JP2003508026A - Human G-protein coupled receptor - Google Patents

Abstract

  (57) [Summary] The present invention relates to newly identified polynucleotides, polypeptides encoded by them and the use of these polynucleotides and polypeptides, and their production. More specifically, the polynucleotides and polypeptides of the present invention relate to a G protein-coupled receptor family called the IGS1-family. The present invention also relates to inhibiting or activating the action of these polynucleotides and polypeptides, vectors containing the polynucleotides, host cells and transgenic animals containing these vectors, wherein the IGS1 gene is overexpressed, non- Either expression, underexpression or suppression (knockout animals). The present invention further provides a method for screening for a compound capable of acting as an agonist or antagonist of the G protein-coupled receptor family IGS1, and an IGS1 polypeptide and polynucleotide and a neuromedicine of an agonist or antagonist for the IGS1 receptor family. And use in the treatment of CNS disorders.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD OF THE INVENTION

The present invention relates to newly identified polynucleotides, the polypeptides encoded thereby and the use of these polynucleotides and polypeptides and their production. More specifically, the polynucleotides and polypeptides of the present invention relate to G protein-coupled receptors (GPCRs), hereinafter referred to as IGS.
Call 1. The present invention also relates to inhibiting or activating the action of these polynucleotides and polypeptides, vectors containing said polynucleotides, host cells containing these vectors and transgenic animals, wherein the IGS1 gene is overexpressed, non-expressed. Either expression, underexpression and / or suppression (knockout animals). The present invention further relates to methods for screening compounds that can act as agonists or antagonists of the G protein-coupled receptor IGS1.

[0002]

BACKGROUND OF THE INVENTION

It is well documented that many medically important biological processes are mediated by proteins involved in signal transduction pathways, including G proteins and / or second messengers such as cAMP (Lefkowitz, Nature 1991, 351). : 353
-354). These proteins are referred to herein as proteins that participate in the pathway along with the G protein. Some examples of these proteins are GPC receptors,
For example, adrenergic agonists and dopamine (Kobilka, BK et al., Proc. Nat
l. Acad, Sci. USA, 1987, 84: 46-50; Kobilka, BK et al., Science, 1987,
238: 650-656; Bunzow, JR, et al., Nature, 1988, 336: 783-787), G protein itself, effector proteins such as phospholipase C, adenylate cyclase, and phosphodiesterase, and actuator proteins such as protein kinases. A and protein kinase C (Simon, MI, et al.,
Science, 1991, 252: 802-8).

For example, in one form of signal transduction upon hormone binding to a GPCR,
The receptor interacts with the heterotrimeric G protein and induces dissociation of GDP from the guanine nucleotide-binding site. At normal cellular concentrations of guanine nucleotides, GTP fills the site immediately. GTP binding to the α-subunit of the G protein results in separation of the G protein from the receptor and in α of the G protein.
And the separation into βγ subunits. The GPT-bearing form then binds and activates adenylate cyclase. Hydrolysis of GTP to GDP, where the α subunit has intrinsic GTPase activity, catalyzed by the G protein itself, returns the G protein to its basal, inactive form. G of α subunit
TPase activity is essentially an internal clock that controls the on / off switch. The GDP-bound form of the α subunit has a high affinity for βγ and αGD
Subsequent recombination of P with βγ returns the system to the ground state. Thus, the G protein has a dual role: as an intermediate that relays the signal from the receptor to the effector (adenylate cyclase in this example) and as a clock that controls the duration of the signal.

The membrane-bound superfamily of G protein-coupled receptors has been characterized as having seven putative transmembrane domains. The domain is believed to exhibit a transmembrane α-helix linked by extracellular or cytoplasmic loops. G-protein coupled receptors include a wide range of biologically active receptors such as hormones, viruses, growth factors and neuroreceptors.

The G protein-coupled receptor family includes dopamine receptors that bind neuroleptic drugs used in the treatment of CNS disorders. Other examples of members of this family include calcitonin, adrenergic agonists, neuropeptide Y, somastatin, neurotensin, neurokinin, capsaicin, VIP, CGRP, CRF,
CCK, bradykinin, galanin, motilin, nociceptin, endothelin, c
AMP, adenosine, muscarinin agonist, acetylcholine, serotonin, histamine, thrombin, quinine, follicle stimulating hormone, opsin, endothelial differentiation gene-
1, but not limited to rhodopsin, odorant, and cytomegalovirus receptors.

Most G protein-coupled receptors contain a single conserved cysteine residue in each of the first two extracellular loops that form a disulfide bond, which is believed to stabilize functional protein structure. Have. The seven transmembrane regions are TM1 and TM
2, TM3, TM4, TM5, TM6 and TM7. TM5 and TM6
The cytoplasmic loop that binds and would be the major component of the G protein binding domain.

Most G protein-coupled receptors contain potential phosphorylation sites within the third cytoplasmic loop and / or the carboxy terminus. Several G-protein coupled receptors,
For example, for β-adrenergic receptors, phosphorylation by protein kinase A and / or specific receptor kinases mediates receptor desensitization.

It has recently been discovered that certain GPCRs, as well as calcitonin receptor-like receptors, are likely to interact with a small, single-pass membrane protein called the receptor activity regulatory protein (RAMP). This interaction of GPCRs with certain RAMPs is crucial for natural ligands to have a relevant affinity for the GPCR-RAMP combination and to regulate the functional signaling activity of the complex (McLathie,
LM et al., Nature (1998) 393: 333-339).

For certain receptors, the ligand binding site of G protein-coupled receptors is
It comprises a hydrophilic socket formed by several G protein-coupled receptor transmembrane domains, which is believed to be surrounded by the hydrophobic residues of the G protein-coupled receptor. The hydrophilic side of each G protein-coupled receptor transmembrane helix is thought to face inward and form a polar ligand-binding site. TM3 is believed to have a ligand-binding site, such as the TM3 aspartic acid residue, on several G protein coupled receptors. TM5 serine,
TM6 asparagine and TM6 and TM7 phenylalanine or tyrosine are also involved in ligand binding.

G protein-coupled receptors can be intracellularly bound to various intracellular enzymes, ion channels and transporters by heterotrimeric G proteins (Johnson
See et al., Endoc. Rev., 1989, 10: 317-331). Different G protein α subunits preferentially stimulate specific effectors to regulate different biological functions intracellularly. Phosphorylation of cytoplasmic residues of G protein coupled receptors has been identified as a key mechanism for the regulation of G protein coupling of certain G protein coupled receptors. G-protein coupled receptors have been found at numerous sites within the mammalian host.

Predominantly GPCR class receptors induce more than half of the currently known drugs (D
rews, Nature Biotechnology, 1996, 14: 1516). This indicates that these receptors have an established and proven history as therapeutic targets. The novel IGS1 GPCRs described in this invention include dysfunctions, disorders, or diseases (hereinafter "
It clearly meets the needs in the art for the identification and characterization of additional receptors that may be useful in the diagnosis, prevention, amelioration or correction of diseases). "Disease" refers to schizophrenia, intermittent seizure anxiety (EPA) disorders such as obsessive-compulsive disorder (O).
CD), post-traumatic stress disorder (PTSD), phobia and panic, severe depressive disorder, bipolar disorder, Parkinson's disease, general anxiety disorder, autism, delirium, multiple sclerosis, Alzheimer's disease / dementia and others Neurodegenerative diseases, severe mental retardation, movement disorders, Huntington's disease, Tourette's syndrome, tics, tremor, dystonia,
Convulsions, loss of appetite, increased appetite, stroke, addiction / addiction / craving, sleep disorders, tencan,
Migraine, psychiatric and CNS disorders including distraction / hyperactivity disorder (ADHD), heart failure, angina, arrhythmia, myocardial infarction, cardiac hypertrophy, hypotension, hypertension-eg essential hypertension, renal hypertension, or pulmonary hypertension. , Thrombosis, arteriosclerosis, cerebral vasospasm, subarachnoid hemorrhage, cerebral ischemia, cerebral infarction, peripheral vascular disease, cardiovascular disease including Raynaud's disease, renal disease-such as renal failure, dyslipidemia, obesity, vomiting , Irritable Bowel Syndrome (IBS), Inflammatory Bowel Disease (IBD), Gastroesophageal Reflex Disease (GERD), Motility Disorders and Delayed Gastrointestinal Symptoms, eg Postoperative or Diabetic Gastroparesis, and Diabetes, Ulcers-eg Gastric Ulcers Gastrointestinal diseases including, diarrhea, other diseases including osteoporosis, inflammation, infections such as bacterial, fungal, protozoal and viral infections, especially HIV-1 or HIV-
2, infection, pain, cancer, chemotherapy-induced disorders, tumor invasion, immune disorders, residual urine, asthma, allergies, arthritis, benign prostatic hypertrophy, endotoxic shock, sepsis, complications of diabetes mellitus, and gynecological disorders , But not limited to these.

[0012] In particular, the novel IGS1 GPCRs according to the invention can be used for psychiatric and CNS deficiencies, disorders and diseases, in particular movement disorders, disorders or diseases such as tics, tremor, Tourette's syndrome, Parkinson's disease, Huntington's disease, Satisfies the need in the art for the identification and characterization of alternative receptors that can play an important role in the diagnosis, prevention, amelioration or correction of movement disorders, dystonia and convulsions.

[0013]

[Summary of Invention]

In one aspect, the invention relates to IGS1 polypeptides and recombinant materials and methods for their production. Another aspect of the invention relates to methods for the use of such IGS1 polypeptides and polynucleotides. Such uses include, but are not limited to, the treatment of one of the "disorders" as described above. In particular use includes the treatment of psychiatric and CNS disorders, in particular movement disorders such as tics, tremors, Tourette's syndrome, Parkinson's disease, Huntington's disease, movement disorders, dystonia and convulsions.

In yet another aspect, the invention provides for the identification of agonists and antagonists using the agents provided by the invention, and IG using the identified compounds.
A method for treating a condition associated with S1 imbalance. Yet another aspect of the invention relates to diagnostic assays for detecting diseases associated with inappropriate IGS1 activity or levels. Yet another aspect of the invention relates to an animal-based system that behaves as a model for disorders resulting from aberrant expression or activity of IGS1.

[0015]

[Table 1]

[0016]

[Table 2]

DETAILED DESCRIPTION OF THE INVENTION Structural similarity within the sequences and motifs is dependent on the IGS1 GPCR of the invention.
And other human GPCRs. Furthermore, IGS1 is expressed in brain tissue, especially in the caudate and putamen. Therefore, IGS1 is, among other things, the above-mentioned “disease”.
Thought to be involved in. In particular, IGS1 is thought to be involved in psychiatric and CNS disorders, especially movement disorders such as tics, tremor, Tourette's syndrome, Parkinson's disease, Huntington's disease, movement disorders, dystonia and convulsions.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods, devices and materials are described herein. All publications cited herein are hereby incorporated by reference as well as incorporated by reference as if all publications were individually and fully described. Be transferred to.

[0019]

[Definition]

The following definitions are provided to aid in the understanding of some terms often used herein.

“IGS1” refers, among other things, to a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 2 or allelic variants thereof.

“Receptor activity” or “receptor biological activity” refers to the metabolic or physiological function of the IGS1 and those activities that have similar or improved activity or reduced undesirable side effects. including. Also included are antigenic and immunogenic activities of said IGS1.

“IGS1 gene” refers to a polynucleotide comprising the nucleotide sequence set forth in SEQ ID NO: 1 or allelic variants thereof and / or their complements.

As used herein, “antibody” includes polyclonal and monoclonal antibodies, chimeric, single chain, and humanized antibodies, and Fab fragments, which include Fab or other immunoglobulins. The products of expression libraries are also included.

“Isolated” means “manually” altered from its original (natural) state and / or separated from its original environment. Thus, an “existing” composition or substance that is naturally present is “isolated” when it undergoes a change or migration or both from its original environment. For example, a polynucleotide or polypeptide that is naturally present in a living animal has not been "isolated", but the same polynucleotide or polypeptide that has been separated from the substances with which it was naturally present is "isolated". “Isolated” and as such the terms are used herein.

“Polynucleotide” generally refers to unmodified RNA or DNA or modified R
Refers to any polyribonucleotide or polydeoxyribonucleotide which may be NA or DNA. “Polynucleotide” refers to single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, and single- and double-stranded. A hybrid molecule comprising DNA and RNA, which may be a mixture of regions, RNA, single-stranded or more typically double-stranded or a mixture of single-stranded and double-stranded regions, Is not limited to. In addition, "polynucleotide" may also include triple-stranded regions comprising RNA or DNA or both RNA and DNA. The term polynucleotide also includes DNA or RNA containing one or more modified bases and DNA or RNA having a backbone modified for stability or for other reasons. "Modified" bases include, for example, tritylated bases and unusual bases such as inosine. Various modifications have been made to DNA and RNA, thus
A "polynucleotide" is a chemically, enzymatically, or metabolically modified form of a polynucleotide typically found in nature, as well as viral and cellular DNA.
And the RNA form of the chemical form of the polynucleotide. "Polynucleotide" also embraces relatively short polynucleotides, often referred to as oligonucleotides.

“Polypeptide” refers to any peptide or protein that comprises two or more amino acids joined together by peptide bonds or modified peptide bonds, ie, peptide isosteres. "Polypeptide" refers to short chains, commonly referred to as peptides, oligopeptides or oligomers, and longer chains, commonly referred to as proteins, and / or combinations thereof. Polypeptides may contain amino acids other than the 20 gene-encoded amino acids. A "polypeptide" comprises an amino acid sequence modified by natural processes, such as post-translational processing, or by chemical modification techniques well known in the art. Such modifications are well documented in basic textbooks and in more lengthy papers and in the vast body of research literature. Modifications can occur anywhere in a polypeptide, including the peptide backbone, the amino acid side-chains and the amino or carboxyl termini. It will be appreciated that the same type of modification may be present in the same or varying degrees at different sites in a given polypeptide. Also, a given polypeptide may contain many types of modifications. Polypeptides may be branched as a result of ubiquitination, and they may be cyclic with or without branching. Cyclic, branched and branched cyclic polypeptides may result from post-translational natural processing or may be made by synthetic methods. Modifications include acetylation, acylation, ADP ribosylation, amidation, covalent attachment of flavins, covalent attachment of heme moieties, covalent attachment of nucleotides or nucleotide derivatives, covalent attachment of lipids or lipid derivatives, phosphotidyl. Covalent addition of inositol, cross-linking, cyclization, disulfide bond formation, demethylation, covalent cross-link formation, cystine formation, pyroglutamic acid formation, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation , Iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer RNA-mediated addition of amino acids to proteins such as arginylation, and ubiquitination. For example, “protein-structure and molecular properties” (PROTEINS-STRUCTURE AND
MOLECULAR PROPERTIES, 2nd Ed., TE Creighton, WH Freeman and Company,
New York, 1993) and "Post-Translational Protein Modifications-Forecasts and Prospects" (Wold, F.,
Posttranslational Protein Modifications: Perspectives and Prospects, pg
s. 1-12 in POSTTRANSLATIONAL COVALENT MODIFICATION OF PROTEINS, BC Joh
nson, Ed. Academic Press, New York, 1983), "Analysis for protein modifications" (Seifter et al., "Analysis for protein modifications")
and nonprotein cofactors ", Meth. Enzymol. (1990) 182: 626-646) and" Protein Synthesis: Post-translational Modification and Aging "(Ratten et al.," Protein Synthesis:
Posttranslational Modifications and Aging ", Ann. NY Acad. Sci. (1992) 66
3: 48-62).

“Variant” as the term is used herein, differs from the polynucleotide or polypeptide to which it is compared, but has essential properties, such as essential biological, structural, regulatory or biochemistry. The property is the polynucleotide or polypeptide that it retains. A typical variant of a polynucleotide differs in nucleotide sequence from another, comparable polynucleotide. Changes in the nucleotide sequence of the variant may or may not change the amino acid sequence of the polypeptide encoded by the comparative polynucleotide. Nucleotide changes may result in amino acid substitutions, additions, deletions, fusions and truncations in the polypeptide encoded by the comparison sequence, which are discussed below. A typical variant of a polypeptide differs in amino acid sequence from another, comparable polypeptide. In general, the differences are limited to the extent that the sequences of the comparison polypeptide and variant are closely similar overall and are consistent in many regions. Variants and comparison polypeptides may differ within the amino acid sequence by any combination of one or more substitutions, additions and deletions. The substituted or inserted amino acid residue may or may not be one encoded by the genetic code. A variant of a polynucleotide or polypeptide may be a naturally occurring, eg, allelic variant, or it may be a variant not known to be naturally occurring. Non-naturally occurring variants of polynucleotides and polypeptides may be made by mutagenesis techniques or direct synthesis.

“Identity” is a measure of the identity of nucleotide or amino acid sequences. Generally, the sequences are aligned for the highest degree of matching. "Identity" itself has its art-recognized meaning and can be calculated using published techniques. See for example: "Computer Molecular Biology" (COMPUTATIONAL MOL
ECULAR BIOLOGY, Lesk, AM, ed., Oxford University Press, New York, 1988
); "Biocomputing: Informatics and Genome Project" (BIOCOMPUT
ING: INFORMATICS AND GENOME PROJECTS, Smith, DW, ed., Academic Press,
New York, 1993); "Computer analysis of sequence data, Part 1" (COMPUTER ANAL
YSIS OF SEQUENCE DATA, PART 1, Griffin, AM, and Griffin, HG, eds., H
umana Press, Newe Jersey, 1994); "Sequence analysis in molecular biology" (SEQUENC
E ANALYSIS IN MOLECULAR BIOLOGY, von Heinje, G. Academic Press, 1987); and "Sequence analysis primer" (SEQUENCE ANALYSIS PRIMER, Gribskov, M. and De.
vereux, J., eds., M. Stockton Press, New York, 1991). Although there are numerous ways to determine the identity between two polynucleotide or polypeptide sequences, the term "identity" is well known to those of skill in the art (Carillo, H.,
and Lipton, D., SIAM J. Applied Math, (1988) 48: 1073). Methods commonly used to determine the identity or similarity between two sequences are described in "Guide to Huge Computers, Martin J. Bishop, ed., Acade.
mic Press, San Diego, 1994) and Carillo, H., and Lipton, D., SIAM J. A
pplied Math, (1988) 48: 1073, but is not limited thereto. Methods to determine identity and similarity are codified in computer programs. A preferred computer program method for determining identity and similarity between sequences is the GCG program package (Devereux, J.
et al., Nucleic Acids Research (1984) 12 (1): 387), BLASTP, BRAS
TN, FASTA (Atschul, SF et al., J. Molec. Biol. (1990) 215: 403), but is not limited thereto. The term "homology" may be replaced with "identity".

By way of explanation, at least eg 9 relative to the comparative nucleotide sequence of SEQ ID NO: 1
A polynucleotide having a nucleotide sequence with 5% “identity” means that the nucleotide sequence of the polynucleotide has 5 or less nucleotide differences with respect to each 100 nucleotides of the comparative nucleotide sequence of SEQ ID NO: 1. It is meant to be identical to the comparison sequence, except that it may be included.
In other words, 5% or less of the nucleotides in the comparison sequence may be deleted or replaced by other nucleotides in order to obtain a polynucleotide having a nucleotide sequence that is at least 95% identical to the comparison nucleotide sequence. Or 5% or less of the total number of nucleotides in the comparison sequence may be inserted in the comparison sequence, or deleted in the number of 5% or less of any of the total nucleotides in the comparison sequence , There may be a combination of insertions and substitutions. These mutations in the comparison sequence may occur at the 5 or 3 terminal positions of the comparison nucleotide sequence or at any position between these terminal positions, individually between the nucleotides in the comparison sequence, or one or more of the nucleotides in the comparison sequence. It may occur scatteredly on any of the nucleotides in the above continuous groups.

Similarly, for example, at least 95% “relative to the comparative amino acid sequence of SEQ ID NO: 2”
A polypeptide having an amino acid sequence with "identity" refers to a polypeptide, except that the polypeptide sequence may contain up to 5 amino acid changes for each of the 100 amino acids in the comparative sequence of SEQ ID NO: 2. It means that the amino acid sequence is identical to the comparison sequence. In other words, 5% or less of the amino acid residues in the comparison sequence may be deleted or replaced by other amino acids in order to obtain a polypeptide having an amino acid sequence that is at least 95% identical to the comparison amino acid sequence. Alternatively, no more than 5% of all amino acid residues in the comparison sequence may be inserted in the comparison sequence. These changes to the comparison sequence may be made at the amino or carboxy terminal position of the comparison amino acid sequence or at any position between these terminal positions, individually between residues within the comparison sequence or one or more within the comparison sequence. May be present scatteredly in any of the adjacent groups. Polypeptides of the Invention In one aspect, the invention relates to IGS1 polypeptides (including IGS1 proteins). The IGS1 polypeptide is the polypeptide of SEQ ID NO: 2 and Cent.
raalbureau voor Schimmelcultures (Baarn, The Netherlands) July 1, 1999
A polypeptide having an amino acid sequence encoded by the DNA insert contained within deposit number CBS102049 deposited on the 5th; and the amino acid sequence of SEQ ID NO: 2 and deposit number CBS102049 in Centraalbureau voor Schimmelcultures (Baarn, The Netherlands). A polypeptide comprising an amino acid sequence encoded by a DNA insert contained within the amino acid sequence, and an amino acid sequence having at least 80% identity to that of SEQ ID NO: 2 and / or Centraalbureau voor Schimmelcultures (Baarn, In the Netherlands) the amino acid sequence encoded by the DNA insert contained within deposit number CBS102049, and more preferably at least 90% identical to said amino acid sequence,
And more preferably further comprising a polypeptide comprising a polypeptide having at least 95% identity. Furthermore, at least 97% identical, in particular at least 9
Most preferred is 9%. The IGS1 polypeptide includes a polypeptide having the amino acid sequence of SEQ ID NO: 2 or Centraalbureau voor Schimmelcultures (Baarn,
In the Netherlands) a polypeptide having an amino acid sequence having at least 80% identity over its entire length to a polypeptide having the amino acid sequence encoded by the DNA insert contained within deposit number CBS102049, and More preferably at least 90% identical to SEQ ID NO: 2, and more preferably at least 95% identical thereto. In addition, at least 97%
Most preferably at least 99%. Preferably, the IGS1 polypeptide exhibits at least one biological activity of the receptor.

The IGS1 polypeptide may be in the form of a “mature” protein or may be part of a larger protein, eg a fusion protein. It is often advantageous to include secretory or leader sequences, prosequences, sequences that aid in purification, such as additional amino acid sequences containing overlapping histidine residues, or additional sequences for stability during recombinant production.

Fragments of IGS1 polypeptides are also included in the invention. A fragment is the above IGS
A polypeptide having an amino acid sequence similar to that of a part but not the whole of the amino acid sequence of one polypeptide. As with IGS1 polypeptides, fragments may be "free-standing," that is, they most preferably comprise a single contiguous region within the larger polypeptide in which they form the part or region. Representative examples of polypeptide fragments of the invention are, for example, amino acid numbers 1-20, 21-40, 41-60, 61-80, 81-100, and 101 of the IGS1 polypeptide.
To the end fragment. Within this range, “about” includes the specific recited range as small or large as several, 5, 4, 3, 2, or 1 amino acid at either or both ends.

Preferred fragments are, for example, deletions of a series of residues containing the amino terminus or a series of residues containing the carboxy terminus, or residues of those containing the amino terminus and those containing the carboxy terminus. A truncated polypeptide having the amino acid sequence of the IGS1 polypeptide, except for the deletion of the two consecutive Structural or functional attributes, such as alpha and alpha helix forming regions, beta sheets and beta sheet forming regions, turns and turn forming regions, coils and coil forming regions, hydrophilic regions, hydrophobic regions, alpha amphoteric Also preferred is a fragment characterized by a fragment comprising a region, a beta amphoteric region, a flexible region, a surface forming region, a substrate binding region, and a high antigen index region. Other preferred fragments are biologically active fragments. Biologically active fragments are those that mediate receptor activity and include those that have a similar or improved activity, or have a decreased undesirable activity. Also preferred are those that are antigenic or immunogenic in animals, especially humans.

Accordingly, a polypeptide of the invention is a polypeptide having an amino acid sequence which is at least 80% identical to that of SEQ ID NO: 2 and / or a Centraalbureau voor.
Deposit number CBS102049 at Schimmelcultures (Baarn, The Netherlands)
Included are those fragments having at least 80% identity to a polypeptide having the amino acid sequence encoded by the DNA insert contained within the signal or the corresponding fragment. Preferably, all of these polypeptide fragments retain the biological activity of the receptor, including antigenic activity. Variants of the defined sequences and fragments also form part of the invention. Preferred variants are those that differ from the comparison by conservative amino acid substitutions. That is, the residue is replaced by another with similar properties. Typical of such substituents are especially Ala, Val, Leu and Lie, especially Se.
r and Thr, especially the acid residues Asp and Glu, especially Asn and Gln, and especially the base residues Lys and Arg, or the aromatic residues Phe and Tyr. Particularly preferred are several, 5-10, 1-5, or 1-, among them.
Variants in which two amino acids are substituted, deleted, or added in any combination.

The IGS1 polypeptide of the present invention can be made by any suitable method. Such polypeptides include isolated naturally occurring polypeptides, recombinantly produced polypeptides, synthetically produced polypeptides, or polypeptides produced by a combination of these methods. Methods for making such polypeptides are well known in the art. Polynucleotides of the Invention Another aspect of the invention pertains to IGS1 polynucleotides. IGS1 polynucleotides include isolated polynucleotides that encode IGS1 polypeptides and fragments, and polynucleotides closely related thereto. More specifically, the IGS1 polynucleotide of the present invention is a polynucleotide comprising the nucleotide sequence contained in SEQ ID NO: 1, eg, one capable of encoding the IGS1 polypeptide of SEQ ID NO: 2, the specific sequence of SEQ ID NO: 1. And a polynucleotide essentially corresponding to the DNA insert contained within deposit number CBS102049 in Centraalbureau voor Schimmelcultures (Baarn, The Netherlands).

IGS1 polynucleotide is a polynucleotide comprising a nucleotide sequence having at least 80% identity over its entire length to a nucleotide sequence encoding the IGS1 polypeptide of SEQ ID NO: 2, of which SEQ ID NO: 1. Further included are a polynucleotide comprising a nucleotide sequence that is at least 80% identical over its entire length and a polynucleotide essentially corresponding to the DNA insert contained within deposit number CBS102049 at Centraalbureau voor Schimmelcultures (Baarn, The Netherlands).

In this regard, polynucleotides that are at least 90% identical are particularly preferred, and polynucleotides that are at least 95% identical are even more preferred. Further, at least 97% is highly preferred, at least 98-99% is most highly preferred, and at least 99% is most preferred. As an IGS1 polynucleotide, for the nucleotide sequence contained within SEQ ID NO: 1 or for Centraalbu
Deposit number CBS1 at reau voor Schimmelcultures (Baarn, The Netherlands)
Nucleotide sequences that have sufficient match to the DNA insert contained within 02049 are also included to hybridize under conditions that can be used for amplification or use as a probe or marker. The present invention uses these IGs
Polynucleotides complementary to the S1 polynucleotides are also provided.

The IGS1 of the present invention is structurally related to other proteins of the G protein coupled receptor family, which is shown by the results of BLAST searches in public databases. The amino acid sequence of Table 2 (SEQ ID NO: 2) has about 30% identity (BLAS).
25. Tuse, Altscul SF et al., [1997], Nucleic Acids Res .. Number # O02824, Miyasa
to et al. RL Life Sci. (1997) 60: 2069-2074) and about 33% identity at amino acid residues 31-220 with the human G protein coupled receptor RE2 (Genebank Accession # AF091890). ) And have. The nucleotide sequence of Table 1 (SEQ ID NO: 1) shows 57% identity with the human α-1a / d adrenergic receptor (accession number # L31722, Bruno JF, et al. Biochem. Biophys.
Res. Commun. (1991) 179: 1485-1490) with 266 nucleotide residues and 44% identity with the human G protein coupled receptor RE2 at the first 1426 nucleotide residues (Genebank Accession # AF091890). In addition, hydropathic analysis of the IGS1 protein sequence (Hoffmann, K., Stoff
el, W (1993) Biol. Chem. Hoppe-Seyler 347: 166) showed the presence of seven transmembrane regions. Accordingly, the IGS1 polypeptides and polynucleotides of the invention are expected to have, among other things, biological functions / properties similar to these homologous polypeptides and polynucleotides, and their utility is found throughout the art. Will be obvious to the skilled person.

The polynucleotides of the present invention can be obtained from natural sources, eg genomic DNA. In particular, degenerate PCR primers can be designed to encode conserved regions within a particular GPCR gene subfamily. A PCR amplification reaction on genomic DNA or cDNA using degenerate primers results in the amplification of several members of the gene family under consideration (both known and novel) (when using a genomic template, degenerate primers). Must be located within the same exon) (Libert et al., Science, 1989, 244: 569-572). The polynucleotides of the present invention can also be synthesized using well known and commercially available techniques.

The nucleotide sequence encoding the IGS1 polypeptide of SEQ ID NO: 2 may be identical to the polypeptide encoding the sequence contained in SEQ ID NO: 1 (nucleotide numbers 36 to 1559) or it may be Duplication of genetic code (degeneracy)
As a result of the changes shown relative to the polypeptide encoding the sequence contained within SEQ ID NO: 1, but may also be different nucleotide sequences encoding the polypeptide of SEQ ID NO: 2.

When the polynucleotide of the present invention is used for the recombinant production of an IGS1 polypeptide, the polypeptide may itself have a coding sequence for the mature polypeptide or a fragment thereof for coding the mature polypeptide or other coding sequences. Fragments within the open reading frame with sequences may be included, such as those encoding leader or secretory sequences, pre-, or pro- or pre-pro-protein sequences or other fusion peptide moieties. For example, a marker sequence that facilitates purification of the fusion polypeptide can be encoded. In one preferred aspect of this practice of the invention, the marker sequence is a hexa-histidine peptide, which is provided as a pQE vector (Qiagen, Inc.), and Gentz et al., Proc.
Natl. Acad. Sci. USA (1989) 86: 821-824 or HA tag. The polynucleotide may also include non-coding 5'and 3'sequences, such as transcribed non-translated sequences, splicing and polyadenylation signals, ribosome binding sites and sequences that stabilize mRNA.

A further preferred embodiment is SEQ ID NO: 2 in which several, 5-10, 1-5, 1-3, 1-2 or 1 amino acid residues are substituted, deleted or added in any combination.
Is a polynucleotide encoding an IGS1 variant comprising the amino acid sequence of the IGS1 polypeptide of.

The polynucleotides of the present invention may be used for a variety of purposes including, but not limited to, cloning, processing, and / or modifying expression of gene products.
Techniques commonly known in the art can be used to modify the GS1 coding sequence. DNA shuffling and gene fragmentation by random fragmentation and PCR reconstruction of synthetic oligonucleotides may be used to manipulate the nucleotide sequences. For example, oligonucleotide-mediated site-directed mutagenesis involves the creation of amino acid substitutions, the creation of new restriction sites, modification of modification (eg, glycosylation or phosphorylation) patterns, changes in codon preference,
It may be used to introduce mutations such as the production of splice variants.

The invention further relates to polynucleotides that hybridize to the sequences described herein above. In this regard, the present invention specifically relates to polynucleotides that hybridize under stringent conditions to the polynucleotides described herein above. As used herein, the term "stringent conditions"
Hybridizes only if there is at least 80%, and preferably at least 90%, and more preferably at least 95%, more preferably at least 97%, and in particular at least 99% identity between the sequences. Means to get up.

The polynucleotide of the present invention, which is identical or sufficiently identical to the nucleotide sequence contained in SEQ ID NO: 1 or a fragment thereof, can be used as a hybridization probe for cDNA and genomic DNA, with a full-length cDNA and a genomic clone encoding IGS1. And a genomic clone of another gene having a high degree of sequence similarity to the IGS1 gene, including genes encoding homologues or orthologs from species other than humans. May be. Those skilled in the art are familiar with such hybridization techniques. Typically these nucleotide sequences are 80% identical, preferably 90% identical, and more preferably 95% identical to that of the comparison. The probe generally comprises at least 5 nucleotides, and preferably at least 8 nucleotides, and more preferably at least 10 nucleotides, more preferably at least 12 nucleotides, especially at least 15 nucleotides. . Most preferably, such a probe may have at least 30 nucleotides and at least 50 nucleotides. Particularly preferred probes are within the range of 30 to 50 nucleotides.

One embodiment for obtaining a polynucleotide encoding an IGS1 polypeptide that includes a homolog or ortholog from a species other than human has SEQ ID NO: 1 or a fragment thereof under stringent hybridization conditions. Screening the appropriate library with labeled probes and isolating the full length DNA as well as the genomic clone containing the polynucleotide sequence. Such hybridization techniques are well known to those of skill in the art. Stringent hybridization conditions are those defined above or at 42 ° C. in 50% formamide, 5 × SSC (150 mM NaCl, 15 mM).
M trisodium citrate), 50 mM sodium phosphate, pH 7.6, 5x Denhardt's solution, 10% dextran sulfate, and 20 μg / ml denatured sheared salmon sperm DNA, incubated overnight and then in 0.1x SSC. Is defined as the condition that the filter is washed at about 65 ° C.

The polynucleotides and polypeptides of the present invention may be used as research reagents and materials for therapeutic and diagnostic discovery of animal and human diseases. Vectors, Host Cells, Expression The present invention provides polynucleotides or vectors comprising the polynucleotides of the invention, and host cells genetically engineered with the vectors of the invention and polypeptides of the invention by recombinant techniques. Also relates to the production of. Cell-free translation systems can also be used to produce such proteins using RNA derived from the DNA constructs of the invention.

For recombinant production, host cells can be genetically engineered to incorporate expression systems or parts thereof for the polynucleotides of the present invention. Introduction of polynucleotides into host cells is accomplished by a number of standard laboratory manuals, such as "Basic Methods in Molecular Biology" (Davis et al., BASIC METHODS IN MOLECULAR BIOLOGY (1986
)) And “Molecular Cloning: Experimental Manual” (Sambrook et al., MOLECULAR
CLONING: A LABORATORY MANUAL, 2nd Ed. Cold Spring Harbor Laboratory Pres
s, Cold Spring Harbor, NY (1989)), eg calcium phosphate transfection, DEAE-dextran mediated transfection, transvection, microinjection, cationic lipid mediated transfection, electroporation, Transduction, scrape loading,
It can be done by ballistic introduction or infection.

Representative examples of suitable hosts are bacterial cells such as Streptococcus, Staphylococcus, Escherichia coli, Streptomyces and Bacillus subtilis cells, fungal cells such as yeast cells and Aspergillus cells, insect cells such as insect cells. Drosophila S2 and Spodoptera Sf9 cells, animal cells such as CHO, COS, Hela, C
127, 3T3, BHK, HEK293 and Bowes melanoma
oma) cells, and plant cells.

A wide variety of expression systems can be used. Such systems include, among others, chromosomal, episomal and viral inducible systems such as from bacterial plasmids, from bacteriophages, from transposons, from yeast episomes, from insert elements, from yeast chromosomal elements, to viruses such as baculovirus, papovavirus. , For example SV4
0, vectors derived from vaccinia virus, adenovirus, fowlpox virus, pseudorabies virus and retrovirus, and vectors derived from combinations thereof, such as plasmids and bacteriophage genetic elements, such as those derived from cosmids and phagemids. . The expression system may include control regions that regulate as well as engender expression. In general, any system or vector suitable for maintaining, propagating or expressing polynucleotides to produce a polypeptide in a host may be used. Appropriate nucleotide sequences may be found in a variety of well-known and conventional techniques, such as "Molecular Cloning: Experimental Manual" (Sambrook et al., MOLECU.
Any of those described in LAR CLONING: A LABORATORY MANUAL, above) may be used for insertion into the expression system.

Appropriate secretion signals may be incorporated into the desired polypeptide for secretion of the translated protein into the lumen of the endoplasmic reticulum, into the periplasmic space or into the extracellular milieu. These signals may be endogenous to the polypeptide or may be heterologous signals.

When the IGS1 polypeptide is expressed for use in a screening assay, it is generally preferred that the polypeptide be produced at the surface of cells. At this event, cells may be harvested prior to use in screening assays. When the affinity or functional activity of the IGS1 polypeptide is modified by the receptor activity modulatory protein (RAMP), co-expression of the relevant RAMPs believed to be at the cell surface is preferred and often required. This event also requires that cells expressing the IGS1 polypeptide and related RAMPs be harvested prior to use in the screening assay. If the IGS1 polypeptide is secreted into the medium, the medium can be recovered to recover and purify the polypeptide. If produced intracellularly, the cells must first be lysed before recovering the polypeptide.

IGS1 polypeptide can be prepared by ammonium sulfate or ethanol precipitation, acid extraction,
It can be recovered and purified from recombinant cell culture by well known methods including anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Most preferably high performance liquid chromatography is used for purification. Well-known techniques for refolding proteins may be used to regenerate the active conformation when the polypeptide is denatured during isolation and / or purification. Diagnostic Assays The present invention also relates to the use of IGS1 polynucleotides for use as diagnostic reagents. Detection of mutant forms of the IGS1 gene associated with dysfunction may be in addition to or may be diagnostic of a disease or susceptibility to a disease resulting from underexpression, overexpression or modified expression of IGS1. I will provide a. Even in this event, co-expression of related receptor activity modulating proteins can be required to obtain a desired quality diagnostic assay. Individuals having a mutation in the IGS1 gene may be detected at the DNA level by various techniques.

Nucleic acid for diagnosis may be obtained from cells of a patient, such as blood, urine, saliva, tissue biopsy or autopsy material. Genomic DNA may be used directly for detection or may be enzymatically amplified using PCR or other amplification techniques prior to analysis.
RNA or cDNA may be used in a similar manner. Deletions and insertions can be detected by changes in the magnitude of amplification as compared to the normal genotype. Point mutations can be identified by hybridizing amplified DNA to labeled IGS1 nucleotide sequences. Perfectly matched sequences can be distinguished from mismatched duplexes by RNase digestion or by differences in melting temperature. DNA sequence differences may be detected by modification of electrophoretic mobility of DNA fragments in gels with or without denaturing agents, or by direct DNA sequencing. For example Myers et al., Science (1985) 230: 1242.
reference. Sequence changes at specific positions can be detected by nuclease protection assays such as RN
May be elucidated by ase and S1 protection or chemical cleavage methods. Cotton et al.,
See Proc. Natl. Acad. Sci. USA (1985) 85: 4397-4401. In another aspect, the IG
Arrays of oligonucleotide probes comprising the S1 nucleotide sequence or fragments thereof can be constructed, eg, for efficient screening of gene mutations. The methods of array technology are well known and have general application and can be used to elucidate various problems in molecular genetics, including gene expression, gene linkage, and gene alteration (eg, M. Chee et al. ., Science, Vol 274, pp
610-613 (1996)).

Diagnostic assays include the detection of mutations in the IGS1 gene by the methods described.
Above all, a method for diagnosing or determining susceptibility to the above-mentioned "diseases" is provided. Diagnostic assays may be used to determine susceptibility to neuromedicine and CNS disorders, particularly movement disorders such as tics, tremors, Tourette's disease, Parkinson's disease, Huntington's disease, dyskinesias, dystonia and convulsions, according to the methods described above. The present invention provides a method for diagnosing or detecting the detection of mutations in

Furthermore, among others, the above-mentioned “disease” can be diagnosed by a method comprising determining from a sample derived from a patient from abnormally decreased or increased levels of IGS1 polypeptide or IGS1 mRNA. Aberrant reduction of IGS1 polypeptide or IGS1 mRNA from samples derived from patients, especially neuromedicine and CNS disorders, in particular movement disorders such as tics, tremors, Tourette's disease, Parkinson's disease, Huntington's disease, movement disorders, dystonia and convulsions or Diagnosis can be made by a method comprising determining from increased levels.

Reduced or increased expression uses any of the methods well known in the art for quantifying polynucleotides, such as PCR, RT-PCR, RNase protection, Northern blotting and other hybridization methods. Can be measured at the RNA level. Assay techniques that can be used to determine the level of a protein, eg, IGS1, in a sample derived from a host are well known to those of skill in the art. Such assay methods include radioimmunoassays, competitive-binding assays, Western Blot analysis and ELISA assays.

In another aspect, the present invention relates to a diagnostic kit for susceptibility to, among other things, a “disease” or one of the above “diseases”, in particular the invention relates to neuromedicine and CNS disorders, in particular exercise. A diagnostic kit for disorders such as tics, tremor, Tourette's disease, Parkinson's disease, Huntington's disease, movement disorders, dystonia and convulsions.

This kit comprises (a) an IGS1 polynucleotide, preferably the nucleotide sequence of SEQ ID NO: 1,
Or a fragment thereof, and / or (b) a nucleotide sequence complementary to that of (a), and / or (c) an IGS1 polypeptide, preferably the polypeptide of SEQ ID NO: 2, or a fragment thereof, and / or (D) an antibody to the IGS1 polypeptide, preferably an antibody to the polypeptide of SEQ ID NO: 2, and / or (e) a RAMP polypeptide necessary for the relevant biological or antigenic properties of the IGS1 polypeptide. May be

In any of such kits, (a), (b), (c), (d) or (e)
It is recognized that) may comprise essential ingredients. Chromosome Assays The nucleotide sequences of the present invention are also valuable for chromosome identification. The sequence is specifically targeted to and can hybridize with a particular location on an individual human chromosome. Mapping the related sequences of chromosomes according to the present invention is an important first step in correlating these sequences with gene-related diseases. The physical location of a sequence on a chromosome can be correlated with genetic map data if the sequence has been localized to a precise chromosomal location. Such data can be found, for example, in V. McKusick, Mendelian Inheritance.
Found in Man (available online from Johns Hopkins University Welch Medical Library). The relationship between the gene localized to the same chromosomal region and the disease is then identified by linkage analysis (co-inheritance of physically adjacent genes).

Differences in cDNA or genomic sequence between affected and unaffected individuals can also be determined.
If the mutation is found in some or all of the affected individuals but not in normal individuals, the mutation may be the causative agent of the disease. Antibodies The polypeptides of the present invention or fragments thereof or analogs thereof, or cells expressing them when required together with a related RAMP are immunogens which produce antibodies immunospecific for the IGS1 polypeptide. May be used as. The term "immunospecificity" means that the antibodies have an affinity for the polypeptides of the invention that is essentially greater than their affinity for other related polypeptides of the prior art.

Antibodies generated against IGS1 polypeptide may be obtained by administering the polypeptide or epitope-bearing fragment, analog or cell to an animal, preferably a human, via conventional protocols. For the production of monoclonal antibodies, any technique that provides antibodies produced by continuous cell-like culture may be used. An example is the hybridoma technology (Kohler, G. and Milstein, C., Nature (1975) 256:
495-497), trioma technology, human B-cell technology (Kozbor et al., Immunology Tod
ay (1983) 4:72) and EBV-hybridoma technology (Cole et al., MONOCLONAL
ANTIBODIES AND CANCER THERAPY, pp77-96, Alan R. Liss, Ic., 1985).

The antibodies described above may be used to isolate or identify clones expressing the polypeptide or to purify the polypeptide by affinity chromatography.

An antibody against the IGS1 polypeptide itself, or IGS1 polypeptide-RA
Antibodies to the MP complex may be used to treat, among other things, the above "diseases". In particular, antibodies against the IGS1 polypeptide itself, or antibodies against the IGS1 polypeptide-RAMP complex, are useful in neuromedicine and CNS disorders, especially movement disorders such as tics, tremors, Tourette's disease, Parkinson's disease, Huntington's disease, dyskinesias. , May be used to treat dystonia and convulsions. Animals Another aspect of the invention relates to non-human animal-based systems that behave as models for disorders resulting from aberrant expression or activity of IGS1. Model systems based on non-human animals may be used to further characterize the activity of the IGS1 gene. Such systems are associated with diseases based on IGS1, such as the “diseases” described above, among others.
May be used as part of a screening strategy designed to identify compounds that can be treated. In particular, this system is based on IGS1-based neuromedicine and CNS
Disorders, especially movement disorders such as tics, tremor, Tourette's disease, Parkinson's disease,
It may be used as part of a screening strategy designed to identify compounds that can be used to treat Huntington's disease, movement disorders, dystonia and convulsions.

In this way, animal-based models may be used to identify drug compounds, treatments and interventions that are effective in treating disorders of aberrant expression or activity of IGS1. In addition, such animal models provide LD ₅₀ and E in animal patients.
It may be used to measure D ₅₀ . These data may be used to determine the in vivo efficacy of potential IGS1 disorder treatment.

Animal-based model systems of IGS1-based disorders based on aberrant IGS1 expression or activity may include both non-recombinant animals as well as recombinant engineered transgenic animals.

Animal models for IGS1 disorders may include, for example, genetic models. I
Animal models exhibiting GS1-based disorder-like syndromes have been engineered using, for example, IGS1 sequences, such as those described above, in combination with techniques well known to those skilled in the art for producing transgenic animals. May be. For example, IGS
1 sequence may be introduced into the genome of the animal concerned and overexpressed and / or deleted, or if the endogenous IGS1 sequence is present, these may be overexpressed or deleted. Alternatively, it may be disrupted to under-express or inactivate IGS1 gene expression.

In order to overexpress or lack expression of the IGS1 gene sequence, the coding portion of the IGS1 gene sequence drives high level gene expression or expression in cell types in which the gene is not normally expressed in the animal species involved. May be linked to a regulatory sequence capable of Such regulatory regions are well known to those of skill in the art,
It may be used without undue experimentation.

Due to underexpression of the endogenous IGS1 gene sequence, such sequences are isolated and engineered to render the endogenous IGS1 gene allele inactive when reintroduced into the genome of the animal concerned. Or "knocked out". Preferably, the engineered IGS1 gene sequence is introduced via gene targeting methods such that the endogenous IGS1 sequence is disrupted upon integration of the engineered IGS1 gene sequence into the animal genome, for example.

Animals of all species, including but not limited to mice, rats, rabbits, squirrels, guinea pigs, pigs, small pigs, goats, and non-human primates, such as baboons, monkeys, and apes, are treated with IGS1. It may be used to make animal models of related disorders.

Any technique known in the art may be used to introduce the IGS1 transgene into an animal to create a founder strain of transgenic animals. Such a technique is described in Pronuclear Microinjection (Hoppe, PC and Wagner, TE 1989, US Patent (US)).
No. 4,873,191); Retrovirus-mediated gene transfer into germline (van der P
utten et al., Proc. Natl. Acad. Sci. USA 82: 6148-6152, 1985); Gene targeting method in embryonic stem cells (Thompson et al., Cell 56: 313-321, 1989); Embryonic electricity Perforation (L
o, Mol. Cell. Biol. 3: 1803-1814, 1983); and sperm-mediated gene transfer (Lavitra
no et al., Cell 57: 717-723, 1989) and the like, but are not limited thereto. A review of such techniques can be found in Gordon, Transgenic Animals, Intl. Rev. Cytrol. 115
: 171-229, 1989.

The present invention provides transgenic animals that carry the IGS1 transgene in all of these cells, as well as animals that carry the transgene in some but not all cells, ie, mosaic animals (eg, Jakobovitz. , Curr. Biol. 4:
761-763, 1994). The transgene is a single transgene,
Alternatively, it may be incorporated into a concatemer, such as a head-to-head tandem or a head-to-tail tandem. Transgenes are described, for example, in the teaching of Lasko et al. (Lasko, M. et al., Proc. N.
Atl. Acad. Sci. USA 89: 6232-6236, 1992), and may be selectively introduced into and activated within a particular cell type.

The regulatory sequences required for such cell type-specific activation will depend on the particular cell type involved and will be apparent to those of skill in the art.

If it is desired to integrate the IGS1 transgene within the chromosomal site of the endogenous IGS1 gene, gene targeting methods are preferred. In summary, when using this technique, a vector containing a nucleotide sequence homologous to the relevant endogenous IGS1 gene (eg, the nucleotide sequence of the mouse IGS1 gene) is transferred to an endogenous endogenous sequence via homologous recombination with a chromosomal sequence. It is designed for the purpose of integrating within the nucleotide sequence of the IGS1 gene or gene allele and destroying its function. The transgene is
It may be selectively introduced into a particular cell type, which inactivates an endogenous gene of interest only within that cell type, which is for example according to the teaching of Gu et al. (Gu, H. et a
L., Science 265: 103-106, 1996). The regulatory sequences required for such cell type-specific inactivation depend on the particular cell type involved and will be apparent to those of skill in the art.

Once a transgenic animal has been produced, recombinant IGS1 gene and protein expression may be assayed using standard techniques. The first screening is
Southern blot analysis or PCR techniques may be used to analyze animal tissue to assay whether transgene integration has occurred. The level of IGS1 transgene mRNA expression in the tissues of transgenic animals was determined using techniques including, but not limited to, Northern blot analysis of tissue samples obtained from the animals, in situ hybridization techniques, and RT-PCR. You may evaluate. Samples of target gene-expressing tissue may be evaluated immunocytochemically using antibody specificity for the target gene transgene product of interest. IG expressing IGS1 gene mRNA or IGS1 transgene peptide at a level that can be easily detected
S1 transgenic animals (immunocytochemically detected using antibodies to target gene product epitopes) are then subsequently evaluated to identify those animals that exhibit a characteristic IGS1-based disorder group. May be.

Once an IGS1 transgenic founder animal (ie, an animal that expresses the IGS1 protein in the cells or tissues of interest, and which preferably exhibits a syndrome of IGS1-based disorders) is produced in a particular animal. These may be bred, inbred, outbred, or crossed to produce colonies. Examples of such breeding strategies are concerned at high levels due to the effect of founder outcrosses with more than one integration site to establish isolated strains, the additive expression of each IGS1 transgene. In order to eliminate the possible need for inbreeding of the isolated strains to produce a composite IGS1 transgenic expressing the IGS1 transgene, increased expression and screening of animals by DNA analysis, to a given integration site. Crosses of heterozygous transgenic animals to produce heterozygous animals, crosses of isolated homozygous strains to produce complex heterozygous or homozygous strains, IGS1 transgene expression and alleles for the development of IGS1-like syndromes Including the breeding of animals to various inbred backgrounds to study the effects of genetic modification Limited et al. Is not. One such method is the crossing of an IGS1 transgenic founder animal with a wild-type strain to generate an IGS1-related disorder-like syndrome, such as the F1 generation exhibiting the above. The F1 generation may then be inbred to generate a heterozygous strain if the heterozygous target gene transgenic animal is viable. Vaccine Another aspect of the invention relates to a method for inducing an immunological response in a mammal,
This involves administering (eg, inoculating) to a mammal an IGS1 polypeptide or fragment thereof, optionally together with a RAMP polypeptide suitable for generating an antibody and / or T cell immune response, among other things as described above. Comprising protecting the animal from one of the "diseases".

Yet another aspect of the invention relates to a method for inducing an immunological response in a mammal for inducing an immunological response that produces antibodies to protect the animal from disease. , Providing the IGS1 polypeptide via a vector that directs the expression of the IGS1 polynucleotide in vivo.

Another aspect of the present invention relates to an immunological / vaccine formulation (composition) that induces an immunological response in a mammal to an IGS1 polypeptide when introduced into a mammalian host, wherein The composition comprises an IGS1 polypeptide or IGS1 gene. Such immunological / vaccine formulation (composition) may be either a therapeutic immunological / vaccine formulation or a prophylactic immunological / vaccine formulation. The vaccine formulation may further comprise a suitable carrier. IGS1
Since the polypeptide is broken down in the stomach, it is preferably administered parenterally (including subcutaneous, intramuscular, intravenous, intradermal etc. injection). Formulations suitable for parenteral administration include sterile aqueous and non-aqueous injection solutions that may contain anti-oxidants, buffers, bacteriostats and solutes to make the formulation isotonic with the blood of the recipient, and suspensions. Includes aqueous and non-aqueous sterile injectable solutions, which may include clouding or thickening agents. The formulations may be presented in unit-dose or multi-dose containers, such as sealed ampoules and vials, and may be stored in lyophilized form, just prior to use with the addition of a sterile liquid carrier. Vaccine formulations may include adjuvant systems to enhance the immunogenicity of the formulation, such as oil-in-water systems and other systems known in the art. The dose depends on the activity of the vaccine and can be easily determined by routine trials. Screening Assays IGS1 polypeptides of the invention may be used in a screening process for compounds that bind receptors and activate (agonists) or inhibit activation (antagonists) of receptor polypeptides of the invention. Thus, the polypeptides of the present invention may be used to assess the binding of ligands within small molecule substrates and eg cells, cell-free preparations, chemical libraries, and natural product mixtures. These substrates and ligands may be natural substrates and ligands or structural or functional mimetics.

IGS1 polypeptides are responsible for biological functions including pathology. Therefore,
It would be desirable to discover compounds or agents that stimulate IGS1 on the one hand and which can inhibit the function of IGS1 on the other. In general, agonists are especially
It is used for the purpose of treatment and prevention of "disease". In particular, the agonists are used for the purpose of treating and preventing neuromedical and CNS disorders, in particular movement disorders such as tics, tremors, Tourette's disease, Parkinson's disease, Huntington's disease, movement disorders, dystonia and convulsions.

Antagonists may be used for various therapeutic and prophylactic purposes, inter alia for the pathologies of the above-mentioned "diseases". In particular, the antagonists are neuromedical and CN
It may be used for various therapeutic and prophylactic purposes for S disorders, in particular movement disorders such as tics, tremors, Tourette's disease, Parkinson's disease, Huntington's disease, movement disorders, dystonia and convulsions.

In general, such screening procedures involve cells suitable for production which express the receptor polypeptides of the invention on their surface and, if necessary, co-express RAMP on their surface. Such cells include cells from mammals, yeast, Drosophila or E. coli. Cells expressing the receptor (or cell membranes containing the expressed receptor) are then contacted with a test compound and binding or stimulation or inhibition of a functional response is observed.

One screening technique is to measure cells that express the receptor of the invention (eg, transfected cells) in a system that measures extracellular pH, intracellular pH, or intracellular calcium changes caused by receptor activation. CHO cells). In this technique, the compound may be contacted with cells expressing the receptor polypeptide of the invention. Second messenger responses, such as signal transduction, pH changes, or changes in calcium levels are then measured to determine if the potential compound activates or inhibits the receptor.

Another method involves screening for receptor inhibitors by determining the regulation of receptor-mediated signals such as cAMP accumulation and / or adenylate cyclase activity. Such a method involves transfecting a eukaryotic cell with a receptor of the present invention to express the receptor on the cell surface. The cells are then exposed to an agonist for the receptor of the invention in the presence of the potential antagonist. If the potential antagonist binds the receptor and thereby inhibits receptor binding, the agonist-mediated signal will be modulated.

Another method for detecting agonists or antagonists for the receptors of the present invention is the yeast-based technique described in US Pat. No. 5,482,835.

The assay may be merely test binding of a candidate compound, where adhesion to cells bearing the receptor uses a label directly or indirectly associated with the candidate compound, or involves competition with a labeled competitor. Detected by assay. In addition, these assays may test whether a candidate compound results in a signal generated by activation of the receptor, using a detection system suitable for cells bearing the receptor on their surface. Inhibitors of activation are generally assayed in the presence of a known agonist and observing the effect on agonist activation by the presence of a candidate compound.

Further, the assay simply comprises mixing a solution containing the IGS1 polypeptide with a candidate compound to form a mixture, measuring IGS1 activity in the mixture, and comparing the IGS1 activity of the mixture to a standard. May include.

IGS1 cDNA, protein and antibodies to the protein are intracellular I
It may be used to form an assay to detect the effect of added compounds on the production of GS1 mRNA and protein. For example, an ELISA may be constructed to measure secretion or cell-associated levels of IGS1 protein using monoclonal and polyclonal antibodies by standard methods known in the art, and which may be used to engineer appropriately engineered cells or IGS1 from the organization
Can be used to discover agents (also called antagonists or agonists, respectively) that would inhibit or promote the production of Standard methods for conducting screening assays are well known in the art.

Examples of potential IGS1 antagonists are antibodies or in some cases oligonucleotides or proteins closely related to ligands of IGS1, eg fragments of the ligand, or small molecules that bind to the receptor but do not react. And block the activity of the receptor.

Accordingly, in another aspect, the invention provides a screening kit for identifying agonists, antagonists, ligands, receptors, substrates, enzymes and the like for an IGS1 polypeptide, or (a) an IGS1 polypeptide, preferably a sequence. No. 2; (b) recombinant cell expressing IGS1 polypeptide, preferably SEQ ID NO: 2; (c) cell membrane expressing IGS1 polypeptide, preferably SEQ ID NO: 2;
Or (d) an antibody against an IGS1 polypeptide, preferably a compound that reduces or promotes the production of an IGS1 polypeptide comprising the one of SEQ ID NO: 2.

It will be appreciated that within any such kit, (a), (b), (c) or (d) may comprise essential components. Prophylactic and Therapeutic Methods The present invention provides methods for treating abnormal conditions associated with both excess and / or inadequate amounts of IGS1 activity.

If the activity of IGS1 is excessive, several methods are available. One way is
The above-described inhibitory compounds (antagonists), together with a pharmaceutically acceptable carrier, by blocking the binding of the ligand to IGS1 or R
Comprising administering to the patient an amount effective to inhibit activation by inhibiting its interaction with the AMP polypeptide or a second signal, thereby alleviating the abnormal condition.

In another method, IGS1 is still able to compete with endogenous IGS1 and still bind ligand
Soluble forms of the polypeptide may be administered. A typical embodiment of such a competitor comprises a fragment of the IGS1 polypeptide.

In yet another method, expression of the gene encoding the endogenous IGS1 can be inhibited using expression blocking techniques. Such known techniques include the use of internally produced or separately administered antisense sequences. For example O'Conner, J. Neurochem. (1991) 5
6: 560 in Oligodeoxynucleotides as Antisense Inhibitors of Gene Expressio
n, CRC Press, Boca Raton, Florida USA (1988). Alternatively, an oligonucleotide can be provided that forms a triple helix with the gene. For example, Lee et al., Nuclei
c Acids Res. (1979) 6: 3073; Cooney et al., Science (1988) 241: 456; Derva
See n et al., Science (1991) 251: 1360. These oligomers can be administered per se or the relevant oligomers can be expressed in vivo. The synthesized antisense or triple helix oligonucleotides may have modified bases or modified backbones. Examples of the latter include methylphosphonate, phosphorothioate or peptide nucleic acid backbones. Such scaffolds have been incorporated into antisense or triple helix oligonucleotides to provide protection from nuclease degradation, which is well known in the art. Synthetic antisense and triple helix molecules with these or other modified backbones form part of the present invention.

Furthermore, expression of the IGS1 polypeptide may be avoided by using a ribozyme specific for the IGS1 mRNA sequence. Ribozymes are catalytically active RNAs that can be natural or synthetic (eg, Usman et al., Curr. Opin. Struct. Bi.
ol. (1996) 6 (4), 527-33). Synthetic ribozymes can be designed to specifically cleave IGS1 mRNA at selected positions, thereby avoiding translation of IGS1 mRNA into a functional polypeptide. Ribozymes may be synthesized using the natural phosphate ribose backbone and natural bases, such as those normally found in RNA molecules. Alternatively, ribozymes may be synthesized using non-natural backbones, such as to 2'-O-methyl RNA, and may contain modified bases to provide protection from ribonuclease degradation.

To treat abnormal conditions associated with low expression of IGS1 and its activity,
Various methods are also available. One method is to administer a therapeutically effective amount of a compound that activates IGS1, ie, an agonist as described above, to a patient together with a pharmaceutically acceptable carrier, thereby alleviating the abnormal condition. Comprises.
Alternatively, gene therapy may be utilized to effect endogenous production of IGS1 by relevant cells within the patient. For example, the polynucleotides of the invention may be engineered for expression in the replication defective retroviral vector discussed above. The retroviral expression construct is then isolated and the RN encoding the polypeptide of the invention.
It may be introduced into packaging cells transduced with a retroviral plasmid vector containing A, which causes the packaging cells to produce infectious viral particles containing the gene of interest. These producer cells may be administered to a patient for engineering cells in vivo and expression of the polypeptide in vivo. For an overview of gene therapy, see Human Molecular Genetics, Strachan.
T. and Read AP, BIOS Scientific Publishers, Ltd (1986), Chapter 20 of “Gene Therapy and Other Molecular Gene-Based Therapy” (Chapter 20, Gene Th
erapy and other Molecular Genetic-based Therapeutic Approaches) and references therein.

Any of the above methods of treatment may be performed, for example, on a mammal such as a dog, cat, cow, horse,
It may be applied to any patient in need of treatment including rabbits, monkeys, and most preferably humans. Formulation and Administration Peptides, eg, soluble forms of IGS1 polypeptides, and agonist and antagonist peptides or small molecules, may be formulated in combination with a suitable drug carrier. Such formulations comprise a therapeutically effective amount of the polypeptide or compound and a pharmaceutically acceptable carrier or excipient. The formulation should suit the mode of administration and is well within the skill of those in the art. The present invention further relates to pharmaceutical packages and kits comprising one or more containers filled with one or more of the ingredients of the above compositions of the invention.

The polypeptides and other compounds of the invention may be used alone or in combination with other compounds, eg therapeutic compounds.

Preferred forms of systemic administration of the pharmaceutical composition include injection, typically intravenous injection.
Other injection routes can be used, such as subcutaneous, intramuscular, or intraperitoneal. Another means of systemic administration includes transmucosal or transdermal administration using penetrants such as bile salts or fusidic acid or other detergents. In addition, oral administration would be possible when properly formulated for oral and encapsulated formulations.

The dosage range required will depend on the choice of peptide or compound, the route of administration, the nature of the formulation, the nature of the patient's medical condition, and the judgment of the attending physician. A suitable dose is 1-100
It is in the range of μg / kg (patient weight). However, a wide variation in the required dosage is also to be expected in view of the different efficiencies of the compounds used and the different routes of administration. For example, oral administration would be expected to require higher doses than administration by intravenous injection. These dose level changes can be adjusted using standard empirical practices for optimization, which are well understood in the art.

The peptides used for treatment can also be generated endogenously in the patient in a treatment modality, often referred to as “gene therapy” as described above. Thus, for example, cells from a patient may be polynucleotides, such as DNA or RNA.
May be engineered to encode the polypeptide in vitro and with, for example, a retroviral plasmid vector. The cells are then introduced into the patient.

The following examples are only intended to illustrate the invention in more detail, and therefore these examples are not to be considered as limiting the invention in any way.

[0102]

【Example】

Example 1. Cloning of CDNA Encoding a Novel G Protein-Coupled Receptor Example 1a. Homologous PCR Cloning of a Genomic Fragment Encoding a Novel G Protein-Coupled Receptor (GPCR) A PCR-based homology cloning strategy to isolate a partial genomic DNA sequence encoding a novel G protein-coupled receptor Used for. The following advance (F
11) and a reverse (R13) degenerate PCR primer were added to the intracellular loop second (TM3 / 12) at the boundary of transmembrane domain 1 (MT1) and transmembrane domain 3 in the conserved region of the neurotensin receptor gene, respectively. It designed using each. F11 (TM1): 5′-CATCTTCGTCGTCCGGCAC (A, C, G or T) G (C or T) (A, C, G or T) GG (A, C, G or T) AA-3 ′ (SEQ ID NO: 3) R13 (TM3 / 12): 5'-GGGTGGGCAGATGGCCA (A or G) (A or G) (C or T) A (A, C, G or T) C (C or T) (C or T) TC -3 ′ (SEQ ID NO: 4) Furthermore, a 3 ′ blocked oligo primer (HNTR1F1STOP) was designed. HNTR1F1STOP: 5'-ACGGTGGGCAACACGGTGACCGGCGTTT-3'-3'-
The dA (SEQ ID NO: 5) 3'blocked primer is specific for the human neurotensin receptor (NTR1) cDNA within the TM1 coding region and partially overlaps (and competes) with the degenerate forward primer. is doing. This 3'end is blocked by a 3'-deoxyadenosine group, avoiding polymerase-catalyzed extension (Euro
gentic, Belgium, catalog number OL-0401-0302).

[0103]   The PCR reaction was performed in a volume of 60 μl and 100 ng human genomic DNA (Clont
ech), 6 μl 10x PCR buffer II (100 mM Tris-HCl pH8.
． 3; 500 mM KCl, Perkin Elmer), 3.6 μl 25 mM MgCl ₂  , 0.36 μl dNTPs (25 mM of each dNTP), 1.5 units
AmpliTaq^TMPolymerase (Perkin Elmer), degenerate forward and backward plies
30 pmol of each mer and 100 pico of 3'blocked primer
It contained moles. The reaction tube is heated at 94 ° C for 2 minutes and then denatured (94 ° C, 30 ° C).
Second), annealing (55 ° C, 1 minute, touchdown-0.25 ° C / cycle)
) And stretching (72 ° C., 1 minute) for 20 cycles, followed by further denaturation (94 ° C.,
2 seconds of annealing (50 ° C, 1 minute) and stretching (72 ° C, 1 minute) for 30 seconds
0 cycles were performed. Finally, the reaction tube was heated at 72 ° C. for 5 minutes. PCR reaction product
The material was size classified on a 2% agarose gel and stained with ethidium bromide.
It was Fragment of expected size (± 300 bp) with Qiaex-II^TMPurification kit (Q
iagen Inc.) and purified from the gel and supplied by the supplier (pGEM-T kit, Promega).
Was ligated into the pGEM-T plasmid according to the procedure recommended by. like this
Competent E. coli SURE^TM2 bacteria
(Stratagene) was used to transform.

The transformed cells were treated with ampicillin (100 μg / ml), IPTG (0.5
mM) and X-gal (50 μg / ml) were plated on LB agar plates. Colonies were lifted onto Hybond N + membranes (Amersham) and the DNA was denatured and the microwave oven method of Buluwela et al.
It was fixed according to ds Research 17, p452; 1989). Colony lift denatured Chu
65 in rch buffer (0.5 M phosphate, 7% SDS, 10 mM EDTA)
Prehybridize for 2 hours at 0 ° C., then equimolar amount of 2 × 10 ⁶ cpm of ³² P-labeled human neurotensin receptor 1 and 2 cDNA probes (NTR1 / 2).
Hybridized overnight at 65 ° C in the same buffer containing / ml. A cDNA probe containing the entire coding sequences of human NTR1 and NTR2 was [α- ³² P] d
Through random initiation recombination of CTP, specific activity up to> 10 ⁹ cpm / μg, P
Radiolabeled using the lime-It II kit ^™ (Stratagene) according to the instructions provided by the supplier. Hybridize the filter with high stringency (2
x 30 minutes, room temperature, 2x SSC / 0.1% SDS, then 40 minutes, 65 x C 0.1x SSc, 2 washes in 0.1% SDS) and autoradiography overnight. . A large number of random white colonies showing no hybridization signal after high stringency washes were selected for DNA sequence analysis.

DNA sequencing reactions were performed using the ABI Prism ^™ BigDye terminator cycle sequencing reaction kit (PE-ABI). Cycle sequencing reaction products were purified via EtOH / NaOAc precipitation and loaded on an ABI373 automated sequencer. Two nearly identical clones (HNT642 and HNT
NT768), which was thought to encode part of a novel member of the GPCR family. We named this new GPCR IGS1.

[0106]

[Table 3]

Example 1b. Cloning of a cDNA fragment containing the complete IGS1 coding sequence The complete coding sequence of the IGS1 cDNA was obtained via rapid amplification of the cDNA ends (RACE analysis). 5'- and 3'-RACE PCR was performed using Ma
Marathon ^™ cDNA on rathon-Ready ^™ human brain cDNA (Clontech, no.7400-1)
Adapter primer provided with A amplification kit (Clontech, K1802-1) (
AP1, SEQ ID NO: 6) and IGS1 specific primer IP11261 (3'R
ACE, SEQ ID NO: 9) and IP11262 and IP11263 (5'RA
CE, SEQ ID NOS: 10 and 11, respectively) and based on the DNA sequences of clones HNT642 and HNT768 (FIG. 1). The nested RACE PCR was then run on adapter primer 2 (AP2; SEQ ID NO: 7) and I
GS1 specific nested primer IP11260 (3'RACE, SEQ ID NO: 8)
) And IP11515 and IP11516 (5′RACE, SEQ ID NOS: 13 and 14, respectively). Primary and nested PCR RACE reactions were performed according to the instructions in the Marathon-Ready ^™ cDNA User Manual provided by Clontech. Nested PCR RACE products were separated on a 1% agarose gel and stained with EtBr. Gels were blotted onto Hybond N ⁺ membranes and hybridized overnight at 65 ° C. with the ³² P-labeled insert of clone HNT642 in Church hybridization buffer.

Southern blot analysis of both AP2 / IP11515 and AP2 / IP11516 5'RACE Nest PCR reactions showed several positive bands (± 2
00bp, ± 250bp, ± 330bp, ± 360bp, ± 400bp and ±
700 bp). Each of these bands was purified from the gel and pGEM-T
Cloned into a plasmid vector (respective PCR fragments from IP11515 and IP11516 Nest 5'RACE reactions were pooled prior to cloning). 3-4 random colonies from each fragment were sequenced (
= Clone HNT1393-1414) (Fig. 1).

The nested AP-2 / IP11260 3'-RACE PCR reaction showed several bands. The largest 3'nested RACE PCR fragment (± 1,550 bp) that hybridized with the IGS1 probe was purified from gel and pGEM-T (P
romega) and used to transform adapted E. coli SURE II cells. IGS1-specific transformants from this ligation were cloned into HNT
Identified after colony hybridization with 642 ³² P-labeled inserts. The colony blot was hybridized to the previously designated probe and high stringency (0.1xSSC 0.1% SDS, 65 ° C for 30 minutes).
Washed with. Hybridization screening of the 3'RACE nested PCR library produced 3 positive clones. These two (HNT14
13-1414) was sequenced.

In two additional experiments, three additional 3'RACE cDNA clones (HB46
86, HB4687 and HB4688) are Marathon-Ready ^™ human brain cDNA (C
lontech) according to the procedure described in the manual provided by the supplier (Clontech PT1156-1). In one experiment, the primary 3'RACE reaction (IG
The product from S1 specific primer IP11261 and adapter primer AP1) was obtained from hemi-nested primer pair IP11260.
/ IP11261 (SEQ ID NOS: 8 and 16, respectively). This generated the expected ± 1400 bp fragment, which was purified from gel and pGE
When cloned into the MT plasmid vector, clones HB4686 and HB4686
B4687 was generated. In a separate experiment, the primary 3'RACE PCR product (I
Nested primer pair IP11260 / I with GS1 specific primer IP11264 (SEQ ID NO: 15) and adapter primer AP1)
It was reamplified using P11261. The ± 1400 bp fragment resulting from this reaction was purified and cloned into the pGEM-T plasmid vector, resulting in clone HB4688.

All isolated IGS1 cDNA clones could be fully sequenced and constructed within a single contig (FIG. 1). A portion of this contiguous cDNA determined from at least four independent cDNA clones is set forth herein as IGS1 DNA (SEQ ID NO: 1). Translation of this contig revealed a long open reading frame, a 508 amino acid protein was predicted, which showed good homology to the GPCR protein (IGS1PROT; SEQ ID NO: 2). Computer-aided homology search of DNA sequences of IGS1 contigs against expressed sequence tag database (dbase) (Blastn; Altschul SF et al., (1997), Nucleic
Acids Res. 25: 3389-3402) is EST 20889 (accession number AA31).
8717) and the presence of EST accession number AI672141, both of which overlap the 3'end of the IGS1 contig but are outside the IGS1 open reading frame (FIG. 1). Example 1c. Isolation of a continuous cDNA fragment containing the complete IGS1 coding sequence A continuous IGS1 cDNA clone was cloned into clones HNT1398 and HNT14.
Made via overlap-PCR on 13 templates. HNT1398 plasmid DNA
And 100 ng of HNT1413 plasmid were PCR amplified (denaturation) in separate reactions (50 μl) with primer pairs IP12264 / IP11264 (SEQ ID NOs: 18 and 12, respectively) and IP11260 / IP12262 (SEQ ID NOs: 8 and 11, respectively). 30 PCR cycles of [94 ° C., 30 seconds], annealing [60 ° C., 30 seconds] and stretching [72 ° C., 1 minute], using Expand ^™ High Fidelity PCR system [Boehringer]). A 1 μl aliquot of each PCR reaction was combined and the primer pair IP12264 / I
P12261 was used and reamplified under the same conditions. This overlap-PCR reaction was
A band of 1730 bp was generated, which was purified from gel and ligated into the pGEM-T plasmid vector. The recombinant plasmid was used to transform the adapted E. coli DH5αF ′ strain. Transformed cells with ampicillin (100 μg
/ Ml) on LB agar plates. Plasmid DNA was generated from a large number of random colonies and the insert size was determined via restriction digestion. Three clones containing the ± 1730 bp insert were sequenced. Clone H
The sequence of B4693 matched perfectly with the consensus IGS1 cDNA sequence (see Figure 1). The cell line containing the plasmid HB4693 was treated with ampicillin at 100 μg / m 2.
replated on LB agar plates containing 1 and then recloned, and Innogenetics strain list (ICCG # 4297) and Centraalbureau voo
r Schimmelculturen (CBS) (Baarn, The Netherlands) (Deposit No. CBS102049)
) Both. The plasmid DNA made from the recloned isolate was resequenced and found to be identical to the previously determined consensus sequence.

Note: We then used the primer IP12262 sequence to clone HNT141.
It was found that the amplicon could not be produced from the HNT1413 template because it was not included in the insert sequence of No. 3 and as a result. Therefore, we presume that successful amplification of overlapping fragments occurred via direct duplication between the HNT1413 plasmid DNA (which was brought into the overlapping PCR reaction tube) and the amplicon generated from the HNT1398 template. Example 2. Northern and "MTE Array" Analysis of IGS1 Example 2a. Construction of pcDNA3.1 (+) huIGS1 expression vector 5 μg of pcDNA3.1 (+) (Invitrogen) was cleaved with HindIII (3 hours, 37 ° C.) and in the presence of dNTPs (0.25 mMfc). Blunted using T4 polymerase and analyzed on gel. The linearized DNA was eluted from the gel (using the Qiaex II extraction kit (Qiagen)) and dissolved in 40 μl H ₂ O. The DNA was digested with NotI and analyzed again on the gel.
The resulting 5364 bp vector fragment was eluted from the gel using the Qiaex II gel extraction kit and dissolved in 40 μl H ₂ O. 5 μl were analyzed on a gel for size, quantity and purity.

The human IGS1 coding sequence was obtained after NaeI / NotI digestion (3 hours, 37 ° C.) of 5 μg pGEM-ThulGS1 plasmid (ICCG # 4297). Digestion is 400bp, 1629bp by agarose gel electrophoresis.
And resulted in 3 fragments of 2702 bp. The 1629 bp fragment was gel (QiaexI
Eluted from I) and redissolved in 40 μl H ₂ O. 5 μl was analyzed on the gel.

1 μl of HindIII digested pcDNA3.1 (+) vector, insert 3
μl and 16 μl H ₂ O were added to Ready-To-Go ligase tubes (T4 DNA ligase, Amersham Pharmacia Biotech) and incubated for 1 hour at room temperature. 2 μl of the ligation mixture was used to transform the chemically adapted DH5αF ′ strain. 200 μl of transformed bacteria were plated on LB plates (100 μg ampicillin / ml) and incubated overnight at 37 ° C. 16 random colonies were picked and grown in 3 ml of LB medium containing ampicillin.
Plasmid DNA was generated using the BioRobot ^™ 9600 nucleic acid purification system (Qiagen) and analyzed via restriction analysis with NotI, PstI and SphI restriction enzymes. DNA from one colony with the correct restriction pattern was partially sequenced to confirm the insertion point and found to have the expected sequence. Partially sequenced colonies were labeled as Innogenetics strainlist (ICCG # 4350).
And large amounts of DNA (MegaPrep, Qiagen 500 kit) were prepared from the deposited strain. Sequence analysis of this large scale DNA prep (500 μl of 3 μg / μl) confirmed the expected sequence. Example 2b. MTE (multi-tissue expression) array analysis 25 ng human IGS1 DNA (pcDNA3.1huIGS1 (ICCG #
4350) to the 1093 bp AatII insert) from (α- ³² P) -dCTP.
Was labeled with. The labeled probe was purified using a Micro Bio-Spin P-30 column (BioRad). 16 × 10 ⁶ cpm of labeled huIGS1 cDNA probe was mixed with 30 μg of C ₀ t-1 DNA, 150 μg of sheared herring sperm DNA and 50 μl 20 × SSC to a total volume of 200 μl, and 5
Heated for 95 minutes at 95 and then incubated for 30 minutes at 68 ° C. This mixture was added to 5 ml of Express Hyb solution and human multiple tissue expression (MTE) array
Evenly distributed over (Clotech # 7775-1). The arrays were hybridized overnight at 68 ° C. Blots were rinsed 4 times for 20 minutes at 65 ° C, 2X SSC / 1% SDS and 2 times for 20 minutes at 55 ° C, 0.1X SSC / 0.5% SDS.
Blots were autoradiographed using X-ray film.

Hybridization of the IGS1 probe on the MTE array showed a strong signal only on the caudate and putamen (FIG. 2). Example 2c. Northern blot analysis 25 ng human IGS1 DNA (pcDNA3.1huIGS1 (ICCG #
4350) to the 1093 bp AatII insert) from (α- ³² P) -dCTP.
Was labeled with. The labeled probe was purified using a Micro Bio-Spin P-30 column (BioRad). 8 × 10 ⁶ cpm of labeled huIGS1 cDNA probe was denatured for 5 minutes at 95 ° C. and 5 ml of Express Hyb solution was added and placed on human brain MTN blot II or IV (Clotech # 7775-1 and # 7769-1 respectively). Distributed evenly. The blot was hybridized overnight at 68 ° C. Blots for 10 minutes at room temperature 4 times in 2X SSC / 0.05% SDS and 40 minutes
Rinse twice in 0.1X SSC / 0.1% SDS at 50 ° C. X blot
Autoradiography was performed using a line film.

Hybridization of the IGS1 probe on Northern blots of RNA from various human brain regions is approximately pharmaceutically acceptable in both the caudate and putamen.
Two strong bands at 400 and 9.000 nucleotides (nt) were shown (Figure 3). This lower band was slightly stronger in the caudate and vice versa in the putamen. The 4.400 and 9.000 nt bands were also seen in the thalamus, but both were very weak. In addition, a very faint 9.000 nt transcript was detected in the substantia nigra but not in the 4.400 band. Finally, an extremely weak 9.000 nt band was observed in the cerebellum, bone marrow and tonsils. The 4.400 nt band was not observable in the thalamus and substantia nigra. These results are identical to those of the MTE analysis in the sense that the strongest expression of IGS1 was observed in the caudate and putamen. However, the presence of two transcripts was not expected. 4.400nt band is IGS1 mRNA
It is most likely to correspond to, but the origin of the 9.000 nt band is not clear.
Since the IGS1 gene does not contain introns (at least not within the coding region), the 9.000nt transcript is probably not due to unspliced or alternatively spliced transcripts. This could be the IGS1 transcript with alternating poly-adenylation sites or it could be the cross-hybridization species itself. We detected only a weak 9.000nt transcript and 4.400n
If no t transcript was detected, this was 4.400n for the 9.000nt transcript.
It is assumed that it is slightly stronger than the t transcript, and therefore this lower band is just below the detection limit of the Northern assay.

These results were confirmed by in situ hybridization analysis of IGS1 in rat brain, where IGS1 expression was detected in the same anatomical region as above. Example 3. Screening of putative ligands for IGS1 Example 3a. Construction of IGS-1 Transfected CHOG 16-Cells To identify ligands for IGS1, Chinese hamster ovary (
CHO) cells were stably transfected with IGS1. IGS1
The G protein-coupling mechanism is unknown and is known as a "universal adapter" for GPCRs (Milligan G. et al., (1996) Trends Pharmacol. Sci. 17: 235-7).
Specific CHO-cells expressing G-protein G16 (CHOG16, Molecular Devices) were used.

Materials used include: IGS1-pREP9 vector; SuperFect Transfection Reagent (Qiagen); Growth-Medium: 10% FCS,
C supplemented with 2 mM L-glutamine, hygromycin B 400 μg / ml
HO-S-SFM II (Gibco BRL), selection-medium; 10% FCS, 2 mM L
-Glutamine, Hygromycin B 400 μg / ml and Geneticin 5
CHO-S-SFM II (Gibco BRL) supplemented with 00 μg / ml; RNeasy Min
i kit (Qiagen), DNase I (Ambion, 2U / μl), SuperScript II (Gibco B
RL), SuperScript II 200U (Gibco BRL), AmpliTaq (Perkin Elmer).

The IGS1 coding sequence was changed to pcDNA3.1huIGS1 (ICCG # 43
50) into pREP9 (Invitrogen) via the XhoI / NheI sites. CHOGα16 cells were transfected with SuperFect (Qiagen) as described by the supplier. Transfection was performed in a T25 flask. After 24 hours in growth-medium, medium was removed and replaced with selection-medium. Selection-Trowing of the polyclonal bodies in T medium after culturing to confluence
It was passed through a 75 flask twice. To obtain monoclonals, the cells are Limi
Inoculated into ted Dilution.

Selection of monoclonals was performed by RT-PCR. Monoclonal body 1
Four were tested. RNeasy Mini Kit (Qiagen) according to the protocol provided
RNA was isolated from the monoclonal using (1 confluent well from a 24-well plate). R using 1 U of DNase I (Ambion, 2 U / μl) per sample
NA was processed. RT of half of RNA sample using SuperScript II (Gibco BRL)
-Used for PCR. Primer annealing was performed using RNA and oligo-dT1.
6 (0.6 μM) for 10 minutes at 65 ° C to 15 ° C. DNH respectively
First Strand Buffer (Gibco BRL) with 0.43 mM of P, DTT 10 mM
, 20U RNasin (Promega, 40U / μl) and SuperScript II 200U (Gibco
BRL, 200 U / μl) was added to a final volume of 30 μl and then incubated at 42 ° C. for 1 hour.

PCR was performed using AmpliTaq (Perkin Elmer) in 25 μl with IGS1 specific internal primers. First, 35 cycles of PCR were performed. Another PCR with a low number of cycles and a higher annealing temperature was performed to confirm positive monoclonals and to select the best one. 2 μl of First Strand cDNA (from 30 μl) was used per PCR reaction.

The 6 best monoclonals were grown to confluence in T75 flasks and frozen in growth medium containing 10% DMSO. Example 3b. Intracellular calcium measurement CHO G 16-IGS1 cells using Fluorometric Imaging Plate Reader (FLIPR)
Functionally screened above to determine the mobility of intracellular calcium in response to putative ligands.

The following materials were used for cell preparation. Clear, flat bottom, black well 96-well plate (Costar); Growth medium: Glutamax supplemented with 10% fetal bovine serum (Gibco)
Nut-Mix F-12 (HAM) with (Gibco); incubator: 5% CO2, 37 ° C
(Nuaire).

Cells were seeded in black-walled microplates 24 or 48 hours before the experiment. Cell density, 0.8 × 10 ^-4 cells / well for 48-hour culture, and was 2.2 × 10 ^-4 cells / well for 24 hours culture. All steps were performed under sterile conditions.

The following materials were used for dye loading: 2 mM dye stock: 443 μl
1 mg Fluo-4 (Molecular Probes) solubilized in low water DMSO (Sigma)
(Aliquots are stored at -20); 20% Pluronic acid solution: 2m
400 mg pluronic acid (S) solubilized at 37 ° C in 1 low water DMSO (Sigma).
igma) (stored at room temperature); Dye / pluronic acid mixture: mixed with equal amount of dye stock and 20% pluronic acid immediately before use (dye and pluronic acid were 1 mM and 10% final concentration respectively); probencid , 250 mM stock solution: 710 mg Provencid (Gibco) mixed with 5 ml Hank's BSS lacking Phenol Red (Gibco) solubilized in 5 ml 1N NaOH solution and supplemented with 20 mM HEPES; Load-buffer: 20 mM HEPES. 10.5ml Hank BSS, 105 lacking Phenol Red (Gibco) supplemented with
μl Provencid, 210 μl 1M HEPES; wash-buffer: 20 mM
Phenol red (Gib supplemented with HEPES and 2.5 mM probencid
Hank BSS lacking co).

The 2 mM dye stock was mixed with an equal volume of 20% (w / v) pluronic acid just before adding to the loading-buffer. The growth-medium was aspirated from the wells without disturbing the confluent cell layer. 100 μl of load-buffer was dispensed into each well using Multidrop (Labsystems). Cells were incubated for 30 minutes in a 37 ° C. incubator with 5% CO ₂ . Some wells were not loaded with dye to calculate background fluorescence. After dye loading, cells are washed 3 times with wash-buffer.
Washed twice (automated Danley cell wash) and background fluorescence over background 20.000
Reduced to ~ 25,000 counts. 100 μl of wash-buffer was added and the cells were incubated at 37 ° C until the start of the experiment.

Compounds to be screened were 20 mM HEPES and 0.1% BSA (Sig
Dilute in Hank BSS lacking Phenol Red (Gibco) supplemented with ma).
Intracellular calcium detection using the FLIPR was performed as described by the manufacturer (Molecular Devices). FLIPR set-up parameters are: exposure length 0.4 sec, filter 1, 50 μl liquid addition, pipetter height 125.
μl, distribution rate 40 μl / sec, no stirring.

[Table 4]

[Table 5]

[Table 6]

[Table 7]

[Sequence list]

[Brief description of drawings]

FIG. 1: Illustration of the relative positions of various clones isolated to produce a common IGS1 contig. The PCR primers used for cloning are indicated by (IP #). Clone HB4693 is an overlapping P of HNT1398 and HNT1413.
Obtained via CR. The position of the IGS1 coding sequence (IGS1PROT) is
It is indicated by a star "*". EST 20889 and EAT accession number A1
The position of 672141 is indicated by "==", and the position of IGS1 DNA is indicated by "++". IGS1 DNA is part of the IGS1 contig and its sequence was determined for at least 4 independent cDNA clones at each nucleotide position.

FIG. 2: Multiplex tissue expression array analysis with human IGS1 probe. There are many trace signals on this membrane. Only the signal indicated by the arrow is the deposited RNA
Is consistent with and specific to.

[Figure 3]   Northern blot analysis of human IGS1 on RNA from various brain regions.

[Procedure amendment]

[Submission date] November 1, 2002 (2002.1.1)

[Procedure Amendment 1]

[Document name to be amended] Statement

[Name of item to be amended] Claims

[Correction method] Change

[Contents of correction]

[Claims]

If the wherein the expression system is present in a compatible host intracellular, IGS1 polypeptide the expression systems comprising an amino acid sequence having at least 80% of the same to the polypeptide of SEQ ID NO: 2 A DNA or RNA molecule comprising said expression system capable of producing

7. A host cell comprising the expression system of claim 6 .

8. The host cell according to claim 7 , which is a yeast cell.

9. an animal cell, according to claim 7, wherein the host cell.

[Claim 1 0] IGS1 receptor membrane preparation derived from cells according to claims 7 to 9.

[Claim 1 1] from culturing a host of claim 7 under conditions sufficient for the production of a polypeptide and the culture comprising recovering the polypeptide IGS1
A method for producing a polypeptide.

In claim 1 2 Suitable culture conditions, so that the cells can produce IGS1 polypeptide, comprising the transforming or transfecting cells with the expression system of claim 6, A method for producing a cell that produces a cellular IGS1 polypeptide.

[Claim 1 3] at least 80% over its entire length to the amino acid sequence of SEQ ID NO: 2 comprises an amino acid sequence identical, IGS1 polypeptide.

[Claim 1 4] comprising the amino acid sequence of SEQ ID NO: 2, claims 1 to 3, wherein the polypeptide.

[Claim 1 5] claim 1 3 immunospecific antibodies against IGS1 polypeptide according.

In claim 1 6. A process for the treatment of patients requiring to enhance the activity or expression of IGS1 polypeptide receptor according to claim 1 3, wherein the therapeutically effective amount of an agonist to (a) receptor Is administered to the patient, and / or
Or (b) a nucleotide sequence having at least 80% identity over its entire length to the nucleotide sequence encoding the IGS1 polypeptide of SEQ ID NO: 2, or in a form such that it produces the receptor activity in vivo. A method comprising providing to a patient an isolated polynucleotide comprising a nucleotide sequence complementary to a nucleotide sequence.

The method according to claim 1 7] claims 1-3 therapeutically effective amount of an antagonist to (a) the receptor in a method for the treatment of patients requiring inhibiting the activity or expression of IGS1 polypeptide receptor according A polypeptide that is administered to a patient and / or (b) a polynucleotide that inhibits the expression of a nucleotide sequence encoding the receptor is administered to the patient, and / or (c) a polypeptide that competes with the receptor for a ligand. A method comprising administering to a patient a therapeutically effective amount of.

[Claim 1 8] The in the genome of claim 1 3 in a method for the diagnosis of susceptibility to a disease or disorder in a patient with respect to the expression or activity of IGS1 polypeptide according (a) said patient in the patient IGS1 Determining the presence or absence of a mutation in the nucleotide sequence encoding the polypeptide, and / or (b) analyzing the presence or amount of IGS1 polypeptide expression in a sample derived from the patient. Method.

19. A method for identifying agonists to IGS1 polypeptide of claim 1 3, wherein, (a) IGS1 polypeptide cells which produce is contacted with a test compound, and (b) the test compound is IGS1 poly A method comprising determining whether a signal generated by activation of a peptide is affected.

2. 0] agonist identified by the method of claim 19, wherein.

2. 1] A method for identifying antagonists to IGS1 polypeptide of claim 1 3, wherein, generated by (a) IGS1 cells producing polypeptides are contacted with an agonist, and (b) the agonist Determining whether the signal is attenuated in the presence of the candidate compound.

2. 2] antagonist identified by the method of claim 2 1, wherein.

2. 3] IGS1 expressing a polypeptide according to claim 1 wherein the method a recombinant host cell or a membrane produced by.

2. 4] non-genetically modified - essentially in a method of creating a human animals, a) a nucleic acid sequence or a biologically active fragment encoding a protein having SEQ ID NO: 2 amino acid sequence A coding sequence of a polynucleotide to be linked to a regulatory sequence capable of driving high level gene expression or expression in a cell type in which the gene is not normally expressed in the animal, or b) the amino acid sequence SEQ ID NO: The coding portion of the polynucleotide consisting essentially of a nucleic acid sequence encoding a protein having 2 or a biologically active fragment thereof is engineered, and the protein having amino acid sequence SEQ ID NO: 2 or biologically active Of the animal in such a way as to completely or partially inactivate the endogenous gene allele encoding the fragment. A method comprising reintroducing the sequence into the genome.

[Procedure Amendment 2]

[Document name to be amended] Statement

[Correction target item name] 0102

[Correction method] Change

[Contents of correction]

[0102]

EXAMPLES Example 1. Cloning Example 1a c DNA encoding the novel G protein-coupled receptors. Homologous PCR Cloning of a Genomic Fragment Encoding a Novel G Protein-Coupled Receptor (GPCR) A PCR-based homology cloning strategy to isolate a partial genomic DNA sequence encoding a novel G protein-coupled receptor Used for. The following advance (F
11) and backward (R13) degenerate PCR primers, a transmembrane domain 1 (TM 1) and in the transmembrane domains 3 and intracellular loop 2 of the boundary it <br/> Each of the (TM3 / 12) Neuro Designed within the conserved region of the tensin receptor gene . F11 (TM1): 5′-CATCTTCGTCGTCCGGCAC (A, C, G or T) G (C or T) (A, C, G or T) GG (A, C, G or T) AA-3 ′ (SEQ ID NO: 3) R13 (TM3 / 12): 5'-GGGTGGGCAGATGGCCA (A or G) (A or G) (C or T) A (A, C, G or T) C (C or T) (C or T) TC -3 ′ (SEQ ID NO: 4) Furthermore, a 3 ′ blocked oligo primer (HNTR1F1STOP) was designed. HNTR1F1STOP: 5'-ACGGTGGGCAACACGGTGACCGGCGTTT-3'-3'-
The dA (SEQ ID NO: 5) 3'blocked primer is specific for the human neurotensin receptor (NTR1) cDNA within the TM1 coding region and partially overlaps (and competes) with the degenerate forward primer. is doing. This 3'end is blocked by a 3'-deoxyadenosine group to avoid polymerase-catalyzed extension (Euro
gentic, Belgium, catalog number OL-0401-0302).

[Procedure 3]

[Document name to be amended] Statement

[Correction target item name] 0103

[Correction method] Change

[Contents of correction]

PCR reactions were performed in a volume of 60 μl and 100 ng human genomic DNA (Clont
ech), 6 μl 10x PCR buffer II (100 mM Tris-HCl pH8.
． 3; 500 mM KCl, Perkin Elmer), 3.6 μl 25 mM MgCl
2, 0.36 μl dNTPs (25 mM of each dNTP), 1.5 units AmpliTaq ™ Polymerase (Perkin Elmer), 30 picomoles of degenerate forward and reverse primers, respectively, and 100 of 3 ′ blocked primer.
It contained picomoles. The reaction tube was heated at 94 ° C for 2 minutes and then denatured (94 ° C,
30 seconds, 20 cycles of annealing (55 ° C, 1 minute, touchdown-0.25 ° C / cycle) and extension (72 ° C, 1 minute), followed by further denaturation (94).
℃, 30 seconds, annealing (50 ℃, 1 minute) and extension (72 ℃, 1 minute)
20 cycles were performed. Finally, the reaction tube was heated at 72 ° C. for 5 minutes. PCR reaction products were size classified on a 2% agarose gel and stained with ethidium bromide. The expected size fragment (± 300 bp) was purified from the gel using the Qiaex-II ™ Purification Kit (Qiagen Inc.) and supplied by the supplier (pGEM-T Kit, Pr.
were ligated into the pGEM-T plasmid according to the procedure recommended by Omega. The recombinant plasmid thus produced was used to transform competent E. coli SURETM2 bacteria (Stratagene).

[Procedure amendment 4]

[Document name to be amended] Statement

[Correction target item name] 0109

[Correction method] Change

[Contents of correction]

The nested AP-2 / IP11260 3'-RACE PCR reaction showed several bands. The largest 3'nested RACE PCR fragment (± 1,550 bp) that hybridized with the IGS1 probe was purified from gel and pGEM-T (P
romega) and used to transform competent E. coli SURE II cells. IGS1-specific transformants from this ligation reaction were identified after colony hybridization with the 32P-labeled insert of clone HNT642. Colony blots were hybridized to previously designated probes and were highly stringent (0.1xSSC 0.1% SDS, 3 ° C at 65 ° C).
For 0 minutes). Hybridization screening of the 3'RACE nested PCR library produced 3 positive clones. These two (H
NT 1413-1414) was sequenced.

[Procedure Amendment 5]

[Document name to be amended] Statement

[Correction target item name] 0111

[Correction method] Change

[Contents of correction]

All isolated IGS1 cDNA clones could be fully sequenced and constructed within a single contig (FIG. 1). A portion of this contiguous cDNA determined from at least four independent cDNA clones is set forth herein as IGS1 DNA (SEQ ID NO: 1). Translation of this contig revealed a long open reading frame, a 508 amino acid protein was predicted, which showed good homology to the GPCR protein (IGS1PROT; SEQ ID NO: 2). Computer-aided homology search of DNA sequences of IGS1 contigs against expressed sequence tag database (dbase) (Blastn; Altschul SF et al., (1997), Nucleic
Acids Res. 25: 3389-3402) is EST 20889 (accession number AA31).
8717) and the presence of EST accession number AI672141, both of which overlap the 3'end of the IGS1 contig but are outside the IGS1 open reading frame (FIG. 1). Example 1c. Isolation of a continuous cDNA fragment containing the complete IGS1 coding sequence A continuous IGS1 cDNA clone was cloned into clones HNT1398 and HNT14.
Made via overlap-PCR on 13 templates. HNT1398 plasmid DNA
And 100 ng of HNT1413 plasmid were PCR amplified (denatured) in separate reactions (50 μl) with primer pairs IP12264 / IP11264 (SEQ ID NOs: 18 and 12, respectively) and IP11260 / IP12262 (SEQ ID NOs: 8 and 11, respectively). 30 PCR cycles of [94 ° C., 30 seconds], annealing [60 ° C., 30 seconds] and extension [72 ° C., 1 minute], using ExpandTM High Fidelity PCR system [Boehringer]). A 1 μl volume of each PCR reaction was combined and the primer pair IP12264 /
IP12261 was used and reamplified under the same conditions. This overlap-PCR reaction
A band of ± 1730 bp was generated, which was purified from gel and ligated into the pGEM-T plasmid vector. The recombinant plasmid was used to transform competent E. coli DH5αF ′ strain. Transformed cells with ampicillin (1
(00 μg / ml) was plated on LB agar plates. Plasmid DNA was generated from a large number of random colonies and the insert size was determined via restriction digestion. Three clones containing the ± 1730 bp insert were sequenced. The sequence of clone HB4693 matched perfectly with the consensus IGS1 cDNA sequence (
(See FIG. 1). A cell line containing the plasmid HB4693 was treated with ampicillin 100
Replating on LB agar plates containing μg / ml followed by recloning, and Innogenetics strain list (ICCG # 4297) and Centraalbu
reau voor Schimmelculturen (CBS) (Baarn, The Netherlands) (Deposit No. CBS10
2049). Plasmid D prepared from recloned isolate
The NA was re-sequenced and found to be identical to the previously determined consensus sequence.

[Procedure correction 6]

[Document name to be amended] Statement

[Correction target item name] 0114

[Correction method] Change

[Contents of correction]

1 μl of HindIII digested pcDNA3.1 (+) vector, insert 3
μl and 16 μl H ₂ O were added to Ready-To-Go ligase tubes (T4 DNA ligase, Amersham Pharmacia Biotech) and incubated for 1 hour at room temperature. 2 μl of the ligation mixture was used to transform the chemically competent DH5αF ′ strain. 200 μl of transformed bacteria were plated on LB plates (100 μg ampicillin / ml) and incubated overnight at 37 ° C. 16 random colonies were picked and grown in 3 ml of LB medium containing ampicillin. Plasmid DNA was generated using the BioRobot ™ 9600 nucleic acid purification system (Qiagen) and analyzed via restriction analysis with NotI, PstI and SphI restriction enzymes. DNA from one colony with the correct restriction pattern was partially sequenced to confirm the insertion point and found to have the expected sequence. The partially sequenced colonies were labeled with the Innogenetics strainlist (ICCG # 4
350) and prepared a large amount of DNA (MegaPrep, Qiagen 500 kit) from the deposited strain. This sequence was expected by sequence analysis of large DNA prep (500 [mu] l of 3 [mu] g / [mu] l) was confirmed.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) A61K 48/00 A61P 1/08 4C084 A61P 1/04 1/12 4C086 1/08 1/14 4H045 1/12 3/04 1/14 3/06 3/04 3/10 3/06 7/02 3/10 9/04 7/02 9/06 9/04 9/10 9/06 101 9/10 9/12 101 11/06 9/12 13/10 11/06 13/12 13/10 19/02 13/12 19/10 19/02 25/00 19/10 25/06 25/00 25/08 25/06 25 / 14 25/08 25/16 25/14 25/18 25/16 25/20 25/18 25/22 25/20 25/24 25/22 25/28 25/24 25/30 25/28 31/04 25 / 30 31/10 31/04 31/12 31/10 31/18 31/12 33/02 31/18 35/00 33/02 37/00 35/00 37/08 37/00 C07K 14/705 37 / 08 16/28 C07K 14/705 C12N 1/15 16/28 1/19 C12N 1/15 1/21 1/19 C12P 21/02 C 1/21 C12Q 1/02 5/10 1/68 A C12P 21 / 02 G01N 33/15 Z C12Q 1/02 33/50 Z 1/68 33/53 D G01N 33/15 M 33/50 33/566 33/53 C12N 15/00 ZNAA 5/00 B 33/566 A61K 37/02 (81) Designated country EP (AT, BE, CH) , CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN , GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, MZ, SD, SL, SZ, TZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CR, CU, CZ, DE, DK, DM, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, K , KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, TZ, UA, UG, US, UZ, VN, YU, ZA, ZW (72) Inventor Tuang, Juan The Netherlands Enuel-1381 C weather-Supu-Shijie Ivan Hou Teng Lahn 36 F-term (reference) 2G045 AA40 BA11 BB50 DA12 DA13 DA14 DA36 FB02 4B024 AA01 AA11 BA61 BA63 CA04 DA02 EA04 GA11 HA01 HA11 HA14 4B063 QA01 QA05 QA18 QA19 QQ42 QQ52 QQ79 QR32 QR35 QR48 QR51 QR55 QR77 QS34 QX07 4B064 AG20 CA05 CA10 CA19 DA01 DA13 4B065 AA26X AA91X AA93Y AB01 BA02 CA24 CA44 CA46 4C084 AA13 AA16 NA14 ZA32 ZA26 ZA26 ZA67 BZA ZA59 ZA67 BZA ZA45 ZA45 ZA42 ZA29 ZB 33 ZB35 ZB38 ZC33 ZC35 ZC39 ZC55 4C086 AA01 AA03 EA16 MA01 NA14 ZA02 ZA05 ZA06 ZA07 ZA08 ZA12 ZA16 ZA32 ZA36 ZA40 BZA ZAB ZA67 BZA ZAB ZA67 BAZ ZAB ZAB ZB38 ZC33 ZC35 ZC55 4H045 AA10 AA11 AA20 AA30 BA10 CA40 DA50 DA75 EA20 FA74

Claims (25)

[Claims] 1. A) a nucleotide sequence encoding the IGS1 polypeptide of SEQ ID NO: 2; b) a polypeptide encoded by the DNA insert contained within deposit number CBS102049 in Centraalbureau voor Schimmelcultures (Baarn, The Netherlands). A nucleotide sequence which encodes, in particular a nucleotide sequence corresponding to SEQ ID NO: 1; c) at least 80% (preferably at least 90%) sequence identity over its entire length to the nucleotide sequence of (a) or (b) D) an isolated polynucleotide comprising a nucleotide sequence selected from the group consisting of nucleotide sequences that are complementary to the nucleotide sequences of (a) or (b) or (c). 2. The polynucleotide of claim 1, wherein the polynucleotide comprises the nucleotide sequence contained within SEQ ID NO: 1 that encodes the IGS1 polypeptide of SEQ ID NO: 2. 3. The polynucleotide of claim 1, wherein the polynucleotide comprises a nucleotide sequence that is at least 80% identical to SEQ ID NO: 1 over its entire length. 4. The polynucleotide according to claim 3, which is the polynucleotide of SEQ ID NO: 1. 5. The polynucleotide according to claim 1-4, which is DNA or RNA. 6. A hybridization probe comprising the polynucleotide of claim 1 or a fragment thereof of at least 5 nucleotides and preferably between 30 and 50 nucleotides. 7. An IGS1 polypeptide comprising an amino acid sequence having at least 80% identity to the polypeptide of SEQ ID NO: 2 when the expression system is present in a compatible host cell. A DNA or RNA molecule comprising said expression system capable of producing 8. A host cell comprising the expression system of claim 7. 9. The host cell according to claim 8, which is a yeast cell. 10. The host cell according to claim 8, which is an animal cell. 11. An IGS1 receptor membrane preparation derived from the cells of claims 8-10. 12. IGS1 comprising culturing the host of claim 8 and recovering the polypeptide from the culture under conditions sufficient for the production of the polypeptide.
A method for producing a polypeptide. 13. A cell comprising transforming or transfecting the cell with the expression system of claim 7 such that the cell is capable of producing an IGS1 polypeptide under suitable culture conditions. A method for producing a cell that produces an IGS1 polypeptide. 14. An IGS1 polypeptide comprising an amino acid sequence that is at least 80% identical over its entire length to the amino acid sequence set forth in SEQ ID NO: 2. 15. The polypeptide of claim 14 which comprises the amino acid sequence of SEQ ID NO: 2. 16. An antibody immunospecific for the IGS1 polypeptide of claim 14. 17. A method for treating a patient in need of enhancing the activity or expression of the IGS1 polypeptide receptor according to claim 14, wherein (a) a therapeutically effective amount of an agonist for the receptor is administered to the patient. , And /
Or (b) a nucleotide sequence having at least 80% identity over its entire length to the nucleotide sequence encoding the IGS1 polypeptide of SEQ ID NO: 2, or in a form such that it produces the receptor activity in vivo. A method comprising providing to a patient an isolated polynucleotide comprising a nucleotide sequence complementary to a nucleotide sequence. 18. A method for the treatment of a patient in need of inhibiting the activity or expression of the IGS1 polypeptide receptor of claim 14, wherein (a) a therapeutically effective amount of an antagonist to the receptor is administered to the patient. And / or (b) administering to the patient a polynucleotide that inhibits expression of a nucleotide sequence encoding the receptor, and / or (c) treatment of a polypeptide that competes with the receptor for a ligand. A method comprising administering to the patient a therapeutically effective amount. 19. A method for diagnosing a disease or susceptibility to a disease in a patient for expression or activity of the IGS1 polypeptide according to claim 14 in the patient (a) said IGS1 polypeptide in the genome of said patient. Determining the presence or absence of a mutation in the nucleotide sequence encoding the gene, and / or (b) analyzing the presence or amount of IGS1 polypeptide expression in a sample from the patient. 20. A method for identifying an agonist for an IGS1 polypeptide according to claim 14, wherein (a) cells producing the IGS1 polypeptide are contacted with a test compound, and (b) the test compound is an IGS1 polypeptide. A method comprising determining whether to affect the signal generated by activation of the. 21. An agonist identified by the method of claim 20. 22. A method for identifying an antagonist to an IGS1 polypeptide according to claim 14, comprising: (a) contacting a cell producing the IGS1 polypeptide with an agonist, and (b) a signal generated by the agonist. Determining whether or not is attenuated in the presence of the candidate compound. 23. An antagonist identified by the method of claim 22. 24. A recombinant host cell or a membrane thereof produced by the method of claim 13, which expresses an IGS1 polypeptide. 25. A method of creating a genetically modified non-human animal comprising: a) consisting essentially of a nucleic acid sequence encoding a protein having the amino acid sequence SEQ ID NO: 2 or a biologically active fragment thereof. A coding portion of a polynucleotide comprising a regulatory sequence capable of driving high level gene expression or expression in a cell type in which the gene is not normally expressed in said animal, or b) the amino acid sequence SEQ ID NO: 2 Engineered a coding portion of a polynucleotide consisting essentially of a nucleic acid sequence encoding a protein having the following: or a biologically active fragment thereof, and having the amino acid sequence SEQ ID NO: 2 or the biologically active fragment Of the animal in such a way that it completely or partially inactivates the endogenous allele that encodes The method comprising the step of reintroducing the sequence in genome.