185 190

Gly Cys Gly lie Arg Leu Pro Asn Pro Asp Thr Asp Leu Tyr Trp Phe 195 200 205

Thr Leu Tyr Gin Phe Phe Leu Ala Phe Ala Leu Pro Phe Val Val lie 210 215 220

Thr Ala Ala Gly Thr Arg Val Phe Gin Glu Ala Lys Arg Gin Leu Gin 225 230 235 240

Lys lie Asp Lys Ser Glu Gly Arg Phe His Val Gin Asn Leu Ser Gin 245 250 255

Val Glu Gin Asp Gly Arg Thr Gly His Gly Leu Arg Arg Ser Ser Lys 260 265 270

Phe Cys Ser Arg Glu Lys Lys Ala Ala Lys Thr Leu Glu Phe lie Cys 275 280 285

Leu Val Phe Phe Val Cys Trp Ala Pro Tyr Tyr Val Leu Gin Leu Thr 290 295 300

Gin Leu Ser lie Ser Arg Pro Thr Leu Thr Phe Val Tyr Leu Tyr Asn 305 310 315 320

Ala Ala lie Ser Leu Gly Tyr Ala Asn Ser Cys Leu Asn Pro Phe Val 325 330 335

Tyr lie Val Leu Cys Glu Thr Phe Arg Lys Arg Leu Val Leu Ser Val 340 345 350

Lys Pro Ala Ala Gin Gly Gin Leu Arg Ala Val Ser Asn Ala Gin Thr 355 360 365 83- 156362 - Sequence Listing.doc 201131168

Ala Asp Glu Glu Arg Thr Glu Ser Lys Gly Thr Glu Asn Ser Met Thr 370 375 380

Ser Lys Val Tyr Asp Pro Glu Gin Arg Lys Arg Met lie Thr Gly Pro 385 390 395 400

Gin Trp Trp Ala Arg Cys Lys Gin Met Asn Val Leu Asp Ser Phe lie 405 410 415

Asn Tyr Tyr Asp Ser Glu Lys His Ala Glu Asn Ala Val lie Phe Leu 420 425 430

His Gly Asn Ala Ala Ser Ser Tyr Leu Trp Arg His Val Val Pro His 435 440 445 lie Glu Pro Val Ala Arg Cys lie lie Pro Asp Leu lie Gly Met Gly 450 455 460

Lys Ser Gly Lys Ser Gly Asn Gly Ser Tyr Arg Leu Leu Asp His Tyr 465 470 475 480

Lys Tyr Leu Thr Ala Trp Phe Glu Leu Leu Asn Leu Pro Lys Lys lie 485 490 495

Asn Phe Val Gly His Asp Trp Gly Ala Cys Leu Ala Phe His Tyr Ser 500 505 510

Tyr Glu His Gin Asp Lys He Lys Ala lie Val His Ala Glu Ser Val 515 520 525

Val Asp Val lie Glu Ser Trp Asp Glu Trp Pro Asp lie Glu Glu Asp 530 535 540 lie Ala Leu lie Lys Ser Glu Glu Gly Glu Lys Met Val Leu Glu Asn 545 550 555 560

Asn Phe Phe Val Glu Thr Met Leu Pro Ser Lys lie Met Arg Lys Leu 565 570 575

Glu Pro Glu Glu Phe Ala Ala Tyr Leu Glu Pro Phe Lys Glu Lys Gly 580 585 590

Glu Val Arg Arg Pro Thr Leu Ser Trp Pro Arg Glu lie Pro Leu Val 595 600 605 84· 156362 - Sequence Listing.doc 201131168

Lys Gly Gly Lys Pro Asp Val Val Gin lie Val Arg Asn Tyr Asn Ala 610 615 620

Tyr Leu Arg Ala Ser Asp Asp Leu Pro Lys Met Phe lie Glu Ser Asp 625 630 635 640

Pro Gly Phe Phe Ser Asn Ala lie Val Glu Gly Ala Lys Lys Phe Pro 645 650 655

Asn Thr Glu Phe Val Lys Val Lys Gly Leu His Phe Ser Gin Glu Asp 660 665 670

Ala Pro Asp Glu Met Gly Lys Tyr lie Lys Ser Phe Val Glu Arg Val 675 680 685

Leu Lys Asn Glu Gin 690 <210> 36 <211> 646 <212> PRT <213> Labor <220><223> Derived from human GPR40 (insulin serotonin) <400>

Met Asp Leu Pro Pro Gin Leu Ser Phe Gly Leu Tyr Val Ala Ala Phe 15 10 15

Ala Leu Gly Phe Pro Leu Asn Val Leu Ala lie Arg Gly Ala Thr Ala 20 25 30

His Ala Arg Leu Arg Leu Thr Pro Ser Leu Val Tyr Ala Leu Asn Leu 35 40 45

Gly Cys Ser Asp Leu Leu Leu Thr Val Ser Leu Pro Leu Lys Ala Val 50 55 60

Glu Ala Leu Ala Ser Gly Ala Trp Pro Leu Pro Ala Ser Leu Cys Pro 65 70 ' 75 80

Val Phe Ala Val Ala His Phe Phe Pro Leu Tyr Ala Gly Gly Gly Phe 85 90 95

Leu Ala Ala Leu Ser Ala Gly Arg Tyr Leu Gly Ala Ala Phe Pro Leu 100 105 110 -85- 156362 · Sequence Listing.doc 201131168

Gly Tyr Gin Ala Phe Arg Arg Pro Cys Tyr Ser Trp Gly Val Cys Ala 115 120 125

Ala lie Trp Ala Leu Val Leu Cys His Leu Gly Leu Val Phe Gly Leu 130 135 140

Glu Ala Pro Gly Gly Trp Leu Asp His Ser Asn Thr Ser Leu Gly lie 145 150 155 160

Asn Thr Pro Val Asn Gly Ser Pro Val Cys Leu Glu Ala Trp Asp Pro 165 170 175

Ala Ser Ala Gly Pro Ala Arg Phe Ser Leu Ser Leu Leu Leu Phe Phe 180 185 190

Leu Pro Leu Ala lie Thr Ala Phe Cys Gly Thr Arg Val Phe Gin Glu 195 200 205

Ala Lys Arg Gin Leu Gin Lys lie Asp Lys Ser Glu Gly Arg Phe His 210 215 220

Val Gin Asn Leu Ser Gin Val Glu Gin Asp Gly Arg Thr Gly His Gly 225 230 235 240

Leu Arg Arg Ser Ser Lys Phe Cys Ser Arg Glu Lys Lys Ala Ala Lys 245 250 255

Thr Leu Glu Phe Ala Leu Leu Thr Leu Leu Leu Cys Val Gly Pro Tyr 260 265 270

Asn Ala Ser Asn Val Ala Ser Phe Leu Tyr Pro Asn Leu Gly Gly Ser 275 280 285

Trp Arg Lys Leu Gly Leu lie Thr Gly Ala Trp Ser Val Val Leu Asn 290 295 300

Pro Leu Val Thr Gly Tyr Leu Gly Arg Gly Pro Gly Leu Lys Thr Val 305 310 315 320

Cys Ala Ala Arg Thr Gin Gly Gly Lys Ser Gin Lys Glu Asn Ser Met 325 330 335

Thr Ser Lys Val Tyr Asp Pro Glu Gin Arg Lys Arg Met lie Thr Gly 340 345 350 • 86· 156362 - Sequence Listing.doc 201131168

Pro Gin Trp Trp Ala Arg Cys Lys Gin Met Asn Val Leu Asp Ser Phe 355 360 365 lie Asn Tyr Tyr Asp Ser Glu Lys His Ala Glu Asn Ala Val lie Phe 370 375 380

Leu His Gly Asn Ala Ala Ser Ser Tyr Leu Trp Arg His Val Val Pro 385 390 395 400

His lie Glu Pro Val Ala Arg Cys lie lie Pro Asp Leu lie Gly Met 405 410 415

Gly Lys Ser Gly Lys Ser Gly Asn Gly Ser Tyr Arg Leu Leu Asp His 420 425 430

Tyr Lys Tyr Leu Thr Ala Trp Phe Glu Leu Leu Asn Leu Pro Lys Lys 435 440 445 lie Asn Phe Val Gly His Asp Trp Gly Ala Cys Leu Ala Phe His Tyr 450 455 460

Ser Tyr Glu His Gin Asp Lys He Lys Ala He Val His Ala Glu Ser 465 470 475 480

Val Val Asp Val lie Glu Ser Trp Asp Glu Trp Pro Asp lie Glu Glu 485 490 495

Asp lie Ala Leu lie Lys Ser Glu Glu Gly Glu Lys Met Val Leu Glu 500 505 510

Asn Asn Phe Phe Val Glu Thr Met Leu Pro Ser Lys lie Met Arg Lys 515 520 525

Leu Glu Pro Glu Glu Phe Ala Ala Tyr Leu Glu Pro Phe Lys Glu Lys 530 535 540

Gly Glu Val Arg Arg Pro Thr Leu Ser Trp Pro Arg Glu lie Pro Leu 545 550 555 560

Val Lys Gly Gly Lys Pro Asp Val Val Gin lie Val Arg Asn Tyr Asn 565 570 575

Ala Tyr Leu Arg Ala Ser Asp Asp Leu Pro Lys Met Phe lie Glu Ser 580 585 590

Asp Pro Gly Phe Phe Ser Asn Ala lie Val Glu Gly Ala Lys Lys Phe 595 600 605 -87 - 156362 · Sequence Listing.doc 201131168

Pro Asn Thr Glu Phe Val Lys Val Lys Gly Leu His Phe Ser Gin Glu 610 615 620

Asp Ala Pro Asp Glu Met Gly Lys Tyr lie Lys Ser Phe Val Glu Arg 625 630 635 640

Val Leu Lys Asn Glu Gin 645 <210> 37 <211> 662 <212> PRT <213> Labor <220><223> Derived from Human GPCR2 (Insulin Serotonin) <400>

Met Glu Ser Ser Phe Ser Phe Gly Val lie Leu Ala Val Leu Ala Ser 15 10 15

Leu He lie Ala Thr Asn Thr Leu Val Ala Val Ala Val Leu Leu Leu 20 25 30 lie His Lys Asn Asp Gly Val Ser Leu Cys Phe Thr Leu Asn Leu Ala 35 40 45

Val Ala Asp Thr Leu lie Gly Val Ala lie Ser Gly Leu Leu Thr Asp 50 55 60

Gin Leu Ser Ser Pro Ser Arg Pro Thr Gin Lys Thr Leu Cys Ser Leu 65 70 75 80

Arg Met Ala Phe Val Thr Ser Ser Ala Ala Ala Ser Val Leu Thr Val 85 90 95

Met Leu lie Thr Phe Asp Arg Tyr Leu Ala lie Lys Gin Pro Phe Arg 100 105 110

Tyr Leu Lys lie Met Ser Gly Phe Val Ala Gly Ala Cys lie Ala Gly 115 120 125

Leu Trp Leu Val Ser Tyr Leu lie Gly Phe Leu Pro Leu Gly lie Pro 130 135 140

Met Phe Gin Gin Thr Ala Tyr Lys Gly Gin Cys Ser Phe Phe Ala Val 145 150 155 160 -88 - 156362 - Sequence Listing.doc 201131168

Phe His Pro His Phe Val Leu Thr Leu Ser Cys Val Gly Phe Phe Pro 165 170 175

Ala Met Leu Leu Phe Val Phe Gly Thr Arg Val Phe Gin Glu Ala Lys 180 185 190

Arg Gin Leu Gin Lys lie Asp Lys Ser Glu Gly Arg Phe His Val Gin 195 200 205

Asn Leu Ser Gin Val Glu Gin Asp Gly Arg Thr Gly His Gly Leu Arg 210 215 220

Arg Ser Ser Lys Phe Cys Ser Arg Glu Lys Lys Ala Ala Lys Thr Leu 225 230 235 240

Glu Phe Leu lie Gly Ser Phe Ala Leu Ser Trp Thr Pro Phe Leu lie 245 250 255

Thr Gly lie Val Gin Val Ala Cys Gin Glu Cys His Leu Tyr Leu Val 260 265 270

Leu Glu Arg Tyr Leu Trp Leu Leu Gly Val Gly Asn Ser Leu Leu Asn 275 280 285

Pro Leu lie Tyr Ala Tyr Trp Gin Lys Glu Val Arg Leu Gin Leu Tyr 290 295 300

His Met Ala Leu Gly Val Lys Lys Val Leu Thr Ser Phe Leu Leu Phe 305 310 315 320

Leu Ser Ala Arg Asn Cys Gly Pro Glu Arg Pro Arg Glu Ser Ser Cys 325 330 335

His lie Val Thr lie Ser Ser Ser Glu Phe Asp Gly Glu Asn Ser Met 340 345 350

Thr Ser Lys Val Tyr Asp Pro Glu Gin Arg Lys Arg Met lie Thr Gly 355 360 365

Pro Gin Trp Trp Ala Arg Cys Lys Gin Met Asn Val Leu Asp Ser Phe 370 375 380 lie Asn Tyr Tyr Asp Ser Glu Lys His Ala Glu Asn Ala Val lie Phe 385 390 395 400 -89- 1563 62 - Sequence Listing.doc 201131168

Leu His Gly Asn Ala Ala Ser Ser Tyr Leu Trp Arg His Val Val Pro 405 410 415

His lie Glu Pro Val Ala Arg Cys lie lie Pro Asp Leu He Gly Met 420 425 430

Gly Lys Ser Gly Lys Ser Gly Asn Gly Ser Tyr Arg Leu Leu Asp His 435 440 445

Tyr Lys Tyr Leu Thr Ala Trp Phe Glu Leu Leu Asn Leu Pro Lys Lys 450 455 460 lie Asn Phe Val Gly His Asp Trp Gly Ala Cys Leu Ala Phe His Tyr 465 470 475 480

Ser Tyr Glu His Gin Asp Lys lie Lys Ala lie Val His Ala Glu Ser 485 490 495

Val Val Asp Val lie Glu Ser Trp Asp Glu Trp Pro Asp lie Glu Glu 500 505 510

Asp lie Ala Leu lie Lys Ser Glu Glu Gly Glu Lys Met Val Leu Glu 515 520 525

Asn Asn Phe Phe Val Glu Thr Met Leu Pro Ser Lys lie Met Arg Lys 530 535 540

Leu Glu Pro Glu Glu Phe Ala Ala Tyr Leu Glu Pro Phe Lys Glu Lys 545 550 555 560

Gly Glu Val Arg Arg Pro Thr Leu Ser Trp Pro Arg Glu lie Pro Leu 565 570 575

Val Lys Gly Gly Lys Pro Asp Val Val Gin lie Val Arg Asn Tyr Asn 580 585 590

Ala Tyr Leu Arg Ala Ser Asp Asp Leu Pro Lys Met Phe lie Glu Ser 595 600 605

Asp Pro Gly Phe Phe Ser Asn Ala lie Val Glu Gly Ala Lys Lys Phe 610 615 620

Pro Asn Thr Glu Phe Val Lys Val Lys Gly Leu His Phe Ser Gin Glu 625 630 635 640 • 90· 156362 - Sequence Listing.doc 201131168

Asp Ala Pro Asp GIu Met Gly Lys Tyr lie Lys Ser Phe Val Glu Arg 645 650 655

Val Leu Lys Asn Glu Gin 660 <210> 38 <211> 2154 <212> DNA <213>manpower <220><223> Modified rat alkaloid receptor, muscarinic 3 ≪ 400 > 38 atgaccttgc acagtaacag tacaacctcg cctttgtttc ccaacatcag ctcttcctgg 60 gtgcacagtc cctcggaggc agggctgccc ttggggacag tcactcagtt gggcagctac 120 aacatttcac aagaaactgg gaatttctcc tcaaacgaca cctccagcga ccctctcggg 180 ggtcacacca tctggcaagt ggtcttcatt gccttcttaa ctggcttcct ggcattggtg 240 accatcattg gcaacatcct tgtcattgtg gccttcaagg tcaacaaaca gctgaagaca 300 gtcaacaact acttcctctt aagcctggcc tgtgcagacc tgatcatcgg ggtcatttcc 360 atgaacctgt tcactaccta catcattatg aaccgttggg cactggggaa cttagcctgc 420 gacctctggc tctccattga ctatgtggcc agcaatgcct ctgtcatgaa tctgctggtc 480 atcagctttg acaggtactt ttccatcact aggccactca cctaccgagc caaaagaaca 540 acaaaacgac gtggtgtgat gattggtctg gcttgggtca tctcctttgt cctatgggct 600 cctgccatct tgttctggca atactttgta gggaagagaa ctgtgccccc aggagaatgt 660 ttcattcagt ttctgagtga gcccaccatc accttcggca cggcgatcgc tgccttttac 720 atgcctgtca ccatcatgac tattttaggt accagggtct ttcaggaggc caaaaggcag 780 ctccagaaga ttgacaaatc tgagggccgc ttccatgtcc Agaaccttag ccaggtggag 840 caggat gggc ggacggggca tggactccgc agatcttcca agttctgctc cagggaaaag 900 aaagcagcca aaaccttgga attcatcttg ctagccttca tcatcacgtg gaccccctac 960 aacatcatgg tcctggtgaa caccttctgt gacagctgca tacccaaaac ctattggaat 1020 ctgggctact ggctgtgcta tatcaacagc accgtgaacc ctgtgtgcta tgccctgtgc 1080 aacaaaacat tcagaaccac cttcaagacg ctcctcttgt gccagtgtga caaaaggaag 1140 aggcgcaaac agcagtacca gcagagacag tcggtcattt ttcacaagcg agtgccggag 1200 caggccttgg agaattccat gacttcgaaa gtttatgatc cagaacaaag gaaacggatg 1260 ataactggtc cgcagtggtg ggccagatgt aaacaaatga atgttcttga ttcatttatt 1320 -91 · 156362- sequence table .doc 1380 201131168 aattattatg attcagaaaa acatgcagaa aatgctgtta tttttttaca tggtaacgcg gcctcttctt atttatggcg acatgttgtg ccacatattg agccagtagc gcggtgtatt ataccagatc ttattggtat gggcaaatca ggcaaatctg gtaatggttc ttataggtta cttgatcatt acaaatatct tactgcatgg tttgaacttc ttaatttacc aaagaagatc aattttgtcg gccatgattg gggtgcttgt ttggcatttc attatagcta tgagcatcaa gataagatca aagcaatagt tcacgctgaa Agtgtagtag atgtgattga atcatgggat ga atggcctg atattgaaga agatattgcg ttgatcaaat ctgaagaagg agaaaaaatg gttttggaga ataacttctt cgtggaaacc atgttgccat caaaaatcat gagaaagtta gaaccagaag aatttgcagc atatcttgaa ccattcaaag agaaaggtga agttcgtcgt ccaacattat catggcctcg tgaaatcccg ttagtaaaag gtggtaaacc tgacgttgta caaattgtta ggaattataa tgcttatcta cgtgcaagtg atgatttacc aaaaatgttt attgaatcgg atccaggatt cttttccaat gctattgttg aaggcgccaa gaagtttcct aatactgaat ttgtcaaagt aaaaggtctt catttttcgc aagaagatgc acctgatgaa atgggaaaat atatcaaatc gttcgttgag cgagttctca aaaatgaaca ataa <210> 39 <211> 2031 <212> DNA <213>manpower <220〉 <223> Modified human melanin agglutinin receptor 2 ≪ 400 > 39 atgaatccat ttcatgcatc ttgttggaac acctctgccg aacttttaaa caaatcctgg aataaagagt ttgcttatca aactgccagt gtggtagata cagtcatcct cccttccatg attgggatta tctgttcaac agggctggtt ggcaacatcc tcattgtatt cactataata agatccagga aaaaaacagt ccctgacatc tatatctgca acctggctgt ggctgatttg gtccacatag ttggaatgcc ttttcttatt caccaatggg cccgaggggg agagtgggtg tttggggggc ctctctgcac catcatcaca tccctggata cttgtaacca atttgcctgt agtgccatca tgactgtaat gagtgtggac aggtactttg ccctcgtcca accatttcga ctgacacgtt ggagaacaag gtacaagacc atccggatca atttgggcct ttgggcagct tcctttatcc tggcattgcc tgtctgggtc tactcgaagg tcatcaaatt taaagacggt gttgagagtt gtgcttttga tttgacatcc cctgacgatg tactctggta tacactttat ttgacgataa caactttttt tttccctcta cccttgattt tggtgtgcta tattggtacc agggtctttc aggaggccaa aaggcagctc cagaagattg acaaatctga gggccgcttc 156362- sequence Listing .doc _92 · 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 2100 2154 60 120 180 240 300 360 420 480 540 600 660 720 780 201131168 catgtccaga accttagcca ggtggagcag gatgggc gga cggggcatgg actccgcaga tcttccaagt tctgctccag ggaaaagaaa gcagccaaaa ccttggaatt cctggtggta gtctttatcc tgagtgctgc cccttatcat gtgatacaac tggtgaactt acagatggaa cagcccacac tggccttcta tgtgggttat tacctctcca tctgtctcag ctatgccagc agcagcatta acccttttct ctacatcctg ctgagtggaa atttccagaa acgtctgcct caaatccaaa gaagagcgac tgagaaggaa atcaacaata tgggaaacac tctgaaatca cactttgaga attccatgac ttcgaaagtt tatgatccag aacaaaggaa acggatgata actggtccgc agtggtgggc cagatgtaaa caaatgaatg ttcttgattc atttattaat tattatgatt cagaaaaaca tgcagaaaat gctgttattt ttttacatgg taacgcggcc tcttcttatt tatggcgaca tgttgtgcca catattgagc cagtagcgcg gtgtattata ccagatctta ttggtatggg caaatcaggc aaatctggta atggttctta taggttactt gatcattaca aatatcttac tgcatggttt gaacttctta atttaccaaa gaagatcaat tttgtcggcc atgattgggg tgcttgtttg gcatttcatt atagctatga gcatcaagat aagatcaaag caatagttca cgctgaaagt gtagtagatg tgattgaatc atgggatgaa tggcctgata ttgaagaaga tattgcgttg atcaaatctg aagaaggaga aaaaatggtt ttggagaata acttcttcgt ggaaaccatg ttgccatcaa aaatca tgag aaagttagaa ccagaagaat ttgcagcata tcttgaacca ttcaaagaga aaggtgaagt tcgtcgtcca acattatcat ggcctcgtga aatcccgtta gtaaaaggtg gtaaacctga cgttgtacaa attgttagga attataatgc ttatctacgt gcaagtgatg atttaccaaa aatgtttatt gaatcggatc caggattctt ttccaatgct attgttgaag gcgccaagaa gtttcctaat actgaatttg tcaaagtaaa aggtcttcat ttttcgcaag aagatgcacc tgatgaaatg ggaaaatata tcaaatcgtt cgttgagcga gttctcaaaa atgaacaata a <210> 40 <211> 1989 <212〉 DNA <213>manpower <220><223> Modified human pickling receptor, muscarinic 4 ≪ 400> 40 atggccaact tcacacctgt caatggcagc tcgggcaatc agtccgtgcg cctggtcacg tcatcatccc acaatcgcta tgagacggtg gaaatggtct tcattgccac agtgacaggc tccctgagcc tggtgactgt cgtgggcaac atcctggtga tgctgtccat caaggtcaac 156362- Sequence Listing .doc -93- 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 I860 1920 1980 2031 60 120 180 201131168 aggcagctgc agacagtcaa caactacttc ctcttcagcc tggcgtgtgc tgatctcatc 240 ataggcgcct tctccatgaa cctctacacc gtgtacatca tcaagggcta ctggcccctg 300 ggcgccgtgg tctgcgacct gtggctggcc ctggactacg tggtgagcaa cgcctccgtc 360 atgaaccttc tcatcatcag ctttgaccgc tacttctgcg tcaccaagcc tctcacctac 420 cctgcccggc gcaccaccaa gatggcaggc ctcatgattg ctgctgcctg ggtactgtcc 480 ttcgtgctct gggcgcctgc catcttgttc tggcagtttg tggtgggtaa gcggacggtg 540 cccgacaacc Agtgcttcat ccagttcctg tccaacccag cagtgacctt tggcacagcc 600 attgctgcct tctacctgcc tgtggtcatc atgacggtgc tgggtaccag ggtctttcag 660 gaggccaaaa ggcagctcca gaagattgac aaatctgagg gccgcttcca tgtccagaac 720 cttagccag g tggagcagga tgggcggacg gggcatggac tccgcagatc ttccaagttc 780 tgctccaggg aaaagaaagc agccaaaacc ttggaattca ttctgctagc cttcatcctc 840 acctggacgc cctacaacgt catggtcctg gtgaacacct tctgccagag ctgcatccct 900 gacacggtgt ggtccattgg ctactggctc tgctacgtca acagcaccat caaccctgcc 960 tgctatgctc tgtgcaacgc cacctttaaa aagaccttcc ggcacctgct gctgtgccag 1020 tatcggaaca tcggcactgc cagggagaat tccatgactt cgaaagttta tgatccagaa 1080 caaaggaaac ggatgataac tggtccgcag tggtgggcca gatgtaaaca aatgaatgtt 1140 cttgattcat ttattaatta ttatgattca gaaaaacatg cagaaaatgc tgttattttt 1200 ttacatggta acgcggcctc ttcttattta tggcgacatg ttgtgccaca tattgagcca 1260 gtagcgcggt gtattatacc agatcttatt ggtatgggca aatcaggcaa atctggtaat 1320 ggttcttata ggttacttga tcattacaaa tatcttactg catggtttga acttcttaat 1380 ttaccaaaga agatcaattt tgtcggccat gattggggtg cttgtttggc atttcattat 1440 agctatgagc atcaagataa gatcaaagca atagttcacg ctgaaagtgt agtagatgtg 1500 attgaatcat gggatgaatg gcctgatatt gaagaagata ttgcgttgat caaatctgaa 1560 gaaggagaaa aaatggtt tt ggagaataac ttcttcgtgg aaaccatgtt gccatcaaaa 1620 atcatgagaa agttagaacc agaagaattt gcagcatatc ttgaaccatt caaagagaaa 1680 ggtgaagttc gtcgtccaac attatcatgg cctcgtgaaa tcccgttagt aaaaggtggt 1740 aaacctgacg ttgtacaaat tgttaggaat tataatgctt atctacgtgc aagtgatgat 1800 ttaccaaaaa tgtttattga atcggatcca ggattctttt ccaatgctat tgttgaaggc 1860 gccaagaagt ttcctaatac tgaatttgtc aaagtaaaag gtcttcattt ttcgcaagaa 1920 gatgcacctg atgaaatggg aaaatatatc aaatcgttcg ttgagcgagt tctcaaaaat 1980 gaacaataa · 1989 •94- 156362·SEQ ID NO.doc 201131168 <210> 41 . <211> 2127 <212> DNA <213>manpower <220〉 <223> Modified human nicotinic acid receptor ≪ 400 > 41 atgaatcggc accatctgca ggatcacttt ctggaaatag acaagaagaa ctgctgtgtg 60 ttccgagatg acttcattgt caaggtgttg ccgccggtgt tggggctgga gtttatcttc 120 gggcttctgg gcaatggcct tgccctgtgg attttctgtt tccacctcaa gtcctggaaa 180 tccagccgga ttttcctgtt caacctggca gtggctgact ttctactgat catctgcctg 240 cccttcctga tggacaacta tgtgaggcgt tgggac.tgga agtttgggga catcccttgc 300 cggctgatgc tcttcatgtt ggctatgaac cgccagggca gcatcatctt cctcacggtg 360 gtggcggtag acaggtattt ccgggtggtc catccccacc acgccctgaa caagatctcc 420 aatcggacag cagccatcat ctcttgcctt ctgtggggca tcactattgg cctgacagtc 480 cacctcctga agaagaagat gccgatccag aatggcggtg caaatttgtg cagcagcttc 540 agcatctgcc ataccttcca gtggcacgaa gccatgttcc tcctggagtt cttcctgccc 600 ctgggcatca tcctgttctg cggtaccagg gtctttcagg aggccaaaag gcagctccag 660 aagattgaca aatctgaggg ccgcttccat gtccagaacc ttagccaggt ggagcaggat 720 gggcggacgg ggcatggact ccgcagatct tccaagttct gctccaggga aaagaaagca 780 gccaaaacct tggaattcgt Ggccatcgtc tttgtcatct gcttccttcc cagcgtggtt 840 gtgcg gatcc gcatcttctg gctcctgcac acttcgggca cgcagaattg tgaagtgtac 900 cgctcggtgg acctggcgtt ctttatcact ctcagcttca cctacatgaa cagcatgctg 960 gaccccgtgg tgtactactt ctccagccca tcctttccca acttcttctc cactttgatc 1020 aaccgctgcc tccagaggaa gatgacaggt gagccagata ataaccgcag cacgagcgtc 1080 gagctcacag gggaccccaa caaaaccaga ggcgctccag aggcgttaat ggccaactcc 1140 ggtgagccat ggagcccctc ttatctgggc ccaacctctc ctgagaattc catgacttcg 1200 aaagtttatg atccagaaca aaggaaacgg atgataactg gtccgcagtg gtgggccaga 1260 tgtaaacaaa tgaatgttct tgattcattt attaattatt atgattcaga aaaacatgca 1320 gaaaatgctg ttattttut acatggtaac gcggcctctt cttatttatg gcgacatgtt 13S0 gtgccacata ttgagccagt agcgcggtgt attataccag atcttattgg tatgggcaaa 1440 tcaggcaaat ctggtaatgg ttcttatagg ttacttgatc attacaaata tcttactgca 1500 -95 · 156362 · sequence Listing .doc 201131168 tggtttgaac ttcttaattt accaaagaag atcaattttg tcggccatga ttggggtgct tgtttggcat ttcattatag ctatgagcat caagataaga tcaaagcaat agttcacgct gaaagtgtag Tagatgtgat tgaatcatgg gatgaatggc ctgatattga ag aagatatt gcgttgatca aatctgaaga aggagaaaaa atggttttgg agaataactt cttcgtggaa accatgttgc catcaaaaat catgagaaag ttagaaccag aagaatttgc agcatatctt gaaccattca aagagaaagg tgaagttcgt cgtccaacat tatcatggcc tcgtgaaatc ccgttagtaa aaggtggtaa acctgacgtt gtacaaattg ttaggaatta taatgcttat ctacgtgcaa gtgatgattt accaaaaatg tttattgaat cggatccagg attcttttcc aatgctattg ttgaaggcgc caagaagttt cctaatactg aatttgtcaa agtaaaaggt cttcattttt cgcaagaaga tgcacctgat gaaatgggaa aatatatcaa atcgttcgtt gagcgagttc tcaaaaatga acaataa <210> 42 <211> 1950 <212> DNA <213>manpower <220><223> Modified human histamine 4 receptor ≪ 400 > 42 atgccagata ctaatagcac aatcaattta tcactaagca ctcgtgttac tttagcattt tttatgtcct tagtagcttt tgctataatg ctaggaaatg ctttggtcat tttagctttt gtggtggaca aaaaccttag acatcgaagt agttattttt ttcttaactt ggccatctct gacttctttg tgggtgtgat ctccattcct ttgtacatcc ctcacacgct gttcgaatgg gattttggaa aggaaatctg tgtattttgg ctcactactg actatctgtt atgtacagca tctgtatata acattgtcct catcagctat gatcgatacc tgtcagtctc aaatgctgtg tcttatagaa ctcaacatac tggggtcttg aagattgtta ctctgatggt ggccgtttgg gtgctggcct tcttagtgaa tgggccaatg attctagttt cagagtcttg gaaggatgaa ggtagtgaat gtgaacctgg atttttttcg gaatggtaca tccttgccat cacatcattc ttggaattcg tgatcccagt catcttagtc gcttatttcg gtaccagggt ctttcaggag gccaaaaggc agctccagaa gattgacaaa tctgagggcc gcttccatgt ccagaacctt agccaggtgg agcaggatgg gcggacgggg catggactcc gcagatcttc caagttctgc tccagggaaa agaaagcagc caaaaccttg gaattcctct taggggtttt tgctgtttgc tgggctccat attctctgtt cacaattgtc ctttcatttt attcctcagc aacaggtcct aaatcagttt ggtatagaat tgcattttgg cttcagtggt tcaattcctt tgtcaa Tcct 156362 - Sequence Listing _doc • 96· 1560 1620 1680 1740 1800 1860 1920 1980 2040 2100 2127 60 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 201131168 cttttgtatc cattgtgtca caagcgcttt caaaaggctt tcttgaaaat attttgtata aaaaagcaac ctctaccatc acaacacagt cggtcagtat cttctgagaa ttccatgact tcgaaagttt atgatccaga acaaaggaaa cggatgataa ctggtccgca gtggtgggcc agatgtaaac aaatgaatgt tcttgattca tttattaatt attatgattc agaaaaacat gcagaaaatg ctgttatttt tttacatggt aacgcggcct cttcttattt atggcgacat gttgtgccac atattgagcc agtagcgcgg tgtattatac cagatcttat tggtatgggc aaatcaggca aatctggtaa tggttcttat aggttacttg atcattacaa atatcttact gcatggtttg aacttcttaa tttaccaaag aagatcaatt ttgtcggcca tgattggggt gcttgtttgg catttcatta tagctatgag catcaagata agatcaaagc aatagttcac gctgaaagtg tagtagatgt gattgaatca tgggatgaat ggcctgatat tgaagaagat attgcgttga tcaaatctga Gaagaggagaa aaaatggttt tggagaataa cttcttcgtg gaaaccatgt tgccatcaaa aatcatgaga aagttagaac cagaagaatt tgcagcatat cttgaaccat tcaaagagaa aggtgaagtt cgtcgtccaa c attatcatg gcctcgtgaa atcccgttag taaaaggtgg taaacctgac gttgtacaaa ttgttaggaa ttataatgct tatctacgtg caagtgatga tttaccaaaa atgtttattg aatcggatcc aggattcttt tccaatgcta ttgttgaagg cgccaagaag tttcctaata ctgaatttgt caaagtaaaa ggtcttcatt tttcgcaaga agatgcacct gatgaaatgg gaaaatatat caaatcgttc gttgagcgag ttctcaaaaa tgaacaataa <210> 43 <211> 2118 <212> DNA <213>manpower <220><223> Modified human polypeptide ghrelin receptor ≪ 400 > 43 atgtggaacg cgacgcccag cgaagagccg gggttcaacc tcacactggc cgacctggac tgggatgctt cccccggcaa cgactcgctg ggcgacgagc tgctgcagct cttccccgcg ccgctgctgg cgggcgtcac agccacctgc gtggcactct tcgtggtggg tatcgctggc aacctgctca ccatgctggt ggtgtcgcgc ttccgcgagc tgcgcaccac caccaacctc tacctgtcca gcatggcctt ctccgatctg ctcatcttcc tctgcatgcc cctggacctc gttcgcctct ggcagtaccg gccctggaac ttcggcgacc tcctctgcaa actcttccaa ttcgtcagtg agagctgcac ctacgccacg gtgctcacca tcacagcgct gagcgtcgag 156362 · Sequence list .doc • 97 · 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1950 60 120 180 240 300 360 420 201131168 cgctacttcg ccatctgctt cccactccgg aagctggtca tcttcgtcat ctgggccgtg ctagtcgggg tggagcacga gaacggcacc accgagtttg cggtgcgctc tggactgctc ttcttccttc ctgtcttctg tctcacggtc aggcagctcc agaagattga caaatctgag gtggagcagg Atgggcggac ggggcatgga gaaaagaaag cagccaaaac cttggaattc tggctcccct tccacgtagg gcgatattta gagattgctc agatcagcca gtactgcaac gctgccatca accccattct gtacaacatc agacttct gg gattcgaacc cttctcccag tctcgggcct ggacagaatc tagtattaat gatccagaac aaaggaaacg gatgataact atgaatgttc ttgattcatt tattaattat gttatttttt tacatggtaa cgcggcctct attgagccag tagcgcggtg tattatacca tctggtaatg gttcttatag gttacttgat cttcttaatt taccaaagaa gatcaatttt tttcattata gctatgagca tcaagataag gtagatgtga ttgaatcatg ggatgaatgg aaatctgaag aaggagaaaa aatggttttg ccatcaaaaa tcatgagaaa gttagaacca aaagagaaag gtgaagttcg tcgtccaaca aaaggtggta aacctgacgt tgtacaaatt agtgatgatt taccaaaaat gtttattgaa gttgaaggcg ccaagaagtt tcctaatact tcgcaagaag atgcacctga Tgaaatggga ctcaaaaatg aacaataa <210> 44 ≪ 211 > 2151 gccaaggtgg tggtcaccaa ggggcgggtg 480 gccttctgca gcgccgggcc catcttcgtg 540 gacccttggg acaccaacga gtgccgcccc 600 acggtcatgg tgtgggtgtc cagcatcttc 660 ctcggtacca gggtctttca ggaggccaaa 720 ggccgcttcc atgtccagaa ccttagccag 780 ctccgcagat cttccaagtt ctgctccagg 840 gctgtagtgg tgtttgcctt catcctctgc 900 ttttccaaat cctttgagcc tggctccttg 960 ctcgtgtcct ttgtcctctt ctacctcagt 1020 atgtccaaga agtaccgggt ggcagtgttc 1080 agaaagctct ccactctgaa agatgaaagt 1140 acagagaatt ccatgacttc gaaagtttat 1200 ggtccgcagt ggtgggccag atgtaaacaa 1260 tatgattcag aaaaacatgc agaaaatgct 1320 tcttatttat ggcgacatgt tgtgccacat 1380 gatcttattg gtatgggcaa atcaggcaaa 1440 cattacaaat atcttactgc atggtttgaa 1500 gtcggccatg attggggtgc ttgtttggca 1560 atcaaagcaa tagttcacgc tgaaagtgta 1620 cctgatattg aagaagatat tgcgttgatc 1680 gagaataact tcttcgtgga aaccatgttg 1740 gaagaatttg cagcatatct tgaaccattc 1800 ttatcatggc ctcgtgaaat cccgttagta 1860 gttaggaatt ataatgctta tctacgtgca 1920 tcggatccag gattct Tttc caatgctatt 1980 gaatttgtca aagtaaaagg tcttcatttt 2040 aaatatatca aatcgttcgt tgagcgagtt 2100 2118 -98· 156362-sequence table.doc 201131168 <212> DNA <213> ΛΧ <220><223> Modified human CXCR3 gonadotropin receptor ≪ 400 > 44 atggtccttg aggtgagtga ccaccaagtg ctaaatgacg ccgaggttgc cgccctcctg 60 gagaacttca gctcttccta tgactatgga gaaaacgaga gtgactcgtg ctgtacctcc 120 ccgccctgcc cacaggactt cagcctgaac ttcgaccggg ccttcctgcc agccctctac 180 agcctcctct ttctgctggg gctgctgggc aacggcgcgg tggcagccgt gctgctgagc 240 cggcggacag ccctgagcag caccgacacc ttcctgctcc acctagctgt agcagacacg 300 ctgctggtgc tgacactgcc gctctgggca gtggacgctg ccgtccagtg ggtctttggc 360 tctggcctct gcaaagtggc aggtgccctc ttcaacatca acttctacgc aggagccctc 420 ctgctggcct gcatcagctt tgaccgctac ctgaacatag ttcatgccac ccagctctac 480 cgccgggggc ccccggcccg cgtgaccctc acctgcctgg ctgtctgggg gctctgcctg 540 cttttcgccc tcccagactt catcttcctg tcggcccacc acgacgagcg cctcaacgcc 600 acccactgcc aatacaactt cccacaggtg ggccgcacgg ctctgcgggt gctgcagctg 660 gtggctggct ttctgctgcc cctgctggtc atggcctact gcggtaccag ggtctttcag 720 gaggccaaaa ggcagctcca gaagattgac aaatctgagg gccgcttcca tgtccagaac 780 cttagccagg tggagcagga tgggcggacg gggcatggac Tccgcagatc ttccaagttc 840 tgctcc aggg aaaagaaagc agccaaaacc ttggaattcg tcgtggtggc ctttgccctc 900 tgctggaccc cctatcacct ggtggtgctg gtggacatcc tcatggacct gggcgctttg 960 gcccgcaact gtggccgaga aagcagggta gacgtggcca agtcggtcac ctcaggcctg 1020 ggctacatgc actgctgcct caacccgctg ctctatgcct ttgtaggggt caagttccgg 1080 gagcggatgt ggatgctgct cttgcgcctg ggctgcccca accagagagg gctccagagg 1140 cagccatcgt cttcccgccg ggattcatcc tggtctgaga cctcagaggc ctcctactcg 1200 ggcttggaga attccatgac ttcgaaagtt tatgatccag aacaaaggaa acggatgata 1260 actggtccgc agtggtgggc cagatgtaaa caaatgaatg ttcttgattc atttattaat 1320 tattatgatt cagaaaaaca tgcagaaaat gctgttattt ttttacatgg taacgcggcc 1380 tcttcttatt tatggcgaca tgttgtgcca catattgagc cagtagcgcg gtgtattata 1440 ccagatctta ttggtatggg caaatcaggc aaatctggta atggttctta taggttactt 1500 gatcattaca aatatcttac tgcatggttt gaacttctta atttaccaaa gaagatcaat 1560 tttgtcggcc atgattgggg tgcttgtttg 1620 -99- 156362- sequence Listing gcatttcatt atagctatga gcatcaagat .doc 201131168 aagatcaaag caatagttca cgctgaaagt gtagtagatg tgatt gaatc atgggatgaa tggcctgata ttgaagaaga tattgcgttg atcaaatctg aagaaggaga aaaaatggtt ttggagaata acttcttcgt ggaaaccatg ttgccatcaa aaatcatgag aaagttagaa ccagaagaat ttgcagcata tcttgaacca ttcaaagaga aaggtgaagt tcgtcgtcca acattatcat ggcctcgtga aatcccgtta gtaaaaggtg gtaaacctga cgttgtacaa attgttagga attataatgc ttatctacgt gcaagtgatg atttaccaaa aatgtttatt gaatcggatc caggattctt ttccaatgct attgttgaag gcgccaagaa gtttcctaat actgaatttg tcaaagtaaa aggtcttcat ttttcgcaag aagatgcacc tgatgaaatg ggaaaatata tcaaatcgtt cgttgagcga gttctcaaaa atgaacaata a <210> 45 <211> 2250 <212> DNA <213〉Artificial <220><223> Modified human enterokin receptor ≪ 400> 45 atgggcagcc cctggaacgg cagcgacggc cccgaggggg cgcgggagcc gccgtggccc gcgctgccgc cttgcgacga gcgccgctgc tcgccctttc ccctgggggc gctggtgccg gtgaccgctg tgtgcctgtg cctgttcgtc gtcggggtga gcggcaacgt ggtgaccgtg atgctgatcg ggcgctaccg ggacatgcgg acttgtacct gggcagcatg gccgtgtccg acctactcat cctgctcggg ctgccgttcg acctgtaccg cctctggcgc tcgcggccct gggtgttcgg gccgctgctc tgccgcctgt ccctctacgt gggcgagggc tgcacctacg ccacgctgct gcacatgacc gcgctcagcg tcgagcgcta cctggccatc tgccgcccgc tccgcgcccg cgtcttggtc accaccacca acccggcgcc gcgtccgcgc gctcatcgct gtgctctggg ccgtggcgct gctctctgcc ggtcccttct tgttcctggt gggcgtcgag caggaccccg gcatctccgt agtcccgggc ctcaatggca ccgcgcggat cgcctcctcg cctctcgcct cgtcgccgcc tctctggctc tcgcgggcgc caccgccgtc cccgccgtcg gggcccgaga ccgcggaggc cgcggcgctg ttcagccgcg aatgccggcc gagccccgcg cagctgggcg cgctgcgtgt catgctgtgg gtcaccaccg cctacttctt cctgcccttt ctgtgcctca gcatcctcgg taccagggtc tttcaggagg ccaaaaggca gctccagaag attgacaaat ctgagggccg cttccatgtc cagaacctta gccaggtgga gcaggat Ggg cggacggggc atggactccg cagatcttcc aagttctgct ccagggaaaa gaaagcagcc 156362-sequence table.doc •100- 1680 1740 1800 1860 1920 1980 2040 2100 2151 60 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 201131168 aaaaccttgg aattcgtggt tctggcattt ataatttgct ggttgccctt ccacgttggc 1020 agaatcattt acataaacac ggaagattcg cggatgatgt acttctctca gtactttaac 1080 atcgtcgctc tgcaactttt ctatctgagc gcatctatca acccaatcct ctacaacctc 1140 atttcaaaga agtacagagc ggcggccttt aaactgctgc tcgcaaggaa gtccaggccg. 1200 agaggcttcc acagaagcag ggacactgcg ggggaagttg caggggacac tggaggagac 1260 acggtgggct acaccgagac aagcgctaac gtgaagacga tgggagagaa ttccatgact 1320 tcgaaagttt atgatccaga acaaaggaaa cggatgataa ctggtccgca gtggtgggcc 1380 agatgtaaac aaatgaatgt tcttgattca tttattaatt attatgattc agaaaaacat 1440 gcagaaaatg Ctgttatttt tttacatggt aacgcggcct cttcttattt atggcgacat 1500 gttgtgccac atattgagcc agtagcgcgg tgtattatac cagatcttat tggtatgggc 1560 aaatcaggca aatctggtaa tggttcttat aggttacttg atcattacaa atatctt act 1620 gcatggtttg aacttcttaa tttaccaaag aagatcaatt ttgtcggcca tgattggggt 1680 gcttgtttgg catttcatta tagctatgag catcaagata agatcaaagc aatagttcac 1740 gctgaaagtg tagtagatgt gattgaatca tgggatgaat ggcctgatat tgaagaagat 1800 attgcgttga tcaaatctga agaaggagaa aaaatggttt tggagaataa cttcttcgtg I860 gaaaccatgt tgccatcaaa aatcatgaga aagttagaac cagaagaatt tgcagcatat 1920 cttgaaccat tcaaagagaa aggtgaagtt cgtcgtccaa cattatcatg gcctcgtgaa 1980 atcccgttag taaaaggtgg taaacctgac gttgtacaaa ttgttaggaa t Tataatgct 2040 tatctacgtg caagtgatga tttaccaaaa atgtttattg aatcggatcc aggattcttt 2100 tccaatgcta ttgttgaagg cgccaagaag tttcctaata ctgaatttgt caaagtaaaa 2160 ggtcttcatt tttcgcaaga agatgcacct gatgaaatgg gaaaatatat caaatcgttc 2220 gttgagcgag ttctcaaaaa tgaacaataa 2250 <210> 46 <211> 2313 <212> DNA <213>manpower <220><223> Modified human 5-hydroxytryptamine (serotonin) receptor 2A ≪ 400 > 46 atggatattc tttgtgaaga aaatacttct ttgagctcaa ctacgaactc cctaatgcaa 60 ttaaatgatg acaccaggct ctacagtaat gactttaact ccggagaagc taacacttct 120 gatgcattta actggacagt cgactctgaa aatcgaacca acctttcctg tgaagggtgc 180 -101 - 156362 · Sequence Listing .doc ctccaggaaa aaaactggtc tgctttactg 240 ggaaacatac tcgtcatcat ggcagtgtcc 300 tatttcctga tgtcacttgc catagctgat 360 tccatgttaa ccatcctgta tgggtaccgg 420 tggatttacc tggacgtgct cttctccacg 480 ctggaccgct acgtcgccat ccagaatccc 540 aaggcatttc tgaaaatcat tgctgtttgg 600 ccagtctttg ggctacagga cgattcgaag 660 gatgataact ttgtcctgat cggctctttt 720 gtgatcaccg gtaccagggt ctttcaggag 780 tctgagggcc gcttccatgt ccagaacctt 840 catggactcc gcagatcttc caagttctgc 900 gaattcgtct tcttcctgtt tgtggtgatg 960 gccgtcatct gcaaagagtc ctgcaatgag 1020 gtttggatcg gttatctctc ttcagcagtc 1080 acctataggt cagccttttc acggtatatt 1140 ttgcagttaa Tittagtgaa cacaataccg 1200 atgggacaaa aaaagaattc aaagcaagat 1260 gttgctctag gaaagcagca ttctgaagag 1320 gaaaaggtga gctgtg tgga gaattccatg 1380 aaacggatga taactggtcc gcagtggtgg 1440 tcatttatta attattatga ttcagaaaaa 1500 ggtaacgcgg cctcttctta tttatggcga 1560 cggtgtatta taccagatct tattggtatg 1620 tataggttac ttgatcatta caaatatctt 1680 aagaagatca attttgtcgg ccatgattgg 1740 gagcatcaag ataagatcaa agcaatagtt 1800 tcatgggatg aatggcctga tattgaagaa 1860 gaaaaaatgg ttttggagaa taacttcttc 1920 agaaagttag aaccagaaga atttgcagca 1980 gttcgtcgtc caacattatc atggcctcgt 2040 201131168 ctctcaccgt cgtgtctctc cttacttcat acagccgtag tgattattct aactattgct ctagagaaaa agctgcagaa tgccaccaac atgctgctgg gtttccttgt catgcccgtg tggcctctgc cgagcaagct ttgtgcagtc gcctccatca tgcacctctg cgccatctcg atccaccaca gccgcttcaa ctccagaact accatatcag taggtatatc catgccaata gtctttaagg aggggagttg cttactcgcc gtgtcatttt tcattccctt aaccatcatg gccaaaaggc agctccagaa gattgacaaa agccaggtgg agcaggatgg gcggacgggg tccagggaaa agaaagcagc caaaaccttg tggtgccctt tcttcatcac aaacatcatg gatgtcattg gggccctgct caatgtgttt aacccactag tctacacact gttcaacaag cagtgtcagt a caaggaaaa caaaaaacca gctttggcct acaagtctag ccaacttcaa gccaagacaa cagataatga ctgctcaatg gcttctaaag acaatagcga cggagtgaat acttcgaaag tttatgatcc agaacaaagg gccagatgta aacaaatgaa tgttcttgat catgcagaaa atgctgttat ttttttacat catgttgtgc cacatattga gccagtagcg ggcaaatcag gcaaatctgg taatggttct actgcatggt ttgaacttct taatttacca ggtgcttgtt tggcatttca ttatagctat cacgctgaaa gtgtagtaga tgtgattgaa gatattgcgt tgatcaaatc tgaagaagga gtggaaacca tgttgccatc aaaaatcatg cattcaaaga gaaaggtgaa 156362- Sequence Listing tatcttgaac .doc • 102- 201131168 gaaatcccgt tagtaaaagg tggtaaacct gacgttgtac aaattgttag gaattataat gcttatctac gtgcaagtga tgatttacca aaaatgttta ttgaatcgga tccaggattc ttttccaatg ctattgttga aggcgccaag aagtttccta atactgaatt tgtcaaagta aaaggtcttc atttttcgca agaagatgca cctgatgaaa tgggaaaata tatcaaatcg ttcgttgagc gagttctcaa aaatgaacaa taa <210〉 47 <211> 2289 <212> DNA <213〉Artificial <220><223> Modified human 5-hydroxytryptamine (serotonin) receptor 2B ≪ 400 > 47 atggctctct cttacagagt gtctgaactt caaagcacaa ttcctgagca cattttgcag agcacctttg ttcacgttat ctcttctaac tggtctggat tacagacaga atcaatacca gaggaaatga aacagattgt tgaggaacag ggaaataaac tgcactgggc agctcttctg atactcatgg tgataatacc cacaattggt ggaaataccc ttgttattct ggctgtttca ctggagaaga agctgcagta tgctactaat tactttctaa tgtccttggc ggtggctgat ttgctggttg gattgtttgt gatgccaatt gccctcttga caataatgtt tgaggctatg tggcccctcc cacttgttct atgtcctgcc tggttatttc ttgacgttct cttttcaacc gcatccatca tgcatctctg tgccatttca gtggatcgtt acatagccat caaaaagcca atccaggcca atcaatataa ctcacgggct acagcattca tcaagattac agtggtgtgg ttaatttcaa taggcattgc cattccagtc cctattaaag ggatagagac tgatgtggac aacccaaaca atatcacttg tgtgctgaca aaggaacgtt ttggcgattt catgctcttt ggctcactgg ctgccttctt cacacctctt gcaattatga ttgtcaccgg taccagggtc tticaggagg ccaaaaggca gctccagaag attgacaaat ctgagggccg cttccatgtc cagaacctta gccaggtgga gcaggatggg cggacggggc atggactccg cagatcttcc aagttctgct ccagggaaaa gaaagcagcc aaaaccttgg aattcgtgtt tttcct cttt ttgcttatgt ggtgtccctt ctttattaca aatataactt tagttttatg tgattcctgt aaccaaacta ctctccaaat gctcctggag atatttgtgt ggataggcta tgtttcctca ggagtgaatc ctttggtcta caccctcttc aataagacat ttcgggatgc atttggccga tatatcacct gcaattaccg ggccacaaag tcagtaaaaa ctctcagaaa acgctccagt aagatctact tccggaatcc aatggcagag aactctaagt ttttcaagaa acatggaatt 156362 · Sequence Listing .doc -103- 2100 2160 2220 2280 2313 60 120 180 240 3⑻ 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 201131168 cgaaatggga ttaaccctgc catgtaccag agtccaatga ggctccgaag ttcaaccatt cagtcttcat caatcattct actagatacg cttctcctca ctgaaaatga aggtgacaaa actgaagagc aagttagtta tgtagagaat tccatgactt cgaaagttta tgatccagaa caaaggaaac ggatgataac tggtccgcag tggtgggcca gatgtaaaca aatgaatgtt cttgattcat ttattaatta ttatgattca gaaaaacatg cagaaaatgc tgttattttt ttacatggta acgcggcctc ttcttattta Tggcgacatg ttgtgccaca tattgagcca gtagcgcggt gtattatacc agatcttatt ggtatgggca aatcaggcaa atctggtaat ggttcttata ggttacttga tcattacaaa tatcttactg catggtt tga acttcttaat ttaccaaaga agatcaattt tgtcggccat gattggggtg cttgtttggc atttcattat agctatgagc atcaagataa gatcaaagca atagttcacg ctgaaagtgt agtagatgtg attgaatcat gggatgaatg gcctgatatt gaagaagata ttgcgttgat caaatctgaa gaaggagaaa aaatggtttt ggagaataac ttcttcgtgg aaaccatgtt gccatcaaaa atcatgagaa agttagaacc agaagaattt gcagcatatc ttgaaccatt caaagagaaa ggtgaagttc gtcgtccaac attatcatgg cctcgtgaaa tcccgttagt aaaaggtggt aaacctgacg ttgtacaaat tgttaggaat tataatgctt atctacgtgc aagtgatgat ttaccaaaaa tgtttattga atcggatcca ggattctttt ccaatgctat tgttgaaggc Gccaagaagt ttcctaatac tgaatttgtc aaagtaaaag gtcttcattt ttcgcaagaa gatgcacctg atgaaatggg aaaatatatc aaatcgttcg ttgagcgagt tctcaaaaat gaacaataa <210> 48 <211> 2250 <212> DNA <213>manpower <220><223> Modified human 5-hydroxytryptamine (serotonin) receptor 2C ≪ 400 > 48 atggtgaacc tgaggaatgc ggtgcattca ttccttgtgc acctaattgg cctattggtt tggcaatgtg atatttctgt gagcccagta gcagctatag taactgacat tttcaatacc tccgatggtg gacgcttcaa attcccagac jggggtacaaa actggccagc actttcaatc gtcatcataa taatcatgac aataggtggc aacatccttg tgatcatggc agtaagcatg gaaaagaaac tgcacaatgc caccaattac ttcttaatgt ccctagccat tgctgatatg ctagtgggac tacttgtcat gcccctgtct ctcctggcaa tcctttatga ttatgtctgg ccactaccta gatatttgtg ccccgtctgg atttctttag atgttttatt ttcaacagcg 156362 · Sequence list .doc -104- 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 2100 2160 2220 2280 2289 60 120 180 240 300 360 420 201131168 tccatcatgc acctctgcgc tatatcgctg gatcggtatg tagcaatacg taatcctatt 480 gagcatagcc gtttcaattc gcggactaag gccatcatga agattgctat tgtttgggca 540 atttctatag gtgtatcagt tcctatccct gtgattggac Tgagggacga agaaaaggtg 600 ttcgtgaaca acacgacgtg cgtgctcaac gacccaaatt tcgttcttat tgggtccttc 660 gtagctttct tcataccgct gacgattatg gtgattacgg gtaccagggt ctttcaggag 720 gccaaaaggc agctcc agaa gattgacaaa tctgagggcc gcttccatgt ccagaacctt 780 agccaggtgg agcaggatgg gcggacgggg catggactcc gcagatcttc caagttctgc 840 tccagggaaa agaaagcagc caaaaccttg gaattcgttt tctttgtgtt tctgatcatg 900 tggtgcccat ttttcattac caatattctg tctgttcttt gtgagaagtc ctgtaaccaa 960 aagctcatgg aaaagcttct gaatgtgttt gtttggattg gctatgtttg ttcaggaatc 1020 aatcctctgg tgtatactct gttcaacaaa atttaccgaa gggcattctc caactatttg 1080 cgttgcaatt ataaggtaga gaaaaagcct cctgtcaggc agattccaag agttgccgcc 1140 actgctttgt ctgggaggga gcttaatgtt aacatttatc ggcataccaa tgaaccggtg 1200 atcgagaaag ccagtgacaa tgagcccggt atagagatgc aagttgagaa tttagagtta 1260 ccagtaaatc cctccagtgt ggttagcgaa aggattagca gtgtggagaa ttccatgact 1320 tcgaaagttt atgatccaga acaaaggaaa cggatgataa ctggtccgca gtggtgggcc 1380 agatgtaaac aaatgaatgt tcttgattca tttattaatt attatgattc agaaaaacat 1440 gcagaaaatg ctgttatttt tttacatggt aacgcggcct cttcttattt atggcgacat 1500 gttgtgccac atattgagcc agtagcgcgg tgtattatac cagatcttat tggtatgggc 1560 aaatcaggca aatctggtaa tggtt cttat aggttacttg atcattacaa atatcttact 1620 gcatggtttg aacttcttaa tttaccaaag aagatcaatt ttgtcggcca tgattggggt 1680 gcttgtttgg catttcatta tagctatgag catcaagata agatcaaagc aatagttcac 1740 gctgaaagtg tagtagatgt gattgaatca tgggatgaat ggcctgatat tgaagaagat 1800 attgcgttga tcaaatctga agaaggagaa aaaatggttt tggagaataa cttcttcgtg 1860 gaaaccatgt tgccatcaaa aatcatgaga aagttagaac cagaagaatt tgcagcatat 1920 cttgaaccat tcaaagagaa aggtgaagtt cgtcgtccaa cattatcatg gcctcgtgaa 1980 atcccgttag taaaaggtgg taaacctgac gttgtacaaa ttgttaggaa ttataatgct 2040 tatctacgtg caagtgatga tttaccaaaa atgtttattg aatcggatcc aggattcttt 2100 tccaatgcta ttgttgaagg cgccaagaag tttcctaata ctgaatttgt caaagtaaaa 2160 ggtcttcatt tttcgcaaga agatgcacct gatgaaatgg gaaaatatat caaatcgttc 2220 -! 05 · 156362 · sequence Listing .doc 201131168 gttgagcgag ttctcaaaaa tgaacaataa <210> 49 <211> 1944 <212> DNA <213〉Artificial <220><223> Modified human dopamine receptor D3 agagctcctg tcggcgcgtg 49 atggcatctc tgagtcagct gagtagccac ctgaactaca cctgtggggc agagaactcc acaggtgcca gccaggcccg cccacatgcc tactatgccc tctcctactg cgcgctcatc ctggccatcg tcttcggcaa tggcctggtg tgcatggctg tgctgaagga gcgggccctg cagactacca ccaactactt agtagtgagc ctggctgtgg cagacttgct ggtggccacc ttggtgatgc cctgggtggt atacctggag gtgacaggtg gagtctggaa tttcagccgc atttgctgtg atgtttttgt caccctggat gtcatgatgt gtacagccag catccttaat ctctgtgcca tcagcataga caggtacact gcagtggtca tgcccgttca ctaccagcat ggcacgggac; < 400 & gt gccctcatga tcacggccgt ctgggtactg gcctttgctg tgtcctgccc tcttctgttt ggctttaata ccacagggga ccccactgtc tgctccatct ccaaccctga ttttgtcatc tactcttcag tggtgtcctt ctacctgccc tttggagtga ctgtccttgt cggtaccagg gtctttcagg aggccaaaag gcagctccag aagattgaca aatctgaggg ccgcttccat gtccagaacc ttagccaggt ggagcaggat gggcggacgg ggcatggact ccgcagatct tccaagttct gctccaggga aaagaaagca gccaaaacct tggaattcgt gcttggggcc ttcattgtct gctggctgcc cttcttcttg acccatgttc tcaataccca ctgccagaca tgccacgtgt ccccagagct ttacag tgcc acgacatggc tgggctacgt gaatagcgcc ctcaaccctg tgatctatac caccttcaat atcgagttcc ggaaagcctt cctcaagatc ctgtcttgcg agaattccal gacttcgaaa gtttatgatc cagaacaaag gaaacggatg ataactggtc cgcagtggtg ggccagatgt aaacaaatga atgttcttga ttcatttatt aattattatg attcagaaaa acatgcagaa aatgctgtta tttttttaca tggtaacgcg gcctcttctt atttatggcg acatgttgtg ccacatattg agccagtagc gcggtgtatt ataccagatc ttattggtat gggcaaatca ggcaaatctg gtaatggttc ttataggtta cttgatcatt acaaatatct tactgcatgg tttgaacttc ttaatttacc aaagaagatc aattttgtcg gccatgattg gggtgcttgt ttggcatttc Attatacta tgagcatcaa gataagatca aagcaatagt tcacgctgaa agtgtagtag atgtgattga atcatgggat gaatggcct atattgaaga agatattgcg 156362·sequence table.doc •106- 2250 60 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 201131168 ttgatcaaat ctgaagaagg agaaaaaatg Gttttggaga ataacttctt cgtggaaacc atgttgccat caaaaatcat gagaaagtta gaaccagaag aatttgcagc atatcttgaa ccattcaaag agaaaggtga agttcgtcgt ccaacattat catggc ctcg tgaaatcccg ttagtaaaag gtggtaaacc tgacgttgta caaattgtta ggaattataa tgcttatcta cgtgcaagtg atgatttacc aaaaatgttt attgaatcgg atccaggatt cttttccaat gctattgttg aaggcgccaa gaagtttcct aatactgaat ttgtcaaagt aaaaggtctt catttttcgc aagaagatgc acctgatgaa atgggaaaat atatcaaatc gttcgttgag cgagttctca aaaatgaaca ataa <210> 50 <211> 1968 <212> DNA <213>manpower <220><223> Modified human dopamine receptor D4 ≪ 400 > 50 atggggaacc gcagcaccgc ggacgcggac gggctgctgg ctgggcgcgg gccggccgcg ggggcatctg cgggggcatc tgcggggctg gctgggcagg gcgcggcggc gctggtgggg ggcgtgctgc tcatcggcgc ggtgctcgcg gggaactcgc tcgtgtgcgt gagcgtggcc accgagcgcg ccctgcagac gcccaccaac tccttcatcg tgagcctggc ggccgccgac ctcctcctcg ctctcctggt gctgccgctc ttcgtctact ccgaggtcca gggtggcgcg tggctgctga gcccccgcct gtgcgacgcc ctcatggcca tggacgtcat gctgtgcacc gcctccatct tcaacctgtg cgccatcagc gtggacaggt tcgtggccgt ggccgtgccg ctgcgctaca accggcaggg tgggagccgc cggcagctgc tgctcatcgg cgccacgtgg ctgctgtccg cggcggtggc ggcgcccgta ctgtgcggcc tcaacgacgt gcgcggccgc gaccccgccg tgtgccgcct ggaggaccgc gactacgtgg tctactcgtc cgtgtgctcc ttcttcctac cctgcccgct catgctgctg ctctactggg gtaccagggt ctttcaggag gccaaaaggc agctccagaa gattgacaaa tctgagggcc gcttccatgt ccagaacctt agccaggtgg agcaggatgg gcggacgggg catggactcc gcagatcttc caagttctgc tccagggaaa agaaagcagc caaaaccttg gaattcgtgg tcggggcctt cctgctgtgc tggacgccct tcttcgtggt gcacatcacg caggcgctgt gtcctgcctg ctccgt Gccc ccgcggctgg tcagcgccgt cacctggctg ggctacgtca acagcgccct caaccccgtc atctacactg tcttcaacgc cgagttccgc aacgtcttcc gcaaggccct gcgtgcctgc 156362·sequence table.doc •107- 1560 1620 1680 1740 1800 1860 1920 1944 60 120 180 240 300 360 420 480 540 600 660 720 *780 840 900 960 1020 1080 201131168 tgcgagaatt ccatgacttc gaaagtttat gatccagaac aaaggaaacg gatgataact ggtccgcagt ggtgggccag atgtaaacaa atgaatgttc ttgattcatt tattaattat tatgattcag aaaaacatgc agaaaatgct gttatttttt tacatggtaa cgcggcctct tcttatttat ggcgacatgt tgtgccacat attgagccag tagcgcggtg tattatacca gatcttattg gtatgggcaa atcaggcaaa tctggtaatg gttcttatag gttacttgat cattacaaat atcttactgc atggtttgaa cttcttaatt taccaaagaa gatcaatttt gtcggccatg attggggtgc ttgtttggca tttcattata gctatgagca tcaagataag atcaaagcaa tagttcacgc tgaaagtgta gtagatgtga ttgaatcatg ggatgaatgg cctgatattg aagaagatat Tgcgttgatc aaatctgaag aaggagaaaa aatggttttg gagaataact tcttcgtgga aaccatgttg ccatcaaaaa tcatgagaaa gttagaacca gaagaatttg cagcatatct tgaaccattc aaagagaaag gtgaa gttcg tcgtccaaca ttatcatggc ctcgtgaaat cccgttagta aaaggtggta aacctgacgt tgtacaaatt gttaggaatt ataatgctta tctacgtgca agtgatgatt taccaaaaat gtttattgaa tcggatccag gattcttttc caatgctatt gttgaaggcg ccaagaagtt tcctaatact gaatttgtca aagtaaaagg tcttcatttt tcgcaagaag atgcacctga tgaaatggga aaatatatca aatcgttcgt tgagcgagtt ctcaaaaatg aacaataa <210> 51 <211> 2271 <212> DNA <213>manpower <220><223> Modified human dopamine receptor D1 ≪ 400 > 51 atgaggactc tgaacacctc tgccatggac gggactgggc tggtggtgga gagggacttc tctgttcgta tcctcactgc ctgtttcctg tcgctgctca tcctgtccac gctcctgggg aacacgctgg tctgtgctgc cgttatcagg ttccgacacc tgcggtccaa ggtgaccaac ttctttgtca tctccttggc tgtgtcagat ctcttggtgg ccgtcctggt catgccctgg aaggcagtgg ctgagattgc tggcttctgg ccctttgggt ccttctgtaa catctgggtg gcctttgaca tcatgtgctc cactgcatcc atcctcaacc tctgtgtgat cagcgtggac aggtattggg ctatctccag ccctttccgg tatgagagaa agatgacccc caaggcagcc ttcatcctga tcagtgtggc atggaccttg Tctgtactca tctccttcat cccagtgcag ctcagctggc acaaggcaaa acccacaagc ccctctgatg gaaatgccac ttccctggct gagaccatag acaactgtga ctccagcctc agcaggacat atgccatctc atcctctgta 156362-sequence table.doc •108- 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1968 60 120 180 240 300 360 420 480 540 600 660 201131168 ataagctttt acatccctgt ggccatcatg attgtcaccg gtaccagggt ctttcaggag gccaaaaggc agctccagaa gattgacaaa tctgagggcc gcttccatgt ccagaacctt agccaggtgg agcaggatgg gcggacgggg cat ggactcc gcagatcttc caagttctgc tccagggaaa agaaagcagc caaaaccttg gaattcatca tgggtgtgtt tgtgtgctgt tcttcatctt gaactgcatt ttgcccttct gtgggtctgg ggagacgcag cccttctgca ttgattccaa cacctttgac gtgtttgtgt ggtttgggtg ggctaattca tccttgaacc ccatcattta tgcctttaat gctgattttc ggaaggcatt ttcaaccctc ttaggatgct acagactttg ccctgcgacg aataatgcca gagtatcaat aacaatgggg ccgcgatgtt ttccagccat catgagccac gaggctccat ctccaaggag tgcaatctgg tttacctgat cccacatgct gtgggctcct ctgaggacct gaaaaaggag gaggcagctg gcatcgccag acccttggag aagctgtccc cagccctatc tggctacctt tagagacggt ggtcatattg gactatgaca ctgacgtctc tctggagaag atccaaccca tgacacaaaa cggtcagcac ccaaccgaga attccatgac ttcgaaagtt tatgatccag aacaaaggaa acggatgata actggtccgc agtggtgggc cagatgtaaa caaatgaatg ttcttgattc atttattaat tattatgatt cagaaaaaca tgcagaaaat gctgttattt ttttacatgg taacgcggcc tcttcttatt tatggcgaca tgttgtgcca catattgagc cagtagcgcg gtgtattata ccagatctta ttggtatggg caaatcaggc aaatctggta atggttctta taggttactt gatcattaca aatatcttac tgcatggttt gaacttctta at ttaccaaa gaagatcaat tttgtcggcc atgattgggg tgcttgtttg gcatttcatt atagctatga gcatcaagat aagatcaaag caatagttca cgctgaaagt gtagtagatg tgattgaatc atgggatgaa tggcctgata ttgaagaaga tattgcgttg atcaaatctg aagaaggaga aaaaatggtt ttggagaata acttcttcgt ggaaaccatg ttgccatcaa aaatcatgag aaagttagaa ccagaagaat ttgcagcata tcttgaacca ttcaaagaga aaggtgaagt tcgtcgtcca acattatcat ggcctcgtga aatcccgtta gtaaaaggtg gtaaacctga cgttgtacaa attgttagga attataatgc ttatctacgt gcaagtgatg atttaccaaa aatgtttatt gaatcggatc caggattctt ttccaatgct attgttgaag gcgccaagaa gtttcctaat Actgaatttg tcaaagtaaa aggtcttcat ttttcgcaag aagatgcacc tgatgaaatg ggaaaatata tcaaatcgtt cgttgagcga gttctcaaaa atgaacaata a

<210> 52 <211> 2088 <212> DNA 156362 - Sequence Listing. doc • 109· 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 2100 2160 2220 2271 201131168 <213>manpower <220><223> Modified human histamine receptor H2 ≪ 400 > 52 atggcaccca atggcacagc ctcttccttt tgcctggact ctaccgcatg caagatcacc 60 atcaccgtgg tccttgcggt cctcatcctc atcaccgttg ctggcaatgt ggtcgtctgt 120 ctggccgtgg gcttgaaccg ccggctccgc aacctgacca attgtttcat cgtgtccttg 180 gctatcactg acctgctcct cggcctcctg gtgctgccct tctctgccat ctaccagctg 240 tcctgcaagt ggagctttgg caaggtcttc tgcaatatct acaccagcct ggatgtgatg 300 ctctgcacag cctccattct taacctcttc atgatcagcc tcgaccggta ctgcgctgtc 360 atggacccac tgcggtaccc ( gtgctggtc accccagttc gggtcgccat ctctctggtc 420 ttaatttggg tcatctccat taccctgtcc tttctgtcta tccacctggg gtggaacagc 480 aggaacgaga ccagcaaggg caatcatacc acctctaagt gcaaagtcca ggtcaatgaa 540 gtgtacgggc tggtggatgg gctggtcacc ttctacctcc cgctactgat catgtgcatc 600 accggtacca gggtctttca ggaggccaaa aggcagctcc agaagattga caaatctgag 660 ggccgcttcc atgtccagaa ccttagccag gtggagcagg atgggcggac ggggcatgga 720 ctccgcagat cttccaagtt ctgctccagg gaaaagaaag cagccaaaac cttggaattc 780 gtcatggggg ccttcatcat ctgctggttt Ccctacttca ccgcgtttgt gtaccgtggg 840 ctgaga gggg atgatgccat caatgaggtg ttagaagcca tcgttctgtg gctgggctat 900 gccaactcag ccctgaaccc catcctgtat gctgcgctga acagagactt ccgcaccggg 960 taccaacagc tcttctgctg caggctggcc aaccgcaact cccacaaaac ttctctgagg 1020 tccaacgcct ctcagctgtc caggacccaa agccgagaac ccaggcaaca ggaagagaaa 1080 cccctgaagc tccaggtgtg gagtgggaca gaagtcacgg ccccccaggg agccacagac 1140 agggagaatt ccatgacttc gaaagtttat gatccagaac aaaggaaacg gatgataact 1200 ggtccgcagt ggtgggccag atgtaaacaa atgaatgttc ttgattcatt tattaattat 1260 tatgattcag aaaaacatgc agaaaatgct gttatttttt tacatggtaa cgcggcctct 1320 tcttatttat ggcgacatgt tgtgccacat attgagccag tagcgcggtg tattatacca 1380 gatcttattg gtatgggcaa atcaggcaaa tctggtaatg gttcttatag gttacttgat 1440 cattacaaat atcttactgc atggtttgaa cttcttaatt taccaaagaa gatcaatttt 1500 gtcggccatg attggggtgc ttgtttggca tttcattata gctatgagca tcaagataag 1560 atcaaagcaa tagttcacgc tgaaagtgta gtagatgtga ttgaatcatg ggatgaatgg 1620 cctgatattg aagaagatat tgcgttgatc aaatctgaag aaggagaaaa aatggttttg 1680 -110 - 156362· SEQUENCE LISTING .doc 201131168 gagaataact tcttcgtgga gaagaatttg cagcatatct ttatcatggc ctcgtgaaat gttaggaatt ataatgctta tcggatccag gattcttttc gaatttgtca aagtaaaagg aaatatatca aatcgttcgt aaccatgttg ccatcaaaaa tgaaccattc aaagagaaag cccgttagta aaaggtggta tctacgtgca agtgatgatt caatgctatt gttgaaggcg tcttcatttt tcgcaagaag tgagcgagtt ctcaaaaatg tcatgagaaa gttagaacca gtgaagttcg tcgtccaaca aacctgacgt tgtacaaatt taccaaaaat gtttattgaa ccaagaagtt tcctaatact atgcacctga tgaaatggga aacaataa 1740 1800 1860 1920 1980 2040 2088 <210> 53 <211> 2022 <212> DNA <213>manpower <220><223> Modified human histamine receptor H3 ≪ 400 > 53 atggagcgcg cgccgcccga cgggccgctg aacgcttcgg gggcgctggc gggcgatgcg gcggcggcgg gcggggcgcg cggcttctcg gcagcctgga ccgcggtgct ggccgcgctc atggcgctgc tcatcgtggc cacggtgctg ggcaacgcgc tggtcatgct cgccttcgtg gccgactcga gcctccgcac ccagaacaac ttcttcctgc tcaacctcgc catctccgac ttcctcgtcg gcgccttctg catcccactg tatgtaccct acgtgctgac aggccgctgg accttcggcc ggggcctctg caagctgtgg ctggtagtgg actacctgct gtgcacctcc tctgccttca acatcgtgct catcagctac gaccgcttcc tgtcggtcac ccgagcggtc tcataccggg cccagcaggg tgacacgcgg cgggcagtgc ggaagatgct gctggtgtgg gtgctggcct tcctgctgta cggaccagcc atcctgagct gggagtacct gtccgggggc agctccatcc ccgagggcca ctgctatgcc gagttcttct acaactggta cttcctcatc acggcttcca ccctggagtt ctttacgccc ttcctcagcg tcaccttctt tggtaccagg gtctttcagg aggccaaaag gcagctccag aagattgaca aatctgaggg ccgcttccat gtccagaacc ttagccaggt ggagcaggat gggcggacgg ggcatggact ccgcagatct tccaagttct gctccaggga aaagaaagca gccaaaacct tggaattcat cgtgagcatc tttgggctct gctgggcccc atacacgctg ctgatgatca tccgggccgc ctgcca tggc cactgcgtcc ctgactactg gtacgaaacc tccttctggc tcctgtgggc caactcggct gtcaaccctg tcctctaccc tctgtgccac cacagcttcc gccgggcctt caccaagctg ctctgccccc agaagctcaa aatccagccc cacagctccc tggagcactg ctggaaggag 156362- Sequence Listing .doc -Ill - 60 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 201131168 aattccatga cttcgaaagt ttatgatcca gaacaaagga aacggatgat aactggtccg cagtggtggg ccagatgtaa acaaatgaat gttcttgatt catttattaa ttattatgat tcagaaaaac atgcagaaaa tgctgttatt tttttacatg gtaacgcggc ctcttcttat ttatggcgac atgttgtgcc acatattgag ccagtagcgc ggtgtattat accagatctt attggtatgg gcaaatcagg caaatctggt aatggttctt ataggttact tgatcattac aaatatctta ctgcatggtt tgaacttctt aatttaccaa agaagatcaa ttttgtcggc catgattggg gtgcttgttt ggcatttcat tatagctatg agcatcaaga taagatcaaa gcaatagttc acgctgaaag tgtagtagat gtgattgaat catgggatga atggcctgat attgaagaag atattgcgtt gatcaaatct gaagaaggag Aaaaaatggt tttggagaat aacttcttcg tggaaaccat gttgccatca aaaatcatga gaaagttaga accagaagaa tttgcagcat atcttgaacc attcaaagag aaaggtgaag ttcgtcgtcc aacattatca tggcctcgtg aaatcccgtt agtaaaaggt ggtaaacctg acgttgtaca aattgttagg aattataatg cttatctacg tgcaagtgat gatttaccaa aaatgtttat tgaatcggat ccaggattct tttccaatgc tattgttgaa ggcgccaaga agtttcctaa tactgaattt gtcaaagtaa aaggtcttca tttttcgcaa gaagatgcac ctgatgaaat gggaaaatat atcaaatcgt tcgttgagcg agttctcaaa aatgaacaat aa <210> 54 <211> 1983 <212> DNA <213>manpower <220><223> Modified Porcine Adrenergic Receptor, alpha-2A ≪ 400 > 54 atgggctccc tgcagccgga agcgggcaac gcgagctgga atgggacaga ggcgccgggg ggcggcgccc gggccacccc ctactccctg caggtgacac tgacgctggt gtgcctggcc ggcctgctca tgctgttcac cgtgttcggc aacgtgcttg tcatcattgc cgtgttcaca agccgcgcgc tcaaggcgcc ccagaacctc ttcctggtgt ctctggcctc ggctgacatc ctagtggcca cgcttgtcat ccctttctcg ctggccaacg aggtcatggg ctactggtac ttcggcaagg cgtggtgtga gatctacctg gcgctcgacg tgctcttctg cacgtcgtcc atcgtgcacc tgtgtgccat cagcttggat cgttactggt ccatcaccca ggccatagag tacaacctga agcgcacgcc acgccgcatc Aaagcaatca tcgtcaccgt gtgggtcatc tcggccgtca tctccttccc gccgctcatc tccatcgaga agaaggcagg cggcggtggc cagcagccgg ccgaaccgcg ctgcgagatc aacgaccaga agtggtacgt catctcgtct 156362-sequence table.doc-112-1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2022 60 120 180 240 300 360 420 480 540 600 660 201131168 tgcatcggct ccttcttcgc gccctgcctc atcatgatcc tggtcggtac cagggtcttt caggaggcca aaaggcagct ccagaagatt gacaaatctg agggccgctt ccatgtccag aaccttagcc aggtggagca ggatgggcgg acggg gcatg gactccgcag atcttccaag ttctgctcca gggaaaagaa agcagccaaa accttggaat tcgtcatagg cgtgttcgtg gtctgttggt tccccttctt cttcacctat acgctcacgg ccgtaggctg ctcggtgccg cccactctct tcaagttctt cttctggttc ggctactgca acagctcgct gaatccggtt atctacacca tcttcaatca cgacttccgc cgcgccttca agaagatcct ctgccgtggg gacaggaaac ggatcgtgga gaattccatg acttcgaaag tttatgatcc agaacaaagg aaacggatga taactggtcc gcagtggtgg gccagatgta aacaaatgaa tgttcttgat tcatttatta attattatga ttcagaaaaa catgcagaaa atgctgttat ttttttacat ggtaacgcgg cctcttctta tttatggcga catgttgtgc cacatattga gccagtagcg cggtgtatta taccagatct tattggtatg ggcaaatcag gcaaatctgg taatggttct tataggttac ttgatcatta caaatatctt actgcatggt ttgaacttct taatttacca aagaagatca attttgtcgg ccatgattgg ggtgcttgtt tggcatttca ttatagctat gagcatcaag ataagatcaa agcaatagtt cacgctgaaa gtgtagtaga. tgtgattgaa tcatgggatg aatggcctga tattgaagaa gatattgcgt tgatcaaatc tgaagaagga gaaaaaatgg ttttggagaa taacttcttc gtggaaacca tgttgccatc aaaaatcatg agaaagttag aaccagaaga atttgcagca tatcttgaac cat tcaaaga gaaaggtgaa gttcgtcgtc caacattatc atggcctcgt gaaatcccgt tagtaaaagg tggtaaacct gacgttgtac aaattgttag gaattataat gcttatctac gtgcaagtga tgatttacca aaaatgttta ttgaatcgga tccaggattc ttttccaatg ctattgttga aggcgccaag aagtttccta atactgaatt tgtcaaagta aaaggtcttc atttttcgca agaagatgca cctgatgaaa tgggaaaata tatcaaatcg ttcgttgagc gagttctcaa aaatgaacaa taa <210> 55 <211> 2073 <212> DNA <213>manpower <220><223> Modified human galanin receptor 1 <400> 55 atggagctgg cggtcgggaa cctcagcgag ggcaacgcga gctgtccgga gccccccgcc 156362. Sequence Listing.doc • 113-720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 I860 1920 1980 1983 gtggagaact tcgtcacgct ggtggtgttc 120 aacagcctag tgatcaccgt gctggcgcgc 180 aacctgttca tcctcaacct gagcatcgcc 240 ttccaggcca ccgtgtacgc gctgcccacc 300 atccactact tcttcaccgt gtccatgctg 360 gtggaccgct acgtggccat cgtgcactcg 420 aacgcgctgc tgggcgtggg ctgcatctgg 480 gcctaccacc agggcctctt ccacccgcgc 540 tggcccgacc ctcgccacaa gaaggcctac 600 ctgccgctcc tgctcatctg cttctgcggt 660 ctccagaaga ttgacaaatc tgagggccgc 720 caggatgggc ggacggggca tggactccgc 780 aaagcagcca aaaccttgga attcgtggtt 840 cacatcatcc atctctgggc tgagtttgga 900 ttcagaatca Ccgcccactg cctggcgtac 960 gcatttctct ctgaaaattt caggaaggcc 1020 aaagattcac acctgagtga tactaaagaa 1080 accaattgta ctcatgtgga gaattccatg 1140 aaacggatga taactggtcc gcagtggtgg 1200 tcatttatta attattatga ttcagaaaaa 1260 ggtaacgcgg cctc ttctta tttatggcga 1320 cggtgtatta taccagatct tattggtatg 1380 tataggttac ttgatcatta caaatatctt 1440 aagaagatca attttgtcgg ccatgattgg 1500 gagcatcaag ataagatcaa agcaatagtt 1560 tcatgggatg aatggcctga tattgaagaa 1620 gaaaaaatgg ttttggagaa taacttcttc 1680 agaaagttag aaccagaaga atttgcagca 1740 gttcgtcgtc caacattatc atggcctcgt 1800 gacgttgtac aaattgttag gaattataat 1860 aaaatgttta ttgaatcgga tccaggattc 1920 201131168 ccggagcccg ggccgctgtt cggcatcggc ggcctgatct tcgcgctggg cgtgctgggc agcaagccgg gcaagccgcg gagcaccacc gacctggcct acctgctctt ctgcatcccc tgggtgctgg gcgccttcat ctgcaagttc gtgagcatct tcaccctggc cgcgatgtcc cggcgctcct cctccctcag ggtgtcccgc gcgctgtcca ttgccatggc ctcgcccgtg gccagcaacc agaccttctg ctgggagcag gtggtgtgca ccttcgtctt cggctacctg accagggtct ttcaggaggc caaaaggcag ttccatgtcc agaaccttag ccaggtggag agatcttcca agttctgctc cagggaaaag gtggtgtttg gaatctcctg gctgccgcac gttttcccgc tgacgccggc ttccttcctc agcaattcct ccgtgaatcc tatcatttat tataaacaag tgttcaagtg tcacattcgc aataaaagtc gaata gacac cccaccatca acttcgaaag tttatgatcc agaacaaagg gccagatgta aacaaatgaa tgttcttgat catgcagaaa atgctgttat ttttttacat catgttgtgc cacatattga gccagtagcg ggcaaatcag gcaaatctgg taatggttct actgcatggt ttgaacttct taatttacca ggtgcttgtt tggcatttca ttatagctat cacgctgaaa gtgtagtaga tgtgattgaa gatattgcgt tgatcaaatc tgaagaagga gtggaaacca tgttgccatc aaaaatcatg tatcttgaac cattcaaaga gaaaggtgaa gaaatcccgt tagtaaaagg tggtaaacct gcttatctac gtgcaagtga tgatttacca 156362- sequence table .doc • 114 · 201131168 ttttccaatg ctattgttga aggcgccaag aagtttccta atactgaatt tgtcaaagta aaaggtcttc atttttcgca agaagatgca cctgatgaaa tgggaaaata tatcaaatcg ttcgttgagc gagttctcaa aaatgaacaa taa <210> 56 <211> 2163 <212> DNA <213>manpower <220><223> Modified human neuropeptide Y receptor Y1 ≪ 400 > 56 atgaattcaa cattattttc ccaggttgaa aatcattcag tccactctaa tttctcagag aagaatgccc agcttctggc ttttgaaaat gatgattgtc atctgccctt ggccatgata tttaccttag ctcttgctta tggagctgtg atcattcttg gtgtctctgg aaacctggcc ttgatcataa tcatcttgaa acaaaaggag atgagaaatg ttaccaacat cctgattgtg aacctttcct tctcagactt gcttgttgcc atcatgtgtc tcccctttac atttgtctac acattaatgg accactgggt ctttggtgag gcgatgtgta agttgaatcc ttttgtgcaa tgtgtttcaa tcactgtgtc cattttctct ctggttctca ttgctgtgga acgacatcag ctgataatca accctcgagg gtggagacca aataatagac atgcttatgt aggtattgct gtgatttggg tccttgctgt ggcttcttct ttgcctttcc tgatctacca agtaatgact gatgagccgt tccaaaatgt aacacttgat gcgtacaaag acaaatacgt gtgctttgat caatttccat cggactctca taggttgtct tataccactc tcctcttggt gctgcagtat tttggtccac tttgttttat atttatttgc ggtaccaggg tctttcagga ggccaaaagg cagctccaga agattgacaa atctgagggc cgcttccatg tccagaacct tagccaggtg gagcaggatg ggcggacggg gcatggactc cgcagatctt ccaagttctg ctccagggaa aagaaagcag ccaaaacctt ggaattcatt gtggtagcat ttgcagtctg ctggct ccct cttaccatct ttaacactgt gtttgattgg aatcatcaga tcattgctac ctgcaaccac aatctgttat tcctgctctg ccacctcaca gcaatgatat ccacttgtgt caaccccata ttttatgggt tcctgaacaa aaacttccag agagacttgc agttcttctt caacttttgt gatttccggt ctcgggatga tgattatgaa acaatagcca tgtccacgat gcacacagat gtttccaaaa cttctttgaa gcaagcaagc ccagtcgcat ttaaaaaaat caacaacaat gatgataatg aaaaaatcga gaattccatg acttcgaaag tttatgatcc agaacaaagg aaacggatga taactggtcc gcagtggtgg gccagatgta aacaaatgaa tgttcttgat 156362- sequence table .doc • 115- 1980 2040 2073 60 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 201131168 tcatttatta attattatga ttcagaaaaa catgcagaaa atgctgttat ttttttacat ggtaacgcgg cctcttctta tttatggcga catgttgtgc cacatattga gccagtagcg cggtgtatta taccagatct tattggtatg ggcaaatcag gcaaatctgg taatggttct tataggttac ttgatcatta caaatatctt Actgcatggt ttgaacttct taatttacca aagaagatca attttgtcgg ccatgattgg ggtgcttgtt tggcatttca ttatagctat gagcatcaag ataagatcaa agcaatagtt cacgctgaaa gtgtagt aga tgtgattgaa tcatgggatg aatggcctga tattgaagaa gatattgcgt tgatcaaatc tgaagaagga gaaaaaatgg ttttggagaa taacttcttc gtggaaacca tgttgccatc aaaaatcatg agaaagttag aaccagaaga atttgcagca tatcttgaac cattcaaaga gaaaggtgaa gttcgtcgtc caacattatc atggcctcgt gaaatcccgt tagtaaaagg tggtaaacct gacgttgtac aaattgttag gaattataat gcttatctac gtgcaagtga tgatttacca aaaatgttta ttgaatcgga tccaggattc ttttccaatg ctattgttga aggcgccaag aagtttccta atactgaatt tgtcaaagta aaaggtcttc atttttcgca agaagatgca cctgatgaaa tgggaaaata tatcaaatcg ttcgttgagc gagttctcaa aaatgaacaa Taa <210> 57 <211> 2109 <212> DNA <213〉Artificial <220〉 <223> Modified human angiotensin 11 receptor 1 ≪ 400 > 57 atgattctca actcttctac tgaagatggt attaaaagaa tccaagatga ttgtcccaaa gctggaaggc ataattacat atttgtcatg attcctactt tatacagtat catctttgtg gtgggaatat ttggaaacag cttggtggtg atagtcattt acttttatat gaagctgaag actgtggcca gtgtttttct tttgaattta gcactggctg acttatgctt tttactgact ttgccactat gggctgtcta cacagctatg gaataccgct ggccctttgg caattaccta tgtaagattg cttcagccag cgtcagtttc aacctgtacg ctagtgtgtt tctactcacg tgtctcagca ttgatcgata cctggctatt gttcacccaa tgaagtcccg ccttcgacgc acaatgcttg tagccaaagt cacctgcatc atcatttggc tgctggcagg cttggccagt ttgccagcta taatccatcg aaatgtattt ttcattgaga acaccaatat tacagtttgt gctttccatt atgagtccca aaattcaacc cttccgatag ggctgggcct gaccaaaaat atactgggtt tcctgtttcc ttttctgatc attcttacaa gtggtaccag ggtctttcag 156362 · sequence Listing .doc • 116 · 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 2100 2160 2163 60 120 180 240 300 360 420 480 540 600 660 720 201131168 gaggccaaaa ggcagctcca gaagattgac aaatctgagg gccgcttcca tgtccagaac ctlagccagg tggagcagga tgggcggacg gg gcatggac tccgcagatc ttccaagttc tgctccaggg aaaagaaagc agccaaaacc ttggaattca ttgtgctttt ctttttcttt tcctggattc cccaccaaat attcactttt ctggatgtat tgattcaact aggcatcata cgtgactgta gaattgcaga tattgtggac acggccatgc ctatcaccat ttgtatagct tattttaaca attgcctgaa tcctcttttt tatggctttc tggggaaaaa atttaaaaga tattttctcc agcttctaaa atatattccc ccaaaagcca aatcccactc aaacctttca acaaaaatga gcacgctttc ctaccgcccc tcagataatg taagctcatc caccaagaag cctgcaccat gttttgaggt tgaggagaat tccatgactt cgaaagttta tgatccagaa caaaggaaac ggatgataac tggtccgcag tggtgggcca gatgtaaaca aatgaatgtt cttgattcat ttattaatta ttatgattca gaaaaacatg cagaaaatgc tgttattttt ttacatggta acgcggcctc ttcttattta tggcgacatg ttgtgccaca agatcttatt tattgagcca gtagcgcggt gtattatacc attgaatcat gggatgaatg gcctgatatt ggtatgggca aatcaggcaa atctggtaat ggttcttata ggttacttga tcattacaaa tatcttactg catggtttga acttcttaat ttaccaaaga agatcaattt tgtcggccat gattggggtg cttgtttggc atttcattat agctatgagc atcaagataa gatcaaagca atagttcacg ctgaaagtgt agtagatgtg gaagaagata t tgcgttgat caaatctgaa gaaggagaaa aaatggtttt ggagaataac ttcttcgtgg aaaccatgtt gccatcaaaa atcatgagaa agttagaacc agaagaattt gcagcatatc ttgaaccatt caaagagaaa ggtgaagttc gtcgtccaac attatcatgg cctcgtgaaa tcccgttagt aaaaggtggt aaacctgacg ttgtacaaat tgttaggaat tataatgctt atctacgtgc aagtgatgat ttaccaaaaa tgtttattga atcggatcca ggattctttt ccaatgctat tgttgaaggc gccaagaagt ttcctaatac tgaatttgtc aaagtaaaag gtcttcattt ttcgcaagaa gatgcacctg atgaaatggg aaaatatatc aaatcgttcg ttgagcgagt tctcaaaaat gaacaataa <210> 58 <211> 2220 <212> DNA <213> ΛΧ <220><223> modified, neuromodulin receptor 1 <400> 58 156362 - Sequence Listing.doc -117-780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 2100 2109 201131168 atgcgcctca acagctccgc gccgggaacc cgggcgcagg ccggactgga ggaggcgctg aacgcgtcgg agcgcgtcct ggcggcaccc tactccaaag tgctggtgac cgccgtgtac aacacggtga cggcgttcac gctggcgcgg gtgcattacc acctgggcag cctggcgctg cccgtggagc tgtacaactt catctgggtg tgccgcggct actacttcct gcgcgacgcc agcctgagtg tggagcgcta cctggccatc tcccgaagcc gcaccaagaa gttcatcagc gtgcctatgc tgttcaccat gggcgagcag ggcctggtgt gcacccccac catccacact accttcatgt ccttcatatt ccccatggtg caggaggcca aaaggcagct ccagaagatt aaccttagcc aggtggagca ggatgggcgg ttctgctcca gggaaaagaa agcagccaaa gtctgctggc tgccctacca cgtgcggcgc tggactccgt tcctctatga cttctaccac tacgtcagct ccaccatcaa ccccatcctg atcttcctgg ccacactggc ctgcctctgc gccttctcga Ggaaggccga cagcgtgtcc cgcgagacgc tgtacgagaa ttccatgact cggatgataa ctggtccgca gtggtgggcc tttattaatt attatgattc agaaaaacat aacgcggcct cttcttattt atggcgacat tgtatta tac cagatcttat tggtatgggc aggttacttg atcattacaa atatcttact aagatcaatt ttgtcggcca tgattggggt catcaagata agatcaaagc aatagttcac tgggatgaat ggcctgatat tgaagaagat aaaatggttt tggagaataa cttcttcgtg ccgggcacgc cggccgccga ccccttccag 60 ctggccccgg gcttcggcaa cgcttcgggc 120 agcagcgagc tggacgtgaa caccgacatc 180 ctggcgctct tcgtggtggg cacggtgggc 240 aagaagtcgc tgcagagcct gcagagcacg 300 tccgacctgc tcaccctgct gctggccatg 360 caccacccct gggccttcgg cgacgccggc 420 tgcacctacg ccacggccct caacgtggcc 480 tgccacccct tcaaggccaa gaccctcatg 540 gccatctggc tcgcctcggc cctgctgacg 600 aaccgcagcg ccgacggcca gcacgccggc 660 gccaccgtca aggtcgtcat acaggtcaac 720 gtcatctcgg tcctgggtac cagggtcttt 780 gacaaatctg agggccgctt ccatgtccag 840 acggggcatg gactccgcag atcttccaag 900 accttggaat tcgtggtcat cgcctttgtg 960 ctcatgttct gctacatctc ggatgagcag 1020 tacttctaca tggtgaccaa cgcactcttc 1080 tacaacctcg tctctgccaa cttccgccac 1140 ccggtgtggc ggcgcaggag gaagaggcca 1200 agcaaccaca ccctctccag Caatgccacc 1260 tcgaaagttt atgatccaga acaaag gaaa 1320 agatgtaaac aaatgaatgt tcttgattca 1380 gcagaaaatg ctgttatttt tttacatggt 1440 gttgtgccac atattgagcc agtagcgcgg 3500 aaatcaggca aatctggtaa tggttcttat 1560 gcatggtttg aacttcttaa tttaccaaag 1620 gcttgtttgg catttcatta tagctatgag 1680 gctgaaagtg tagtagatgt gattgaatca 1740 attgcgttga tcaaatctga agaaggagaa 1800 gaaaccatgt tgccatcaaa aatcatgaga 1860 • 118 · 156362 · Sequence Listing .doc 201131168 aagttagaac cagaagaatt tgcagcatat cttgaaccat tcaaagagaa aggtgaagtt cgtcgtccaa cattatcatg gcctcgtgaa atcccgttag taaaaggtgg taaacctgac gttgtacaaa ttgttaggaa ttataatgct tatctacgtg caagtgatga tttaccaaaa atgtttattg aatcggatcc aggattcttt tccaatgcta ttgttgaagg cgccaagaag tttcctaata ctgaatttgt caaagtaaaa ggtcttcatt tttcgcaaga agatgcacct gatgaaatgg gaaaatatat caaatcgttc gttgagcgag ttctcaaaaa tgaacaataa <210> 59 <211> 2022 <212> DNA <213>manual+ <220><223> Human Melanocortin 4 Receptor ≪ 400 > 59 atggtgaact ccacccaccg tgggatgcac acttctctgc acctctggaa ccgcagcagt tacagactgc acagcaatgc cagtgagtcc cttggaaaag gctactctga tggagggtgc tacgagcaac tttttgtctc tcctgaggtg tttgtgactc tgggtgtcat cagcttgttg gagaatatct tagtgattgt ggcaatagcc aagaacaaga atctgcattc acccatgtac tttttcatct gcagcttggc tgtggctgat atgctggtga gcgtttcaaa tggatcagaa accattatca tcaccctatt aaacagtaca gatacggatg cacagagttt cacagtgaat attgataatg tcattgactc ggtgatctgt agctccttgc ttgcatccat ttgcagcctg ctttcaattg cagtggacag gtactttact atcttctatg ctctccagta ccataacatt atgacagtta agcgggttgg gatcagcata agttgtatct gggcagcttg cacggtttca ggcattttgt tcatcattta ctcagatagt agtgctgtca tcatctgcct catcaccatg ttcttcacca tgctggctct catggcttct ctctatgtcc acatgttcct gggtaccagg gtctttcagg aggccaaaag gcagctccag aagattgaca aatctgaggg ccgcttccat gtccagaacc ttagccaggt ggagcaggat gggcggacgg ggcatggact ccgcagatct tccaagttct gctccaggga aaagaaagca gccaaaacct tggaattcct gattggcgtc tttgttgtct gctgggcccc attcttcctc cacttaatat tctacatctc ttgtcc tcag aatccatatt gtgtgtgctt catgtctcac tttaacttgt atctcatact gatcatgtgt aattcaatca tcgatcctct gatttatgca ctccggagtc aagaactgag gaaaaccttc aaagagatca tctgttgcta tcccctggga ggcctttgtg acttgtctag cagatatgag aattccatga cttcgaaagt ttatgatcca gaacaaagga aacggatgat aactggtccg 156362- Sequence Listing .doc 119- 1920 1980 2040 2100 2160 2220 60 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 201131168 cagtggtggg ccagatgtaa acaaatgaat gttcttgatt catttattaa ttattatgat tcagaaaaac atgcagaaaa tgctgttatt tttttacatg gtaacgcggc ctcttcttat ttatggcgac atgttgtgcc acatattgag ccagtagcgc ggtgtattat accagatctt attggtatgg gcaaatcagg caaatctggt aatggttctt ataggttact tgatcattac aaatatctta ctgcatggtt tgaacttctt aatttaccaa agaagatcaa ttttgtcggc catgattggg gtgcttgttt ggcatttcat tatagctatg agcatcaaga taagatcaaa gcaatagttc acgctgaaag tgtagtagat gtgattgaat Catgggatga atggcctgat attgaagaag atattgcgtt gatcaaatct gaagaaggag aaaaaatggt tttggagaat aacttcttcg tggaaaccat gttgccatca aaaatcatga gaaagttaga accagaagaa tttgcagcat atcttgaacc attcaaagag aaaggtgaag ttcgtcgtcc aacattatca tggcctcgtg aaatcccgtt agtaaaaggt ggtaaacctg acgttgtaca aattgttagg aattataatg cttatctacg tgcaagtgat gatttaccaa aaatgtttat tgaatcggat ccaggattct tttccaatgc tattgttgaa ggcgccaaga agtttcctaa tactgaattt gtcaaagtaa aaggtcttca tttttcgcaa gaagatgcac ctgatgaaat gggaaaatat atcaaatcgt tcgttgagcg agttctcaaa aatgaacaat aa <210> 60 <211> 2457 <212〉 DNA <213>manpower <220><223> Modified human glycoprotein-like peptide 1 receptor ≪ 400 > 60 atggccggcg cccccggccc gctgcgcctt gcgctgctgc tgctcgggat ggtgggcagg gccggccccc gcccccaggg tgccactgtg tccctctggg agacggtgca gaaatggcga gaataccgac gccagtgcca gcgctccctg actgaggatc cacctcctgc cacagacttg ttctgcaacc ggaccttcga tgaatacgcc tgctggccag atggggagcc aggctcgttc gtgaatgtca gctgcccctg gtacctgccc tgggccagca gtgtgccgca gggccacgtg taccggttct gcacagctga aggcctctgg ctgcagaagg acaactccag cctgccctgg agggacttgt cggagtgcga ggagtccaag cgaggggaga gaagctcccc ggaggagcag ctcctgttcc tctacatcat ctacacggtg ggctacgcac tctccttctc tgctctggtt atcgcctctg cgatcctcct cggcttcaga cacctgcact gcaccaggaa ctacatccac ctgaacctgt ttgcatcctt catcctgcga gcattgtccg tcttcatcaa ggacgcagcc ctgaagtgga tgtatagcac agccgcccag cagcaccagt gggatgggct cctctcctac 156362- sequence Listing .doc • 120 · 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2022 60 120 180 240 300 360 420 480 540 600 660 720 201131168 caggactctc tgagctgccg cctggtgttt ctgctcatgc agtactgtgt ggcggccaat tactactggc tcttggtgga gggcgtgtac ctg tacacac tgctggcctt ctcggtcttc tctgagcaat ggatcttcag gctctacgtg agcataggct ggggtgttcc cctgctgttt gttgtcccct ggggcattgt caagtacctc tatgaggacg agggctgctg gaccaggaac tccaacatga actactggct cattatccgg ctgcccattc tctttggcat tggggtgaac ttcctcatct ttgttcgggt catctgcggt accagggtct ttcaggaggc caaaaggcag ctccagaaga ttgacaaatc tgagggccgc ttccatgtcc agaaccttag ccaggtggag caggatgggc ggacggggca tggactccgc agatcttcca agttctgctc cagggaaaag aaagcagcca aaaccttgga attcagactt gccaagtcca cgctgacact catccccctg ctggggactc atgaggtcat ctttgccttt gtgatggacg agcacgcccg ggggaccctg cgcttcatca agctgtttac agagctctcc ttcacctcct tccaggggct gatggtggcc atcttatact gctttgtcaa caatgaggtc cagctggaat ttcggaagag ctgggagcgc tggcggcttg agcacttgca catccagagg gacagcagca tgaagcccct caagtgtccc accagcagcc tgagcagtgg agccacggcg ggcagcagca tgtacacagc cacttgccag gcctcctgca gcgagaattc catgacttcg aaagtttatg atccagaaca aaggaaacgg atgataactg gtccgcagtg gtgggccaga tgtaaacaaa tgaatgttct tgattcattt attaattatt atgattcaga aaaacatgca gaaaatgctg tt attttttt acatggtaac gcggcctctt cttatttatg gcgacatgtt gtgccacata ttgagccagt agcgcggtgt attataccag atcttattgg tatgggcaaa tcaggcaaat ctggtaatgg ttcttatagg ttacttgatc attacaaata tcttactgca tggtttgaac ttcttaattt accaaagaag atcaattttg tcggccatga ttggggtgct tgtttggcat ttcattatag ctatgagcat caagataaga tcaaagcaat agttcacgct gaaagtgtag tagatgtgat tgaatcatgg gatgaatggc ctgatattga agaagatatt gcgttgatca aatctgaaga aggagaaaaa atggttttgg agaataactt cttcgtggaa accatgttgc catcaaaaat catgagaaag ttagaaccag aagaatttgc agcatatctt gaaccattca aagagaaagg tgaagttcgt cgtccaacat tatcatggcc tcgtgaaatc ccgttagtaa aaggtggtaa acctgacgtt gtacaaattg ttaggaatta taatgcttat ctacgtgcaa gtgatgattt accaaaaatg tttattgaat cggatccagg attcttttcc aatgctattg ttgaaggcgc caagaagttt cctaatactg aatttgtcaa agtaaaaggt cttcattttt cgcaagaaga tgcacctgat gaaatgggaa aatatatcaa atcgttcgtt gagcgagttc tcaaaaatga acaataa 156362- sequence Listing .doc • 121- 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 2100 2160 2220 2280 2340 2400 2457 201131168 <210> 61 <211> 1983 <212> DNA <213>manpower <220〉 <223> Modified human adenosine A1 receptor ≪ 400 > 61 atgccgccct ccatctcagc tttccaggcc gcctacatcg gcatcgaggt gctcatcgcc 60 ctggtctctg tgcccgggaa cgtgctggtg atctgggcgg tgaaggtgaa ccaggcgctg 120 cgggatgcca ccttctgctt catcgtgtcg ctggcggtgg ctgatgtggc cgtgggtgcc 180 ctggtcatcc ccctcgccat cctcatcaac attgggccac agacctactt ccacacctgc 240 ctcatggttg cctgtccggt cctcatcctc acccagagct ccatcctggc cctgctggca 300 attgctgtgg accgctacct ccgggtcaag atccctctcc ggtacaagat ggtggtgacc 360 ccccggaggg cggcggtggc catagccggc tgctggatcc tctccttcgt ggtgggactg 420 acccctatgt ttggctggaa caatctgagt gcggtggagc gggcctgggc agccaacggc 480 agcatggggg agcccgtgat caagtgcgag ttcgagaagg tcatcagcat ggagtacatg 540 gtctacttca acttctttgt gtgggtgctg cccccgcttc tcctcatggt cctcatcggt 600 accagggtct ttcaggaggc caaaaggcag ctccagaaga ttgacaaatc tgagggccgc 660 ttccatgtcc agaaccttag ccaggtggag caggatgggc ggacggggca tggactccgc 720 agatcttcca agttctgctc cagggaaaag aaagcagcca aaaccttgga attcatcctc 780 ttcctctttg ccctcagctg gctgcctttg cacatcctca Actgcatcac cctcttctgc 840 ccgtcc tgcc acaagcccag catccttacc tacattgcca tcttcctcac gcacggcaac 900 tcggccatga accccattgt ctatgccttc cgcatccaga agttccgcgt caccttcctt 960 aagatttgga atgaccattt ccgctgccag cctgcacctc ccattgacga ggatctccca 1020 gaagagaggc ctgatgacga gaattccatg acttcgaaag tttatgatcc agaacaaagg 1080 aaacggatga taactggtcc gcagtggtgg gccagatgta aacaaatgaa tgttcttgat 1140 tcatttatta attattatga ttcagaaaaa catgcagaaa atgctgttat ttttttacat 1200 ggtaacgcgg cctcttctta tttatggcga catgttgtgc cacatattga gccagtagcg 1260 cggtgtatta taccagatct tattggtatg ggcaaatcag gcaaatctgg taatggttct 1320 tataggttac ttgatcatta caaatatctt actgcatggt ttgaacttct taatttacca 1380 aagaagatca attttgtcgg ccatgattgg ggtgcttgtt tggcatttca ttatagctat 1440 gagcatcaag ataagatcaa agcaatagtt cacgctgaaa gtgtagtaga tgtgattgaa 1500 tcatgggatg aatggcctga tattgaagaa gatattgcgt tgatcaaatc tgaagaagga 1560 -122 · 156362- sequence table .doc 201131168 gaaaaaatgg ttttggagaa taacttcttc gtggaaacca tgttgccatc aaaaatcatg Agaaagttag aaccagaaga atttgcagca tatcttgaac cattcaaa ga gaaaggtgaa gttcgtcgtc caacattatc atggcctcgt gaaatcccgt tagtaaaagg tggtaaacct gacgttgtac aaattgttag gaattataat gcttatctac gtgcaagtga tgatttacca aaaatgttta ttgaatcgga tccaggattc ttttccaatg ctattgttga aggcgccaag aagtttccta atactgaatt tgtcaaagta aaaggtcttc atttttcgca agaagatgca cctgatgaaa tgggaaaata tatcaaatcg ttcgttgagc gagttctcaa aaatgaacaa taa <210> 62 <211> 2370 <212> DNA <213> ΛΧ <220><223> Modified human cannabinoid receptor 1 aaggtgacat ggcatccaaa ttagggtact tcccacagaa attcccttta acttccttta ggggaagtcc cttccaagag aagatgactg cgggagacaa cccccagcta gtcccagcag accaggtgaa cattacagaa ttttacaaca agtctctctc gtccttcaag gagaatgagg agaacatcca gtgtggggag aacttcatgg acatagagtg tttcatggtc ctgaacccca gccagcagct ggccattgca gtcctgtccc tcacgctggg caccttcacg gtcctggaga acctcctggt gctgtgcgtc atcctccact cccgcagcct ccgctgcagg 62 atgaagtcga tcctagatgg ccttgcagat accaccttcc gcaccatcac cactgacctc ctgtacgtgg gctcaaatga cattcagtac gaagacatca; < 400 & gt ccttcctacc acttcatcgg cagcctggcg gtggcagacc tcctggggag tgtcattttt gtctacagct tcattgactt ccacgtgttc caccgcaaag atagccgcaa cgtgtttctg ttcaaactgg gtggggtcac ggcctccttc actgcctccg tgggcagcct gttcctcaca gccatcgaca ggtacatatc cattcacagg cccctggcct ataagaggat tgtcaccagg cccaaggccg tggtagcgtt ttgcctgatg tggaccatag ccattgtgat cgccgtgctg cctctcctgg gctggaactg cgagaaactg caatctgttt gctcagacat tttcccacac attgatgaaa cctacctgat gttctggatc ggggtcacca gcgtactgct tctgttcatc gtgggtacca gggtctttca ggaggc Caaa aggcagctcc agaagattga caaatctgag ggccgcttcc atgtccagaa ccttagccag gtggagcagg atgggcggac ggggcatgga ctccgcagat cttccaagtt ctgctccagg 156362·sequence table.doc -123- 1620 1680 1740 1800 1860 1920 1980 1983 60 120 180 240 300 360 420 4S0 540 600 660 720 780 840 900 960 1020 1080 201131168 gaaaagaaag cagccaaaac cttggaattc atcctggtgg tgttgatcat ctgctggggc cctctgcttg caatcatggt gtatgatgtc tttgggaaga tgaacaagct cattaagacg gtgtttgcat tctgcagtat gctctgcctg ctgaactcca ccgtgaaccc catcatctat gctctgagga gtaaggacct gcgacacgct ttccggagca tgtttccctc ttgtgaaggc actgcgcagc ctctggataa cagcatgggg gactcggact gcctgcacaa acacgcaaac aatgcagcca gtgttcacag ggccgcagaa agctgcatca agagcacggt caagattgcc aaggtaacca tgtctgtgtc cacagacacg tctgccgagg ctctggagaa ttccatgact tcgaaagttt atgatccaga acaaaggaaa cggatgataa ctggtccgca gtggtgggcc agatgtaaac aaatgaatgt tcttgattca Tttattaatt attatgattc agaaaaacat gcagaaaatg ctgttatttt tttacatggt aacgcggcct cttcttattt atggcgacat gttgtgccac atattgagcc agtagcgcgg tgtattatac cagatctt at tggtatgggc aaatcaggca aatctggtaa tggttcttat aggttacttg atcattacaa atatcttact gcatggtttg aacttcttaa tttaccaaag aagatcaatt ttgtcggcca tgattggggt gcttgtttgg catttcatta tagctatgag catcaagata agatcaaagc aatagttcac gctgaaagtg tagtagatgt gattgaatca tgggatgaat ggcctgatat tgaagaagat attgcgttga tcaaatctga agaaggagaa aaaatggttt tggagaataa cttcttcgtg gaaaccatgt tgccatcaaa aatcatgaga aagttagaac cagaagaatt tgcagcatat cttgaaccat tcaaagagaa aggtgaagtt cgtcgtccaa cattatcatg gcctcgtgaa atcccgttag taaaaggtgg taaacctgac gttgtacaaa ttgttaggaa ttataatgct Tatctacgtg caagtgatga tttdccaaad atgtttattg aatcggatcc aggattcttt tccaatgcta ttgttgaagg cgccaagaag tttcctaata ctgaatttgt caaagtaaaa ggtcttcatt tttcgcaaga agatgcacct gatgaaatgg gaaaatatat caaatcgttc gttgagcgag ttctcaaaaa tgaacaataa <210> 63 <211> 2082 <212> DNA <213>manpower <220><223> Modified human melanin agglutinin receptor 1 ≪ 400 > 63 atggacctgg aagcctcgct gctgcccact ggtcccaatg ccagcaacac ctctgatggc cccgataacc tcacttcggc aggatcacct cctcgcacgg ggagcatcic ctacatcaac atcatcatgc cttcggtgtt cggcaccatc tgcctcctgg gcatcatcgg gaactccacg 156362- Sequence Listing .doc -124- 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 2100 2160 2220 2280 2340 2370 60 120 180 201131168 gtcatcttcg cggtcgtgaa gaagtccaag ctgcactggt gcaacaacgt ccccgacatc 240 ttcatcatca acctctcggt agtagatctc ctctttctcc tgggcatgcc cttcatgatc 300 caccagctca tgggcaatgg ggtgtggcac tttggggaga ccatgtgcac cctcatcacg 360 gccatggatg ccaatagtca gttcaccagc acctacatcc tgaccgccat ggccattgac 420 cgctacctgg ccactgtcca ccccatctct tccacgaagt tccggaagcc ctctgtggcc 480 accctggtga tctgcctcct gtgggccctc tccttcatca gcatcacccc tgtgtggctg 540 Tatgccagac tcatcccctt cccaggaggt gcagtgggct gcggcatacg cctgcccaac 600 ccagacactg acctctactg gttcaccctg taccagtttt tcctggcctt tgccctgcct 660 tttgtggtca tcacagccgc aggtaccagg gtctttcagg aggccaaaag gcagctccag 720 aagattgaca aatctgaggg ccgcttccat gtccagaacc ttagccaggt ggagcaggat 780 gggcggacgg ggcatggact ccgcagatct tccaagttct gctccaggga aaagaaagca 840 gccaaaacct tggaattcat ctgtctggtc ttctttgtgt gctgggcacc ctactatgtg 900 ctacagctga cccagttgtc catcagccgc ccgaccctca cctttgtcta cttatacaat 960 gcggccatca gcttgggcta tgccaacagc tgcctcaacc cctttgtgta catcgtgctc 1020 tgtgagacgt tccgcaaacg cttggtcctg tcggtgaagc ctgcagccca ggggcagctt 1080 cgcgctgtca gcaacgctca gacggctgac gaggagagga cagaaagcaa aggcaccgag 1140 aattccatga cttcgaaagt ttatgatcca gaacaaagga aacggatgat aactggtccg 1200 cagtggtggg ccagatgtaa acaaatgaat gttcttgatt catttattaa ttattatgat 1260 tcagaaaaac atgcagaaaa tgctgttatt tttttacatg gtaacgcggc ctcttcttat 1320 ttatggcgac atgttgtgcc acatattgag ccagtagcgc ggtgtattat accagatctt 1380 attggtatgg gcaaatcagg caaatctggt aatggttctt ataggttact tgatcattac 1440 aaatatctta ctgcatggtt tgaacttctt aatttaccaa agaagatcaa ttttgtcggc 1500 catgattggg gtgcttgttt ggcatttcat tatagctatg agcatcaaga taagatcaaa 1560 gcaatagtt c acgctgaaag tgtagtagat gtgattgaat catgggatga atggcctgat 1620 attgaagaag atattgcgtt gatcaaatct gaagaaggag aaaaaatggt tttggagaat 1680 aacttcttcg tggaaaccat gttgccatca aaaatcatga gaaagttaga accagaagaa 1740 tttgcagcat atcttgaacc attcaaagag aaaggtgaag ttcgtcgtcc aacattatca 1800 tggcctcgtg aaatcccgtt agtaaaaggt ggtaaacctg acgttgtaca aattgttagg 1860 aattataatg cttatctacg tgcaagtgat gatttaccaa aaatgtttat tgaatcggat 1920 ccaggattct tttccaatgc tattgttgaa ggcgccaaga agtttcctaa tactgaattt 1980 • 125- 156362-SEQ ID NO.doc 201131168 gtcaaagtaa aaggtcttca tttttcgcaa gaagatgcac ctgatgaaat gggaaaatat atcaaatcgt tcgttgagcg agttctcaaa aatgaacaat aa <210> 64 <211> 1941 <212〉 DNA <213〉Artificial <220><223> Modified human GPR40 (insulin serotonin) ≪ 400 > 64 atggacctgc ccccgcagct ctccttcggc ctctatgtgg ccgcctttgc gctgggcttc ccgctcaacg tcctggccat ccgaggcgcg acggcccacg cccggctccg tctcacccct agcctggtct acgccctgaa cctgggctgc tccgacctgc tgctgacagt ctctctgccc ctgaaggcgg tggaggcgct agcctccggg gcctggcctc tgccggcctc gctgtgcccc gtcttcgcgg tggcccactt cttcccactc tatgccggcg ggggcttcct ggccgccctg agtgcaggcc gctacctggg agcagccttc cccttgggct accaagcctt ccggaggccg tgctattcct ggggggtgtg cgcggccatc tgggccctcg tcctgtgtca cctgggtctg gtctttgggt tggaggctcc aggaggctgg ctggaccaca gcaacacctc cctgggcatc aacacaccgg tcaacggctc tccggtctgc ctggaggcct gggacccggc ctctgccggc ccggcccgct tcagcctctc tctcctgctc ttttttctgc ccttggccat cacagccttc tgcggtacca gggtctttca ggaggccaaa aggcagctcc agaagattga caaatctgag ggccgcttcc atgtccagaa ccttagccag gtggagcagg atgggcggac ggggcatgga ctccgcagat cttccaagtt ctgctccagg gaaaagaaag cagccaaaac cttggaattc gccctcctca cgctgctgct ctgcgtagga ccctacaacg cctccaacgt ggccagcttc ctgtacccca atctaggagg ctcctggcgg aagctggggc tcatcacggg tgcctg gagt gtggtgctta atccgctggt gaccggttac ttgggaaggg gtcctggcct gaagacagtg tgtgcggcaa gaacgcaagg gggcaagtcc cagaaggaga attccatgac ttcgaaagtt tatgatccag aacaaaggaa acggatgata actggtccgc agtggtgggc cagatgtaaa caaatgaatg ttcttgattc atttattaat tattatgatt cagaaaaaca tgcagaaaat gctgttattt ttttacatgg taacgcggcc tcttcttatt tatggcgaca tgttgtgcca catattgagc cagtagcgcg gtgtattata ccagatctta ttggtatggg caaatcaggc aaatctggta atggttctta taggttactt gatcattaca aatatcttac tgcatggttt gaacttctta atttaccaaa gaagatcaat tttgtcggcc atgattgggg tgcttgtttg gcatttcatt Atagctatga gcatcaagat aagatcaaag caatagttca cgctgaaagt 156362 - sequence listing.doc -126· 2040 2082 60 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 201131168 gtagtagatg tgattgaatc atgggatgaa tggcctgata ttgaagaaga tattgcgttg atcaaatctg aagaaggaga Aaaaatggtt ttggagaata acttcttcgt ggaaaccatg ttgccatcaa aaatcatgag aaagttagaa ccagaagaat ttgcagcata tcttgaacca ttcaaagaga aaggtgaagt tcgtcgtcca acattatcat ggc ctcgtga aatcccgtta gtaaaaggtg gtaaacctga cgttgtacaa attgttagga attataatgc ttatctacgt gcaagtgatg atttaccaaa aatgtttatt gaatcggatc caggattctt ttccaatgct attgttgaag gcgccaagaa gtttcctaat actgaatttg tcaaagtaaa aggtcttcat ttttcgcaag aagatgcacc tgatgaaatg ggaaaatata tcaaatcgtt cgttgagcga gttctcaaaa atgaacaata a <210> 65 <211> 1989 <212> DNA <213>manpower <220><223> Modified human GPCR2 (insulin serotonin > ≪ 400 > 65 atggaatcat ctttctcatt tggagtgatc cttgctgtcc tggcctccct catcattgct actaacacac tagtggctgt ggctgtgctg ctgttgatcc acaagaatga tggtgtcagt ctctgcttca ccttgaatct ggctgtggct gacaccttga ttggtgtggc catctctggc ctactcacag accagctctc cagcccttct cggcccacac agaagaccct gtgcagcctg cggatggcat ttgtcacttc ctccgcagct gcctctgtcc tcacggtcat gctgatcacc tttgacaggt accttgccat caagcagccc ttccgctact tgaagatcat gagtgggttc gtggccgggg cctgcattgc cgggctgtgg ttagtgtctt acctcattgg cttcctccca ctcggaatcc ccatgttcca gcagactgcc tacaaagggc agtgcagctt ctttgctgta tttcaccctc acttcgtgct gaccctctcc tgcgttggct tcttcccagc catgctcctc tttgtcttcg gtaccagggt ctttcaggag gccaaaaggc agctccagaa gattgacaaa tctgagggcc gcttccatgt ccagaacctt agccaggtgg agcaggatgg gcggacgggg catggactcc gcagatcttc caagttctgc tccagggaaa agaaagcagc caaaaccttg gaattcctca ttgggagctt tgctctatcc tggaccccct tccttatcac tggcattgtg caggtggcct gccaggagtg tcacctctac ctagtgctgg aacggtacct gtggctgctc ggcgtgggca actccctgct caacccactc atctatgcct attggcagaa ggaggt Gcga ctgcagctct accacatggc cctaggagtg aagaaggtgc tcacctcatt cctcctcttt 156362-sequence table.doc •127· 1560 1620 1680 1740 1800 1860 1920 1941 60 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 201131168 ctctcggcca ggaattgtgg cccagagagg atctccagct cagagtttga tggcgagaat caaaggaaac ggatgataac tggtccgcag cttgattcat ttattaatta ttatgattca ttacatggta acgcggcctc ttcttattta gtagcgcggt gtattatacc agatcttatt ggttcttata ggttacttga tcattacaaa ttaccaaaga agatcaattt tgtcggccat agctatgagc atcaagataa gatcaaagca attgaatcat gggatgaatg gcctgatatt gaaggagaaa aaatggtttt ggagaataac atcatgagaa agttagaacc agaagaattt ggtgaagttc gtcgtccaac attatcatgg aaacctgacg ttgtacaaat tgttaggaat ttaccaaaaa tgtttattga atcggatcca gccaagaagt ttcctaatac tgaatttgtc gatgcacctg atgaaatggg aaaatatatc gaacaataa cccagggaaa gttcctgtca catcgtcact 1020 tccatgactt cgaaagttta tgatccagaa 1080 tggtgggcca gatgtaaaca aatgaatgtt 1140 gaaaaacatg cagaaaatgc tgttattttt 1200 tggcgacatg ttgtgccaca tattgagcca 1260 ggtatgggca aatcaggca a atctggtaat 1320 tatcttactg catggtttga acttcttaat 1380 gattggggtg cttgtttggc atttcattat 1440 atagttcacg ctgaaagtgt agtagatgtg 15⑻ gaagaagata ttgcgttgat caaatctgaa 1560 ttcttcgtgg aaaccatgtt gccatcaaaa 1620 gcagcatatc ttgaaccatt caaagagaaa 1680 cctcgtgaaa tcccgttagt aaaaggtggt 1740 tataatgctt atctacgtgc aagtgatgat 1800 ggattctttt ccaatgctat tgttgaaggc 1860 aaagtaaaag gtcttcattt ttcgcaagaa 1920 aaatcgttcg ttgagcgagt tctcaaaaat 1980 1989 128- 156362-Sequence table.doc

Claims (1)

201131168 VII. Scope of application: 1. A G-protein coupled receptor (GPCR) that can be regulated by a ligand, wherein the GPCR which can be up-regulated by a ligand is a parental GPCR, which contains a GPCR-containing a higher amino acid sequence substituted TM5-IC3-TM6, wherein the amino acid sequence substitution is at the C-terminus of TM5 and the N-terminus of TM6, wherein the GPCR upregl comprises at least 90% of SEQ ID NO: a sequence of amino acid sequence, and comprising a murine muscarinic acetylcholine receptor 3 (Rm3) pro of TM5-IC3-TM6 substituted with an amino acid sequence of the GPCR Generation GPCRs can be upgraded by atropine. 2. A GPCR as claimed in claim 1 which is upregulated by a ligand, wherein the parental GPCR is a native or altered known GPCR. 3. A GPCR which can be subjected to ligand upregulation as claimed in claim 2, wherein the parental GPCR is known to be human dopamine receptor D3. 4. A GPCR which can be subjected to ligand upregulation as claimed in claim 1, wherein the parental GPCR is a native or altered orphan ligand GPCR. 5. A GPCR which can be subjected to ligand upregulation as in claim 4, wherein the parental GPCR is human GPR40. 6. A GPCR which is isolated by a ligand as claimed in claim 1 wherein the parental GPCR comprises an operably linked reporter protein. 7. The isolated PCR-receivable GPCR of claim 1, wherein the GPCR upregl comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 2. 8. The GPCR of claim 1, wherein the GPCR upregl comprises an amino acid sequence 156362.doc 201131168 column consistent with the sequence of SEQ ID NO: 2. 9. A composition comprising an isolated G-protein coupled receptor (GPCR) that is upregulated by a ligand, wherein the ligand-approved GPCR is a parental GPCR comprising a GPCR upregulated Amino acid sequence substituted TM5-IC3-TM6, wherein the amino acid sequence substitution is at the C-terminus of TM5 and the N-terminus of TM6, wherein the GPCR upregl comprises at least 90% sequence with SEQ ID NO: A homologous amino acid sequence, and comprising a murine muscarinic acetylcholine receptor 3 (Rm3) parental GPCR comprising the amino acid sequence substituted by the GPCR upreglase of TM5-IC3-TM6 Increased by atropine. 10. The composition of claim 9, wherein the parental GPCR is a native or altered known GPCR. 11. The composition of claim 10, wherein the parental GPCR is known to be human dopamine receptor D3. 1 2. The composition of claim 9, wherein the parental GPCR is a native or altered orphan ligand GPCR. 13. The composition of claim 12, wherein the parental GPCR is human GPR40. 1 4. The composition of claim 9, wherein the parental GPCR comprises an operably linked reporter protein. 15. The composition of claim 9, wherein the GPCR upregl comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 2. 16. The composition of claim 9, wherein the GPCR upregl comprises an amino acid sequence consistent with the sequence of SEQ ID NO: 2. 17. An isolated polynucleotide comprising a nucleic acid encoding a 156362.doc 201131168 GPCR which is upregulated by a ligand, wherein the ligand-approvable GPCR is a parental GPCR comprising a GPCR-containing a raised amino acid sequence substituted TM5-IC3-TM6, wherein the amino acid sequence substitution is at the C-terminus of TM5 and the N-terminus of TM6, wherein the GPCR upregl comprises at least 90% with SEQ ID NO: A sequence-equilibrium amino acid sequence comprising a murine muscarinic acetylcholine receptor 3 (Rm3) parental GPCR comprising the amino acid sequence substituted by the GPCR-tune-suppressing TM5-IC3-TM6 Can be upgraded by atropine. 18. The isolated polynucleotide of claim 17, wherein the parental GPCR is a native or altered known GPCR. 19. The isolated polynucleotide of claim 18, wherein the known parental GPCR is human dopamine receptor D3. 20. The isolated polynucleotide of claim 17, wherein the parental GPCR is a native or altered orphan ligand GPCR. 21. The isolated polynucleotide of claim 20, wherein the parental GPCR is human GPR40. 22. The isolated polynucleotide of claim 17, wherein the parental GPCR comprises an operably linked reporter protein. 23. The isolated polynucleotide of claim 17, wherein the GPCR upregl comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 2. 24. The isolated polynucleotide of claim 17, wherein the GPCR upregl comprises an amino acid sequence consistent with the sequence of SEQ ID NO: 2. 25. A vector comprising the polynucleoside 156362.doc 201131168 acid of any one of claims 17 to 24. 26. The carrier of claim 25, wherein the vector is a performance vector and wherein the polynucleotide is operably linked to a promoter. A recombinant sputum host cell comprising the vector of claim 25. A recombinant injured host cell comprising the vector of claim 26. A method of making a recombinant host cell comprising: (a) introducing a vector according to claim 26 into a suitable host cell; and (b) cultivating the host under conditions permitting the GPCR expression of the ligand to be upregulated cell. A cell membrane of a recombinant host cell prepared by the method of claim 29, wherein the isolated cell raft comprises a GPCR which is subject to ligand upregulation. . 156362.doc