CA2402147A1 - Melanin concentrating hormone receptor chimeric and fusion proteins - Google Patents

Abstract

The present invention features melanin concentrating hormone receptor (MCH-R ) chimeric and fusion proteins. MCH-R chimeric proteins comprise an MCH-R polypeptide region made up of at least two or more polypeptide regions characteristic of MCH-R found in different species. MCH-R fusion proteins comprise an MCH-R polypeptide region and a fluorescent protein region.</SDOA B>

Description

TITLE OF THE INVENTION
MELANIN CONCENTRATING-HORMONE RECEPTOR CHIMERIC AND
FUSION PROTEINS
CROSS-REFERENCE TO RELATED APPLICATIONS
The present application claims priority to provisional application U.S.
Serial No. 601189,698, filed March 15, 2000, hereby incorporated by reference herein.
BACKGROUND OF THE INVENTION
The references cited herein are not admitted to be prior art to the claimed invention.
Neuropeptides present in the hypothalamus play a major role in mediating the control of body weight. (Flier et al., 1998. Cell, 92, 437-440.) Melanin-concentrating hormone (MCH) is a cyclic 19-amino acid neuropeptide synthesized as part of a larger pre-prohormone precursor in the hypothalamus which also encodes neuropeptides NEI and NGE. (Nation et al., 1990. Mol. Endocrifzol. 4, 632-637.) MCH was first identified in salmon pituitary, and in fish MCH affects melanin aggregation thus affecting skin pigmentation. In trout and in eels MCH has also been shown to be involved in stress induced or CRF-stimulated ACTH release.
(Kawauchi et al., 1983. NatuYe 305, 321-323.) In humans two genes encoding MCH have been identified that are expressed in the brain. (Breton et al., 1993. Mol. Brain Res. 18, 297-310.) In mammals MCH has been localized primarily to neuronal cell bodies of the hypothalamus which are implicated in the control of food intake, including perikarya of the lateral hypothalamus and zona inertia. (Knigge et al., 1996. Peptides 17, 1063-1073.) Pharmacological and genetic evidence suggest that the primary mode of MCH action is to promote feeding (orexigenic). MCH mRNA is up regulated in fasted mice and rats and in the oblob mouse. (Qu et al., 1996. Nature 380, 243-247.) Injection of MCH centrally (ICV) stimulates food intake and MCH antagonizes the hypophagic effects seen with oc-melanocyte stimulating hormone (aMSH). (Qu et al., 1996. Nature 380, 243-247.) MCH-deficient mice are lean, hypophagic, and have increased metabolic rate. (Shimada et al., 1998. Nature 396, 670-673.) MCH action is not limited to modulation of food intake as effects on the hypothalamic-pituitary-axis have been reported. (Nation 1994. Critical Rev. ifz Neurobiol. 8, 221-262.) MCH may be involved in the body response to stress as MCH can modulate the stress-induced release of CRF from the hypothalamus and ACTH from the pituitary. In addition, MCH neuronal systems may be involved in reproductive or maternal function.
Several references describe a receptor that is indicated to bind MCH.
(Chambers et al., 1999. Nature 400, 261-265; Saito et al., 1999. Nature 400, 265-269;
Bachner et al., 1999. FEBS Letters 457:522-524; Shimomura et al., 1999.
Biochemical and Biophysical Researcla Comnzunicatiofzs 261, 622-626; and Lembo et al., 1999. Nat. Cell Biol. 1, 267-271.) SUMMARY OF THE INVENTION
The present invention features melanin concentrating hormone receptor (MCH-R) chimeric and fusion proteins. MCH-R chimeric proteins comprise an MCH-R polypeptide region made up of at least two or more polypeptide regions characteristic of MCH-R found in different species. MCH-R fusion proteins comprise an MCH-R polypeptide region and a fluorescent protein region.
An MCH-R polypeptide region provides a functional G-protein coupled receptor region able to bind MCH and transduce an intracellular signal.
Examples of MCH-R polypeptide regions include naturally occurring MCH-R, chimeric MCH-R containing two or more regions from naturally occurring MCH-R, and functional derivatives thereof.
Reference to the terms "characteristic" and "derivatives thereof describe a relationship to a reference sequence. In both cases, there is at least about 75°70 sequence similarity to the reference sequence.
Thus, a first aspect of the present invention describes a fusion protein comprising (a) an MCH-R polypeptide region and (b) a fluorescent polypeptide region. The fluorescent polypeptide region is joined directly, or though a polypeptide linker, to the carboxy side of the MCH-R polypeptide region.
Another aspect of the present invention describes an MCH-R chimeric protein. The protein comprises: (a) an MCH-R binding region characteristic of a human MCH-R, (b) a transmembrane domain characteristic of a non-human MCH-R, and (c) an intracellular domain characteristic of a non-human MCH-R.
Another aspect of the present invention describes a nucleic acid encoding for an MCH-R fusion protein or an MCH-R chimeric protein described herein. Such nucleic acid comprises either a contiguous nucleotide sequence that _2_ codes for the protein or a sequence that is processed by a host cell to produce a contiguous nucleotide sequence encoding for the protein. Processing of a nucleic acid sequence to produce a contiguous nucleotide sequence encoding for a protein can occur by the splicing together of exons resulting in intron removal.
Another aspect of the present invention describes an expression vector comprising a nucleic acid encoding for an MCH-R fusion protein or an MCH-R
chimeric protein described herein.
Another aspect of the present invention describes a recombinant cell comprising nucleic acid encoding for an MCH-R fusion protein or an MCH-R
chimeric protein described herein. The nucleic acid may be part of the host genome or may exist independently of the host genome.
Another aspect of the present invention describes a non-human transgenic animal comprising nucleic acid encoding for an MCH-R fusion protein or an MCH-R chimeric protein described herein.
Another aspect of the present invention describes a method for assaying for MCH-R active compounds by measuring the effect of a test preparation on one or more MCH-R activities. The method is performed using either an MCH-R
fusion protein or an MCH-R chimeric protein described herein.
Other features and advantages of the present invention are apparent from the additional descriptions provided herein including the different examples.
The provided examples illustrate different components and methodology useful in practicing the present invention. The examples do not limit the claimed invention.
Based on the present disclosure the skilled artisan can identify and employ other components and methodology useful for practicing the present invention.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 illustrates aequorin assay results comparing a mouse MCH-R
fusion with a human wild type MCH-R and a CMV-EGFP control.
Figure 2 illustrates a cAMP flashplate assay of CHO cell clones stably expressing mMCH-1R-EGFP. Cells from individual clones were dissociated in enzyme free media and stimulated for 15 minutes at 37°C with human MCH
at the indicated concentrations in the presence of 10 ~M forskolin. Cells were then lysed and assayed for bound ~l2sl]CAMP. Mouse MCH-1R-EGFP clones exhibited EC50 values (0.1111, 0.1255, 0.1291, or 0.2304 nM) indistinguishable from that of a CHO
cell clone expressing the wild-type human short isoform of MCH-1R ( 0.1282 nM).

Figure 3 illustrates a cAMP flashplate assay of CHO cell clones stably expressing human short/mouse species chimeric MCH-1R-EGFP. Cells from individual clones were dissociated in enzyme free media and stimulated for 15 minutes at 37°C with human MCH at the indicated concentrations in the presence of ~M forskolin. Cells were then lysed and assayed for bound [12~I]CAMP. Human short/mouse species chimeric MCH-1R-EGFP clones exhibited EC50 values (0.0366, 0.0462, 0.2117, or 0.2499 nM) indistinguishable from that of a CHO cell clone expressing the wild-type human short isoform of MCH-1R (0.1137 nM).

The present invention features MCH-R chimeric and fusion proteins.
Such proteins have a variety of different uses including being used as a research tool to study MCH-R function and dynamics, and being used to screen for MCH-R
agonists and antagonists.
The MCH-R provides a target to achieve different beneficial effects in a patient. Preferably, MCH-R activity is modulated to achieve one or more of the following: weight loss, weight gain, treat cancer (e.g., colon or breast), reduce pain, treat diabetes, reduce stress, or teat sexual dysfunction.
Modulation of MCH-R activity can be achieved by evoking a response at the MCH receptor or by altering a response evoked by an MCH receptor agonist or antagonist. Compounds modulating MCH-R receptor activity include agonists, antagonists, and allosteric modulators. Generally, MCH-R antagonists and allosteric modulators negatively affecting activity will be used to achieve weight loss, treat cancer (e.g., colon or breast), reduce pain, reduce stress, or teat sexual dysfunction;
and MCH-R agonists and allosteric modulators positively affecting activity will be used to produce a weight gain.
Preferably, MCH-R activity is modulated to achieve a weight loss or to treat diabetes in a patient. Diabetes mellitus can be treated by modulating MCH-R
activity to achieve, for example, one or both of the following: enhancing glucose tolerance or decreasing insulin resistance.
Excessive body weight is a contributing factor to different diseases, including hypertension, diabetes, dyslipidemias, cardiovascular disease, gall stones, osteoarthritis, and certain forms of cancers. Bringing about a weight loss can be used, for example, to reduce the likelihood of such diseases and as part of a treatment for such diseases. Weight reduction can be achieved by modulating MCH-R activity to obtain, for example, one or more of the following effects: reducing appetite, increasing metabolic rate, reducing fat intake, or reducing carbohydrate craving.
Increasing body weight is particularly useful for a patient having a disease or disorder, or under going a treatment, accompanied by weight loss.
Examples of diseases or disorders accompanied by weight loss include anorexia, AIDS, wasting, cachexia, and frail elderly. Examples of treatments accompanied by weight Ioss include chemotherapy and radiation therapy.
MCH-R Chimeric Proteins MCH-R chimeric proteins contain an MCH-R polypeptide region made up by at least two or more polypeptide regions characteristic of MCH-R found in different species. The different polypeptide regions that are present provide for an N-terminal extracellular domain; a transmembrane domain made up of transmembrane regions, extracellular loop regions, and intracellular loop regions; and an intracellular carboxy terminus domain. Examples of MCH-R amino acid sequences include the following: SEQ. ID. NO. 1 (human MCH1R long form), SEQ. ID. NO. 2 (human MCH1R short form), and SEQ. ID. NO. 3 (mouse MCH1R).
Preferably, the MCH-R chimeric protein comprises an MCH-R binding region characteristic of a human MCH-R along with transmembrane and intracellular domains characteristic of a non-human MCH-R. There are substantial amino acid differences between the N-terminus of the MCH-R present in humans and that present in other species such as mice. Such differences could result in, for example, the mouse MCH-R having different intrinsic properties and responsiveness to agonists and/or antagonists than the human MCH-R. The presence of a human MCH-R
binding region provides for a "humanized" MCH-R chimeric receptor.
The transmembrane and intracellular domains characteristic of a non-human MCH-R can be used in conjunction with a non-human host to provide a more naturally occurring environment for these regions. For example, an MCH-R
chimeric having mouse transmembrane and intracellular domains are preferably used in murine cells lines or in transgenic mice.
MCH-R chimeric proteins may contain regions other than extracellular, transmembrane, and intracellular domains that do not substantially decrease the activity of the protein. Preferably, additional regions do not cause a decrease of more than about 25°70 of MCH-R activity as measured using one or more of the assays described in the examples provided below. Examples of additional regions that may be present include fluorescent protein regions and linker regions.
In an embodiment of the present invention, the MCH-R chimeric protein comprises: (a) an MCH binding region characteristic of a first species and (b) a transmembrane and intracellular domain region characteristic of a second species joined directly, or though a linker, to the carboxy side of the MCH binding region.
Preferably, the protein comprises, consists, or consists essentially of an MCH-R
polypeptide having a sequence similarity of at least about 75%, at least 85%, or at least 95% with either SEQ.1D. NO. 4 (human short form/mouse species chimeric MCH1R) or SEQ. ID. NO. 5 (human Long form/mouse species chimeric). Even more preferably, the protein comprises, consists essentially of, or consists of, SEQ. )D. NO.
4 or SEQ. ID. NO. 5.
Sequence similarity for polypeptides can be determined by BLAST.
(Altschul et al., 1997. Nucleic Acids Res. 25, 3389-3402, hereby incorporated by reference herein.) In an embodiment of the present invention, sequence similarity is determined using tBLASTn search program with the following parameters:
MATRIX:BLOSLTM62, PER RESIDUE GAP COST: 11, and Lambda ratio: 1.
Differences in naturally occurring amino acids are due to different R
groups. An R group effects different properties of the amino acid such as physical size, charge, and hydrophobicity. Amino acids can be divided into different groups as follows: neutral and hydrophobic (alanine, valine, leucine, isoleucine, proline, tryptophan, phenylalanine, and methionine); neutral and polar (glycine, serine, threonine, tyrosine, cysteine, asparagine, and glutamine); basic (lysine, arginine, and histidine); and acidic (aspartic acid and glutamic acid).
Generally, in substituting different amino acids it is preferable to exchange amino acids having similar properties. Substituting different amino acids within a particular group, such as substituting valine for leucine, arginine for lysine, and asparagine for glutamine are good candidates for not causing a change in polypeptide functioning.
Changes outside of different amino acids groups can also be made.
Preferably, such changes are made taking into account the position of the amino acid to be substituted in the polypeptide. For example, arginine can substitute more freely for nonpolor amino acids in the interior of a polypeptide then glutamate because of its long aliphatic side chain. (See, Ausubel, Current Protocols in Molecular Biology, John Wiley, 1987-1998, Supplement 33 Appendix 1C.) MCH-R Fusion Proteins MCH-R fusion proteins contain an MCH-R polypeptide region and a fluorescent protein region either directly joined together or joined together through a linker. These regions provide MCH-R activity and a marker for evaluating MCH-R
dynamics.
An MCH-R polypeptide region provides functional MCH-R activity and includes naturally occurring MCH-R, chimeric MCH-R, and derivatives thereof.
Preferred derivatives thereof have a sequence similarity of at least about 75%, at least about 85%, or at least about 95% to a naturally occurring MCH-R or a chimeric MCH-R described herein.
A fluorescent protein region contains a chromophore that fluoresces.
Preferably, the fluorescent protein region is the green fluorescent protein of the jellyfish Aequorea victories or a derivative thereof. Preferred derivatives have a sequence similarity of at least about 75%, at least about 85%, or at least about 95% to the Aequorea victories green fluorescent protein (GFP). The Aequorea victories green fluorescent protein and examples of derivatives thereof are described by Cormack et al., 1996. Gene 17, 33-38; Yang et al., 1996. Nucleic Acids Research 24, 4592-4593;
Tsien et al., U.S. Patent No. 5,625,048; Tsien et al., U.S. Patent No.
5,777,079; and Cormack et al., U.S. Patent No. 5,804,387 (each of which are hereby incorporated by reference herein).
In different embodiments the MCH-R polypeptide region comprises, consists essentially of, or consists of, a sequence selected from the group consisting of: SEQ. m. NO. 1, SEQ. m. NO. 2, SEQ.1D. NO. 3, SEQ. m. NO. 4, and SEQ. m.
NO. 5; and the fluorescent polypeptide region comprises, consists essentially of, or consists of, an amino acid sequence selected from the group consisting of SEQ.
m.
NO. 6 (GFP), SEQ. ID. NO. 7 (EGFP), SEQ. m. NO. 8 (Emerald), SEQ. m. NO. 9 (Topaz), and SEQ. )D. NO. 10 (Wlb). EGFP, Emerald, Topaz, and Wlb are derivatives of GFP.
The optionally present linker is a polypeptide region that is preferably from 1 to about 100 amino acids in length. In different embodiments the linker is up to 75, 50 or 25 amino acids in length.
Preferably, the MCH-R fusion protein comprises, consists essentially of, or consists of, the MCH-R polypeptide region and the fluorescent polypeptide region. More preferably, the protein comprises, consists essentially of, or consists of, _7_ an amino acid sequence selected from the group consisting of: SEQ. ID. NO. 11 (mouse MCH1R-linker-EGFP), SEQ. ID. NO. 12 (mouse MCH1R/EGFP direct fusion), SEQ. ID. NO. 13 (human short form/mouse species chimeric MCH1R-linker-EGFP), or SEQ. ID. NO. 14 (human long form/mouse species chimeric MCH1R-linker-EGFP).
MCH-R Chimeric and Fusion Proteins Nucleic Acid and Expression MCH-R chimeric and fusion proteins can be produced using techniques well known in the art. Preferably, such proteins are produced by recombinant expression inside a host cell by way of an expression vector or by way of nucleic acid integrated into the host genome. Examples of nucleic acid sequences encoding for MCH-R polypeptide regions, fluorescent protein regions, MCH-R
chimeric proteins, and MCH-R fusion proteins are provided for by SEQ. ID. NOs.

29 (see Example 1, infra).
Starting with a particular amino acid sequence and the known degeneracy of the genetic code, a large number of different encoding nucleic acid sequences can be obtained. The degeneracy of the genetic code arises because almost all amino acids are encoded for by different combinations of nucleotide triplets or codons. The translation of a particular codon into a particular amino acid is well known in the art (see, e.g., Lewin GENES IV, p. 119, Oxford University Press, 1990).
Amino acids are encoded for by codons as follows:
A=Ala=Alanine: codons GCA, GCC, GCG, GCU
C=Cys=Cysteine: codons UGC, UGU
D=Asp=Aspartic acid: codons GAC, GAU
E=Glu=Glutamic acid: codons GAA, GAG
F=Phe=Phenylalanine: codons UUC, UUU
G=Gly=Glycine: codons GGA, GGC, GGG, GGU
H=His=Histidine: codons CAC, CAU
I=Ile=Isoleucine: codons AUA, AUC, AUU
K=Lys=Lysine: codons AAA, AAG
L=Leu=Leucine: codons UUA, UUG, CUA, CUC, CUG, CUU
M=Met=Methionine: codon AUG
N=Asn=Asparagine: codons AAC, AAU
P=Pro=Proline: codons CCA, CCC, CCG, CCU
Q=Gln=Glutamine: codons CAA, CAG
_g_ R=Arg=Arginine: codons AGA, AGG, CGA, CGC, CGG, CGU
S=Ser=Serine: codons AGC, AGU, UCA, UCC, UCG, UCU
T=Thr=Threonine: codons ACA, ACC, ACG, ACU
V=Val=Valine: codons GUA, GUC, GUG, GUU
W=Trp=Tryptophan: codon UGG
Y=Tyr=Tyrosine: codons UAC, UAU
Examples of techniques for introducing nucleic acid into a cell and expressing the nucleic acid to produce protein are provided in references such as Ausubel, Current Protocols ifa Molecular Biology, John Wiley, 1987-1998, and Sambrook, et al., in Molecular Cloni~eg, A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, 1989.
An expression vector contains recombinant nucleic acid encoding for a polypeptide along with regulatory elements for proper transcription and processing.
The recombinant nucleic acid contains two or more nucleic acid regions not naturally associated with each other. Exogenous regulatory elements such as an exogenous promoter can be useful for expressing recombinant nucleic acid in a particular host.
Examples of expression vectors are cloning vectors, modified cloning vectors, specifically designed plasmids, and viruses.
Generally, the regulatory elements that are present in an expression vector include a transcriptional promoter, a ribosome binding site, a terminator, and an optionally present operator. Another preferred element is a polyadenylation signal providing for processing in eukaryotic cells. Preferably, an expression vector also contains an origin of replication for autonomous replication in a host cell, a selectable marker, a limited number of useful restriction enzyme sites, and a potential for high copy number.
Expression vectors providing suitable levels of polypeptide expression in different hosts are well known in the art. Mammalian expression vectors well known in the art include pcDNA3 (Invitrogen), pMClneo (Stratagene), pXT1 (Stratagene), pSGS (Stratagene), EBO-pSV2-neo (ATCC 37593), pBPV-1(8-2) (ATCC 37110), pdBPV-MMTneo(342-12) (ATCC 37224), pRSVgpt (ATCC 37199), pRSVneo (ATCC 37198), pSV2-dhfr (ATCC 37146), pUCTag (ATCC 37460), pCI-neo (Promega) and .lambda.ZD35 (ATCC 37565). Bacterial expression vectors well known in the art include pETlla (Novagen), lambda gtll (Invitrogen), pcDNAII
(Invitrogen), and pKK223-3 (Pharmacia). Fungal cell expression vectors well known _g_ in the art include pYES2 (Invitrogen) and Pichia expression vector (Invitrogen).
Insect cell expression vectors well known in the art include Blue Bac III
(Invitrogen).
Recombinant host cells may be prokaryotic or eukaryotic. Examples of recombinant host cells include the following: bacteria such as E. coli;
fungal cells such as yeast; mammalian cells such as human, bovine, porcine, monkey, hampster, and rodent; and insect cells such as Drosophila and silkworm derived cell lines.
Commercially available mammalian cell lines include L cells L-M(TI~^-) (ATCC
CCL 1.3), L cells L-M (ATCC CCL 1.2), 293 (ATCC CRL 1573), Raji (ATCC CCL
86), CV-1 (ATCC CCL 70),,COS-1 (ATCC CRL 1650), COS-7 (ATCC CRL 1651), CHO-K1 (ATCC CCL 61), 3T3 (ATCC CCL 92), NIH/3T3 (ATCC CRL 1658), HeLa (ATCC CCL 2), C127I (ATCC CRL 1616), BS-C-1 (ATCC CCL 26) and MRC-5 (ATCC CCL 171).
To enhance expression in a particular host it may be useful to modify the sequence to take into account codon usage of the host. Codon usage of different organisms are well known in the art. (See, Ausubel, Current Protocols ih Molecular Biology, John Wiley, 1987-1998, Supplement 33 Appendix 1C.) Expression vectors may be introduced into host cells using standard techniques. Examples of such techniques include transformation, transfection, lipofection, protoplast fusion, and electroporation.
Nucleic acid encoding for a polypeptide can be expressed in a cell without the use of an expression vector employing, for example, synthetic mRNA
or native mRNA. Additionally, mRNA can be translated in various cell-free systems such as wheat germ extracts and reticulocyte extracts, as well as in cell based systems, such as frog oocytes. Introduction of mRNA into cell based systems can be achieved, for example, by microinjection.
Techniques for producing transgenic animals are well known in the art.
Examples of such techniques are provided for by Teratocarcinomas and embryonic stem cells: a practical approach. Ed. By E. J. Robertson, IRL Press Limited, Oxford, England (1987); and Gene Targeting: a practical approach. Ed. By A. L. Joyner, Oxford University Press Inc. New York, NY (1993).
G-Protein Coupled Receptor Assaxs MCH-R is G-protein coupled receptor. Techniques for measuring different G-protein activities, such as Gi/o, Gs, and Gq are well known in the art.
MCH-R activity is preferably assayed for by measuring either Gi/o or Gq.

Gi/o and Gs activity can be measured using techniques such as a melonaphore assay, measuring cAMP production, measuring inhibition of cAMP
accumulation, and measuring binding of 35S-GTP. cAMP can be measured using different techniques such as radioimmunoassay and indirectly by cAMP
responsive gene reporter proteins.
Gq activity can be measured using techniques such as those measuring intracellular Ca2+. Examples of techniques well known in the art that can be employed to measure Ca2+ include the use of dyes such as Fura-2 and the use of Ca2+-bioluminescent sensitive reporter proteins such as aequorin. An example of a cell line employing aequorin to measure G-protein activity is HEK293/aeql7. (Button et al.,1993. Cell Calcium 14, 663-671, and Feighner et al., 1999. Scief2ce 284, 2188, both of which are hereby incorporated by reference herein.) Functional assays can be performed using individual compounds or preparations containing different compounds. A preparation containing different compounds where one or more compounds affect MCH-R chimeric or fusion protein activity can be divided into smaller groups of compounds to identify the compounds) affecting MCH-R chimeric or fusion protein activity. In an embodiment of the present invention a test preparation containing at least 10 compounds is used in a functional assay.
Functional assays can be performed using recombinantly produced MCH-R chimeric or fusion protein present in different environments. Such environments include, for example, cell extracts and purified cell extracts containing the MCH-R chimeric or fusion protein expressed from recombinant nucleic acid and an appropriate membrane for the polypeptide; and the use of a purified MCH-R
chimeric or fusion protein produced by recombinant means that is introduced into a different environment suitable for measuring G-protein activity.
Fluorescent Protein Assays Fluorescent protein joined to an MCH receptor can be employed to study different aspects of receptor dynamics including receptor sequestration, receptor densitization, and receptor localization. The fluorescent protein can be used in ira vitro or in vivo systems.
ha vitro applications of fluorescent proteins can be performed using techniques well known in the art. Examples of such techniques are provided by Barak et al., 1997. Mol PlaaYm. S, 177-184; Tarasova et al., 1997. J. Biol. Chem.
272, 14817-14824; Lin et al., 1998. Mol. Cell. Endo. 146, 27-37; Tarasova et al., 1998. J.
Biol. Chenz. 273, 15883-15886; Kallal et al., 1998. J. Biol. Chem. 273, 322-328;
Groake et al., 1999. J. Biol. Chem. 274, 23263-23269; Doherty et al., 1999.
Biochem. J. 341, 415-422; Brock et al., 1999. Proc. Natl. Acad. Sci. USA 96, 10128; Cornea et al., 1999. Endocri~zology 140, 4272-4280; and Lembo et al., 1999.
Nat. Cell Biol. 1, 267-271 (these references are not admitted to be prior art to the claimed invention).
IfZ vivo applications of fluorescent proteins can be performed using techniques well known in the art. Examples of such techniques are provided by Mombaerts et al., 1996. Cell 87, 675-686; Rodriquez et al., 1999. Cell 97, 199-208;
Spergel et al., 1999. J. Neurosci. l, 2037-2050; and Zuo et al., 1999. Proc.
Natl.
Acad. Sci. USA 96, 14100-14105 (these references are not admitted to be prior art to the claimed invention).
EXAMPLES
Examples are provided below to further illustrate different features and advantages of the present invention. The examples also illustrate useful methodology for practicing the invention. These examples do not limit the claimed invention.
Example 1:, Amino acid and nucleic acid sequence information for SEQ..ID. NOs.
1-29 are provided below. SEQ. ~. NOs. 1-29 include examples of polypeptide and encoding nucleic acid sequences for MCH-R polypeptide regions, fluorescent polypeptide regions, fusion proteins and chimeric proteins. In some cases the encoding nucleic acid is shown with additional nucleic acid upsteam or downstream from an open reading frame.
SEQ. ID. N0.1: Human long form MCH1R
MS VGAMKKGVGRAVGLGGGSGCQATEEDPLPNCGACAPGQGGRRWRLPQP
AWVEGSSARLWEQATGTGWMDLEASLLPTGPNASNTSDGPDNLTSAGSPPR

LLFLLGMPFMIHQLMGNGV WHFGETMCTLITAMDANS QFTSTYILTAMAmR
YLATVHPISSTKFRKPSVATLVICLLWALSFISITPVWLYARLIPFPGGAVGCGI
RLPNPDTDLYWFTLYQFFLAFALPFV VITAAYVRILQRMTS S VAPAS QRSIRLR

TKRVTRTAIAICLVFFVCWAPYYVLQLTQLSISRPTLTFVYLYNAAISLGYANS
CLNPFVYIVLCETFRKRLVLS VKPAAQGQLRAVSNAQTADEERTESKGT
SEQ. ID. NO. 2: Human short form MCH1R
MDLEASLLPTGPNASNTSDGPDNLTSAGSPPRTGSISYINmVIPSVFGTICLLGIIG
NSTVIFAV VKKSKLHW CNNVPDIFBNLS V VDLLFLLGMPFMIHQLMGNGV WH
FGETMCTLITAMDANSQFTSTYILTAMAlDRYLATVHPISSTKFRKPSVATLVI
CLLWALSFISITPVWLYARLIPFPGGAVGCGIRLPNPDTDLYWFTLYQFFLAFA
LPFV VITAAYVRILQRMTS S VAPAS QRSIRLRTKRVTRTAIAICLVFFV C WAPY
YVLQLTQLSISRPTLTFVYLYNAAISLGYANSCLNPFVYIVLCETFRKRLVLSV
KPAAQGQLRAVSNAQTADEERTESKGT
SEQ. ID. NO. 3: Mouse MCH1R
MDLQASLLS TGPNASNISDGQDNFTLAGPPPRTRS VS YIIVIIMPS VFGTICLLGI
VGNSTVIFAVVKKSKLHWCSNVPDIFIINLSVVDLLFLLGMPFMIHQLMGNGV
WHFGETMCTLITAMDANSQFTSTYILTAMAIDRYLATVHPISSTKFRKPSMAT
LVICLLWALSFISITPVWLYARLIPFPGGAVGCGIRLPNPDTDLYWF TLYQFFLA
FALPFV VITAAYVKILQRMTS S VAPAS QRSIRLRTKRVTRTAIAICLVFFV C WA
PYYVLQLTQLSISRPTLTFVYLYNAAISLGYANSCLNPFVYIVLCETFRKRLVLS
VKPAAQGQLRTVSNAQTADEERTESKGT
SEQ. ID. NO. 4: Human short form/mouse species chimeric MCH1R
MDLEASLLPTGPNASNTSDGPDNLTSAGSPPRTGSISYIIVI>MPS VFGTICLLGIIG

FGETMCTLITAMDANSQFTSTYILTAMAIDRYLATVHPISSTKFRKPSMATLVI
CLLWALSFISITPVWLYARLIPFPGGAVGCGIRLPNPDTDLYWFTLYQFFLAFA

YVLQLTQLSISRPTLTFVYLYNAAISLGYANSCLNPFVYIVLCETFRKRLVLSV
KPAAQGQLRTVSNAQTADEERTESKGT
SEQ. TD. NO. 5: Human long form/mouse species chimeric MCH1R
MSVGAMKKGVGRAVGLGGGSGCQATEEDPLPNCGACAPGQGGRRWRLPQP
AWVEGSSARLWEQATGTGWMDLEASLLPTGPNASNTSDGPDNLTSAGSPPR
TGSISYINIIMPSVFGTICLLGIIGNSTVIFAVVKKSKLHWCNNVPDIFIINLSVVD
T.T.FT-T.GMPFM113QLMGNGVWHFGETMCTLITAMDANSQFTSTYILTAMAIDR

YLATVHPIS STKFRKPSMATLVICLLWALSFISITPV WLYARLIPFPGGAV GCGI
RLPNPDTDLYWFTLYQFFLAFALPFV VITAAYVKILQRMTS S VAPAS QRSIRLR
TKRVTRTAIAICLVFFVCWAPYYVLQLTQLSISRPTLTFVYLYNAAISLGYANS
CLNPFVYIVLCETFRKRLVLS VKPAAQGQLRTVSNAQTADEERTESKGT
SEQ. ID. NO. 6: GFP
MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKL
PVPWPTLVTTFSYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGN

KNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKD
PNEKRDHMVLLEFVTAAGITHGMDELYK
SEQ. ID. NO. 7: EGFP
MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGK
LPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADK
QKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSK
DPNEKRDHMVLLEFVTAAGITLGMDELYK
SEQ. ID. NO. 8: Emerald Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Leu Thr Tyr Gly Val Gln Cys Phe Ala Arg Tyr Pro Asp His Met Lys Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu VaI Lys Phe Glu GIy Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile Asp Phe Lys Glu Asp Gly Asn lle Leu Gly His Lys Leu Glu Tyr Asn Tyr Asn Ser His Lys Val Tyr Ile Thr Ala Asp Lys Gln Lys Asn Gly Ile Lys Val Asn Phe Lys Thr Arg His Asn Ile Glu Asp Gly Ser Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro lle Gly Asp Gly Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Ala Leu Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu Tyr Lys SEQ. ID. NO. 9: Topaz Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Phe Gly Tyr Gly Val Gln Cys Phe Ala Arg Tyr Pro Asp His Met Arg Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn Tyr Asn Ser His Asn Va1 Tyr Ile Met Ala Asp Lys Gln Lys Asn Gly Ile Lys Val Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly Ser Val Gln Leu Ala Asp His Tyr GIn Gln Asn Thr Pro Ile Gly Asp Gly Pro Va1 Leu Leu Pro Asp Asn His Tyr Leu Ser Tyr Gln Sex Ala Leu Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu Tyr Lys SEQ. ID. N0.10: W1B
Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val Glu Leu Asp Gly Asp Val Asn Gly His Arg Phe Ser Val Ser Gly Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Leu Thr Trp Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg Thr lle Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn Tyr Ile Ser His Asn Val Tyr Ile Thr Ala Asp Lys GIn Lys Asn Gly Ile Lys Ala His Phe Lys Ile Arg His Asn Ile Glu Asp Gly Ser Val Gln Leu AIa Asp His Tyr GIn Gln Asn Thr Pro Ile GIy Asp Gly Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Ala Leu Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu Tyr Lys SEQ. ID. N0.11: Mouse MCH1R-linker-EGFP
MDLQASLLSTGPNASNISDGQDNFTLAGPPPRTRSVSYINmVIPSVFGTICLLGI
VGNSTVIFAVVKKSKLHWCSNVPDIFaNLSVVDLLFLLGMPFMIHQLMGNGV
WHFGETMCTLITAMDANS QFTSTYILTAMAIDRYLATVHPIS STI~FRRKPSMAT
LVICLLWALSFISITPVWLYARLIPFPGGAVGCGIRLPNPDTDLYWF'TLYQFFLA
FALPFVVITAAYVKILQRMTSSVAPASQRSIRLRTKRVTRTAIAICLVFFVCWA
PYYVLQLTQLSISRPTLTFVYLYNAAISLGYANSCLNPFVYIVLCETFRKRLVLS

VKPAAQGQLRTVSNAQTADEERTESKGTVDGTAGPGSIATMVSKGEELFTGV
VPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTL
TYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFE

HNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVL
LEFVTAAGITLGMDELYK
SEQ. ID. N0.12: Mouse MCH1R/EGFP direct fusion MDLQASLLSTGPNASNISDGQDNFTLAGPPPRTRS V S YIIVIIMPS VFGTICLLGI

WHFGETMCTLITAMDANSQFTSTYILTAMA>DRYLATVHPISSTKFRKPSMAT
LVICLLWALSFISITPVWLYARLIPFPGGAVGCGIRLPNPDTDLYWFTLYQFFLA
FALPFVVITAAYVKILQRMTSSVAPASQRSIRLRTKRVTRTAIAICLVFFVCWA
PYYVLQLTQLSISRPTLTFVYLYNAAISLGYANSCLNPFVYIVLCETFRKRLVLS
VKPAAQGQLRTVSNAQTADEERTESKGTMVSKGEELFTGVVPILVELDGDVN
GHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPD

HYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLG
MDELYK
SEQ. ID. N0.13: Human short form/mouse species chimeric MCH1R-linker-EGFP
MDLEASLLPTGPNASNTSDGPDNLTSAGSPPRTGSISYINIIn~IPSVFGTICLLGIIG

FGETMCTLITAMDANSQFTSTYII,TAMAIDRYLATVHPISSTKFRKPSMATLVI
CLLWALSFISITPVWLYARLIPFPGGAVGCGIRLPNPDTDLYWF'TLYQFFLAFA
LPFVVITAAYVKILQRMTSS VAPASQRSIRLRTKRVTRTAIAICLVFFVCWAPY
YVLQLTQLSISRPTLTFVYLYNAAISLGYANSCLNPFVYIVLCETFRKRLVLS V
KPAAQGQLRTVSNAQTADEERTESKGTVDGTAGPGSIATMVSKGEELFTGVV
PILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLT

NIEDGS VQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
EFVTAAGITLGMDELYK

SEQ. ID. N0.14: Human Iong form/mouse species chimeric MCHIR-linker-EGFP
MS VGAMKKGVGRAVGLGGGSGCQATEEDPLPNCGACAPGQGGRRWRLPQP
AWVEGSSARLWEQATGTGWMDLEASLLPTGPNASNTSDGPDNLTSAGSPPR
TGSISYINInVIPSVFGTICLLGIIGNSTVIFAVVKKSKLHWCNNVPDIFaNLSVVD
LLFLLGMPFMIHQLMGNGVWHFGETMCTLITAMDANSQFTSTYILTAMAIDR
YLATVHPISSTKFRKPSMATLVICLLWALSFISITPVWLYARLIPFPGGAVGCGI
RLPNPDTDLYWFTLYQFFLAFALPFV VITAAYVKILQRMTS S VAPAS QRSIRLR
TKRVTRTAIAICLVFFVCWAPYYVLQLTQLSISRPTLTFVYLYNAAISLGYANS
CLNPFVYIVLCETFRKRLVLSVKPAAQGQLRTVSNAQTADEERTESKGTVDGT
AGPGSIATMVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLT
LKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQE
RTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSH

LSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK
SEQ. ID. N0.15: Human long form MCH1R cDNA
ATGTCAGTGGGAGCCATGAAGAAGGGAGTGGGGAGGGCAGTTGGGCTTG
GAGGCGGCAGCGGCTGCCAGGCTACGGAGGAAGACCCCCTTCCCAACTGC
GGGGCTTGCGCTCCGGGACAAGGTGGCAGGCGCTGGAGGCTGCCGCAGC
CTGCGTGGGTGGAGGGGAGCTCAGCTCGGTTGTGGGAGCAGGCGACCGG
CACTGGCTGGATGGACCTGGAAGCCTCGCTGCTGCCCACTGGTCCCAACG
CCAGCAACACCTCTGATGGCCCCGATAACCTCACTTCGGCAGGATCACCT
CCTCGCACGGGGAGCATCTCCTACATCAACATCATCATGCCTTCGGTGTTC
GGCACCATCTGCCTCCTGGGCATCATCGGGAACTCCACGGTCATCTTCGCG
GTCGTGAAGAAGTCCAAGCTGCACTGGTGCAACAACGTCCCCGACATCTT
CATCATCAACCTCTCGGTAGTAGATCTCCTCTTTCTCCTGGGCATGCCCTT
CATGATCCACCAGCTCATGGGCAATGGGGTGTGGCACTTTGGGGAGACCA
TGTGCACCCTCATCACGGCCATGGATGCCAATAGTCAGTTCACCAGCACC
TACATCCTGACCGCCATGGCCATTGACCGCTACCTGGCCACTGTCCACCCC
ATCTCTTCCACGAAGTTCCGGAAGCCCTCTGTGGCCACCCTGGTGATCTGC
CTCCTGTGGGCCCTCTCCTTCATCAGCATCACCCCTGTGTGGCTGTATGCC
AGACTCATCCCCTTCCCAGGAGGTGCAGTGGGCTGCGGCATACGCCTGCC
CAACCCAGACACTGACCTCTACTGGTTCACCCTGTACCAGTTTTTCCTGGC

CTTTGCCCTGCCT'TTTGTGGTCATCACAGCCGCATACGTGAGGATCCTGCA
GCGCATGACGTCCTCAGTGGCCCCCGCCTCCCAGCGCAGCATCCGGCTGC
GGACAAAGAGGGTGACCCGCACAGCCATCGCCATCTGTCTGGTCTTCTTT
GTGTGCTGGGCACCCTACTATGTGCTACAGCTGACCCAGTTGTCCATCAGC
CGCCCGACCCTCACCTTTGTCTACTTATACAATGCGGCCATCAGCTTGGGC
TATGCCAACAGCTGCCTCAACCCCTTTGTGTACATCGTGCTCTGTGAGACG
TTCCGCAAACGCTTGGTCCTGTCGGTGAAGCCTGCAGCCCAGGGGCAGCT
TCGCGCTGTCAGCAACGCTCAGACGGCTGACGAGGAGAGGACAGAAAGC
AAAGGCACCTGA
SEQ. ID. N0.16: Human short form MCH1R cDNA
ATGGACCTGGAAGCCTCGCTGCTGCCCACTGGTCCCAATGCCAGCAACAC
CTCTGATGGCCCCGATAACCTCACTTCGGCAGGATCACCTCCTCGCACGG
GGAGCATCTCCTACATCAACATCATCATGCCTTCGGTGTTCGGCACCATCT
GCCTCCTGGGCATCATCGGGAACTCCACGGTCATCTTCGCGGTCGTGAAG
AAGTCCAAGCTGCACTGGTGCAACAACGTCCCCGACATCTTCATCATCAA
CCTCTCGGTAGTAGATCTCCTCTTTCTCCTGGGCATGCCCTTCATGATCCA
CCAGCTCATGGGCAATGGGGTGTGGCACTTTGGGGAGACCATGTGCACCC
TCATCACGGCCATGGATGCCAATAGTCAGTTCACCAGCACCTACATCCTG
ACCGCCATGGCCATTGACCGCTACCTGGCCACTGTCCACCCCATCTCTTCC
ACGAAGTTCCGGAAGCCCTCTGTGGCCACCCTGGTGATCTGCCTCCTGTGG
GCCCTCTCCTTCATCAGCATCACCCCTGTGTGGCTGTATGCCAGACTCATC
CCCTTCCCAGGAGGTGCAGTGGGCTGCGGCATACGCCTGCCCAACCCAGA
CACTGACCTCTACTGGTTCACCCTGTACCAGTTTTTCCTGGCCTTTGCCCTG
CCTTTTGTGGTCATCACAGCCGCATACGTGAGGATCCTGCAGCGCATGAC
GTCCTCAGTGGCCCCCGCCTCCCAGCGCAGCATCCGGCTGCGGACAAAGA
GGGTGACCCGCACAGCCATCGCCATCTGTCTGGTCTTCTTTGTGTGCTGGG
CACCCTACTATGTGCTACAGCTGACCCAGTTGTCCATCAGCCGCCCGACCC
TCACCTTTGTCTACTTATACAATGCGGCCATCAGCTTGGGCTATGCCAACA
GCTGCCTCAACCCCTTTGTGTACATCGTGCTCTGTGAGACGTTCCGCAAAC
GCTTGGTCCTGTCGGTGAAGCCTGCAGCCCAGGGGCAGCTTCGCGCTGTC
AGCAACGCTCAGACGGCTGACGAGGAGAGGACAGAAAGCAAAGGCACCT
GA
_18_ SEQ. ID. N0.17: Mouse MCH1R cDNA
Nucleic acid sequence start and stop codons are highlighted:
GGCGGTAGAGGAAGACCCTTTTCTGGACTGCGGGGCTCAAGCTCCGGACA
AGGCGGTGGAGGGCGCTGGAGGCTGCCGCAGCCTGCGTGGGTGGACGGG
CGCTCCACTCCAGGGAGCAGGCGACCTGCACCGGCTGCATGGATCTGCAA
GCCTCGTTGCTGTCCACTGGCCCCAATGCCAGCAACATCTCCGATGGCCA
GGATAATTTCACATTGGCGGGGCCACCTCCTCGCACAAGGAGTGTCTCCT
ACATCAACATCATCATGCCTTCAGTGTTTGGTACCATCTGTCTCCTGGGCA
TTGTGGGAAACTCCACAGTCATTTTTGCCGTGGTGAAGAAATCCAAGCTG
CACTGGTGCAGCAACGTCCCTGACATCTTCATCATCAACCTCTCTGTGGTG
GATCTGCTTTTCCTGCTGGGCATGCCTTTCATGATCCACCAGCTCATGGGT
AATGGTGTCTGGCACTTTGGGGAAACCATGTGCACCCTCATCACAGCCAT
GGACGCCAACAGTCAGTTCACCAGCACCTACATCCTGACTGCTATGGCCA
TTGACCGCTACTTGGCCACCGTCCATCCCATCTCCTCCACCAAGTTCCGGA
AGCCCTCCATGGCCACCCTGGTGATCTGCCTCCTGTGGGCTCTCTCGTTCA
TTAGCATCACTCCTGTGTGGCTCTATGCCAGGCTTATCCCCTTCCCAGGGG
GTGCTGTGGGCTGTGGCATCCGCCTACCAAACCCAGATACTGATCTTTACT
GGTTCACTCTGTATCAGTTTTTCCTGGCCTTCGCCCTTCCGTTTGTGGTCAT
CACTGCTGCGTACGTGAAAATACTACAGCGCATGACGTCTTCGGTGGCCC
CAGCCTCTCAACGCAGCATCCGGCTTCGGACAAAGAGGGTGACCCGCACA
GCCATTGCCATCTGTCTGGTCTTCTTTGTGTGCTGGGCGCCCTACTACGTG
CTGCAGCTGACCCAGTTGTCCATCAGCCGCCCGACCCTCACATTCGTCTAC
CTGTACAATGCGGCCATCAGCTTGGGCTATGCCAACAGCTGCCTCAATCC
CTTTGTGTACATAGTACTCTGTGAGACCTTTCGAAAACGCTTGGTGCTGTC
GGTGAAGCCCGCGGCCCAGGGGCAGCTTCGCACGGTCAGCAATGCTCAGA
CAGCTGACGAGGAGAGGACAGAAAGCAAAGGCACCTGACAATCCCCCCC
GGTCACCTCCAAGTCAGGTCACCGCATCAAACCATGGGGAGAGATACTGA
GATAAACCCGGGGCTACCCTGGGAGGATGCAGAAGCTGGAGGCTGGGGG
CTTGTAGCAAACCACATTCCACGGGGCCCACAAATTGCTAGGGAGGCTTG
CAGCCTGGTTTGGGGGGGAAGCCTCAGACTGCAGGGATCCCCTTGACAGA
ATAGAAGCGGAGCAAGAAGGAAAGGGTGGTTTGACTGGTTCTCGGGGTCT
GTATCTGTTGGCTCGCATATATCTTTCTCTCAAGGGAAGAAGGCGGAGGT
GCCTAGCTGGGTTCCTTTAAAACTAGGCAGGGCTAGGATCTGAGCAGCTA
GGGCTCTACTGTGAGACTGGGCAAGCCGAGCGTTCCCTCCCATCTCTCATT
GGTGTTGATAGAAGGCAGTCTTTCTCCCAAGCTGGTGGATCTCCTGAAGC

ACGCTGCCTGGGCTCCAGCATCCTGTGCGGATTTCACGTTCTCTTTAGGGG
ATGCATGTTGACACTGGGGTGTGGGCTCTGAGCCCACAGGAGTTTAAAAA
ACCAAAAGAGCTCAGAGTGTCGAGAGAGACCCAATCACCGAGAATGACA
AGGCAACCTGGGGTGGATGTGGATCTTGAAACTAATAAAAAGGGGTTTTC
ACAGTGACAGCGACATTCTCTTCATAGGGCACAGCTGTCAGTCTATGGCT
GATCCAGAGCGAGCATCCATGAATTCTGCATGTGCAGGGGTCACTCTAAT
ACCTGATATGTTGGCATCATCTTTGTGCTTGAGCCTTCCNCTCCCAAATGG
GAATGAAATAAAGGCAAATTCCCNCCCCCCCCAAAAAAGGGGNAAAAAA
AAAAAAAAAAAAAAAAAAAA
SEQ. ID. N0.18: Mouse MCH1R genomic DNA
Nucleic acid sequence start and stop codons, as well as intron borders, are highlighted:
GGCGGTAGAGGAAGACCCTTTTCTGGACTGCGGGGCTCAAGCTCCGGACA
AGGCGGTGGAGGGCGCTGGAGGCTGCCGCAGCCTGCGTGGGTGGACGGG
CGCTCCACTCCAGGGAGCAGGCGACCTGCACCGGCTGCATGGATCTGCAA
GCCTCGTTGCTGTCCACTGGCCCCAATGCCAGCAACATCTCCGATGGCCA
GGATAATTTCACATTGGCGGGTGAGTCGAGTTGGAGTCCTCCCTCCTCCG
GGATGGGTGTGGAAAATGGGAAGGTTTCACCTCCCAAGCCAAACTGCCTG
GGAAACTTTATCTTACAGTTCTTGGTGATAAGATCTGCAGTCGGCTTTGCC
TGAAGAGGAAGAGGAGAGGAGGGGACACCAGCTAGGACAGAAGGGGCA
GGGAGGAATAGAGATGGGGCAGAGGCACATTTAGAAACAACAAGGGTTG
GTGACAAGACGTGAGGCAGGCTTGAGGGGAAAGCTTGCTGATGAGTCCCA
AATATGCTTTGCAGGGGGGGGGGGGGGGGAATCAAGGCTGGAGAAGCAA
GCAAGCAAGACAGCAAGACAGCGGGCGGGTAGTATGTGGGAGCCAGCAG
AAGCGCTTTGATTCACCGCTATCCTGGGCTCAATCCTCTGGCCTCGCACTG
GGGAAATGGGGTCTGAGTGGTCCTTGCTGTCTTCTGGCAAAGGCTGCTGG
GAGCAAAAGACTTCACAGGGCGTGAGAGGATTAACTTTTCTGGTGAATTA
AGCTTCTTGACATTTGCAGAACGTCAATGCCTTAAAATTCTAGCTCTGAAG
GAGAAGGGAATGAAGGGGAAAGAGGGAAGGTTGGTGTGGAGAAATTCCC
AAGCTTCTGGGGTGTAACACAGCTCCAGTCCCTACCCTATTGGGAAAGCC
CAGACTCAGGAGACATGGTCCAAGGAAATCCCTGACAGAAAACCGGGAG
AGGGCAGGGCTGTGGAGCCTGAAACACACCCCACACCCATGGTGACAGTC
ACTTCTCACATATGCCTAGGAACCTATCTGAAACCTTTGGCCATCTCTCTC
TGAAAAGATGAGGCTGCAAATACACACACACACACACACACACACACAC

ACACACACACACACACACACACACACACACACACAAATGTCCTTCAAGCC
TTTT'TGACAAGGTTTTCTGGTGGATCCCGGGGATATGAAGTTGTTCTCAGC
AGATATCTGGGAGTCTTGACTCCTGGCCCTCTGAGTAAATGGATGAAGCG
AAGAAGAATGGGGTCCTCTGAGTAACAGGTGGATCTAGAAAATCCTATAG
GAGTCACCAGGGCACGGTGGAGGAGGGTAAGGTACAGAACTAACAATAG
CCCGAGAAGGGGAAACAGCAGGAGATGATTCCAGAGACGTAGTGACCCC
AAGCTGCAAGGGAAAGCATGAGGGGCCAGCAGGAAGGCCGACATGGCAG
GTTGTCAGCTTCTAGATCGGAAGGCGGGTCACACTTGCTCTTTCTATCCTC
AGGGCCACCTCCTCGCACAAGGAGTGTCTCCTACATCAACATCATCATGC
CTTCAGTGTTTGGTACCATCTGTCTCCTGGGCATTGTGGGAAACTCCACAG
TCATTTTTGCCGTGGTGAAGAAATCCAAGCTGCACTGGTGCAGCAACGTC
CCTGACATCTTCATCATCAACCTCTCTGTGGTGGATCTGCTTTTCCTGCTGG
GCATGCCTTTCATGATCCACCAGCTCATGGGTAATGGTGTCTGGCACTTTG
GGGAAACCATGTGCACCCTCATCACAGCCATGGACGCCAACAGTCAGTTC
ACCAGCACCTACATCCTGACTGCTATGGCCATTGACCGCTACTTGGCCACC
GTCCATCCCATCTCCTCCACCAAGTTCCGGAAGCCCTCCATGGCCACCCTG
GTGATCTGCCTCCTGTGGGCTCTCTCGTTCATTAGCATCACTCCTGTGTGG
CTCTATGCCAGGCTTATCCCCTTCCCAGGGGGTGCTGTGGGCTGTGGCATC
CGCCTACCAAACCCAGATACTGATCTTTACTGGTTCACTCTGTATCAGTTT
TTCCTGGCCTTCGCCCTTCCGTTTGTGGTCATCACTGCTGCGTACGTGAAA
ATACTACAGCGCATGACGTCTTCGGTGGCCCCAGCCTCTCAACGCAGCAT
CCGGCTTCGGACAAAGAGGGTGACCCGCACAGCCATTGCCATCTGTCTGG
TCTTCTTTGTGTGCTGGGCGCCCTACTACGTGCTGCAGCTGACCCAGTTGT
CCATCAGCCGCCCGACCCTCACATTCGTCTACCTGTACAATGCGGCCATCA
GCTTGGGCTATGCCAACAGCTGCCTCAATCCCTTTGTGTACATAGTACTCT
GTGAGACCTTTCGAAAACGCTTGGTGCTGTCGGTGAAGCCCGCGGCCCAG
GGGCAGCTTCGCACGGTCAGCAATGCTCAGACAGCTGACGAGGAGAGGA
CAGAAAGCAAAGGCACCTGACAATCCCCCCCGGTCACCTCCAAGTCAGGT
CACCGCATCAAACCATGGGGAGAGATACTGAGATAAACCCGGGGCTACC
CTGGGAGGATGCAGAAGCTGGAGGCTGGGGGCTTGTAGCAAACCACATTC
CACGGGGCCCACAAATTGCTAGGGAGGCTTGCAGCCTGGTTTGGGGGGGA
AGCCTCAGACTGCAGGGATCCCCTTGACAGAATAGAAGCGGAGCAAGAA
GGAAAGGGTGGTTTGACTGGTTCTCGGGGTCTGTATCTGTTGGCTCGCATA
TATCTTTCTCTCAAGGGAAGAAGGCGGAGGTGCCTAGCTGGGTTCCTTTA
AAACTAGGCAGGGCTAGGATCTGAGCAGCTAGGGCTCTACTGTGAGACTG

GGCAAGCCGAGCGTTCCCTCCCATCTCTCATTGGTGTTGATAGAAGGCAG
TCTTTCTCCCAAGCTGGTGGATCTCCTGAAGCACGCTGCCTGGGCTCCAGC
ATCCTGTGCGGATTTCACGTTCTCTTTAGGGGATGCATGTTGACACTGGGG
TGTGGGCTCTGAGCCCACAGGAGTTTAAAAAACCAAAAGAGCTCAGAGTG
TCGAGAGAGACCCAATCACCGAGAATGACAAGGCAACCTGGGGTGGATG
TGGATCTTGAAACTAATAAAAAGGGGTTTTCACAGTGACAGCGACATTCT
CTTCATAGGGCACAGCTGTCAGTCTATGGCTGATCCAGAGCGAGCATCCA
TGAATTCTGCATGTGCAGGGGTCACTCTAATACCTGATATGTTGGCATCAT
CTTTGTGCTTGAGCCTTCCNCTCCCAAATGGGAATGAAATAAAGGCAAAT
TCCCNCCCCCCCCAAAAAAGGGGNAAAAAAAAAAAAAAAAAAAAAAAA
AA
SEQ. ID. N0.19: Human short form/mouse species chimeric MCH1R
ATGGACCTGGAAGCCTCGCTGCTGCCCACTGGTCCCAATGCCAGCAACAC
CTCTGATGGCCCCGATAACCTCACTTCGGCAGGATCACCTCCTCGCACGG
GGAGCATCTCCTACATCAACATCATCATGCCTTCGGTGTTCGGCACCATCT
GCCTCCTGGGCATCATCGGGAACTCCACGGTCATCTTCGCGGTCGTGAAG
AAGTCCAAGCTGCACTGGTGCAACAACGTCCCCGACATCTTCATCATCAA
CCTCTCGGTAGTAGATCTCCTCTTTCTCCTGGGCATGCCCTTCATGATCCA
CCAGCTCATGGGCAATGGGGTGTGGCACTTTGGGGAGACCATGTGCACCC
TCATCACGGCCATGGATGCCAATAGTCAGTTCACCAGCACCTACATCCTG
ACCGCCATGGCCATTGACCGCTACCTGGCCACTGTCCACCCCATCTCTTCC
ACGAAGTTCCGGAAGCCCTCCATGGCCACCCTGGTGATCTGCCTCCTGTG
GGCTCTCTCGTTCATTAGCATCACTCCTGTGTGGCTCTATGCCAGGCTTAT
CCCCTTCCCAGGGGGTGCTGTGGGCTGTGGCATCCGCCTACCAAACCCAG
ATACTGATCTTTACTGGTTCACTCTGTATCAGTTTTTCCTGGCCTTCGCCCT
TCCGTTTGTGGTCATCACTGCTGCGTACGTGAAAATACTACAGCGCATGAC
GTCTTCGGTGGCCCCAGCCTCTCAACGCAGCATCCGGCTTCGGACAAAGA
GGGTGACCCGCACAGCCATTGCCATCTGTCTGGTCTTCTTTGTGTGCTGGG
CGCCCTACTACGTGCTGCAGCTGACCCAGTTGTCCATCAGCCGCCCGACC
CTCACATTCGTCTACCTGTACAATGCGGCCATCAGCTTGGGCTATGCCAAC
AGCTGCCTCAATCCCTTTGTGTACATAGTACTCTGTGAGACCTTTCGAAAA
CGCTTGGTGCTGTCGGTGAAGCCCGCGGCCCAGGGGCAGCTTCGCACGGT
CAGCAATGCTCAGACAGCTGACGAGGAGAGGACAGAAAGCAAAGGCACC
TGA

SEQ. ID. NO. 20: Human long form/mouse species chimeric MCH1R
ATGTCAGTGGGAGCCATGAAGAAGGGAGTGGGGAGGGCAGTTGGGCTTG
GAGGCGGCAGCGGCTGCCAGGCTACGGAGGAAGACCCCCTTCCCAACTGC
GGGGCTTGCGCTCCGGGACAAGGTGGCAGGCGCTGGAGGCTGCCGCAGC
CTGCGTGGGTGGAGGGGAGCTCAGCTCGGTTGTGGGAGCAGGCGACCGG
CACTGGCTGGATGGACCTGGAAGCCTCGCTGCTGCCCACTGGTCCCAACG
CCAGCAACACCTCTGATGGCCCCGATAACCTCACTTCGGCAGGATCACCT
CCTCGCACGGGGAGCATCTCCTACATCAACATCATCATGCCTTCGGTGTTC
GGCACCATCTGCCTCCTGGGCATCATCGGGAACTCCACGGTCATCTTCGCG
GTCGTGAAGAAGTCCAAGCTGCACTGGTGCAACAACGTCCCCGACATCTT
CATCATCAACCTCTCGGTAGTAGATCTCCTCTTTCTCCTGGGCATGCCCTT
CATGATCCACCAGCTCATGGGCAATGGGGTGTGGCACTTTGGGGAGACCA
TGTGCACCCTCATCACGGCCATGGATGCCAATAGTCAGTTCACCAGCACC
TACATCCTGACCGCCATGGCCATTGACCGCTACCTGGCCACTGTCCACCCC
ATCTCTTCCACGAAGTTCCGGAAGCCCTCCATGGCCACCCTGGTGATCTGC
CTCCTGTGGGCTCTCTCGTTCATTAGCATCACTCCTGTGTGGCTCTATGCC
AGGCTTATCCCCTTCCCAGGGGGTGCTGTGGGCTGTGGCATCCGCCTACCA
AACCCAGATACTGATCTTTACTGGTTCACTCTGTATCAGTTTTTCCTGGCCT
TCGCCCTTCCGTTTGTGGTCATCACTGCTGCGTACGTGAAAATACTACAGC
GCATGACGTCTTCGGTGGCCCCAGCCTCTCAACGCAGCATCCGGCTTCGG
ACAAAGAGGGTGACCCGCACAGCCATTGCCATCTGTCTGGTCTTCTTTGTG
TGCTGGGCGCCCTACTACGTGCTGCAGCTGACCCAGTTGTCCATCAGCCGC
CCGACCCTCACATTCGTCTACCTGTACAATGCGGCCATCAGCTTGGGCTAT
GCCAACAGCTGCCTCAATCCCTTTGTGTACATAGTACTCTGTGAGACCTTT
CGAAAACGCTTGGTGCTGTCGGTGAAGCCCGCGGCCCAGGGGCAGCTTCG
CACGGTCAGCAATGCTCAGACAGCTGACGAGGAGAGGACAGAAAGCAAA
GGCACCTGA
SEQ. ID. NO. 21: Aequorea victoria Green Fluorescent Protein (GFP) cDNA
Nucleic acid sequence start and stop codons are highlighted:
TACACACGAATAAAAGATAACAAAGATGAGTAAAGGAGAAGAACTTTTC
ACTGGAGTTGTCCCAATTCTTGTTGAATTAGATGGTGATGTTAATGGGCAC
AAATTTTCTGTCAGTGGAGAGGGTGAAGGTGATGCAACATACGGAAAACT
TACCCTTAAATTTATTTGCACTACTGGAAAACTACCTGTTCCATGGCCAAC

ACTTGTCACTACTTTCTCTTATGGTGTTCAATGCTTTTCAAGATACCCAGAT
CATATGAAACAGCATGACTTTTTCAAGAGTGCCATGCCCGAAGGTTATGT
ACAGGAAAGAACTATATT'TTTCAAAGATGACGGGAACTACAAGACACGTG
CTGAAGTCAAGTTTGAAGGTGATACCCTTGTTAATAGAATCGAGTTAAAA
GGTATTGATTTTAAAGAAGATGGAAACATTCTTGGACACAAATTGGAATA
CAACTATAACTCACACAATGTATACATCATGGCAGACAAACAAAAGAATG
GAATCAAAGTTAACTTCAAAATTAGACACAACATTGAAGATGGAAGCGTT
CAACTAGCAGACCATTATCAACAAAATACTCCAATTGGCGATGGCCCTGT
CCTTTTACCAGACAACCATTACCTGTCCACACAATCTGCCCTTTCGAAAGA
TCCCAACGAAAAGAGAGACCACATGGTCCTTCTTGAGTTTGTAACAGCTG
CTGGGATTACACATGGCATGGATGAACTATACAAATAAATGTCCAGACTT
CCAATTGACACTAAAGTGTCCGAACAATTACTAAAATCTCAGGGTTCCTG
GTTAAATTCAGGCTGAGATATTATTTATATATTTATAGATTCATTAAAATT
GTATGAATAATTTATTGATGTTATTGATAGAGGTTATTTTCTTATTAAACA
GGCTACTTGGAGTGTATTCTTAATTCTATATTAATTACAATTTGATTTGACT
TGCTCAAA
SEQ. TD. NO. 22: EGFP + Linker Nucleic acid sequence start and stop codons are highlighted and a 12 amino acid linker sequence is denoted in lower case:
gtcgacggtaccgcgggcccgggatccatcgccaccATGGTGAGCAAGGGCGAGGAGCTGTT
CACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCC
ACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAA
GCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGC
CCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTAC
CCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGG
CTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGA
CCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAG
CTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGC
TGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAG
AAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACG
GCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGAC
GGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCT
GAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCG

TGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAAAGC
GGCCGC
SEQ. ID. NO. 23: Emerald ATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGT
CGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAG
GGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCAC
CACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCTTGACCT
ACGGCGTGCAGTGCTTCGCCCGCTACCCCGACCACATGAAGCAGCACGAC
TTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTC
TTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGG
GCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAG
GACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACA
AGGTCTATATCACCGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTC
AAGACCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACT
ACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAAC
CACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCG
CGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCG
GCATGGACGAGCTGTACAAGTAA
SEQ. ID. NO. 24: Topaz ATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGT
CGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAG
GGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCAC
CACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCTTCGGCT
ACGGCGTGCAGTGCTTCGCCCGCTACCCCGACCACATGCGCCAGCACGAC
TTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTC
TTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGG
GCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAG
GACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACA
ACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTC
AAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTA
CCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACC
ACTACCTGAGCTACCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGC

GATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGG
CATGGACGAGCTGTACAAGTAA
SEQ. ID. NO. 25: W1B
ATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGT
CGAGCTGGACGGCGACGTAAACGGCCACAGGTTCAGCGTGTCCGGCGAG
GGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCAC
CACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCT
GGGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGAC
TTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGTACCATCTTC
TTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGG
GCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAG
GACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACATCAGCCACA
ACGTCTATATCACCGCCGACAAGCAGAAGAACGGCATCAAGGCCCACTTC
AAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTA
CCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACC
ACTACC'TGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGC
GATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGG
CATGGACGAGCTGTACAAGTAA
SEQ. ID. NO. 26: Mouse MCH1R-linker-EGFP
Nucleic acid sequence start codon and start and stop codons for mouse MCH1R
and EGFP, respectively, as well as intron borders, are highlighted and a 12 amino acid linker sequence is denoted in lower case:
ATGGATCTGCAAGCCTCGTTGCTGTCCACTGGCCCCAATGCCAGCAACAT
CTCCGATGGCCAGGATAATTTCACATTGGCGGGTGAGTCGAGTTGGAGTC
CTCCCTCCTCCGGGATGGGTGTGGAAAATGGGAAGGTTTCACCTCCCAAG
CCAAACTGCCTGGGAAACTTTATCTTACAGTTCTTGGTGATAAGATCTGCA
GTCGGCTTTGCCTGAAGAGGAAGAGGAGAGGAGGGGACACCAGCTAGGA
CAGAAGGGGCAGGGAGGAATAGAGATGGGGCAGAGGCACATTTAGAAAC
AACAAGGGTTGGTGACAAGACGTGAGGCAGGCTTGAGGGGAAAGCTTGC
TGATGAGTCCCAAATATGCTTTGCAGGGGGGGGGGGGGGGGAATCAAGG
CTGGAGAAGCAAGCAAGCAAGACAGCAAGACAGCGGGCGGGTAGTATGT
GGGAGCCAGCAGAAGCGCTTTGATTCACCGCTATCCTGGGCTCAATCCTC
TGGCCTCGCACTGGGGAAATGGGGTCTGAGTGGTCCTTGCTGTCTTCTGGC

AAAGGCTGCTGGGAGCAAAAGACTTCACAGGGCGTGAGAGGATTAACTTT
TCTGGTGAATTAAGCTTCTTGACATTTGCAGAACGTCAATGCCTTAAAATT
CTAGCTCTGAAGGAGAAGGGAATGAAGGGGAAAGAGGGAAGGTTGGTGT
GGAGAAATTCCCAAGCTTCTGGGGTGTAACACAGCTCCAGTCCCTACCCT
ATTGGGAAAGCCCAGACTCAGGAGACATGGTCCAAGGAAATCCCTGACA
GAAAACCGGGAGAGGGCAGGGCTGTGGAGCCTGAAACACACCCCACACC
CATGGTGACAGTCACTTCTCACATATGCCTAGGAACCTATCTGAAACCTTT
GGCCATCTCTCTCTGAAAAGATGAGGCTGCAAATACACACACACACACAC
ACACACACACACACACACACACACACACACACACACACACACACACAAA
TGTCCTTCAAGCCTTTTTGACAAGGTTTTCTGGTGGATCCCGGGGATATGA
AGTTGTTCTCAGCAGATATCTGGGAGTCTTGACTCCTGGCCCTCTGAGTAA
ATGGATGAAGCGAAGAAGAATGGGGTCCTCTGAGTAACAGGTGGATCTA
GAAAATCCTATAGGAGTCACCAGGGCACGGTGGAGGAGGGTAAGGTACA
GAACTAACAATAGCCCGAGAAGGGGAAACAGCAGGAGATGATTCCAGAG
ACGTAGTGACCCCAAGCTGCAAGGGAAAGCATGAGGGGCCAGCAGGAAG
GCCGACATGGCAGGTTGTCAGCTTCTAGATCGGAAGGCGGGTCACACTTG
CTCTTTCTATCCTCAGGGCCACCTCCTCGCACAAGGAGTGTCTCCTACATC
AACATCATCATGCCTTCAGTGTTTGGTACCATCTGTCTCCTGGGCATTGTG
GGAAACTCCACAGTCATTTTTGCCGTGGTGAAGAAATCCAAGCTGCACTG
GTGCAGCAACGTCCCTGACATCTTCATCATCAACCTCTCTGTGGTGGATCT
GCTTTTCCTGCTGGGCATGCCTTTCATGATCCACCAGCTCATGGGTAATGG
TGTCTGGCACTTTGGGGAAACCATGTGCACCCTCATCACAGCCATGGACG
CCAACAGTCAGTTCACCAGCACCTACATCCTGACTGCTATGGCCATTGACC
GCTACTTGGCCACCGTCCATCCCATCTCCTCCACCAAGTTCCGGAAGCCCT
CCATGGCCACCCTGGTGATCTGCCTCCTGTGGGCTCTCTCGTTCATTAGCA
TCACTCCTGTGTGGCTCTATGCCAGGCTTATCCCCTTCCCAGGGGGTGCTG
TGGGCTGTGGCATCCGCCTACCAAACCCAGATACTGATCTTTACTGGTTCA
CTCTGTATCAGTTTTTCCTGGCCTTCGCCCTTCCGTTTGTGGTCATCACTGC
TGCGTACGTGAAAATACTACAGCGCATGACGTCTTCGGTGGCCCCAGCCT
CTCAACGCAGCATCCGGCTTCGGACAAAGAGGGTGACCCGCACAGCCATT
GCCATCTGTCTGGTCTTCTTTGTGTGCTGGGCGCCCTACTACGTGCTGCAG
CTGACCCAGTTGTCCATCAGCCGCCCGACCCTCACATTCGTCTACCTGTAC
AATGCGGCCATCAGCTTGGGCTATGCCAACAGCTGCCTCAATCCCTTTGTG
TACATAGTACTCTGTGAGACCTTTCGAAAACGCTTGGTGCTGTCGGTGAA
GCCCGCGGCCCAGGGGCAGCTTCGCACGGTCAGCAATGCTCAGACAGCTG

ACGAGGAGAGGACAGAAAGCAAAGGCACCgtcgacggtaccgcgggcccgggatccatcg ccaccATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCC
TGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGC
GAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTG
CACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGA
CCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCAC
GACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCAT
CTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCG
AGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAG
GAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCC
ACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAA
CTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACC
ACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGAC
AACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAA
GCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTC
TCGGCATGGACGAGCTGTACAAGTAA
SEQ. ID. NO. 27: Mouse MCH1R/EGFP direct fusion Nucleic acid sequence start codon and start and stop codons for mouse MCH1R
and ~ EGFP, respectively, as well as intron borders, are highlighted:
ATGGATCTGCAAGCCTCGTTGCTGTCCACTGGCCCCAATGCCAGCAACAT
CTCCGATGGCCAGGATAATTTCACATTGGCGGGTGAGTCGAGTTGGAGTC
CTCCCTCCTCCGGGATGGGTGTGGAAAATGGGAAGGTTTCACCTCCCAAG
CCAAACTGCCTGGGAAACTTTATCTTACAGTTCTTGGTGATAAGATCTGCA
GTCGGCTTTGCCTGAAGAGGAAGAGGAGAGGAGGGGACACCAGCTAGGA
CAGAAGGGGCAGGGAGGAATAGAGATGGGGCAGAGGCACATTTAGAAAC
AACAAGGGTTGGTGACAAGACGTGAGGCAGGCTTGAGGGGAAAGCTTGC
TGATGAGTCCCAAATATGCTTTGCAGGGGGGGGGGGGGGGGAATCAAGG
CTGGAGAAGCAAGCAAGCAAGACAGCAAGACAGCGGGCGGGTAGTATGT
GGGAGCCAGCAGAAGCGCTTTGATTCACCGCTATCCTGGGCTCAATCCTC
TGGCCTCGCACTGGGGAAATGGGGTCTGAGTGGTCCTTGCTGTCTTCTGGC
AAAGGCTGCTGGGAGCAAAAGACTTCACAGGGCGTGAGAGGATTAACTTT
TCTGGTGAATTAAGCTTCTTGACATTTGCAGAACGTCAATGCCTTAAAATT
CTAGCTCTGAAGGAGAAGGGAATGAAGGGGAAAGAGGGAAGGTTGGTGT
GGAGAAATTCCCAAGCTTCTGGGGTGTAACACAGCTCCAGTCCCTACCCT

ATTGGGAAAGCCCAGACTCAGGAGACATGGTCCAAGGAAATCCCTGACA
GAAAACCGGGAGAGGGCAGGGCTGTGGAGCCTGAAACACACCCCACACC
CATGGTGACAGTCACTTCTCACATATGCCTAGGAACCTATCTGAAACCTTT
GGCCATCTCTCTCTGAAAAGATGAGGCTGCAAATACACACACACACACAC
ACACACACACACACACACACACACACACACACACACACACACACACAAA
TGTCCTTCAAGCCTTTTTGACAAGGTTTTCTGGTGGATCCCGGGGATATGA
AGTTGTTCTCAGCAGATATCTGGGAGTCTTGACTCCTGGCCCTCTGAGTAA
ATGGATGAAGCGAAGAAGAATGGGGTCCTCTGAGTAACAGGTGGATCTA
GAAAATCCTATAGGAGTCACCAGGGCACGGTGGAGGAGGGTAAGGTACA
GAACTAACAATAGCCCGAGAAGGGGAAACAGCAGGAGATGATTCCAGAG
ACGTAGTGACCCCAAGCTGCAAGGGAAAGCATGAGGGGCCAGCAGGAAG
GCCGACATGGCAGGTTGTCAGCTTCTAGATCGGAAGGCGGGTCACACTTG
CTCTTTCTATCCTCAGGGCCACCTCCTCGCACAAGGAGTGTCTCCTACATC
AACATCATCATGCCTTCAGTGTTTGGTACCATCTGTCTCCTGGGCATTGTG
GGAAACTCCACAGTCATTTTTGCCGTGGTGAAGAAATCCAAGCTGCACTG
GTGCAGCAACGTCCCTGACATCTTCATCATCAACCTCTCTGTGGTGGATCT
GCTTTTCCTGCTGGGCATGCCTTTCATGATCCACCAGCTCATGGGTAATGG
TGTCTGGCACTTTGGGGAAACCATGTGCACCCTCATCACAGCCATGGACG
CCAACAGTCAGTTCACCAGCACCTACATCCTGACTGCTATGGCCATTGACC
GCTACTTGGCCACCGTCCATCCCATCTCCTCCACCAAGTTCCGGAAGCCCT
CCATGGCCACCCTGGTGATCTGCCTCCTGTGGGCTCTCTCGTTCATTAGCA
TCACTCCTGTGTGGCTCTATGCCAGGCTTATCCCCTTCCCAGGGGGTGCTG
TGGGCTGTGGCATCCGCCTACCAAACCCAGATACTGATCTTTACTGGTTCA
CTCTGTATCAGTTTTTCCTGGCCTTCGCCCTTCCGTTTGTGGTCATCACTGC
TGCGTACGTGAAAATACTACAGCGCATGACGTCTTCGGTGGCCCCAGCCT
CTCAACGCAGCATCCGGCTTCGGACAAAGAGGGTGACCCGCACAGCCATT
GCCATCTGTCTGGTCTTCTTTGTGTGCTGGGCGCCCTACTACGTGCTGCAG
CTGACCCAGTTGTCCATCAGCCGCCCGACCCTCACATTCGTCTACCTGTAC
AATGCGGCCATCAGCTTGGGCTATGCCAACAGCTGCCTCAATCCCTTTGTG
TACATAGTACTCTGTGAGACCTTTCGAAAACGCTTGGTGCTGTCGGTGAA
GCCCGCGGCCCAGGGGCAGCTTCGCACGGTCAGCAATGCTCAGACAGCTG
ACGAGGAGAGGACAGAAAGCAAAGGCACCATGGTGAGCAAGGGCGAGG
AGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTA
AACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCT
ACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTG

CCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAG
CCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGC
CCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAAC
TACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACC
GCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGG
GCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCG
ACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACAT
CGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCC
ATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCA
GTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGC
TGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTAC
AAGTAA
SEQ. ID. NO. 28: Human short form/mouse species chimeric MCH1R-linker-EGFP
Nucleic acid sequence start codon and start and stop codons for mouse MCH1R
and EGFP, respectively, are highlighted and a 12 amino acid linker sequence is denoted in lower case:
ATGGACCTGGAAGCCTCGCTGCTGCCCACTGGTCCCAATGCCAGCAACAC
CTCTGATGGCCCCGATAACCTCACTTCGGCAGGATCACCTCCTCGCACGG
GGAGCATCTCCTACATCAACATCATCATGCCTTCGGTGTTCGGCACCATCT
GCCTCCTGGGCATCATCGGGAACTCCACGGTCATCTTCGCGGTCGTGAAG
AAGTCCAAGCTGCACTGGTGCAACAACGTCCCCGACATCTTCATCATCAA
CCTCTCGGTAGTAGATCTCCTCTTTCTCCTGGGCATGCCCTTCATGATCCA
CCAGCTCATGGGCAATGGGGTGTGGCACTTTGGGGAGACCATGTGCACCC
TCATCACGGCCATGGATGCCAATAGTCAGTTCACCAGCACCTACATCCTG
ACCGCCATGGCCATTGACCGCTACCTGGCCACTGTCCACCCCATCTCTTCC
ACGAAGTTCCGGAAGCCCTCCATGGCCACCCTGGTGATCTGCCTCCTGTG
GGCTCTCTCGTTCATTAGCATCACTCCTGTGTGGCTCTATGCCAGGCTTAT
CCCCTTCCCAGGGGGTGCTGTGGGCTGTGGCATCCGCCTACCAAACCCAG
ATACTGATCTTTACTGGTTCACTCTGTATCAGTTTTTCCTGGCCTTCGCCCT
TCCGTTTGTGGTCATCACTGCTGCGTACGTGAAAATACTACAGCGCATGAC
GTCTTCGGTGGCCCCAGCCTCTCAACGCAGCATCCGGCTTCGGACAAAGA
GGGTGACCCGCACAGCCATTGCCATCTGTCTGGTCTTCTTTGTGTGCTGGG
CGCCCTACTACGTGCTGCAGCTGACCCAGTTGTCCATCAGCCGCCCGACC

CTCACATTCGTCTACCTGTACAATGCGGCCATCAGCTTGGGCTATGCCAAC
AGCTGCCTCAATCCCTTTGTGTACATAGTACTCTGTGAGACCTTTCGAAAA
CGCTTGGTGCTGTCGGTGAAGCCCGCGGCCCAGGGGCAGCTTCGCACGGT
CAGCAATGCTCAGACAGCTGACGAGGAGAGGACAGAAAGCAAAGGCACC
gtcgacggtaccgcgggcccgggatccatcgccaccATGGTGAGCAAGGGCGAGGAGCTGTT
CACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCC
ACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAA
GCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGC
CCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTAC
CCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGG
CTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGA
CCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAG
CTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGC
TGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAG
AAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACG
GCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGAC
GGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCT
GAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCG
TGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAA
SEQ. ID. NO. 29: Human long form/mouse species chimeric MCH1R-linker-EGFP
Nucleic acid sequence start codon and start and stop codons for mouse MCH1R
and EGFP, respectively, are highlighted and a 12 amino acid linker sequence is denoted in lower case:
ATGTCAGTGGGAGCCATGAAGAAGGGAGTGGGGAGGGCAGTTGGGCTTG
GAGGCGGCAGCGGCTGCCAGGCTACGGAGGAAGACCCCCTTCCCAACTGC
GGGGCTTGCGCTCCGGGACAAGGTGGCAGGCGCTGGAGGCTGCCGCAGC
CTGCGTGGGTGGAGGGGAGCTCAGCTCGGTTGTGGGAGCAGGCGACCGG
CACTGGCTGGATGGACCTGGAAGCCTCGCTGCTGCCCACTGGTCCCAACG
CCAGCAACACCTCTGATGGCCCCGATAACCTCACTTCGGCAGGATCACCT
CCTCGCACGGGGAGCATCTCCTACATCAACATCATCATGCCTTCGGTGTTC
GGCACCATCTGCCTCCTGGGCATCATCGGGAACTCCACGGTCATCTTCGCG
GTCGTGAAGAAGTCCAAGCTGCACTGGTGCAACAACGTCCCCGACATCTT
CATCATCAACCTCTCGGTAGTAGATCTCCTCTTTCTCCTGGGCATGCCCTT

CATGATCCACCAGCTCATGGGCAATGGGGTGTGGCACTTTGGGGAGACCA
TGTGCACCCTCATCACGGCCATGGATGCCAATAGTCAGTTCACCAGCACC
TACATCCTGACCGCCATGGCCATTGACCGCTACCTGGCCACTGTCCACCCC
ATCTCTTCCACGAAGTTCCGGAAGCCCTCCATGGCCACCCTGGTGATCTGC
CTCCTGTGGGCTCTCTCGTTCATTAGCATCACTCCTGTGTGGCTCTATGCC
AGGCTTATCCCCTTCCCAGGGGGTGCTGTGGGCTGTGGCATCCGCCTACCA
AACCCAGATACTGATCTT'TACTGGTTCACTCTGTATCAGTTTTTCCTGGCCT
TCGCCCTTCCGTTTGTGGTCATCACTGCTGCGTACGTGAAAATACTACAGC
GCATGACGTCTTCGGTGGCCCCAGCCTCTCAACGCAGCATCCGGCTTCGG
ACAAAGAGGGTGACCCGCACAGCCATTGCCATCTGTCTGGTCTTCTTTGTG
TGCTGGGCGCCCTACTACGTGCTGCAGCTGACCCAGTTGTCCATCAGCCGC
CCGACCCTCACATTCGTCTACCTGTACAATGCGGCCATCAGCTTGGGCTAT
GCCAACAGCTGCCTCAATCCCTTTGTGTACATAGTACTCTGTGAGACCTTT
CGAAAACGCTTGGTGCTGTCGGTGAAGCCCGCGGCCCAGGGGCAGCTTCG
CACGGTCAGCAATGCTCAGACAGCTGACGAGGAGAGGACAGAAAGCAAA
GGCACCgtcgacggtaccgcgggcccgggatccatcgccaccATGGTGAGCAAGGGCGAGGA
GCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAA
ACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTAC
GGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCC
CTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCC
GCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCC
GAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTA
CAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGC
ATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGC
ACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGAC
AAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCG
AGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATC
GGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTC
CGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGG
AGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAG
TAA
Example 2: Generation of Chimeric and Fusion Proteins DNA vectors encoding fusion proteins between a MCH-R receptor (MCH1R) and several different superbright variants of Green Fluorescent Protein (GFP) were generated. GFP variants were fused either via a 12 amino acid linker:
TCGACGGTACCGCGGGCCCGGGATCCATCGCCACC (SEQ. ID. NO. 30), amino acid sequence: VDGTAGPGSIAT (SEQ. ID. NO. 31) (linker fusions) or directly to the C-terminus of MCH1R (direct fusions).
Mouse MCH1R-linker-GFP Variant Fusion Constructs Initially, mouse MCH1R was fused in frame via the linker to Enhanced Green Fluorescent Protein (EGFP). MCH1R was PCR-amplified (95°C
for 5 minutes; 95°C for 30 seconds, 60°C for 45 seconds, 68°C
for 3.5 minutes, for 15 cycles; 68°C for 7 minutes) from a full-length mouse MCH1R genomic DNA
lambda clone utilizing a high fidelity polymerase mix (Expand High Fidelity PCR
System from Boehringer Mannheim) and PCR primers [MCH1R (Eco RI) 5':
GCGAATTCACCATGGATCTGCAAGCCTCG (SEQ. ID. NO. 32), MCH1R (Sal I) 3': GCGTCGACGGTGCCTTTGCTTTCTGTCC (SEQ. m. NO. 33)] that generated Eco RI and Sal I enzymatic restriction sites at the N- and C-terminus, respectively.
The MCH1R N-terminal PCR primer was also designed to introduce a Kozak consensus sequence for translation which contained an Nco I site (5'-ACCATGG-3'), and the MCH1R C-terminal PCR primer was also designed to eliminate the endogenous stop codon present in the mouse MCH1R gene. The resulting PCR
product was phenol/chloroform extracted, restriction digested with Eco RI and Sal I, gel purified, and subcloned in frame into the multicloning site of Clontech's pEGFP-N3 vector between Eco RI and Sal I sites. Several resulting clones for this construct were sequenced to identify a clone with an entirely correct nucleotide sequence. This clone was named mMCHIR-1-EGFP for mouse MCH1R-linker-EGFP.
An approximately 760 by Sal I to Not I fragment of mMCHIR-1-EGFP
was excised, gel purified, and subcloned into the multicloning site of pBluescript (SK+) (Stratagene) between Sal1 and Not I sites. An approximately 710 by Nco I
to Bsr G1 fragment of EGFP was excised from the resulting pBluescript-EGFP vector and replaced with the corresponding Nco I to Bsr G1 fragment of either Emerald, Topaz, or W1B (other superbright GFP variants), which were excised from vectors pRSET-Emerald, pRSET-Topaz, and pRSET-W1B, respectively. pRSET-Emerald, pRSET-Topaz, and pRSET-W 1B were obtained from Aurora Biosciences Co. Sal I to Not I fragments containing either Emerald, Topaz, or W 1B were excised from the resulting pBluescript-Emerald, pBluescript-Topaz, and pBluescript-W1B vectors, respectively. Appropriate fragments were gel purified and subcloned into mMCHIR-1-EGFP digested with Sal I and Not I, replacing the Sal I to Not I EGFP
fragment with the corresponding Sal I to Not I fragment from either Emerald, Topaz, or W1B.
Several clones for each construct were sequenced to confirm the presence of the appropriate GFP variant. The resulting vectors were named mMCHIR-1-Emerald, mMCHIR-1-Topaz, and mMCHIR-1-W 1B for mouse MCH1R-linker-Emerald, mouse MCH1R-linker-Topaz, and mouse MCH1R-linker-W1B, respectively.
Mouse MCH1R/GFP Variant Direct Fusion Constructs A two step PCR strategy was employed to generate the direct fusion constructs. First, mouse MCH1R, EGFP, and Emerald were PCR-amplified from a full-length mouse MCH1R genomic DNA lambda clone, Clontech's pEGFP-N3 vector, and Aurora's pRSET-Emerald vector, respectively. Mouse MCH1R was PCR-amplified according to the previously mentioned conditions utilizing the same N-terminal PCR primer [MCH1R (Eco RI) 5': GCGAATTCACCATGGATCTGCA
AGCCTCG (SEQ. ID. NO. 32)], but in this case a different C-terminal PCR primer was employed. The C-terminal PCR primer [MCH1R (EGFP/Emerald) 3' CCTTGCTCACCATGGTGCCTTTGCTTTCTGTCC (SEQ. ID. NO. 34)] eliminated the endogenous stop codon of mouse MCH1R as before and introduced a region of nucleotide sequence complementary to the nucleotide sequence of the N-terminus of EGFP.
EGFP and Emerald were PCR-amplified (95°C for 5 minutes;
95°C for seconds, 60°C for 45 seconds, 68°C for 1.5 minutes, for 15 cycles; 68°C for 7 minutes) separately with a high fidelity polymerase mix (Advantage HF-2 from Clontech) from their respective templates utilizing a common N-terminal PCR
primer 25 [EGFP/Emerald (MCH1R) 5': CAGAAAGCAAAGGCACCATGGTGAGCAA
GGGCGAGGAGC (SEQ. ID. NO. 35)] that generated a region of nucleotide sequence complementary to the C-terminus of mouse MCH1R and C-terminal PCR
primers [EGFP 3' : GGCGGATCCTCTAGAGTCGCGGCC (SEQ. ID. NO. 36), or Emerald (EGFP) 3' : GCTCTAGAGTCGCGGCCGCTTACTTGTACAGCTCGTCC
30 (SEQ. ID. NO. 37)] that generated a Not I site at the C-terminus. The resulting PCR
products were electrophoresed on an agarose gel and the appropriate fragments were gel purified.
In a second PCR step, PCR reactions were set up between the previously generated mouse MCH1R and EGFP, or mouse MCH1R and Emerald PCR products. Following an initial 5 minute denaturation step at 95°C, two rounds of thermocycling (95°C for 30 seconds, 60°C for 45 seconds, 68°C for 4 minutes) were performed in the absence of PCR primers. This allowed the mouse MCH1R and GFP
variants to anneal at their complementary regions and to be filled in by the high fidelity polymerase mix (Expand High Fidelity PCR System from Boehringer Mannheim), yielding double stranded template DNA.
Subsequently, the common N-terminal mouse MCH1R [MCH1R (Eco RI) 5' : GCGAATTCACCATGGATCTGCAAGCCTCG (SEQ. ID. NO. 32)] and appropriate C-terminal PCR primers [EGFP 3': GGCGGATCCTCTAGAGTC
GCGGCC (SEQ. ll~. NO. 36) or Emerald (EGFP) 3': GCTCTAGAGTCGCGG
CCGCTTACTTGTACAGCTCGTCC (SEQ. ID. NO. 37)] were added to the reactions and thermocycling was continued for an additional fifteen cycles followed by a final extension at 68°C for 7 minutes. The resulting PCR products were phenol/chloroform extracted, restriction digested with Eco RI and Not I, electrophoresed on an agarose gel, and appropriate fragments were gel purified.
These Eco RI to Not I fragments represent direct fusions between either mouse MCH1R and EGFP, or mouse MCH1R and Emerald. Clontech's pEGFP-N3 vector was restriction digested with Eco RI and Not I liberating an approximately 780 by Eco RI to Not I EGFP fragment. This restriction digest was electrophoresed on an agarose gel and the approximately 3.9 Kb pEGFP-N3 vector backbone was gel purified. Eco RI to Not I mouse MCH1R/EGFP or mouse MCH1R/Emerald direct fusion fragments were subcloned into the pEGFP-N3 vector backbone between Eco RI and Not I sites. Several resulting clones for each of these two constructs were sequenced to identify clones with correct nucleotide sequence;
however, no clones with entirely correct nucleotide sequences were identified.
Fortunately, several clones for each of the two constructs only had nucleotide mismatches in the intron region of mouse MCH1R, and therefore, were not expected to effect the functionality of the resulting fusion proteins. These clones were named mMCHIR/EGFP and mMCHIR/Emerald for mouse MCHIRIEGFP direct fusion and mouse MCH1R/Emerald direct fusion, respectively.
Human Short and Long Form/Mouse Species Chimeric MCH1R-linker-GFP Variant Fusion Constructs The initial mouse MCH1R-linker-GFP variant fusion constructs were modified to generate both human short form and human long form/mouse species chimeric MCH1R-linker-GFP variant fusion constructs. An approximately 1.7 kb Hind III to Bsp EI fragment of the mouse MCH1R gene containing exon l, the intron, and 127 amino acids of exon 2 was excised from the various mouse MCH1R-linker-GFP variant fusion constructs and replaced by either an approximately 470 by Hind III to Bsp EI fragment from the wild-type human MCH1R short form or an approximately 670 by Hind III to Bsp EI fragment from the wild-type human long form.
Several clones for each construct were sequenced to confirm the presence of the N-terminal region of either the human MCH1R short or long forms.
These clones were named hshort/mMCHIR-1-GFP variant or hlong/mMCHIR-1-GFP
variant for human short form/mouse species chimeric MCH1R-linker-GFP variant and human long form/mouse species chimeric MCH1R-linker-GFP variant, respectively.
Example 3: Functional Evaluation of MCH1R/GFP Variant Fusion Proteins Both HEK293 Aequorin (National Institutes of Health) and CHO
mammalian cell lines were transiently transfected with the various MCH1R/GFP
variant fusion constructs, as well as the appropriate control constructs.
Transfection was performed using Lipofectamine 2000 (Gibco BRL) per the manufacturer recommended protocol. Approximately 48 hours after transfection cells were harvested, stimulated with various concentrations of human MCH, and assayed for either aequorin bioluminescence (HEK293 Aequorin cells) or cAMP production (CHO cells). Aequorin bioluminescence is a representative measure of intracellular Ca2+mobilization. cAMP production was measured with the Adenylyl Cyclase Activation FlashPlate Assay (NEN Life Science Products, Inc.).
Following transient transfection of the mMCHIR-linker-EGFP
construct (MCH-R-1-EGFP) into HEK293 Aequorin cells, the resulting fusion protein exhibited functional activity comparable to that of the wild-type human MCH1R
short form (MCH-R wt). By this functional assay, the EC50 value for mMCHIR-1-EGFP
was nearly identical to that of the wild-type human short form receptor (Figure 1).
Following transient transfections of the mMCHIR-1-EGFP and mMCHIR/EGFP fusion constructs into CHO cells, the resulting fusion proteins exhibited functional activity comparable to that of the wild-type human MCH1R
short form. By this functional assay, the EC50 values for mMCHIR-1-EGFP and mMCHIR/EGFP were comparable to that of the wild-type human receptor (Table 1).

Transient transfections with the corresponding Emerald constructs yielded similar results (data not shown).
Table 1 Rece for EC50 (nM) Wild-t a Human MCH1R Short 2.166 Form Mouse MCH1R/EGFP 0.819 Mouse MCH1R-1-EGFP 3.199 Following transient transfections of the human short form/mouse species chimeric MCH1R-1-EGFP (HuShort/mMCHIR-1-EGFP) and human long form/mouse species chimeric MCH1R-1-EGFP (HuLong/mMCHIR-1-EGFP) constructs into HEK293 cells, the resulting fusion proteins exhibited functional activity comparable to that of the wild-type human MHC1R short and long forms, respectively. By this functional assay, the EC50 value for each fusion proteins was nearly identical to that of the corresponding wild-type human receptor (Table 2).
Table 2 Rece for ECSp (nM) Wild-t a Human MCH1R Short 22.27 Form HuShort/mMCHIR-1-EGFP 19.54 Wild-t a Human MCH1R Lon 196.7 Form HuLon mMCHIR-1-EGFP Form 217.5 Following transient transfections of the human short form/mouse species chimeric MCH1R-1-EGFP (HuShort/mMCHIR-1-EGFP) and human long form/mouse species chimeric MCH1R-1-EGFP (HuLong/mMCHIR-1-EGFP) constructs into CHO cells, the resulting fusion proteins exhibited functional activity comparable to or less than that of the wild-type human MHC1R short and long forms, respectively (Table 3). By this functional assay, the EC50 value for the human short form/mouse species chimeric MCH1R-1-EGFP fusion protein was comparable to that of the corresponding wild-type human receptor, whereas, the human long form/mouse species chimeric MCH1R-1-EGFP fusion protein had an EC50 value approximately 7.5-fold higher than that of its corresponding wild-type control.
Table 3 Rece for EC50 (nM) Wild-t a Human MCH1R Short 1.029 Form Wild-t a Human MCH1R Lon 1.515 Form HuShort/mMCHIR-1-EFGP 1.565 HuLon mMCHIR-1-EGFP 11.580 Transient expression of all the MCH1R/GFP variant fusion proteins that underwent functional evaluation resulted in fluorescence primarily associated with the plasma membrane in both HEK293 and CHO cells (data not shown). This pattern of fluorescence is consistent with a predominant membrane associated localization.
Example 4: Generation of Stable Cell Lines Wild-type CHO cells were transfected using SuperFect (Qiagen) and either mouse MCH-1R-EGFP or human short/mouse species chimeric MCH-1R-EGFP. Forty-eight hours after transfection, transfected cells were subjected to positive selection for approximately ten days in media containing 6418.
Following selection, MCH-1R-EGFP expressing CHO cells were bulk sorted by Fluorescence Assisted Cell Sorting (FACS) for one or two rounds on the basis of fluorescence intensity to increase the population of cells expressing EGFP. Following bulk sorts, individual clones of varying fluorescence intensities were isolated by FAGS
and expanded.
Fluorometric Microvolume Assay Technology (FMAT) was initially employed to screen a large number of stable clones by whole cell binding with a fluorescently labeled MCH derivative (SymJz-MCH, PE Biosystems) to identify those clones with good specific binding windows. Several clones exhibiting specific binding windows greater than 3-fold were further evaluated for MCH binding with the SPA-based Binding Assay. Cells from individual clones were dissociated in enzyme free dissociation media and cell membranes were prepared and subsequently tested for their ability to bind [lzsl]Phel3Tyr19-MCH in the presence of human MCH. CHO
cell lines expressing either mouse MCH-1R-EGFP or human short/mouse species chimeric MCH-1R-EGFP (Figure 4) displayed IC50 values with MCH that were indistinguishable from the corresponding IC50 values obtained with a CHO cell line expressing the wild-type human short isoform of MCH-1R.
The functional activity of these clones was evaluated with the cAMP
Flashplate Assay (Figures 2 and 3). CHO cell lines expressing either mouse MCH-1R-EGFP (Figure 2) or human short/mouse species chimeric MCH-1R-EGFP (Figure 3) displayed EC50 values with human MCH that were indistinguishable from the EC50 value obtained with a CHO cell line expressing the wild-type human short isoform of MCH-1R.
The subcellular localization of the MCH-1R-EGFP fusion proteins were determined by confocal microscopy utilizing EGFP fluorescence as a marker for MCH-1R expression. CHO cell lines stably expressing either mouse MCH-1R-EGFP
or human short/mouse species chimeric MCH-1R-EGFP displayed EGFP
fluorescence primarily associated with the plasma membrane, demonstrating that these MCH-1R-EGFP fusion proteins are primarily associated with the plasma membrane.
Other embodiments are within the following claims. While several embodiments have been shown and described, various modifications may be made without departing from the spirit and scope of the present invention.

SEQUENCE LISTING
<110> Merck & Co., Inc.
<120> MELANIN CONCENTRATING HORMONE RECEPTOR
CHIMERIC AND FUSTON PROTEINS
<130> 20652 PCT
<150> 60/189,698 <151> 2000-03-15 <160> 37 <170> FastSEQ for Windows Version 4.0 <210> 1 <211> 422 <212> PRT
<213> Human <400> 1 Met Ser Val Gly Ala Met Lys Lys Gly Val Gly Arg Ala Val Gly Leu Gly Gly Gly Ser Gly Cys Gln Ala Thr Glu Glu Asp Pro Leu Pro Asn Cys Gly Ala Cys Ala Pro Gly Gln Gly Gly Arg Arg Trp Arg Leu Pro Gln Pro Ala Trp Val Glu Gly Ser Ser Ala Arg Leu Trp Glu Gln Ala Thr Gly Thr Gly Trp Met Asp Leu Glu Ala Ser Leu Leu Pro Thr Gly Pro Asn Ala Ser Asn Thr Ser Asp Gly Pro Asp Asn Leu Thr Ser Ala Gly Ser Pro Pro Arg Thr Gly Ser Ile Ser Tyr Ile Asn Ile Ile Met Pro Ser Val Phe Gly Thr Ile Cys Leu Leu Gly Ile Ile Gly Asn Ser Thr Val Ile Phe Ala Val Va1 Lys Lys Ser Lys Leu His Trp Cys Asn Asn Val Pro Asp Ile Phe Ile Ile Asn Leu Ser Val Val Asp Leu Leu Phe Leu Leu Gly Met Pro Phe Met Ile His Gln Leu Met Gly Asn Gly Val Trp His Phe Gly Glu Thr Met Cys Thr Leu Ile Thr Ala Met Asp Ala Asn Ser Gln Phe Thr Ser Thr Tyr Ile Leu Thr Ala Met Ala Ile Asp Arg Tyr Leu Ala Thr Val His Pro Ile Ser Ser Thr Lys Phe Arg Lys Pro Ser Val Ala Thr Leu Val Ile Cys Leu Leu Trp Ala Leu Ser Phe Ile Ser Ile Thr Pro Val Trp Leu Tyr Ala Arg Leu Ile Pro Phe Pro Gly Gly Ala Val Gly Cys Gly Ile Arg Leu Pro Asn Pro Asp Thr Asp Leu Tyr Trp Phe Thr Leu Tyr Gln Phe Phe Leu Ala Phe Ala Leu Pro Phe Val Val Ile Thr Ala Ala Tyr Val Arg Ile Leu Gln Arg Met Thr Ser Ser Val Ala Pro Ala Ser Gln Arg Ser Ile Arg Leu Arg Thr Lys Arg Val Thr Arg Thr Ala Ile Ala Ile Cys Leu Val Phe Phe Val Cys Trp Ala Pro Tyr Tyr Val Leu Gln Leu Thr Gln Leu Ser Ile Ser Arg Pro Thr Leu Thr Phe Val Tyr Leu Tyr Asn Ala Ala Ile Ser Leu Gly Tyr Ala Asn Ser Cys Leu Asn Pro Phe Val Tyr Ile Val Leu Cys Glu Thr Phe Arg Lys Arg Leu Val Leu Ser Val Lys Pro Ala Ala Gln Gly Gln Leu Arg Ala Val Ser Asn Ala Gln Thr Ala Asp Glu Glu Arg Thr Glu Ser Lys Gly Thr <210> 2 <211> 353 <212> PRT
<213> Human <400> 2 Met Asp Leu Glu Ala Ser Leu Leu Pro Thr Gly Pro Asn Ala Ser Asn Thr Ser Asp Gly Pro Asp Asn Leu Thr Ser Ala Gly Ser Pro Pro Arg Thr Gly Ser Ile Ser Tyr Ile Asn Ile Ile Met Pro Ser Val Phe Gly Thr Ile Cys Leu Leu Gly Ile Ile Gly Asn Ser Thr Val Ile Phe Ala Val Val Lys Lys Ser Lys Leu His Trp Cys Asn Asn Val Pro Asp Ile Phe Ile Ile Asn Leu Ser Val Val Asp Leu Leu Phe Leu Leu Gly Met Pro Phe Met Ile His Gln Leu Met Gly Asn Gly Val Trp His Phe Gly Glu Thr Met Cys Thr Leu Ile Thr Ala Met Asp Ala Asn Ser Gln Phe Thr Ser Thr Tyr Ile Leu Thr Ala Met Ala Ile Asp Arg Tyr Leu Ala Thr Val His Pro Ile Ser Ser Thr Lys Phe Arg Lys Pro Ser Val A1a Thr Leu Val Ile Cys Leu Leu Trp Ala Leu Ser Phe Ile Ser Ile Thr Pro Val Trp Leu Tyr Ala Arg Leu Ile Pro Phe Pro Gly Gly Ala Val Gly Cys Gly Ile Arg Leu Pro Asn Pro Asp Thr Asp Leu Tyr Trp Phe Thr Leu Tyr Gln Phe Phe Leu Ala Phe Ala Leu Pro Phe Val Val Ile Thr Ala Ala Tyr Val Arg Ile Leu Gln Arg Met Thr Ser Ser Val Ala Pro Ala Ser Gln Arg Ser Ile Arg Leu Arg Thr Lys Arg Val Thr Arg Thr Ala Ile Ala Ile Cys Leu Val Phe Phe Val Cys Trp Ala Pro Tyr Tyr Val Leu Gln Leu Thr Gln Leu Ser Ile Ser Arg Pro Thr Leu Thr Phe Val Tyr Leu Tyr Asn Ala Ala Ile Ser Leu Gly Tyr Ala Asn Ser Cys Leu Asn Pro Phe Val Tyr Ile Val Leu Cys Glu Thr Phe Arg Lys Arg Leu Val Leu Ser Val Lys Pro Ala Ala Gln Gly Gln Leu Arg Ala Val Ser Asn Ala Gln Thr Ala Asp Glu Glu Arg Thr Glu Ser Lys Gly Thr <210> 3 <211> 353 <212> PRT
<213> Mouse <400> 3 Met Asp Leu Gln Ala Ser Leu Leu Ser Thr Gly Pro Asn Ala Ser Asn Ile Ser Asp Gly Gln Asp Asn Phe Thr Leu Ala Gly Pro Pro Pro Arg Thr Arg Ser Val Ser Tyr Ile Asn Ile Ile Met Pro Ser Val Phe Gly Thr Ile Cys Leu Leu Gly Ile Val Gly Asn Ser Thr Val Ile Phe Ala Val Val Lys Lys Ser Lys Leu His Trp Cys Ser Asn Val Pro Asp Ile Phe Ile Ile Asn Leu Ser Val Val Asp Leu Leu Phe Leu Leu Gly Met Pro Phe Met Ile His Gln Leu Met Gly Asn Gly Val Trp His Phe Gly Glu Thr Met Cys Thr Leu Ile Thr Ala Met Asp Ala Asn Ser Gln Phe Thr Ser Thr Tyr Ile Leu Thr Ala Met Ala Ile Asp Arg Tyr Leu Ala Thr Val His Pro Ile Ser Ser Thr Lys Phe Arg Lys Pro Ser Met Ala Thr Leu Val Ile Cys Leu Leu Trp Ala Leu Ser Phe Ile Ser Ile Thr Pro Val Trp Leu Tyr Ala Arg Leu Tle Pro Phe Pro Gly Gly Ala Val Gly Cys Gly Ile Arg Leu Pro Asn Pro Asp Thr Asp Leu Tyr Trp Phe Thr Leu Tyr Gln Phe Phe Leu Ala Phe Ala Leu Pro Phe Val Val Ile Thr Ala Ala Tyr Val Lys Ile Leu Gln Arg Met Thr Ser Ser Val Ala Pro Ala Ser Gln Arg Ser Ile Arg Leu Arg Thr Lys Arg Val Thr Arg Thr Ala Ile Ala Ile Cys Leu Val Phe Phe Val Cys Trp Ala Pro Tyr Tyr Val Leu Gln Leu Thr Gln Leu Ser Ile Ser Arg Pro Thr Leu Thr Phe Val Tyr Leu Tyr Asn Ala Ala Ile Ser Leu Gly Tyr Ala Asn Ser Cys Leu Asn Pro Phe Val Tyr Ile Val Leu Cys Glu Thr Phe Arg Lys Arg Leu Val Leu Ser Val Lys Pro Ala Ala Gln Gly Gln Leu Arg Thr Val Ser Asn Ala Gln Thr Ala Asp Glu Glu Arg Thr Glu Ser Lys Gly Thr <210> 4 <211> 353 <212> PRT
<213> Artificial Sequence <220>
<223> Human short form/mouse species chimeric MCH1R
<400> 4 Met Asp Leu Glu Ala Ser Leu Leu Pro Thr Gly Pro Asn Ala Ser Asn Thr Ser Asp Gly Pro Asp Asn Leu Thr Ser Ala Gly Ser Pro Pro Arg Thr Gly Ser Ile Ser Tyr Ile Asn Ile Ile Met Pro Ser Val Phe Gly Thr Ile Cys Leu Leu Gly Ile Ile Gly Asn Ser Thr Val Ile Phe Ala Val Val Lys Lys Ser Lys Leu His Trp Cys Asn Asn Val Pro Asp Ile Phe Ile Ile Asn Leu Ser Val Val Asp Leu Leu Phe Leu Leu Gly Met Pro Phe Met Ile His Gln Leu Met Gly Asn Gly Val Trp His Phe Gly Glu Thr Met Cys Thr Leu Ile Thr Ala Met Asp Ala Asn Ser Gln Phe Thr Ser Thr Tyr Ile Leu Thr Ala Met Ala Ile Asp Arg Tyr Leu Ala Thr Val His Pro Ile Ser Ser Thr Lys Phe Arg Lys Pro Ser Met Ala Thr Leu Val Ile Cys Leu Leu Trp Ala Leu Ser Phe Ile Ser IIe Thr Pro Val Trp Leu Tyr Ala Arg Leu Ile Pro Phe Pro Gly Gly Ala Val Gly Cys Gly Ile Arg Leu Pro Asn Pro Asp Thr Asp Leu Tyr Trp Phe Thr Leu Tyr Gln Phe Phe Leu Ala Phe Ala Leu Pro Phe Val Val Ile Thr Ala Ala Tyr Val Lys Ile Leu Gln Arg Met Thr Ser Ser Val Ala Pro Ala Ser Gln Arg Ser Ile Arg Leu Arg Thr Lys Arg Val Thr Arg Thr Ala Ile Ala Ile Cys Leu Val Phe Phe Val Cys Trp Ala Pro Tyr Tyr Val Leu Gln Leu Thr Gln Leu Ser Ile Ser Arg Pro Thr Leu Thr Phe Val Tyr Leu Tyr Asn Ala Ala Tle Ser Leu Gly Tyr Ala Asn Ser Cys Leu Asn Pro Phe Val Tyr Ile Val Leu Cys Glu Thr Phe Arg Lys Arg Leu Val Leu Ser Val Lys Pro Ala Ala Gln Gly Gln Leu Arg Thr Val Ser Asn Ala Gln Thr Ala Asp Glu Glu Arg Thr Glu Ser Lys Gly Thr <210> 5 <211> 422 <212> PRT
<213> Artificial Sequence <220>

<223> Human long form/mouse species chimeric MCH1R
<400> 5 Met Ser Val Gly Ala Met Lys Lys Gly Val Gly Arg Ala Val Gly Leu Gly Gly Gly Ser Gly Cys Gln Ala Thr Glu Glu Asp Pro Leu Pro Asn Cys Gly Ala Cys Ala Pro Gly Gln Gly Gly Arg Arg Trp Arg Leu Pro Gln Pro Ala Trp Val Glu Gly Ser Ser Ala Arg Leu Trp Glu Gln Ala Thr Gly Thr Gly Trp Met Asp Leu Glu Ala Ser Leu Leu Pro Thr Gly Pro Asn Ala Ser Asn Thr Ser Asp Gly Pro Asp Asn Leu Thr Ser Ala Gly Ser Pro Pro Arg Thr Gly Ser Ile Ser Tyr Ile Asn Ile Ile Met Pro Ser Val Phe Gly Thr Ile Cys Leu Leu Gly Ile Ile Gly Asn Ser Thr Val Ile Phe Ala Val Val Lys Lys Ser Lys Leu His Trp Cys Asn Asn Val Pro Asp Ile Phe Ile Ile Asn Leu Ser Val Val Asp Leu Leu Phe Leu Leu Gly Met Pro Phe Met Ile His Gln Leu Met Gly Asn Gly Val Trp His Phe Gly Glu Thr Met Cys Thr Leu Ile Thr Ala Met Asp Ala Asn Ser Gln Phe Thr Ser Thr Tyr Ile Leu Thr Ala Met Ala Ile Asp Arg Tyr Leu Ala Thr Val His Pro Ile Ser Ser Thr Lys Phe Arg Lys Pro Ser Met Ala Thr Leu Val Ile Cys Leu Leu Trp Ala Leu Ser Phe Ile Ser Ile Thr Pro Val Trp Leu Tyr Ala Arg Leu Ile Pro Phe Pro Gly Gly Ala Val Gly Cys Gly Ile Arg Leu Pro Asn Pro Asp Thr Asp Leu Tyr Trp Phe Thr Leu Tyr Gln Phe Phe Leu Ala Phe Ala Leu Pro Phe Val Val Ile Thr Ala Ala Tyr Val Lys Ile Leu Gln Arg Met Thr Ser Ser Val Ala Pro Ala Ser Gln Arg Ser Ile Arg Leu Arg Thr Lys Arg Val Thr Arg Thr Ala Ile Ala Ile Cys Leu Val Phe Phe Val Cys Trp Ala Pro Tyr Tyr Val Leu Gln Leu Thr Gln Leu Ser Ile Ser Arg Pro Thr Leu Thr Phe Val Tyr Leu Tyr Asn Ala Ala Ile Ser Leu Gly Tyr Ala Asn Ser Cys Leu Asn Pro Phe Val Tyr Ile Val Leu Cys Glu Thr Phe Arg Lys Arg Leu Val Leu Ser Val Lys Pro Ala Ala Gln Gly Gln Leu Arg Thr Val Ser Asn Ala Gln Thr Ala Asp Glu Glu Arg Thr Glu Ser Lys Gly Thr <210> 6 <211> 238 <212> PRT
<213> Aequorea Victoria <400> 6 Met Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Phe Ser Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn Tyr Asn Ser His Asn Val Tyr Ile Met Ala Asp Lys Gln Lys Asn Gly Ile Lys Val Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly Ser Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Ala Leu Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly Ile Thr His G1y Met Asp Glu Leu Tyr Lys <210> 7 <211> 239 <212> PRT
<213> Artificial Sequence <220>
<223> GFP derivative <400> 7 Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly Glu Gly G1u Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Leu Thr Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn Tyr Asn Ser His Asn Val Tyr Ile Met Ala Asp Lys Gln Lys Asn Gly Ile Lys Val Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly Ser Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Ala Leu 195 200 . 205 Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu Tyr Lys <210> 8 <211> 239 <212> PRT
<213> Artificial Sequence <220>
<223> GFP derivative <400> 8 Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Leu Thr Tyr Gly Val Gln Cys Phe Ala Arg Tyr Pro Asp His Met Lys Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn Tyr Asn Ser His Lys Val Tyr Ile Thr Ala Asp Lys Gln Lys Asn Gly Ile Lys Val Asn Phe Lys Thr Arg His Asn Ile Glu Asp Gly Ser Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Ala Leu Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu Tyr Lys <210> 9 <211> 239 <212> PRT
<213> Artificial Sequence <220>
<223> GFP derivative <400> 9 Met.Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Phe Gly Tyr Gly Val Gln Cys Phe Ala Arg Tyr Pro Asp His Met Arg Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn Tyr Asn Ser His Asn Val Tyr Ile Met Ala Asp Lys Gln Lys Asn Gly Ile Lys Val Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly Ser Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Tyr Gln Ser Ala Leu Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu Tyr Lys <210> 10 <211> 239 <212> PRT
<213> Artificial Sequence <220>
<223> GFP derivative <400> 10 Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val Glu Leu Asp Gly Asp Val Asn Gly His Arg Phe Ser Val Ser Gly Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Leu Thr Trp Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly l15 120 125 Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn Tyr Ile Ser His Asn Val Tyr Ile Thr Ala Asp Lys Gln Lys Asn Gly Ile Lys Ala His Phe Lys Ile Arg His Asn Ile Glu Asp Gly Ser Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly _g_ Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Ala Leu Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu Tyr Lys <210> 11 <211> 604 <212> PRT
<213> Artificial Sequence <220>
<223> Mouse MCH1R-linker-EGFP
<400> 11 Met Asp Leu Gln Ala Ser Leu Leu Ser Thr Gly Pro Asn Ala Ser Asn Ile Ser Asp Gly Gln Asp Asn Phe Thr Leu Ala Gly Pro Pro Pro Arg Thr Arg Ser Val Ser Tyr Ile Asn Ile Ile Met Pro Ser Val Phe Gly Thr Ile Cys Leu Leu Gly Ile Val Gly Asn Ser Thr Val Ile Phe Ala Val Val Lys Lys Ser Lys Leu His Trp Cys Ser Asn Val Pro Asp Ile Phe Ile Ile Asn Leu Ser Val Val Asp Leu Leu Phe Leu Leu Gly Met Pro Phe Met Ile His Gln Leu Met Gly Asn Gly Val Trp His Phe Gly Glu Thr Met Cys Thr Leu Ile Thr Ala Met Asp Ala Asn Ser Gln Phe Thr Ser Thr Tyr Ile Leu Thr Ala Met Ala Ile Asp Arg Tyr Leu Ala Thr Val His Pro Ile Ser Ser Thr Lys Phe Arg Lys Pro Ser Met Ala Thr Leu Val Ile Cys Leu Leu Trp Ala Leu Ser Phe Ile Ser Ile Thr Pro Val Trp Leu Tyr Ala Arg Leu Ile Pro Phe Pro Gly Gly Ala Val Gly Cys Gly Ile Arg Leu Pro Asn Pro Asp Thr Asp Leu Tyr Trp Phe Thr Leu Tyr Gln Phe Phe Leu Ala Phe Ala Leu Pro Phe Val Val Ile Thr Ala Ala Tyr Val Lys Ile Leu Gln Arg Met Thr Ser Ser Val Ala Pro Ala Ser Gln Arg Ser Ile Arg Leu Arg Thr Lys Arg Val Thr Arg Thr Ala Ile Ala Ile Cys Leu Val Phe Phe Val Cys Trp Ala Pro Tyr Tyr Val Leu Gln Leu Thr Gln Leu Ser Ile Ser Arg Pro Thr Leu Thr Phe Val Tyr Leu Tyr Asn Ala Ala Ile Ser Leu Gly Tyr Ala Asn Ser Cys Leu Asn Pro Phe Val Tyr Ile Val Leu Cys Glu Thr Phe Arg Lys Arg Leu Val Leu Ser Val Lys Pro Ala Ala Gln Gly Gln Leu Arg Thr Val Ser Asn Ala Gln Thr Ala Asp Glu Glu Arg Thr Glu Ser Lys Gly Thr Val Asp Gly Thr Ala Gly Pro Gly Ser Ile Ala Thr Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Leu Thr Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn Tyr Asn Ser His Asn Val Tyr Ile Met Ala Asp Lys Gln Lys Asn Gly Ile Lys Val Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly Ser Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Ala Leu Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu Tyr Lys <210> 12 <211> 592 <212> PRT
<213> Artificial Sequence <220>
<223> Mouse MCH1R/EGFP
<400> 12 Met Asp Leu Gln Ala Ser Leu Leu Ser Thr Gly Pro Asn Ala Ser Asn Ile Ser Asp Gly Gln Asp Asn Phe Thr Leu Ala Gly Pro Pro Pro Arg Thr Arg Ser Val Ser Tyr Ile Asn Ile Ile Met Pro Ser Val Phe Gly Thr Ile Cys Leu Leu Gly Ile Val Gly Asn Ser Thr Val Ile Phe Ala Val Val Lys Lys Ser Lys Leu His Trp Cys Ser Asn Val Pro Asp Ile Phe Ile Ile Asn Leu Ser Val Val Asp Leu Leu Phe Leu Leu Gly Met Pro Phe Met Ile His Gln Leu Met Gly Asn Gly Val Trp His Phe Gly Glu Thr Met Cys Thr Leu Ile Thr Ala Met Asp Ala Asn Ser Gln Phe Thr Ser Thr Tyr Ile Leu Thr Ala Met Ala Ile Asp Arg Tyr Leu Ala Thr Val His Pro Ile Ser Ser Thr Lys Phe Arg Lys Pro Ser Met Ala Thr Leu Val Ile Cys Leu Leu Trp Ala Leu Ser Phe Ile Ser Ile Thr Pro Val Trp Leu Tyr Ala Arg Leu Ile Pro Phe Pro Gly Gly Ala Val Gly Cys Gly I1e Arg Leu Pro Asn Pro Asp Thr Asp Leu Tyr Trp Phe Thr Leu Tyr Gln Phe Phe Leu Ala Phe Ala Leu Pro Phe Val Val Ile Thr Ala Ala Tyr Val Lys Ile Leu Gln Arg Met Thr Ser Ser Val Ala Pro Ala Ser Gln Arg Ser Ile Arg Leu Arg Thr Lys Arg Val Thr Arg Thr Ala Ile Ala Ile Cys Leu Val Phe Phe Val Cys Trp Ala Pro Tyr Tyr Val Leu Gln Leu Thr Gln Leu Ser Ile Ser Arg Pro Thr Leu Thr Phe Val Tyr Leu Tyr Asn Ala Ala Ile Ser Leu Gly Tyr Ala Asn Ser Cys Leu Asn Pro Phe Val Tyr Ile Val Leu Cys Glu Thr Phe Arg Lys Arg Leu Val Leu Ser Val Lys Pro Ala Ala Gln Gly Gln Leu Arg Thr Val Ser Asn Ala Gln Thr Ala Asp Glu Glu Arg Thr Glu Ser Lys Gly Thr Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Leu Thr Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn Tyr Asn Ser His Asn Val Tyr Ile Met Ala Asp Lys Gln Lys Asn Gly Ile Lys Val Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly Ser Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro Va1 Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Ala Leu Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu Tyr Lys <210> 13 <211> 604 <212> PRT
<213> Artificial Sequence <220>
<223> MCH1R-linker-EGFP
<400> 13 Met Asp Leu Glu Ala Ser Leu Leu Pro Thr Gly Pro Asn Ala Ser Asn Thr Ser Asp Gly Pro Asp Asn Leu Thr Ser Ala Gly Ser Pro Pro Arg Thr Gly Ser Ile Ser Tyr Ile Asn Ile Ile Met Pro Ser Val Phe Gly Thr Ile Cys Leu Leu Gly Ile Ile Gly Asn Ser Thr Val Ile Phe Ala Val Val Lys Lys Ser Lys Leu His Trp Cys Asn Asn Val Pro Asp Ile Phe Ile Ile Asn Leu Ser Val Val Asp Leu Leu Phe Leu Leu Gly Met Pro Phe Met Ile His Gln Leu Met Gly Asn Gly Val Trp His Phe Gly Glu Thr Met Cys Thr Leu Ile Thr Ala Met Asp Ala Asn Ser Gln Phe Thr Ser Thr Tyr Ile Leu Thr Ala Met Ala Ile Asp Arg Tyr Leu Ala Thr Val His Pro Ile Ser Ser Thr Lys Phe Arg Lys Pro Ser Met Ala Thr Leu Val Ile Cys Leu Leu Trp Ala Leu Ser Phe Ile Ser Ile Thr Pro Val Trp Leu Tyr Ala Arg Leu Ile Pro Phe Pro Gly Gly Ala Val Gly Cys Gly Ile Arg Leu Pro Asn Pro Asp Thr Asp Leu Tyr Trp Phe Thr Leu Tyr Gln Phe Phe Leu Ala Phe Ala Leu Pro Phe Val Val Ile Thr Ala Ala Tyr Val Lys Ile Leu Gln Arg Met Thr Ser Ser Val Ala Pro Ala Ser Gln Arg Ser Ile Arg Leu Arg Thr Lys Arg Val Thr Arg Thr Ala Ile Ala Ile Cys Leu Val Phe Phe Val Cys Trp Ala Pro Tyr Tyr Val Leu Gln Leu Thr Gln Leu Ser Ile Ser Arg Pro Thr Leu Thr Phe Val Tyr Leu Tyr Asn Ala Ala Ile Ser Leu Gly Tyr Ala Asn Ser Cys Leu Asn Pro Phe Val Tyr Ile Val Leu Cys Glu Thr Phe Arg Lys Arg Leu Val Leu Ser Val Lys Pro Ala Ala Gln Gly Gln Leu Arg Thr Val Ser Asn Ala Gln Thr Ala Asp Glu Glu Arg Thr Glu Ser Lys Gly Thr Val Asp Gly Thr Ala Gly Pro Gly Ser Ile Ala Thr Met Val Ser Lys Gly Glu Glu Leu Phe Thr G1y Val Val Pro Ile Leu Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Leu Thr Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg Thr Tle Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val Lys Phe Glu Gly Asp Thr Leu Va1 Asn Arg Ile Glu Leu Lys Gly Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr-Asn Tyr Asn Ser His Asn Val Tyr Ile Met Ala Asp Lys Gln Lys Asn Gly Ile Lys Val Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly Ser Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Ala Leu Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu Tyr Lys <210> 14 <211> 673 <212> PRT
<213> Artificial Sequence <220>
<223> MCH1R-linker-EGFP
<400> 14 Met Ser Val Gly Ala Met Lys Lys Gly Val Gly Arg Ala Val Gly Leu Gly Gly Gly Ser Gly Cys Gln Ala Thr Glu Glu Asp Pro Leu Pro Asn Cys Gly Ala Cys Ala Pro Gly Gln Gly Gly Arg Arg Trp Arg Leu Pro Gln Pro Ala Trp Val Glu Gly Ser Ser Ala Arg Leu Trp Glu Gln Ala Thr Gly Thr Gly Trp Met Asp Leu Glu Ala Ser Leu Leu Pro Thr Gly Pro Asn Ala Ser Asn Thr Ser Asp Gly Pro Asp Asn Leu Thr Ser Ala Gly Ser Pro Pro Arg Thr Gly Ser Ile Ser Tyr Ile Asn Ile Ile Met Pro Ser Val Phe Gly Thr Ile Cys Leu Leu Gly Ile Ile Gly Asn Ser Thr Val Ile Phe Ala Val Val Lys Lys Ser Lys Leu His Trp Cys Asn Asn Val Pro Asp Ile Phe Ile Ile Asn Leu Ser Val Val Asp Leu Leu Phe Leu Leu Gly Met Pro Phe Met Ile His Gln Leu Met Gly Asn Gly Val Trp His Phe Gly Glu Thr Met Cys Thr Leu Ile Thr Ala Met Asp Ala Asn Ser Gln Phe Thr Ser Thr Tyr Ile Leu Thr Ala Met Ala Ile Asp Arg Tyr Leu Ala Thr Val His Pro Ile Ser Ser Thr Lys Phe Arg Lys Pro Ser Met Ala Thr Leu Val Ile Cys Leu Leu Trp Ala Leu Ser Phe Ile Ser Ile Thr Pro Val Trp Leu Tyr Ala Arg Leu Ile Pro Phe Pro Gly Gly Ala Val Gly Cys Gly Ile Arg Leu Pro Asn Pro Asp Thr Asp Leu Tyr Trp Phe Thr Leu Tyr Gln Phe Phe Leu Ala Phe Ala Leu Pro Phe Val Val Ile Thr Ala Ala Tyr Val Lys Ile Leu Gln Arg Met Thr Ser Ser Val Ala Pro Ala Ser Gln Arg Ser Ile Arg Leu Arg Thr Lys Arg Val Thr Arg Thr Ala Ile Ala Ile Cys Leu Val Phe Phe Val Cys Trp Ala Pro Tyr Tyr Val Leu Gln Leu Thr Gln Leu Ser Ile Ser Arg Pro Thr Leu Thr Phe Val Tyr Leu Tyr Asn Ala Ala Ile Ser Leu Gly Tyr Ala Asn Ser Cys Leu Asn Pro Phe Val Tyr Ile Val Leu Cys Glu Thr Phe Arg Lys Arg Leu Val Leu Ser Val Lys Pro Ala Ala Gln Gly Gln Leu Arg Thr Val Ser Asn Ala Gln Thr Ala Asp Glu Glu Arg Thr Glu Ser Lys Gly Thr Val Asp Gly Thr Ala Gly Pro Gly Ser Ile Ala Thr Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys 465 470 475 ' 480 Phe Ile Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Leu Thr Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile Asp Phe Lys Glu Asp G1y Asn Ile Leu Gly His Lys Leu Glu Tyr Asn Tyr Asn Ser His Asn Val Tyr Ile Met Ala Asp Lys Gln Lys Asn Gly Ile Lys Val Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly Ser Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Ala Leu Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu Tyr Lys <210> 15 <211> 1269 <212> DNA
<213> Human <400>

atgtcagtgggagccatgaagaagggagtggggagggcagttgggcttggaggcggcagc 60 ggctgccaggctacggaggaagacccccttcccaactgcggggcttgcgctccgggacaa 120 ggtggcaggcgctggaggctgccgcagcctgcgtgggtggaggggagctcagctcggttg 180 tgggagcaggcgaccggcactggctggatggacctggaagcctcgctgctgcccactggt 240 cccaacgccagcaacacctctgatggccccgataacctcacttcggcaggatcacctcct 300 cgcacggggagcatctcctacatcaacatcatcatgccttcggtgttcggcaccatctgc 360 ctcctgggcatcatcgggaactccacggtcatcttcgcggtcgtgaagaagtccaagctg 420 cactggtgcaacaacgtccccgacatcttcatcatcaacctctcggtagtagatctcctc480 tttctcctgggcatgcccttcatgatccaccagctcatgggcaatggggtgtggcacttt540 ggggagaccatgtgcaccctcatcacggccatggatgccaatagtcagttcaccagcacc600 tacatcctgaccgccatggccattgaccgctacctggccactgtccaccccatctcttcc660 acgaagttccggaagccctctgtggccaccctggtgatctgcctcctgtgggccctctcc720 ttcatcagcatcacccctgtgtggctgtatgccagactcatccccttcccaggaggtgca780 gtgggctgcggcatacgcctgcccaacccagacactgacctctactggttcaccctgtac840 cagtttttcctggcctttgccctgccttttgtggtcatcacagccgcatacgtgaggatc900 ctgcagcgcatgacgtcctcagtggcccccgcctcccagcgcagcatccggctgcggaca960 aagagggtgacccgcacagccatcgccatctgtctggtcttctttgtgtgctgggcaccc1020 tactatgtgctacagctgacccagttgtccatcagccgcccgaccctcacctttgtctac1080 ttatacaatgcggccatcagcttgggctatgccaacagctgcctcaacccctttgtgtac1140 atcgtgctctgtgagacgttccgcaaacgcttggtcctgtcggtgaagcctgcagcccag1200 gggcagcttcgcgctgtcagcaacgctcagacggctgacgaggagaggacagaaagcaaa1260 ggcacctga <210>

<211>

<212>
DNA

<213>
Human <400>

atggacctggaagcctcgctgctgcccactggtcccaatgccagcaacacctctgatggc60 cccgataacctcacttcggcaggatcacctcctcgcacggggagcatctcctacatcaac120 atcatcatgccttcggtgttcggcaccatctgcctcctgggcatcatcgggaactccacg180 gtcatcttcgcggtcgtgaagaagtccaagctgcactggtgcaacaacgtccccgacatc240 ttcatcatcaacctctcggtagtagatctcctctttctcctgggcatgcccttcatgatc300 caccagctcatgggcaatggggtgtggcactttggggagaccatgtgcaccctcatcacg360 gccatggatgccaatagtcagttcaccagcacctacatcctgaccgccatggccattgac420 cgctacctggccactgtccaccccatctcttccacgaagttccggaagccctctgtggcc480 accctggtgatctgcctcctgtgggccctctccttcatcagcatcacccctgtgtggctg540 tatgccagactcatccccttcccaggaggtgcagtgggctgcggcatacgcctgcccaac600 ccagacactgacctctactggttcaccctgtaccagtttttcctggcctttgccctgcct660 tttgtggtcatcacagccgcatacgtgaggatcctgcagcgcatgacgtcctcagtggcc720 cccgcctcccagcgcagcatccggctgcggacaaagagggtgacccgcacagccatcgcc780 atctgtctggtcttctttgtgtgctgggcaccctactatgtgctacagctgacccagttg840 tccatcagccgcccgaccctcacctttgtctacttatacaatgcggccatcagcttgggc900 tatgccaacagctgcctcaacccctttgtgtacatcgtgctctgtgagacgttccgcaaa960 cgcttggtcctgtcggtgaagcctgcagcccaggggcagcttcgcgctgtcagcaacgct1020 cagacggctgacgaggagaggacagaaagcaaaggcacctga 1062 <210>

<211>

<212>
DNA

<213>
Mouse <220>

<221> feature misc_ <222>
(1)...(2080) <223>
n = A,T,C
or G

<400>

ggcggtagaggaagacccttttctggactgcggggctcaagctccggacaaggcggtgga 60 gggcgctggaggctgccgcagcctgcgtgggtggacgggcgctccactccagggagcagg 120 cgacctgcaccggctgcatggatctgcaagcctcgttgctgtccactggccccaatgcca 180 gcaacatctccgatggccaggataatttcacattggcggggccacctcctcgcacaagga 240 gtgtctcctacatcaacatcatcatgccttcagtgtttggtaccatctgtctcctgggca 300 ttgtgggaaactccacagtcatttttgccgtggtgaagaaatccaagctgcactggtgca 360 gcaacgtccctgacatcttcatcatcaacctctctgtggtggatctgcttttcctgctgg 420 gcatgcctttcatgatccaccagctcatgggtaatggtgtctggcactttggggaaacca 480 tgtgcaccctcatcacagccatggacgccaacagtcagttcaccagcacctacatcctga 540 ctgctatggccattgaccgctacttggccaccgtccatcccatctcctccaccaagttcc 600 ggaagccctccatggccaccctggtgatctgcctcctgtgggctctctcgttcattagca660 tcactcctgtgtggctctatgccaggcttatccccttcccagggggtgctgtgggctgtg720 gcatccgcctaccaaacccagatactgatctttactggttcactctgtatcagtttttcc780 tggccttcgcccttccgtttgtggtcatcactgctgcgtacgtgaaaatactacagcgca840 tgacgtcttcggtggccccagcctctcaacgcagcatccggcttcggacaaagagggtga900 cccgcacagccattgccatctgtctggtcttctttgtgtgctgggcgccctactacgtgc960 tgcagctgacccagttgtccatcagccgcccgaccctcacattcgtctacctgtacaatg1020 cggccatcagcttgggctatgccaacagctgcctcaatccctttgtgtacatagtactct1080 gtgagacctttcgaaaacgcttggtgctgtcggtgaagcccgcggcccaggggcagcttc1140 gcacggtcagcaatgctcagacagctgacgaggagaggacagaaagcaaaggcacctgac1200 aatcccccccggtcacctccaagtcaggtcaccgcatcaaaccatggggagagatactga1260 gataaacccggggctaccctgggaggatgcagaagctggaggctgggggcttgtagcaaa1320 ccacattccacggggcccacaaattgctagggaggcttgcagcctggtttgggggggaag1380 cctcagactgcagggatccccttgacagaatagaagcggagcaagaaggaaagggtggtt1440 tgactggttctcggggtctgtatctgttggctcgcatatatctttctctcaagggaagaa1500 ggcggaggtgcctagctgggttcctttaaaactaggcagggctaggatctgagcagctag1560 ggctctactgtgagactgggcaagccgagcgttccctcccatctctcattggtgttgata1620 gaaggcagtctttctcccaagctggtggatctcctgaagcacgctgcctgggctccagca1680 tcctgtgcggatttcacgttctctttaggggatgcatgttgacactggggtgtgggctct1740 gagcccacaggagtttaaaaaaccaaaagagctcagagtgtcgagagagacccaatcacc1800 gagaatgacaaggcaacctggggtggatgtggatcttgaaactaataaaaaggggttttc1860 acagtgacagcgacattctcttcatagggcacagctgtcagtctatggctgatccagagc1920 gagcatccatgaattctgcatgtgcaggggtcactctaatacctgatatgttggcatcat1980 ctttgtgcttgagccttccnctcccaaatgggaatgaaataaaggcaaattcccnccccc2040 cccaaaaaaggggnaaaaaaaaaaaaaaaaaaaaaaaaaa 2080 <210>

<211>

<212>
DNA

<213>
Mouse <220>

<221> feature misc _ <222>
(1).
.(3357) <223>
n = A,T,C
or G

<400>

ggcggtagaggaagacccttttctggactgcggggctcaagctccggacaaggcggtgga60 gggcgctggaggctgccgcagcctgcgtgggtggacgggcgctccactccagggagcagg120 cgacctgcaccggctgcatggatctgcaagCCtCgttgCtgtCCaCtggCCCCaatgCCa180 gcaacatctccgatggccaggataatttcacattggcgggtgagtcgagttggagtcctc240 cctcctccgggatgggtgtggaaaatgggaaggtttcacctcccaagccaaactgcctgg300 gaaactttatcttacagttcttggtgataagatctgcagtcggctttgcctgaagaggaa360 gaggagaggaggggacaccagctaggacagaaggggcagggaggaatagagatggggcag420 aggcacatttagaaacaacaagggttggtgacaagacgtgaggcaggcttgaggggaaag480 cttgctgatgagtcccaaatatgctttgcaggggggggggggggggaatcaaggctggag540 aagcaagcaagcaagacagcaagacagcgggcgggtagtatgtgggagccagcagaagcg600 ctttgattcaccgctatcctgggctcaatcctctggcctcgcactggggaaatggggtct660 gagtggtccttgctgtcttctggcaaaggctgctgggagcaaaagacttcacagggcgtg720 agaggattaacttttctggtgaattaagcttcttgacatttgcagaacgtcaatgcctta780 aaattctagctctgaaggagaagggaatgaaggggaaagagggaaggttggtgtggagaa840 attcccaagcttctggggtgtaacacagctccagtccctaccctattgggaaagcccaga900 ctcaggagacatggtccaaggaaatccctgacagaaaaccgggagagggcagggctgtgg960 agcctgaaacacaccccacacccatggtgacagtcacttctcacatatgcctaggaacct1020 atctgaaacctttggccatctctctctgaaaagatgaggctgcaaatacacacacacaca1080 cacacacacacacacacacacacacacacacacacacacacacacacacaaatgtccttc1140 aagcctttttgacaaggttttctggtggatcccggggatatgaagttgttctcagcagat1200 atctgggagtcttgactcctggccctctgagtaaatggatgaagcgaagaagaatggggt1260 cctctgagtaacaggtggatctagaaaatcctataggagtcaccagggcacggtggagga1320 gggtaaggtacagaactaacaatagcccgagaaggggaaacagcaggagatgattccaga1380 gacgtagtgaccccaagctgcaagggaaagcatgaggggccagcaggaaggccgacatgg1440 caggttgtcagcttctagatcggaaggcgggtcacacttgctctttctatcctcagggcc1500 acctcctcgcacaaggagtgtctcctacatcaacatcatcatgccttcagtgtttggtac1560 catctgtctcctgggcattgtgggaaactccacagtcatttttgccgtggtgaagaaatc1620 caagctgcactggtgcagcaacgtccctgacatcttcatcatcaacctctctgtggtgga1680 tctgcttttcctgctgggcatg'cctttcatgatccaccagctcatgggtaatggtgtctg1740 gcactttggggaaaccatgtgcaccctcatcacagccatggacgccaacagtcagttcac1800 cagcacctacatcctgactgctatggccattgaccgctacttggccaccgtccatcccat1860 ctcctccaccaagttccggaagccctccatggccaccctggtgatctgcctcctgtgggc1920 tctctcgttcattagcatcactcctgtgtggctctatgccaggcttatccccttcccagg1980 gggtgctgtgggctgtggcatccgcctaccaaacccagatactgatctttactggttcac2040 tctgtatcagtttttcctggccttcgcccttccgtttgtggtcatcactgctgcgtacgt2100 gaaaatactacagcgcatgacgtcttcggtggccccagcctctcaacgcagcatccggct2160 tcggacaaagagggtgacccgcacagccattgccatctgtctggtcttctttgtgtgctg2220 ggcgccctactacgtgctgcagctgacccagttgtccatcagccgcccgaccctcacatt2280 cgtctacctgtacaatgcggccatcagcttgggctatgccaacagctgcctcaatccctt2340 tgtgtacatagtactctgtgagacctttcgaaaacgcttggtgctgtcggtgaagcccgc2400 ggcccaggggcagcttcgcacggtcagcaatgctcagacagctgacgaggagaggacaga2460 aagcaaaggcacctgacaatcccccccggtcacctccaagtcaggtcaccgcatcaaacc2520 atggggagagatactgagataaacccggggctaccctgggaggatgcagaagctggaggc2580 tgggggcttgtagcaaaccacattccacggggcccacaaattgctagggaggcttgcagc2640 ctggtttgggggggaagcctcagactgcagggatccccttgacagaatagaagcggagca2700 agaaggaaagggtggtttgactggttctcggggtctgtatctgttggctcgcatatatct2760 ttctctcaagggaagaaggcggaggtgcctagctgggttcctttaaaactaggcagggct2820 aggatctgagcagctagggctctactgtgagactgggcaagccgagcgttCCCtCCCatC2880 tctcattggtgttgatagaaggcagtctttctcccaagctggtggatctcctgaagcacg2940 ctgcctgggctccagcatcctgtgcggatttcacgttctctttaggggatgcatgttgac3000 actggggtgtgggctctgagcccacaggagtttaaaaaaccaaaagagctcagagtgtcg3060 agagagacccaatcaccgagaatgacaaggcaacctggggtggatgtggatcttgaaact3120 aataaaaaggggttttcacagtgacagcgacattctcttcatagggcacagctgtcagtc3180 tatggctgatccagagcgagcatccatgaattctgcatgtgcaggggtcactctaatacc3240 tgatatgttggcatcatctttgtgcttgagccttccnctcccaaatgggaatgaaataaa3300 ggcaaattcccnccccccccaaaaaaggggnaaaaaaaaaaaaaaaaaaaaaaaaaa 3357 <210>

<211>

<212>
DNA

<213> icial Artif Sequence <220>

<223> short Human form/mouse species chimeric <400>

atggacctggaagcctcgctgctgcccactggtcccaatgccagcaacacctctgatggc60 cccgataacctcacttcggcaggatcacctcctcgcacggggagcatctcctacatcaac120 atcatcatgccttcggtgttcggcaccatctgcctcctgggcatcatcgggaactccacg180 gtcatcttcgcggtcgtgaagaagtccaagctgcactggtgcaacaacgtccccgacatc240 ttcatcatcaacctctcggtagtagatctcctctttctcctgggcatgcccttcatgatc300 caccagctcatgggcaatggggtgtggcactttggggagaccatgtgcaccctcatcacg360 gccatggatgccaatagtcagttcaccagcacctacatcctgaccgccatggccattgac420 cgctacctggccactgtccaccccatctcttccacgaagttccggaagccctccatggcc480 accctggtgatctgcctcctgtgggctctctcgttcattagcatcactcctgtgtggctc540 tatgccaggcttatccccttcccagggggtgctgtgggctgtggcatccgcctaccaaac600 ccagatactgatctttactggttcactctgtatcagtttttcctggccttcgcccttccg660 tttgtggtcatcactgctgcgtacgtgaaaatactacagcgcatgacgtcttcggtggcc720 ccagcctctcaacgcagcatccggcttcggacaaagagggtgacccgcacagccattgcc780 atctgtctggtcttctttgtgtgctgggcgccctactacgtgctgcagctgacccagttg840 tccatcagccgcccgaccctcacattcgtctacctgtacaatgcggccatcagcttgggc900 tatgccaacagctgcctcaatccctttgtgtacatagtactctgtgagacctttcgaaaa960 cgcttggtgctgtcggtgaagcccgcggcccaggggcagcttcgcacggtcagcaatgct1020 cagacagctgacgaggagaggacagaaagcaaaggcacctga 1062 <210>

<211>

<212>
DNA

<213>
Artificial Sequence <220>

<223> MCH1R
Human long form/mouse species chimeric <400>

atgtcagtgggagccatgaagaagggagtggggagggcagttgggcttggaggcggcagc60 ggctgccaggctacggaggaagacccccttcccaactgcggggcttgcgctccgggacaa120 ggtggcaggcgctggaggctgccgcagcctgcgtgggtggaggggagctcagctcggttg180 tgggagcaggcgaccggcactggctggatggacctggaagcctcgctgctgcccactggt240 cccaacgccagcaacacctctgatggccccgataacctcacttcggcaggatcacctcct300 cgcacggggagcatctcctacatcaacatcatcatgccttcggtgttcggcaccatctgc360 ctcctgggcatcatcgggaactccacggtcatcttcgcggtcgtgaagaagtccaagctg420 cactggtgcaacaacgtccccgacatcttcatcatcaacctctcggtagtagatctcctc480 tttctcctgggcatgcccttcatgatccaccagctcatgggcaatggggtgtggcacttt540 ggggagaccatgtgcaccctcatcacggccatggatgccaatagtcagttcaccagcacc600 tacatcctgaccgccatggccattgaccgctacctggccactgtccaccccatctcttcc660 acgaagttccggaagccctccatggccaccctggtgatctgcctcctgtgggctctctcg720 ttcattagcatcactcctgtgtggctctatgccaggcttatccccttcccagggggtgct780 gtgggctgtggcatccgcctaccaaacccagatactgatctttactggttcactctgtat840 cagtttttcctggccttcgcccttccgtttgtggtcatcactgctgcgtacgtgaaaata900 ctacagcgcatgacgtcttcggtggccccagcctctcaacgcagcatccggcttcggaca960 aagagggtgacccgcacagccattgccatctgtctggtcttctttgtgtgctgggcgccc1020 tactacgtgctgcagctgacccagttgtccatcagccgcccgaccctcacattcgtctac1080 ctgtacaatgcggccatcagcttgggctatgccaacagctgcctcaatccctttgtgtac1140 atagtactctgtgagacctttcgaaaacgcttggtgctgtcggtgaagcccgcggcccag1200 gggcagcttcgcacggtcagcaatgctcagacagctgacgaggagaggacagaaagcaaa1260 ggcacctga 1269 <210>

<211>

<212>
DNA

<213>
Aequorea Victoria <400>

tacacacgaataaaagataacaaagatgagtaaaggagaagaacttttcactggagttgt60 cccaattcttgttgaattagatggtgatgttaatgggcacaaattttctgtcagtggaga120 gggtgaaggtgatgcaacatacggaaaacttacccttaaatttatttgcactactggaaa180 actacctgttccatggccaacacttgtcactactttctcttatggtgttcaatgcttttc240 aagatacccagatcatatgaaacagcatgactttttcaagagtgccatgcccgaaggtta300 tgtacaggaaagaactatatttttcaaagatgacgggaactacaagacacgtgctgaagt360 caagtttgaaggtgatacccttgttaatagaatcgagttaaaaggtattgattttaaaga420 agatggaaacattcttggacacaaattggaatacaactataactcacacaatgtatacat480 catggcagacaaacaaaagaatggaatcaaagttaacttcaaaattagacacaacattga540 agatggaagcgttcaactagcagaccattatcaacaaaatactccaattggcgatggccc600 tgtccttttaccagacaaccattacctgtccacacaatctgccctttcgaaagatcccaa660 cgaaaagagagaccacatggtccttcttgagtttgtaacagctgctgggattacacatgg720 catggatgaactatacaaataaatgtccagacttccaattgacactaaagtgtccgaaca780 attactaaaatctcagggttcctggttaaattcaggctgagatattatttatatatttat840 agattcattaaaattgtatgaataatttattgatgttattgatagaggttattttcttat900 taaacaggctacttggagtgtattcttaattctatattaattacaatttgatttgacttg960 ctcaaa 966 <210> 22 <211> 765 <212> DNA
<213> Artificial Sequence <220>
<223> GFP derivative <400> 22 gtcgacggtaccgcgggcccgggatccatcgccaccatggtgagcaagggcgaggagctg 60 ttcaccggggtggtgcccatcctggtcgagctggacggcgacgtaaacggccacaagttc 120 agcgtgtccggcgagggcgagggcgatgccacctacggcaagctgaccctgaagttcatc 180 tgcaccaccggcaagctgcccgtgccctggcccaccctcgtgaccaccctgacctacggc 240 gtgcagtgcttcagccgctaccccgaccacatgaagcagcacgacttcttcaagtccgcc 300 atgcccgaaggctacgtccaggagcgcaccatcttcttcaaggacgacggcaactacaag 360 acccgcgccgaggtgaagttcgagggcgacaccctggtgaaccgcatcgagctgaagggc 420 atcgacttcaaggaggacggcaacatcctggggcacaagctggagtacaactacaacagc 480 cacaacgtctatatcatggccgacaagcagaagaacggcatcaaggtgaacttcaagatc 540 cgccacaacatcgaggacggcagcgtgcagctcgccgaccactaccagcagaacaccccc 600 atcggcgacggccccgtgctgctgcccgacaaccactacctgagcacccagtccgccctg 660 agcaaagaccccaacgagaagcgcgatcacatggtcctgctggagttcgtgaccgccgcc 720 gggatcactctcggcatggacgagctgtacaagtaaagcggccgc 765 <210>

<211>

<212>
DNA

<213> ficial Arti Sequence <220>

<223> derivative GFP

<400>

atggtgagcaagggcgaggagctgttcaccggggtggtgcccatcctggtcgagctggac 60 ggcgacgtaaacggccacaagttcagcgtgtccggcgagggcgagggcgatgccacctac 120 ggcaagctgaccctgaagttcatctgcaccaccggcaagctgcccgtgccctggcccacc 180 ctcgtgaccaccttgacctacggcgtgcagtgcttcgcccgctaccccgaccacatgaag 240 cagcacgacttcttcaagtccgccatgcccgaaggctacgtccaggagcgcaccatcttc 300 ttcaaggacgacggcaactacaagacccgcgccgaggtgaagttcgagggcgacaccctg 360 gtgaaccgcatcgagctgaagggcatcgacttcaaggaggacggcaacatcctggggcac 420 aagctggagtacaactacaacagccacaaggtctatatcaccgccgacaagcagaagaac 480 ggcatcaaggtgaacttcaagacccgccacaacatcgaggacggcagcgtgcagctcgcc 540 gaccactaccagcagaacacccccatcggcgacggccccgtgctgctgcccgacaaccac 600 tacctgagcacccagtccgccctgagcaaagaccccaacgagaagcgcgatcacatggtc 660 ctgctggagttcgtgaccgccgccgggatcactctcggcatggacgagctgtacaagtaa 720 <210>

<211>

<212>
DNA

<213> ficial Arti Sequence <220>

<223> derivative GFP

<400>

atggtgagcaagggcgaggagctgttcaccggggtggtgcccatcctggtcgagctggac60 ggcgacgtaaacggccacaagttcagcgtgtccggcgagggcgagggcgatgccacctac120 ggcaagctgaccctgaagttcatctgcaccaccggcaagctgcccgtgccctggcccacc180 ctcgtgaccaccttcggctacggcgtgcagtgcttcgcccgctaccccgaccacatgcgc240 cagcacgacttcttcaagtccgccatgcccgaaggctacgtccaggagcgcaccatcttc300 ttcaaggacgacggcaactacaagacccgcgccgaggtgaagttcgagggcgacaccctg360 gtgaaccgcatcgagctgaagggcatcgacttcaaggaggacggcaacatcctggggcac420 aagctggagtacaactacaacagccacaacgtctatatcatggccgacaagcagaagaac480 ggcatcaaggtgaacttcaagatccgccacaacatcgaggacggcagcgtgcagctcgcc540 gaccactaccagcagaacacccccatcggcgacggccccgtgctgctgcccgacaaccac600 tacctgagctaccagtccgccctgagcaaagaccccaacgagaagcgcgatcacatggtc660 ctgctggagttcgtgaccgccgccgggatcactctcggcatggacgagctgtacaagtaa720 <210>

<211>

<212>
DNA

<213> Artificial Sequence <220>
<223> GFP derivative <400> 25 atggtgagcaagggcgaggagctgttcaccggggtggtgcccatcctggtcgagctggac60 ggcgacgtaaacggccacaggttcagcgtgtccggcgagggcgagggcgatgccacctac120 ggcaagctgaccctgaagttcatctgcaccaccggcaagctgcccgtgccctggcccacc180 ctcgtgaccaccctgacctggggcgtgcagtgcttcagccgctaccccgaccacatgaag240 cagcacgacttcttcaagtccgccatgcccgaaggctacgtccaggagcgtaccatcttc300 ttcaaggacgacggcaactacaagacccgcgccgaggtgaagttcgagggcgacaccctg360 gtgaaccgcatcgagctgaagggcatcgacttcaaggaggacggcaacatcctggggcac420 aagctggagtacaactacatcagccacaacgtctatatcaccgccgacaagcagaagaac480 ggcatcaaggcccacttcaagatccgccacaacatcgaggacggcagcgtgcagctcgcc540 gaccactaccagcagaacacccccatcggcgacggccccgtgctgctgcccgacaaccac600 tacctgagcacccagtccgccctgagcaaagaccccaacgagaagcgcgatcacatggtc660 ctgctggagttcgtgaccgccgccgggatcactctcggcatggacgagctgtacaagtaa720 <210>

<211>

<212>
DNA

<213>
Artificial Sequence <220>

<223> MCH1R-linker-EGFP
Mouse <400> 26 atggatctgcaagcctcgttgctgtccactggccccaatgccagcaacatctccgatggc 60 caggataatttcacattggcgggtgagtcgagttggagtcctccctcctccgggatgggt 120 gtggaaaatgggaaggtttcacctcccaagccaaactgcctgggaaactttatcttacag 180 ttcttggtgataagatctgcagtcggctttgcctgaagaggaagaggagaggaggggaca 240 ccagctaggacagaaggggcagggaggaatagagatggggcagaggcacatttagaaaca 300 acaagggttggtgacaagacgtgaggcaggcttgaggggaaagcttgctgatgagtccca 360 aatatgctttgcaggggggggggggggggaatcaaggctggagaagcaagcaagcaagac 420 agcaagacagcgggcgggtagtatgtgggagccagcagaagcgctttgattcaccgctat 480 cctgggctcaatcctctggcctcgcactggggaaatggggtctgagtggtccttgctgtc 540 ttctggcaaaggctgctgggagcaaaagacttcacagggcgtgagaggattaacttttct 600 ggtgaattaagcttcttgacatttgcagaacgtcaatgccttaaaattctagctctgaag 660 gagaagggaatgaaggggaaagagggaaggttggtgtggagaaattcccaagcttctggg 720 gtgtaacacagctccagtccctaccctattgggaaagcccagactcaggagacatggtcc 780 aaggaaatccctgacagaaaaccgggagagggcagggctgtggagcctgaaacacacccc 840 acacccatggtgacagtcacttctcacatatgcctaggaacctatctgaaacctttggcc 900 atctctctctgaaaagatgaggctgcaaatacacacacacacacacacacacacacacac 960 acacacacacacacacacacacacacacacacaaatgtccttcaagcctttttgacaagg 1020 ttttctggtggatcccggggatatgaagttgttctcagcagatatctgggagtcttgact 1080 cctggccctctgagtaaatggatgaagcgaagaagaatggggtcctctgagtaacaggtg 1140 gatctagaaaatcctataggagtcaccagggcacggtggaggagggtaaggtacagaact 1200 aacaatagcccgagaaggggaaacagcaggagatgattccagagacgtagtgaccccaag 1260 ctgcaagggaaagcatgaggggccagcaggaaggccgacatggcaggttgtcagcttcta 1320 gatcggaaggcgggtcacacttgctctttctatcctcagggccacctcctcgcacaagga 1380 gtgtctcctacatcaacatcatcatgccttcagtgtttggtaccatctgtctcctgggca 1440 ttgtgggaaactccacagtcatttttgccgtggtgaagaaatccaagctgcactggtgca 1500 gcaacgtccctgacatcttcatcatcaacctctctgtggtggatctgcttttcctgctgg 1560 gcatgccttcatgatccact gtaatggtgtctggcactttggggaaacca 1620 cagctcatgg tgtgcaccctcatcacagccatggacgccaacagtcagttcaccagcacctacatcctga 1680 ctgctatggccattgaccgctacttggccaccgtccatcccatctcctccaccaagttcc 1740 ggaagccctccatggccaccctggtgatctgcctcctgtgggctctctcgttcattagca 1800 tcactcctgtgtggctctatgccaggcttatccccttcccagggggtgctgtgggctgtg 1860 gcatccgcctaccaaacccagatactgatctttactggttcactctgtatcagtttttcc 1920 tggccttcgcccttccgtttgtggtcatcactgctgcgtacgtgaaaatactacagcgca 1980 tgacgtcttcggtggccccagcctctcaacgcagcatccggcttcggacaaagagggtga 2040 cccgcacagccattgccatctgtctggtcttctttgtgtgctgggcgccctactacgtgc2100 tgcagctgacccagttgtccatcagccgcccgaccctcacattcgtctacctgtacaatg2160 cggccatcagcttgggctatgccaacagctgcctcaatccctttgtgtacatagtactct2220 gtgagacctttcgaaaacgcttggtgctgtcggtgaagcccgcggcccaggggcagcttc2280 gcacggtcagcaatgctcagacagctgacgaggagaggacagaaagcaaaggcaccgtcg2340 acggtaccgcgggcccgggatccatcgccaccatggtgagcaagggcgaggagctgttca2400 ccggggtggtgcccatcctggtcgagctggacggcgacgtaaacggccacaagttcagcg2460 tgtccggcgagggcgagggcgatgccacctacggcaagctgaccctgaagttcatctgca2520 ccaccggcaagctgcccgtgccctggcccaccctcgtgaccaccctgacctacggcgtgc2580 agtgcttcagccgctaccccgaccacatgaagcagcacgacttcttcaagtccgccatgc2640 ccgaaggctacgtccaggagcgcaccatcttcttcaaggacgacggcaactacaagaccc2700 gcgccgaggtgaagttcgagggcgacaccctggtgaaccgcatcgagctgaagggcatcg2760 acttcaaggaggacggcaacatcctggggcacaagctggagtacaactacaacagccaca2820 acgtctatatcatggccgacaagcagaagaacggcatcaaggtgaacttcaagatccgcc2880 acaacatcgaggacggcagcgtgcagctcgccgaccactaccagcagaacacccccatcg2940 gcgacggccccgtgctgctgcccgacaaccactacctgagcacccagtccgccctgagca3000 aagaccccaacgagaagcgcgatcacatggtcctgctggagttcgtgaccgccgccggga3060 tcactctcggcatggacgagctgtacaagtas 3092 <210> 27 <211> 3056 <212> DNA
<213> Artificial Sequence <220>
<223> Mouse MCH1R/EGFP direct fusion <400>

atggatctgcaagcctcgttgctgtccactggccccaatgccagcaacatctccgatggc60 caggataatttcacattggcgggtgagtcgagttggagtcctccctcctccgggatgggt120 gtggaaaatgggaaggtttcacctcccaagccaaactgcctgggaaactttatcttacag180 ttcttggtgataagatctgcagtcggctttgcctgaagaggaagaggagaggaggggaca240 ccagctaggacagaaggggcagggaggaatagagatggggcagaggcacatttagaaaca300 acaagggttggtgacaagacgtgaggcaggcttgaggggaaagcttgctgatgagtccca360 aatatgctttgcaggggggggggggggggaatcaaggctggagaagcaagcaagcaagac420 agcaagacagcgggcgggtagtatgtgggagccagcagaagcgctttgattcaccgctat480 cctgggctcaatcctctggcctcgcactggggaaatggggtctgagtggtccttgctgtc540 ttctggcaaaggctgctgggagcaaaagacttcacagggcgtgagaggattaacttttct600 ggtgaattaagcttcttgacatttgcagaacgtcaatgccttaaaattctagctctgaag660 gagaagggaatgaaggggaaagagggaaggttggtgtggagaaattcccaagcttctggg720 gtgtaacacagctccagtccctaccctattgggaaagcccagactcaggagacatggtcc780 aaggaaatccctgacagaaaaccgggagagggcagggctgtggagcctgaaacacacccc840 acacccatggtgacagtcacttctcacatatgcctaggaacctatctgaaacctttggcc900 atctctctctgaaaagatgaggctgcaaatacacacacacacacacacacacacacacac960 acacacacacacacacacacacacacacacacaaatgtccttcaagcctttttgacaagg1020 ttttctggtggatcccggggatatgaagttgttctcagcagatatctgggagtcttgact1080 cctggccctctgagtaaatggatgaagcgaagaagaatggggtcctctgagtaacaggtg1140 gatctagaaaatcctataggagtcaccagggcacggtggaggagggtaaggtacagaact1200 aacaatagcccgagaaggggaaacagcaggagatgattccagagacgtagtgaccccaag1260 ctgcaagggaaagcatgaggggccagcaggaaggccgacatggcaggttgtcagcttcta1320 gatcggaaggcgggtcacacttgctctttctatcctcagggccacctcctcgcacaagga1380 gtgtctcctacatcaacatcatcatgccttcagtgtttggtaccatctgtctcctgggca1440 ttgtgggaaactccacagtcatttttgccgtggtgaagaaatccaagctgcactggtgca1500 gcaacgtccctgacatcttcatcatcaacctctctgtggtggatctgcttttcctgctgg1560 gcatgcctttcatgatccaccagctcatgggtaatggtgtctggcactttggggaaacca1620 tgtgcaccctcatcacagccatggacgccaacagtcagttcaccagcacctacatcctga1680 ctgctatggccattgaccgctacttggccaccgtccatcccatctcctccaccaagttcc1740 ggaagccctccatggccaccctggtgatctgcctcctgtgggctctctcgttcattagca1800 tcactcctgtgtggctctatgccaggcttatccccttcccagggggtgctgtgggctgtg1860 gcatccgcctaccaaacccagatactgatctttactggttcactctgtatcagtttttcc1920 tggccttcgcccttccgtttgtggtcatcactgctgcgtacgtgaaaatactacagcgca1980 tgacgtcttcggtggccccagcctctcaacgcagcatccggcttcggacaaagagggtga2040 cccgcacagccattgccatctgtctggtcttctttgtgtgctgggcgccctactacgtgc2100 tgcagctgacccagttgtccatcagccgcccgaccctcacattcgtctacctgtacaatg2160 cggccatcagcttgggctatgccaacagctgcctcaatccctttgtgtacatagtactct2220 gtgagacctttcgaaaacgcttggtgctgtcggtgaagcccgcggcccaggggcagcttc2280 gcacggtcagcaatgctcagacagctgacgaggagaggacagaaagcaaaggcaccatgg2340 tgagcaagggcgaggagctgttcaccggggtggtgcccatcctggtcgagctggacggcg2400 acgtaaacggccacaagttcagcgtgtccggcgagggcgagggcgatgccacctacggca2460 agctgaccctgaagttcatctgcaccaccggcaagctgcccgtgccctggcccaccctcg2520 tgaccaccctgacctacggcgtgcagtgcttcagccgctaccccgaccacatgaagcagc2580 acgacttcttcaagtccgccatgcccgaaggctacgtccaggagcgcaccatcttcttca2640 aggacgacggcaactacaagacccgcgccgaggtgaagttcgagggcgacaccctggtga2700 accgcatcgagctgaagggcatcgacttcaaggaggacggcaacatcctggggcacaagc2760 tggagtacaactacaacagccacaacgtctatatcatggccgacaagcagaagaacggca2820 tcaaggtgaacttcaagatccgccacaacatcgaggacggcagcgtgcagctcgccgacc2880 actaccagcagaacacccccatcggcgacggccccgtgctgctgcccgacaaccactacc2940 tgagcacccagtccgccctgagcaaagaccccaacgagaagcgcgatcacatggtcctgc3000 tggagttcgtgaccgccgccgggatcactctcggcatggacgagctgtacaagtaa 3056 <210> 28 <211> 1815 <212> DNA
<213> Artificial Sequence <220>
<223> Human short form/mouse species chimeric MCH1R-linker-EGFP
<400> 28 atggacctggaagcctcgctgctgcccactggtcccaatgccagcaacacctctgatggc60 cccgataacctcacttcggcaggatcacctcctcgcacggggagcatctcctacatcaac120 atcatcatgccttcggtgttcggcaccatctgcctcctgggcatcatcgggaactccacg180 gtcatcttcgcggtcgtgaagaagtccaagctgcactggtgcaacaacgtccccgacatc240 ttcatcatcaacctctcggtagtagatctcctctttctcctgggcatgcccttcatgatc300 caccagctcatgggcaatggggtgtggcactttggggagaccatgtgcaccctcatcacg360 gccatggatgccaatagtcagttcaccagcacctacatcctgaccgccatggccattgac420 cgctacctggccactgtccaccccatctcttccacgaagttccggaagccctccatggcc480 accctggtgatctgcctcctgtgggctctctcgttcattagcatcactcctgtgtggctc540 tatgccaggcttatccccttcccagggggtgctgtgggctgtggcatccgcctaccaaac600 ccagatactgatctttactggttcactctgtatcagtttttcctggccttcgcccttccg660 tttgtggtcatcactgctgcgtacgtgaaaatactacagcgcatgacgtcttcggtggcc720 ccagcctctcaacgcagcatccggcttcggacaaagagggtgacccgcacagccattgcc780 atctgtctggtcttctttgtgtgctgggcgccctactacgtgctgcagctgacccagttg840 tccatcagccgcccgaccctcacattcgtctacctgtacaatgcggccatcagcttgggc900 tatgccaacagctgcctcaatccctttgtgtacatagtactctgtgagacctttcgaaaa960 cgcttggtgctgtcggtgaagcccgcggcccaggggcagcttcgcacggtcagcaatgct1020 cagacagctgacgaggagaggacagaaagcaaaggcaccgtcgacggtaccgcgggcccg1080 ggatccatcgccaccatggtgagcaagggcgaggagctgttcaccggggtggtgcccatc1140 ctggtcgagctggacggcgacgtaaacggccacaagttcagcgtgtccggcgagggcgag1200 ggcgatgccacctacggcaagctgaccctgaagttcatctgcaccaccggcaagctgccc1260 gtgccctggcccaccctcgtgaccaccctgacctacggcgtgcagtgcttcagccgctac1320 cccgaccacatgaagcagcacgacttcttcaagtccgccatgcccgaaggctacgtccag1380 gagcgcaccatcttcttcaaggacgacggcaactacaagacccgcgccgaggtgaagttc1440 gagggcgacaccctggtgaaccgcatcgagctgaagggcatcgacttcaaggaggacggc1500 aacatcctggggcacaagctggagtacaactacaacagccacaacgtctatatcatggcc1560 gacaagcagaagaacggcatcaaggtgaacttcaagatccgccacaacatcgaggacggc1620 agcgtgcagctcgccgaccactaccagcagaacacccccatcggcgacggccccgtgctg1680 ctgcccgacaaccactacctgagcacccagtccgccctgagcaaagaccccaacgagaag1740 cgcgatcacatggtcctgctggagttcgtgaccgccgccgggatcactctcggcatggac1800 gagctgtacaagtaa 1815 <210> 29 <211> 2022 <212> DNA
<213> Artificial Sequence <220>
<223> Human long form/mouse species chimeric MCH1R-linker-EGFP
<400>

atgtcagtgggagccatgaagaagggagtggggagggcagttgggcttggaggcggcagc60 ggctgccaggctacggaggaagacccccttcccaactgcggggcttgcgctccgggacaa120 ggtggcaggcgctggaggctgccgcagcctgcgtgggtggaggggagctcagctcggttg180 tgggagcaggcgaccggcactggctggatggacctggaagcctcgctgctgcccactggt240 cccaacgccagcaacacctctgatggccccgataacctcacttcggcaggatcacctcct300 cgcacggggagcatctcctacatcaacatcatcatgccttcggtgttcggcaccatctgc360 ctcctgggcatcatcgggaactccacggtcatcttcgcggtcgtgaagaagtccaagctg420 cactggtgcaacaacgtccccgacatcttcatcatcaacctctcggtagtagatctcctc480 tttctcctgggcatgcccttcatgatccaccagctcatgggcaatggggtgtggcacttt540 ggggagaccatgtgcaccctcatcacggccatggatgccaatagtcagttcaccagcacc600 tacatcctgaccgccatggccattgaccgctaCCtggCCaCtgtCCaCCCCatCtCttCC660 acgaagttccggaagccctccatggccaccctggtgatctgcctcctgtgggctctctcg720 ttcattagcatcactcctgtgtggctctatgccaggcttatccccttcccagggggtgct780 gtgggctgtggcatccgcctaccaaacccagatactgatctttactggttcactctgtat840 cagtttttcctggccttcgcccttccgtttgtggtcatcactgctgcgtacgtgaaaata900 ctacagcgcatgacgtcttcggtggccccagcctctcaacgcagcatccggcttcggaca960 aagagggtgacccgcacagccattgccatctgtctggtcttctttgtgtgctgggcgccc1020 tactacgtgctgCagCtgaCCCagttgtCCatCagCCgCCCgaCCCtC2.CattCgtCtaC1080 ctgtacaatgcggccatcagcttgggctatgccaacagctgcctcaatccctttgtgtac1140 atagtactctgtgagacctttcgaaaacgcttggtgctgtcggtgaagcccgcggcccag1200 gggcagcttcgcacggtcagcaatgctcagacagctgacgaggagaggacagaaagcaaa1260 ggcaccgtcgacggtaccgcgggcccgggatccatcgccaccatggtgagcaagggcgag1320 gagctgttcaccggggtggtgcccatcctggtcgagctggacggcgacgtaaacggccac1380 aagttcagcgtgtccggcgagggcgagggcgatgccacctacggcaagctgaccctgaag1440 ttcatctgcaccaccggcaagctgcccgtgccctggcccaccctcgtgaccaccctgacc1500 tacggcgtgcagtgcttcagccgctaccccgaccacatgaagcagcacgacttcttcaag1560 tccgccatgcccgaaggctacgtccaggagcgcaccatcttcttcaaggacgacggcaac1620 tacaagacccgcgccgaggtgaagttcgagggcgacaccctggtgaaccgcatcgagctg1680 aagggcatcgacttcaaggaggacggcaacatcctggggcacaagctggagtacaactac1740 aacagccacaacgtctatatcatggccgacaagcagaagaacggcatcaaggtgaacttc1800 aagatccgccacaacatcgaggacggcagcgtgcagctcgccgaccactaccagcagaac1860 acccccatcggcgacggccccgtgctgctgcccgacaaccactacctgagcacccagtcc1920 gccctgagcaaagaccccaacgagaagcgcgatcacatggtcctgctggagttcgtgacc1980 gccgccgggatcactctcggcatggacgagctgtacaagtas 2022 <210> 30 <211> 35 <212> DNA
<213> Artificial Sequence <220>
<223> Linker <400> 30 tcgacggtac cgcgggcccg ggatccatcg ccacc 35 <210> 31 <211> 12 <212> PRT
<213> Artificial Sequence <220>
<223> Linker <400> 31 Val Asp Gly Thr Ala Gly Pro Gly Ser Ile Ala Thr <210> 32 <211> 29 <212> DNA

<213> Artificial Sequence <220>

<223> PCR primer <400> 32 gcgaattcac catggatctg caagcctcg 29 <210> 33 <211> 28 <212> DNA

<213> Artificial Sequence <220>

<223> PCR primer <400> 33 gcgtcgacgg tgcctttgct ttctgtcc 28 <210> 34 <211> 33 <212> DNA

<213> Artificial Sequence <220>

<223> PCR primer <400> 34 ccttgctcac catggtgcct ttgctttctg tcc 33 <210> 35 <211> 39 <212> DNA

<213> Artificial Sequence <220>

<223> PCR primer <400> 35 cagaaagcaa aggcaccatg gtgagcaagg gcgaggagc 39 <210> 36 <211> 24 <212> DNA

<213> Artificial Sequence <220>

<223> PCR primer <400> 36 ggcggatcct ctagagtcgc ggcc 24 <210> 37 <211> 38 <212> DNA

<213> Artificial Sequence <220>
<223> PCR primer <400> 37 gctctagagt cgcggccgct tacttgtaca gctcgtcc 38

Claims (30)

WHAT IS CLAIMED IS:

1. A fusion protein comprising:
a) a melanin concentrating hormone receptor polypeptide region comprising a sequence selected from the group consisting of: SEQ. ID.
NO. 1, SEQ. ID. NO. 2, SEQ. SEQ. ID. NO. 3, SEQ. ID. NO. 4, and SEQ. ID. NO. 5; and b) a fluorescent polypeptide region joined directly, or though a linker, to the carboxy side of said melanin concentrating hormone receptor polypeptide region.

2. The protein of claim 1, wherein said fluorescent polypeptide region consists of an amino acid sequence selected from the group consisting of SEQ.
ID. NO. 6, SEQ. ID. NO. 7, SEQ. ID. NO. 8, SEQ. ID. NO. 9, and SEQ. ID. NO.
10.

3. The protein of claim 2, wherein said melanin concentrating hormone polypeptide region consists of a sequence selected from the group consisting of: SEQ. ID. NO. 1, SEQ. ID. NO. 2, SEQ. ID. NO. 3, SEQ. ID. NO. 4, and SEQ.
ID.
NO. 5.

4. The protein of claim 3, wherein said protein consists essentially of said melanin concentrating hormone receptor polypeptide region and said fluorescent polypeptide region.

5. The protein of claim 4, wherein said protein consists of the amino acid sequence of SEQ. ID. NO. 11 or SEQ. ID. NO. 12.

6. The protein of claim 1, wherein said melanin concentrating hormone polypeptide region is a chimeric polypeptide comprising (a) an MCH
binding region from a first species and (b) a transmembrane and intracellular domain region from a second species joined directly, or though a linker, to the carboxy side of said MCH binding region.

7. The protein of claim 6, wherein said fluorescent polypeptide region consists of an amino acid sequence selected from the group consisting of: SEQ.
ID. NO. 6, SEQ. ID. NO. 7, SEQ. ID. NO. 8, SEQ. ID. NO. 9, and SEQ. ID. NO.
10.

8. The protein of claim 7, wherein said protein consists of the amino acid sequence of SEQ. ID. NO. 13 or SEQ. ID. NO. 14.

9. A chimeric melanin concentrating hormone protein comprising:
a) a melanin concentrating hormone binding region characteristic of a human melanin concentrating hormone receptor;
b) a transmembrane domain characteristic of a non-human melanin concentrating hormone receptor; and c) an intracellular domain characteristic of a non-human melanin concentrating hormone receptor.

10. The protein of claim 9, wherein said protein comprises a melanin concentrating hormone receptor polypeptide having a sequence similarity of at least 75% with either SEQ. ID. NO. 4 or SEQ. ID. NO. 5.

11. The protein of claim 10, wherein said protein comprises the sequence of SEQ. ID. NO. 4 or SEQ. ID. NO. 5.

12. The protein of claim 11, wherein said protein consists of the sequence of SEQ. ID. NO. 4 or SEQ. ID. NO. 5.

13. A nucleic acid comprising a nucleotide sequence encoding for the protein of claim 1.

14. The nucleic acid of claim 13, wherein said nucleotide sequence is a contiguous sequence.

15. The nucleic acid of claim 13, wherein said nucleotide sequence is selected from the group consisting of SEQ. ID. NO. 26, SEQ. ID. NO. 27, SEQ. ID.
NO. 28 and SEQ. ID. NO. 29.

16. A nucleic acid comprising a nucleotide sequence encoding for the protein of claim 9.

17. The nucleic acid of claim 16, wherein said nucleotide sequence is a contiguous sequence.

18. The nucleic acid of claim 16, wherein said nucleotide sequence is selected from the group consisting of SEQ. ID. NO. 19 and SEQ. ID. NO. 20.

19. An expression vector comprising the nucleic acid of claim 13.

20. An expression vector comprising the nucleic acid of claim 16.

21. A recombinant cell comprising the nucleic acid of claim 13.

22. The recombinant cell of claim 21, wherein said nucleic acid is present in an expression vector.

23. The recombinant cell of claim 21, wherein said nucleic acid is present in the genome of said cell.

24. A recombinant cell comprising the nucleic acid of claim 16.

25. The recombinant cell of claim 24, wherein said nucleic acid is present in an expression vector.

26. The recombinant cell of claim 24, wherein said nucleic acid is present in the genome of said cell.

27. A non-human transgenic animal comprising the nucleic acid of claim 13.

28. A non-human transgenic animal comprising the nucleic acid of claim 16.

29. A method for assaying for melanin concentrating hormone receptor active compounds comprising the steps of:

a) contacting the cell of claim 21 with a test preparation comprising one or more test compounds; and b) measuring the effect of said test preparation on one or more melanin concentrating hormone receptor activities.

30. A method for assaying for melanin concentrating hormone receptor active compounds comprising the steps of:
a) contacting the cell of claim 24 with a test preparation comprising one or more test compounds; and b) measuring the effect of said test preparation on one or more melanin concentrating hormone receptor activities.