CN1371390A

CN1371390A - Human G-protein coupled receptor

Info

Publication number: CN1371390A
Application number: CN00808983A
Authority: CN
Inventors: W·德里尔斯尼德尔; G·尼斯; 张帆
Original assignee: Solvay Pharmaceuticals GmbH
Current assignee: Abbott Products GmbH; Abbott Healthcare Products BV
Priority date: 1999-07-15
Filing date: 2000-07-17
Publication date: 2002-09-25
Also published as: AU764310B2; CA2379360A1; IL146151A0; JP2003508026A; AU5985800A; EP1200474A1; WO2001009184A1; HK1042099A1

Abstract

The present invention relates to novel identified polynucleotides, polypeptides encoded by them and to the use of such polynucleotides and polypeptides, and to their production. More particularly, the polynucleotides and polypeptides of the present invention relate to the G-protein coupled receptor family, referred to as IGS1-family. The invention also relates to inhibiting or activating the action of such polynucleotides and polypeptides, to a vector containing said polynucleotides, a host cell containing such vector and transgenic animals where the IGS1-gene is either overexpressed, misexpressed, underexpressed or suppressed (knock-out animals). The invention further relates to a method for screening compounds capable to act as an agonist or an antagonist of said G-protein coupled receptor family IGS1 and the use of IGS1 polypeptides and polynucleotides and agonists or antagonists to the IGS1 receptor family in the treatment of psychiatric and CNS disorders.

Description

Human G-protein coupled receptor

Describe

The present invention relates to new identified polynucleotides, by the purposes of their encoded polypeptides and this type of polynucleotide and polypeptide, and relate to their generation.More clearly, polynucleotide of the present invention and polypeptide relate to G-protein linked receptor (GPCR), after this are referred to as IGS1.The present invention also relates to inhibition or activation to the effect of these type of polynucleotide and polypeptide, relate to the carrier that contains these polynucleotide, the host cell that contains this carrier and transgenic animal, wherein IGS1 gene overexpression, mistake are expressed, are expressed not enough and/or be suppressed (knock-out animal).The invention further relates to the method for SCREENED COMPOUND, wherein said compound can be as agonist or the antagonist of this G-protein linked receptor IGS1.

Background of invention

Fully determine many medically important biological processess by the protein mediation that participates in signal transduction pathway, wherein signal transduction pathway relates to G-albumen and/or second messenger; For example cAMP (Lefkowitz, the nature (Nature), 1991,351:353-354).These protein refer to the protein that participates in G-albumen approach herein.These more proteinic examples comprise that (for example (institute of NAS reports (Proc.Natl.Acad.Sci.USA), 1987,84:46-50 to those acceptors of the adrenergic factor and Dopamine HCL to gpc receptor for Kobilka, people such as B.K.; Kobilka, people such as B.K., science (Science), 1987,238:650-656; Bunzow, J.R. wait the people, nature, 1988,336:783-787)), G-albumen self, effect protein matter (for example Phospholipase C, adenylate cyclase and phosphodiesterase) and regulate (actuator) protein (protein kinase A and protein kinase C (Simon, people such as M.I., science for example, 1991,252:802-8)).

For example, in a kind of form of signal transduction, hormone is with after GPCR combines, the G-protein-interacting of this receptor and heterotrimer, induce GDP from guanylic acid binding site position from.Under normal guanylic acid cell concn, GTP fills this site immediately.GTP combines with the proteic α subunit of G-and causes G-albumen to dissociate from acceptor, and the G-proteolysis is α and β _γSubunit.The form that carries GTP is bonded to the activatory adenylate cyclase then.GTP is hydrolyzed to GDP, and this process makes G-albumen get back to its basic disactivation form by G-albumen autocatalysis (the α subunit has intrinsic GTPase activity).The GTPase activity of α subunit comes down to the internal clocking of controlled opening/pass swap switch.The GDP combining form of α subunit is to β _γTool high-affinity, next α GDP and β _γRecombine makes this system get back to base state.Like this, G-albumen plays dual function, promptly as intermediate signal is passed to effector (being adenylate cyclase) from acceptor this example, and as the clock of control signal time length.

The film of G-protein linked receptor has been characterized by in conjunction with superfamily has seven membrane spaning domains of inferring.These structural domains are considered to represent transmembrane spanning, and it is outer or kytoplasm ring connection by born of the same parents.The G-protein linked receptor comprises the biologic activity acceptor of going up, for example hormone, virus, somatomedin and neuroreceptor on a large scale.

G-protein linked receptor family comprises Dopamine Receptors, and it is in conjunction with the psychosis medicine that is used for the treatment of the CNS disorder.Other example of this family member includes, but is not limited to thyrocalcitonin, adrenergic, neuropeptide tyrosine, somastotatin, neurotensin, neurokinin, capsaicine, VIP, CGRP, CRF, CCK, bradykinin, galanin, motilin, nociception element (nociceptin), endothelin, cAMP, adenosine, muscarine, vagusstoff, serotonin, histamine, zymoplasm, kassinin kinin, follicle stimulating hormone, opsin, endothelial differentiation gene-1, Visual purple, taste-additive and cytomegalovirus acceptor.

Respectively have single conserved cysteine residue in two born of the same parents' outer shrouds of most of G-protein linked receptor, it can form disulfide linkage, but this disulfide linkage is thought the protein structure of stabilization function.Stride the film district with these 7 and be appointed as TM1, TM2, TM3, TM4, TM5, TM6 and TM7.The kytoplasm ring that connects TM5 and TM6 may be a chief component of G-protein binding structural domain.

Most of G-protein linked receptor comprises potential phosphorylation site, and it is positioned at the 3rd kytoplasm ring and/or C-terminal.For some G-protein linked receptors (as receptor), the phosphorylation of protein kinase A and/or special receptor kinase can mediate the desensitization of acceptor.

Recently find that some GPCR (as Calcitonin Receptor sample acceptor) can interact with little single transmembrane protein, the latter is called as receptor active modifying protein (RAMP ' s).Which native ligand the interaction of GPCR and certain RAMP has determined the combination of GPCR-RAMP is had suitable affinity, and the function signal that can regulate this complex body is transmitted active (McLathie, people such as L.M., nature (1988) 393:333-339).

To some acceptors, it is believed that the ligand-binding site point of G-protein linked receptor comprises hydrophilic pocket, its membrane spaning domain by several G-protein linked receptors forms, and this pocket is surrounded by the hydrophobic residue of G-protein linked receptor.In the hydrophilic site of the transbilayer helix of each G-protein linked receptor is inferred and faced, and form polar ligand-binding site point.Some G-protein linked receptors involve TM3, because it has the ligand-binding site point, as the TM3 asparagicacid residue.The phenylalanine of the Serine of TM5, the l-asparagine of TM6 and TM6 or TM7 or tyrosine also participate in the combination of part.

The G-protein linked receptor can be by heterotrimer G-albumen and multiple intracellular enzyme, ionic channel and translocator carry out coupling in the born of the same parents (see people such as Johnson, internal secretion comment (Endoc.Rev.), 1989,10:317-331).Different G-protein alphas-subunit dominance stimulates specific effector, to regulate multiple biological function in the cell.The phosphorylation of having determined G-protein linked receptor kytoplasm residue is the important mechanisms of regulating some G-protein linked receptors and G-albumen coupling.Found the G-protein linked receptor in many places of mammalian hosts.

The a greater part of present known medicine of acceptor-mainly be GPCR class-caused (Drews, Nature Biotechnol (Nature Biotechnology), 1996,14:1516).This shows that these acceptors are as existing determine, the attested history of treatment target position.The new IGS1 GPCR that the present invention describes has satisfied this area undoubtedly to identifying and characterize the demand of further acceptor, and wherein said further acceptor is for can be at the acceptor of diagnosing, preventing, play a role in improvement or rectifier dysfunction, disorder or the disease (after this being commonly referred to as " disease ").Disease includes, but is not limited to psychiatric and the CNS disorder, comprises schizophrenia, paroxysmal anxiety (EPA) disorder is obsessive-compulsive disorder (OCD) for example, posttraumatic stress disorder (PTSD), phobia and HA, most depressive disorder, bipolar disorder, parkinsons disease, general anxiety disorder, lonely tinea, delirious speech, multiple sclerosis, Alzheimer disease/dementia and other neurodegenerative disease, serious mental retardation, dyskinesia, Huntington Chorea, Tourette's syndrome, twitch, tremble, dystonia, spasm, apositia, exessive appetite, cerebral apoplexy, habituation/dependence/serious hope, somnopathy, epilepsy, migraine, attention deficit disorder (ADD) (companion is how moving) (ADHD); Cardiovascular disorder comprises the renal failure of heart failure, stenocardia, irregular pulse, myocardial infarction, cardiac hypertrophy, ypotension, hypertension-for example essential hypertension, kidney hypertension or pulmonary hypertension, thrombosis, arteriosclerosis, cerebral vasospasm, subarachnoid hemorrhage, cerebral ischemia, cerebral infarction, peripheral vascular disease, Raynaud disease, kidney disease-for example; Dyslipidemias; Obesity; Vomiting; Gastrointestinal disturbance comprises the stomach emptying situation of irritable bowel syndrome (IBS), inflammatory bowel (IBD), gastroesophageal reflux disease (GERD), motility obstacle, delay, as perform the operation back or diabetic gastroparesis and diabetes, the stomach ulcer of ulcer-for example; Diarrhoea; Other disease comprises osteoporosis; Inflammation; Infect, as bacterium, fungi, protozoon and virus infection, the especially infection that causes of HIV-1 or HIV-2; Pain; Tumour; The damage that chemotherapy is brought out; Tumor invasion; Immunologic derangement; Uroschesis; Asthma; Transformation reactions; Sacroiliitis; Benign prostatauxe; Endotoxin shock; Septicemia; Diabetic complication and gynaecopathia.

Especially, the new IGS1 GPCR that the present invention describes has satisfied this area undoubtedly to identifying and characterize the demand of further acceptor, the acceptor of wherein said further acceptor for can in diagnosing, prevent, improve or correct psychiatric and CNS dysfunction, disorder or disease, playing a role, particularly motion function obstacle, ataxia or disease are for example twitched, are trembled, Tourette's syndrome, parkinsons disease, huntington disease, dyskinesia, dystonia and spasm.

Summary of the invention

The method that one aspect of the present invention relates to the IGS1 polypeptide and recombinates thing and produce them.The present invention relates to the method for utilizing this IGS1 polypeptide and polynucleotide on the other hand.This type of purposes includes, but is not limited to the treatment to one of above-mentioned disease.Especially, this purposes comprises psychiatric and treatment, especially ataxia the CNS obstacle, for example twitches, trembles, Tourette's syndrome, parkinsons disease, huntington disease, dyskinesia, dystonia and spasm.

Moreover another aspect of the present invention relates to the method for using material provided by the invention to identify agonist and antagonist, and treats the imbalance situation relevant with IGS1 with compounds identified.Also have another aspect of the present invention to relate to the diagnostic method that detects disease, wherein disease is relevant with unsuitable IGS1 activity or level.The further aspect of the present invention relates to the system based on animal, and it is as IGS1 abnormal expression or the active improper and disorderly model that causes.

The accompanying drawing summary

Fig. 1. the graphic representation of different clone's relative positions, isolating these different clones are used to produce the IGS1 contig of consensus sequence.The PCR primer that is used to clone is expressed as (IP#).Can obtain to clone HB4693 by overlapping PCR to clone HNT1398 and HNT1413.(" * ") represented with asterisk in the position of IGS1 encoding sequence (IGS1 PROT).The location of EST20889 and EST registration number AI672141 uses " ++ " to represent with "=" expression, the location of IGS1 dna sequence dna.IGS1 DNA is the part of IGS1 contig, wherein on each nucleotide position, at least to 4 independently the cDNA clone carried out sequencing.

Fig. 2. many tissue expressions array analysis that personnel selection IGS1 probe carries out.Many spurious responsees are arranged on the film.The signal that has only the arrow indication is identical with the RNA position of institute preservation and be special.

Fig. 3. use the Northern engram analysis that carries out people IGS1 from the RNA in different brains district.

The IGS1-DNA of table 1:SEQ ID NO:1

5’- GCCTGCAACCTGTCYCACGCCCTCTGGCTGTTGCCATGACGTCCACCTGCACCAACAGCA CGCGCGAGAGTAACAGCAGCCACACGTGCATGCCCCTCTCCAAAATGCCCATCAGCCTGG CCCACGGCATCATCCGCTCAACCGTGCTGGTTATCTTCCTCGCCGCCTCTTTCGTCGGCA ACATAGTGCTGGCGCTAGTGTTGCAGCGCAAGCCGCAGCTGCTGCAGGTGACCAACCGTT TTATCTTTAACCTCCTCGTCACCGACCTGCTGCAGATTTCGCTCGTGGCCCCCTGGGTGG TGGCCACCTCTGTGCCTCTCTTCTGGCCCCTCAACAGCCACTTCTGCACGGCCCTGGTTA GCCTCACCCACCTGTTCGCCTTCGCCAGCGTCAACACCATTGTCTTGGTGTCAGTGGATC GCTACTTGTCCATCATCCACCCTCTCTCCTACCCGTCCAAGATGACCCAGCGCCGCGGTT ACCTGCTCCTCTATGGCACCTGGATTGTGGCCATCCTGCAGAGCACTCCTCCACTCTACG GCTGGGGCCAGGCTGCCTTTGATGAGCGCAATGCTCTCTGCTCCATGATCTGGGGGGCCA GCCCCAGCTACACTATTCTCAGCGTGGTGTCCTTCATCGTCATTCCACTGATTGTCATGA TTGCCTGCTACTCCGTGGTGTTCTGTGCAGCCCGGAGGCAGCATGCTCTGCTGTACAATG TCAAGAGACACAGCTTGGAAGTGCGAGTCAAGGACTGTGTGGAGAATGAGGATGAAGAGG GAGCAGAGAAGAAGGAGGAGTTCCAGGATGAGAGTGAGTTTCGCCGCCAGCATGAAGGTG AGGTCAAGGCCAAGGAGGGCAGAATGGAAGCCAAGGACGGCAGCCTGAAGGCCAAGGAAG GAAGCACGGGGACCAGTGAGAGTAGTGTAGAGGCCAGGGGCAGCGAGGAGGTCAGAGAGA GCAGCACGGTGGCCAGCGACGGCAGCATGGAGGGTAAGGAAGGCAGCACCAAAGTTGAGG AGAACAGCATGAAGGCAGACAAGGGTCGCACAGAGGTCAACCAGTGCAGCATTGACTTGG GTGAAGATGGCATGGAGTTTGGTGAAGACGACATCAATTTCAGTGAGGATGACGTCGAGG CAGTGAACATCCCGGAGAGCCTCCCACCCAGTCGTCGTAACAGCAACAGCAACCCTCCTC TGCCCAGGTGCTACCAGTGCAAAGCTGCTAAAGTGATCTTCATCATCATTTTCTCCTATG TGCTATCCCTGGGGCCCTACTGCTTTTTAGCAGTCCTGGCCGTGTGGGTGGATGTCGAAA CCCAGGTACCCCAGTGGGTGATCACCATAATCATCTGGCTTTTCTTCCTGCAGTGCTGCA TCCACCCCTATGTCTATGGCTACATGCACAAGACCATTAAGAAGGAAATCCAGGACATGC TGAAGAAGTTCTTCTGCAAGGAAAAGCCCCCGAAAGAAGATAGCCACCCAGACCTGCCCG GAACAGAGGGTGGGACTGAAGGCAAGATTGTCCCTTCCTACGATTCTGCTACTTTTCCTT GAAGTTAGTTCTAAGGCAAACCTTGAAAATCAGTCCTTCAGCCACAGCTATTTAGAGCTT TAAAACTACCAGGTTCAATCACTGGTTATGCTTTCTGTG-3’

The IGS1-protein of table 2:SEQ ID NO:2

MTSTCTNSTRESNSSHTCMPLSKMPISLAHGIIRSTVLVIFLAASFVGNIVLALVLQRKP QLLQVTNRFIFNLLVIDLLQISLVAPWVVATSVPLFWPLNSHFCTALVSLTHLFAFASVN TIVLVSVDRYLSIIHPLSYPSKMTQRRGYLLLYGTWIVAILQSTPPLYGWGQAAFDERNA LCSMIWGASPSYTILSVVSFIVIPLIVMIACYSVVFCAARRQHALLYNVKRHSLEVRVKD CVENEDEEGAEKKEEFQDESEFRRQHEGEVKAKEGRMEAKDGSLKAKEGSTGTSESSVEA RGSEEVRESSTVASDGSMEGKEGSTKVEENSMKADKGRTEVNQCSIDLGEDGMEFGEDDI NFSEDDVEAVNIPESLPPSRRNSNSNPPLPRCYQCKAAKVIFIIIFSYVLSLGPYCFLAV LAVWVDVETQVPQWVITIIIWLFFLQCCIHPYVYGYMHKTIKKEIQDMLKKFFCKEKPPK EDSHPDLPGTEGGTEGKIVPSYDSATFP

Detailed Description Of The Invention

There is structural similarity in sequence and motif between IGS1 GPCR of the present invention and other the human GPCR. In addition, IGS1 expresses in brain tissue, particularly expresses in caudate nucleus and lenticular nucleus. Therefore hinted that IGS1 works in especially above mentioned disease. Hinted that IGS1 especially acts in psychiatric and CNS obstacle, ataxia is particularly for example twitched, is trembled, Tourette's syndrome, parkinsons disease, Huntingdon disease, dyskinesia, dystonia and spasm.

Unless defined in addition, what those skilled in the art understood usually in the field under whole technology used herein and scientific terminology and the present invention is equivalent in meaning. Although similar or be equal to either method described herein and material and can be used for still preferred method, equipment and material being described now in practice of the present invention or the test. All publications in the specification are quoted as a reference herein, although each independent publication cited herein all ad hoc, point out individually. Definition

Provide to give a definition, be beneficial to understanding herein some term commonly used.

" IGS1 " is meant the polypeptide that comprises aminoacid sequence shown in the SEQ ID NO:2 especially, or its allele variant.

" receptor active " or " biological activity of acceptor " refers to metabolic function or the physiological function of this IGS1, comprises the active of similar activity or raising or has these activity reduction, undesirable side effect.The antigenic activity and the immunogenicity activity that also comprise this IGS1.

" IGS1-gene " refers to comprise the polynucleotide of nucleotide sequence shown in the SEQ ID NO:1, or its allele variant, and/or their complement.

" antibody " used herein comprises polyclone and monoclonal antibody, chimeric, strand and humanized antibody, and the Fab fragment, comprises the product in Fab or other immunoglobulin expression library.

" isolating " refers to that " by artificial " changes and/or separate from natural surroundings from native state.Like this, if be present in natural " isolating " composition or material quilt " separation ", it has been changed or has been removed from its initial environment so, or the both has.For example, natural polynucleotide or the polypeptide that is present in the animal alive is not " separation ", but isolating same polynucleotide or polypeptide are " isolating " from the coexisting substances of its native state, just as used herein this term.

" polynucleotide " are often referred to arbitrary polybribonucleotide or polydeoxyribonucleotide, and it can be not modified RNA or DNA or modified RNA or DNA." polynucleotide " include, but is not limited to strand and double-stranded DNA, strand district and double stranded region blended DNA, strand and double-stranded RNA, and strand district and double stranded region blended RNA, comprise the hybrid molecule of DNA and RNA, it can be strand or more generally, can be the heterozygote of two strands or strand district and double stranded region.In addition, " polynucleotide " also can comprise three sequences, and it comprises RNA or DNA or comprises RNA and DNA simultaneously.The term polynucleotide also comprise DNA or the RNA that contains one or more modified bases, and because stability or because other DNA or RNA former thereby that main chain has been modified." modification " base for example comprises, tritylation base and unusual base (as Trophicardyl).Can carry out multiple modification to DNA and RNA; Like this, " polynucleotide " comprise the form of polynucleotide after chemically modified, enzyme modification or metabolism are modified, and they generally can be found at occurring in nature, and the DNA and the distinctive chemical species of RNA of virus and cell." polynucleotide " also comprise short relatively polynucleotide, are commonly referred to oligonucleotide.

" polypeptide " refers to arbitrary peptide or protein, and it comprises interconnection two or more amino acid (being the peptide isostere) by the peptide bond of peptide bond or modification." polypeptide " refers to short chain and long chain, and wherein the former is commonly referred to peptide, oligopeptides or oligomer, and the latter is commonly referred to protein and/or its composition.Polypeptide can comprise other amino acid except 20 kinds of gene coding amino acids." polypeptide " comprises modified aminoacid sequence, described modification or modified (as the translation post-treatment) by natural method, or modified by chemical modification technology well known in the art.This type of is modified in basic textbook and the more detailed monograph, and in a large amount of research documents sufficient description is arranged.Modification can occur in arbitrary position of polypeptide, comprises peptide main chain, amino acid side chain and amino or C-terminal.Should recognize some sites at specific polypeptide, the modification of same type can occur equally or in various degree.Equally, specific polypeptide also can contain the modification of many types.Polypeptide can be because of ubiquitination branch, and they can be with or without the ramose ring-type.Cyclic, ramose and branch's annular polypeptide can maybe can produce by synthesis method from translation back natural process.Modification comprises covalently bound, phosphatidylinositols covalently bound of covalently bound, the lipid of covalently bound, the Nucleotide of covalently bound, heme moiety of acetylizing, acylation, ADP-ribosylation, amidation, flavine or nucleotide derivative or lipid derivant; Crosslinked; cyclic action; disulfide linkage forms; the demethylation effect; the formation of covalent cross-linking; the formation of Gelucystine; the formation of Pyrrolidonecarboxylic acid; formylation; the gamma-carboxylation effect; glycosylation; the formation of GPI anchor; hydroxylation; iodization; methylation; the Semen Myristicae acylation; oxygenizement; proteolysis processing; phosphorylation; the isoprenylation effect; racemization; the selenizing effect; sulfation; what transfer RNA (tRNA) mediated adds amino acid (turning usefulness and ubiquitination into as arginyl) to protein.For example see, protein-structure and molecular characterization, second edition (PROTEIN-STRUCTURE AND MOLECULAR PROPERTIES, 2nd Ed.) T.E.Creighton, W.H.Freeman and Company, New York, 1993 and, proteinic translation back covalent modification (POSTTRANSLATIONAL COVALENTMODIFICATION OF PROTEINS), B.C.Johnson, Ed., academic press, New York, Wold in 1,983 one books, F., the protein modification after the translation: prospect and prospect (Posttranslational Protein Modifications:Perspectives and Prospects), 1-12 page or leaf; People such as Seifter, " analysis of protein modification and nonprotein cofactor " (" Analysisfor protein modifications and nonprotein cofactors "), people such as Enzymology method (Meth.Enzymol.) (1990) 182:626-646 and Rattan, " protein synthesis: posttranslational modification and aging " (" Protein Synthesis:Posttranslational Modificationsand aging "), record event (Ann.NY Acad.Sci.) (1992) 663:48-62 of association of New York section.

Term used herein " variant " is meant polynucleotide or polypeptide, and it is different from the polynucleotide or the polypeptide of reference respectively, but has kept basic characteristic, as basic biological nature, structural performance, control characteristic or biochemical characteristic.The nucleotide sequence of the general variant of polynucleotide is different from another, reference nucleotide.The variation of variant nucleotide sequence can change or not change amino acid sequence of polypeptide, and wherein said polypeptide is by the reference polynucleotide encoding.The variation of Nucleotide can cause amino acid whose replacement, interpolation, deletion, fusion and brachymemma in the reference sequences encoded polypeptide, and this is discussed below.The aminoacid sequence of the general variant of polypeptide is different from another, reference polypeptide.Usually, difference is limited, so that the sequence of reference polypeptide and variant are closely similar generally, and is identical in many zones.Difference on variant and the reference polypeptide aminoacid sequence can be replacement, interpolation and the deletions of one or more amino acid with arbitrary combination.The amino-acid residue of replacing or inserting can be encoded by genetic code, also can can't help the genetic code coding.Polynucleotide or variant polypeptides can produce (as allele variant) naturally, and perhaps it can the spontaneous variant of right and wrong.The non-spontaneous variant of polynucleotide and polypeptide can be by induced-mutation technique or directly synthetic the generation.

" identity " is measuring the identity of nucleotide sequence or aminoacid sequence.Usually series arrangement is got up, to obtain coupling to greatest extent." identity " itself has the meaning and the available disclosed technique computes of this area cognition.For example see: (calculate molecular biology (COMPUTATIONAL MOLECULAR BIOLOGY), Lesk, A.M., ed., Oxford University Press, New York, 1988; Biocomputer is learned: information and genome plan (BIOCOMPUTING:INFORMATICS AND GENOME PROJECTS), Smith, D.W., ed., academic press, New York, 1993; The Computer Analysis of sequence data, and first part (COMPUTER ANALYSIS OF SEQUENCE DATA, PARTI), Griffin, A.M., and Griffin, H.G., eds., Humana Press, New Jersey, 1994; Sequential analysis in the molecular biology (SEQUENCE ANALYSIS IN MOLECULARBIOLOGY), von Heinje, G., academic press, 1987; And sequential analysis guiding (SEQUENCE ANALYSIS PRIMER), Gribskov, M. and Devereux, J., eds., MStockton Press, New York, 1991).Though there are many methods that can be used for measuring identity between two polynucleotide or polypeptide, this term " identity " is known (Carillo, H., and Lipton of technician, D., industry and applied mathematics meeting applied mathematics magazine (SIAM J.Applied Math.) (1988) 48:1073).Measure the identity between two sequences or the common method of similarity and include, but is not limited to be disclosed in super large computer guide (Guide to Huge Computers), MartinJ.Bishop, ed., the academic press, San Diego, 1994 and Carillo, H., and Lipton, D., the method among industry and applied mathematics meeting applied mathematics magazine (1988) 48:1073.The method of measuring identity or similarity is organized in the computer program by rule.Preferably, be used to measure two between sequence identity or the computer program means of similarity include, but is not limited to GCG routine package (Devereux, J., Deng the people, 387), BLASTP, BLASTN, FASTA (Atschul nucleic acids research (Nucleic Acids Research) (1984) 12 (1):, S.F. wait the people, molecular biology magazine (J.Molec.Biol.) (1990) 215:403).The alternative word of word " homology " " identity ".

Describe by a kind of polynucleotide, the nucleotide sequence that it had for example with the reference nucleotide sequence of SEQ IDNO:1 at least " identity " of tool 95% be meant: in per 100 Nucleotide of the reference nucleotide sequence of SEQ ID NO:1, the nucleotide sequence of these polynucleotide is except containing the difference that reaches 5 Nucleotide, and the nucleotide sequence of these polynucleotide is identical with reference sequences.In other words, in order to obtain the identical polynucleotide of nucleotide sequence and reference nucleotide sequence at least 95%, nearly 5% Nucleotide can be deleted or by another nucleotide substitution in the reference sequences; Maybe some Nucleotide can be inserted in reference sequences, wherein the Nucleotide of Cha Ruing can reach reference sequences total nucleotide 5%; Or in some Nucleotide, the combination of have deletion, inserting and replacing, wherein said Nucleotide reach reference sequences total nucleotide 5%.These sudden changes of reference sequences can occur in 5 or 3 terminal positions of reference nucleotide sequence, or any place between these terminal positions, they or be dispersed in separately in the Nucleotide of reference sequences, or be present in the reference sequences with the group of one or more vicinities.

Similarly, one peptide species, the aminoacid sequence that it had for example with the reference amino acid sequence of SEQ ID NO:2 at least " identity " of tool 95% be meant: in per 100 amino acid of the reference amino acid sequence of SEQ ID NO:2, the aminoacid sequence of this polypeptide reaches 5 amino acid whose variations except containing, and the aminoacid sequence of this polypeptide is identical with reference sequences.In other words, in order to obtain the identical polypeptide of aminoacid sequence and reference amino acid sequence at least 95%, nearly 5% amino-acid residue can be deleted or by another amino acid replacement in the reference sequences; Maybe can be with in some aminoacid insertion reference sequences, wherein the amino acid of Cha Ruing reach reference sequences total amino acid residue 5%.These sudden changes of reference sequences can occur in the amino or the C-terminal position of reference amino acid sequence, or any place between these terminal positions, they or be dispersed in separately in the residue of reference sequences, or be present in the reference sequences with the group of one or more vicinities.Polypeptide of the present invention

One aspect of the present invention relates to IGS1 polypeptide (comprising IGS1 protein).The IGS1 polypeptide comprises the polypeptide of SEQ ID NO:2 and has peptide more than the aminoacid sequence of DNA insertion fragment coding that wherein DNA inserted fragment and is included in preserving number CBS 102049, was deposited in the fungi strain preservation center of Holland on July 15th, 1999; Also comprise the polypeptide of the aminoacid sequence that comprises SEQ ID NO:2 and comprise to have peptide more than the aminoacid sequence of DNA insertion fragment coding, wherein DNA inserts fragment and is included in preserving number CBS 102049, is preserved in the fungi strain preservation center of Holland; And comprise with SEQ ID NO:2 and/or have the polypeptide that inserts the aminoacid sequence of peptide at least 80% identity more than the aminoacid sequence of fragment coding by DNA, wherein DNA inserts fragment and is included in preserving number CBS 102049, be preserved in the fungi strain preservation center of Holland, and at least 90%, more preferably at least 95% identity preferably.Further, the polypeptide of those identity of at least 97%, particularly at least 99% is highly preferred.Also comprise in the IGS1 polypeptide have with the polypeptide of the aminoacid sequence that comprises SEQ ID NO:2 or with have the amino acid whose polypeptide that inserts peptide at least 80% identity more than the aminoacid sequence of fragment coding by DNA, wherein DNA inserts fragment and is included in preserving number CBS 102049, be preserved in the fungi strain preservation center of Holland, and preferably at least with SEQ ID NO:2 90% identity, more preferably at least 95%.Further, the polypeptide of those at least 97%, particularly at least 99% identity is highly preferred.Preferably, the IGS1 polypeptide shows at least a biological activity of this receptor.

The IGS1 polypeptide can be " maturation " protein form or is the part than larger protein (as fused protein).It is normally favourable to contain extra aminoacid sequence, and wherein said extra aminoacid sequence comprises secretion or leader sequence, presequence, be beneficial to the sequence of purifying (as a plurality of histidine residues) or play the additional sequences of stabilization in the recombinant chou production process.

The IGS1 polypeptide fragment is also included among the present invention.Fragment is the polypeptide with aminoacid sequence, and it has and the identical aminoacid sequence of the part of the aminoacid sequence of above-mentioned IGS1 polypeptide (non-all).The same with the IGS1 polypeptide, fragment can " independently exist " or be included in the bigger polypeptide, and fragment forms a part or a zone therein, most preferably is as single successive zone.The representative example of peptide fragment for example comprises more than the present invention, and amino acid no is from the fragment of about 1-20,21-40,41-60,61-80,81-100 and 101 to IGS1 polypeptide ends.In the literary composition " pact " be included in arbitrary end of the scope of refering in particular to or two ends have more or lacked several, 5,4,3,2 or 1 amino acid.

Preferred fragment for example comprises, brachymemma polypeptide with aminoacid sequence of IGS1 polypeptide, wherein do not comprise comprising the deletion of aminoterminal continuous residue sequence, or to the deletion of the continuous residue sequence that comprises C-terminal, or to the deletion of two continuous residue sequence, one of them comprises N-terminal, and one comprises C-terminal.The fragment that characterizes by structure or functional attributes also is preferred, for example contains alpha-helix and alpha-helix and forms the fragment that district, β-lamella and β-lamella formation district, corner and corner form the district, curl and curl into district, hydrophilic area, hydrophobic region, α amphiphilic district, β amphiphilic district, deformable zone, surface formation district, substrate land and high antigen coefficient district.Other preferred fragment is a bioactive fragment.Bioactive fragment is the fragment of those mediation receptor actives, comprises the activity that similar activity of those tools or tool raise, or tool fragment that reduce, undesirable activity.Also comprising those to animal, is antigen or immunogenic fragment to the people especially.

Like this, polypeptide of the present invention comprises the polypeptide of aminoacid sequence at least 80% identity with aminoacid sequence and SEQ ID NO:2 and/or has peptide more than the aminoacid sequence of DNA insertion fragment coding, wherein DNA inserts fragment and is included in preserving number CBS 102049, be preserved in the fungi strain preservation center of Holland, or its fragment, wherein said fragment and homologous segment have at least 80% identity.Preferably, all these polypeptide fragments have kept the biological activity of acceptor, comprise antigenic activity.Defined sequence and segmental variant have also constituted a part of the present invention.To be those replace the variant with the residue replacement of another similar characteristics of the variant that is different from reference sequences-promptly by conserved amino acid to preferred variant.General this type of replaces with between Ala, Val, Leu and the Ile; Between Ser and the Thr; Between acidic residues Asp and the Glu; Between Asn and the Gln; And between alkaline residue Lys and the Arg; Or between aromatic residue Phe or the Tyr.Particularly preferred variant be wherein have several, 5-10,1-5 or 1-2 amino acid is replaced with arbitrary combination, deletion or interpolation.

IGS1 polypeptide of the present invention can arbitrary suitable manner preparation.This type of polypeptide comprises the isolating natural polypeptide in conjunction with generation that has polypeptide, the synthetic polypeptide that produces of polypeptide, reorganization generation or pass through these methods.The method for preparing this type of polypeptide is known in this field.Polynucleotide of the present invention

The further aspect of the present invention relates to the IGS1 polynucleotide.The IGS1 polynucleotide comprise coding IGS1 polypeptide and segmental isolating polynucleotide, and polynucleotide closely-related with it.More clearly, IGS1 polynucleotide of the present invention comprise such polynucleotide, its nucleotide sequence that comprises is contained among the SEQ ID NO:1, Nucleotide more than the IGS1 polypeptide of codified SEQ ID NO:2 for example, polynucleotide with SEQ ID NO:1 particular sequence, and correspond essentially to DNA and insert segmental polynucleotide, wherein DNA inserts fragment and is included in preserving number CBS102049, is preserved in the fungi strain preservation center of Holland.

The IGS1 polynucleotide also comprise following polynucleotide: comprise its total length Nucleotide more than the nucleotide sequence of nucleotide sequence 80% identity more than the coding IGS1 of SEQ ID NO:2 at least, comprise Nucleotide more than the nucleotide sequence of its full length sequence and SEQ ID NO:1 at least 80% identity, and insert segmental polynucleotide corresponding to the DNA that is included in the preservation thing (CBS 102049) that is preserved in Dutch fungi strain preservation center basically.

In this, the polynucleotide of tool at least 90% identity are particularly preferred, and the polynucleotide of those tool at least 95% identity are especially preferred.And the polynucleotide of tool at least 97% identity are highly preferred, and those tools at least the polynucleotide of 98-99% identity be that topnotch is preferred, the polynucleotide of tool at least 99% identity are most preferred.It is such being also included within IGS1 polynucleotide bar nucleotide sequence now, wherein said nucleotides sequence is classified as and can be under certain condition can be inserted one section nucleotide sequence that the enough identity of the nucleotide sequence tool that contained in the fragment can be hybridized with it with SEQ ID NO:1 or DNA, wherein DNA inserts fragment and is included in preserving number CBS 102049, be preserved in the fungi strain preservation center of Holland, described condition can be used for amplification or is used as probe or mark.The present invention also provides and these type of IGS1 polynucleotide complementary polynucleotide.

Other protein of IGS1 of the present invention and G-protein linked receptor family has structural dependence, and this point can show by BLAST result of study in the public database.The major portion (amino-acid residue 7-222 and 396-470) of the aminoacid sequence of table 2 (SEQ ID NO:2) and rabbit α-1c adrenergic receptor (registration number #O02824, people such as Miyamoto S, RL life Sci. (1997) 60:2069-2074) has about 30% identity and (use BLAST, AltschulS.F. wait the people, [1997], nucleic acids research 25:3389-3402), and the amino-acid residue of 31-220 and human G-protein coupled receptor RE2 (GenBank registration number #AF091890) have about 33% identity.266 nucleotide residue (registration number #L31722 in the nucleotide sequence of table 1 (SEQ ID NO:1) and the people α-1a/d adrenergic receptor, people such as Bruno J.F., biological chemistry and biophysical studies news flash (Biochem.Biophys.Res.Commun.) (1991) 179:1485-1490) 57% identical, identical with preceding 1426 nucleotide residues (GenBank registration number #AF091890) 44% of human G-protein coupled receptor RE2.Further, the hydrophilicity analysis of IGS1 protein sequence (Hofmann, K., Stoffel, W. (1993) Biol.Chem.Hoppe-Seyler 347:166) shows 7 membrane spaning domains of existence.Like this, can estimate that IGS1 polypeptide of the present invention and polynucleotide especially have and they homologous polypeptide and the similar biological function/characteristic of polynucleotide, and their application is conspicuous to arbitrary those skilled in the art.

Polynucleotide of the present invention can obtain from natural resources, as genomic dna.Especially, can design the degenerate pcr primer, the conserved regions in its specific gpcr gene subfamily of encoding.Can cause some members' of this target gene family amplification (comprising known and new) (when being genomic templates, degenerated primer must be positioned at same exon when employed) to the pcr amplification reaction of genomic dna or cDNA with degenerated primer.(people such as Libert, science, 1989,244:569-572).The also available known and commercial available technology of polynucleotide of the present invention is synthetic.

The nucleotide sequence of the IGS1 polypeptide of coding SEQ ID NO:2 can identical with the polypeptid coding sequence in being included in SEQ IDNO:1 (few nucleotide 36 to 1559), perhaps it can be a different nucleotide sequence, because the redundancy of gene codon (degeneracy), it is compared with the polypeptid coding sequence that contains among the SEQ IDNO:1, also can show variation, but still peptide more than the coding SEQ ID NO:2.

When polynucleotide of the present invention are used as the recombinant chou that produces the IGS1 polypeptide, but these polynucleotide self comprise mature polypeptide or its segmental encoding sequence; Also can meet and comprise mature polypeptide or its segmental encoding sequence and other encoding sequence with reading, for example those coding leader sequence or secretion sequence, preceding albumen, former albumen or preceding crude protein sequence or other fusogenic peptide parts.For example, codified promotes the flag sequence of fusion polypeptide purifying.In the present invention's some embodiment preferred in this respect, flag sequence is 6 Histidine peptides or HA label, and wherein (Qiagen Inc.) can provide 6 Histidine peptides to the pQE carrier, and people such as Gentz, newspaper (1989) 86:821-824 of NAS is described.Polynucleotide also can comprise 5 ' and 3 ' non-coding sequence, for example transcribe, the sequence of non-translational region, shearing and poly-adenosine signal, ribosome binding site and stable mRNA.

Further preferred embodiment is the polynucleotide of coding IGS1 variant, wherein the IGS1 variant comprises the aminoacid sequence of the IGS1 polypeptide of SEQ ID NO:2, and several, 5-10 wherein, 1-5 or 1-2 amino-acid residue are replaced, delete or add with arbitrary combination.

The method genetically engineered of available this area common general knowledge designs polynucleotide of the present invention, so that for some purposes change the IGS1 encoding sequence, it includes, but is not limited to clone, the change of the expression of processing and/or gene product.DNA reorganization by random fragmentation and the PCR of gene fragment re-assemblies and synthetic oligonucleotide can be used to genetically engineered nucleotide sequence.For example oligonucleotide mediated site-directed mutagenesis can be used for importing sudden change, replaces, produces new restriction site to produce amino acid, changes modification (for example glycosylation or phosphorylation) pattern, changes codon preference, produces splice variant etc.

The invention further relates to can with the polynucleotide of above-mentioned sequence hybridization.In this respect, present invention is specifically related under stringent condition can with the polynucleotide of above-mentioned multi-nucleotide hybrid.Term used herein " stringent condition " is meant: as long as have at least 80% between sequence, and preferably at least 90%, preferably at least 95%, more preferably at least 97%, particularly preferably at least 99% identity then can be hybridized.

Polynucleotide of the present invention can be used as the hybridization probe of cDNA and genomic dna, to separate full-length cDNA and the genomic clone of coding IGS1, and separation and IGS1 gene have the cDNA and the genomic clone of other gene (comprising that coding is from the isoplassont of inhuman species or the gene of lineal homology evolution thing) of high sequence similarity, the nucleotide sequence that contains among wherein said polynucleotide and the SEQ ID NO:1 or its fragment is identical or very identical.Known this type of hybridization technique of those skilled in the art.Usually, the nucleotide sequence 80% of these nucleotide sequences and reference, preferably 90%, more preferably 95% is identical.Probe comprises at least 5 Nucleotide usually, and preferably at least 8 Nucleotide, preferably at least 10 Nucleotide, more preferably at least 12 Nucleotide, particularly at least 15 Nucleotide.This type of probe is most preferred for having at least 30 Nucleotide, and has at least 50 Nucleotide.Particularly preferred probe is between the scope of 30 to 50 Nucleotide.

For Nucleotide more than the IGS1 polypeptide that obtains to encode, it comprises from the isoplassont of inhuman species or lineal homology evolution thing, an embodiment comprises following steps: under tight hybridization conditions, with having the suitable library of the segmental label probe screening of SEQ ID NO:1 or its, and separate full-length cDNA and the genomic clone that contains this nucleotide sequence.This type of hybridization technique is that those skilled in the art are known.Tight hybridization conditions is as defined above or the condition through changing, 42 ℃ of incubations that spend the night in solution, then under about 65 ℃, wash film with 0.1 * SSC, wherein said hybridization solution contains: the cut-out salmon sperm DNA of 50% methane amide, 5 * SSC (150mM NaCl, 15mM trisodium citrate), 50mM sodium phosphate (pH7.6), 5 * Denhardt ' s solution, 10% T 500 and 20 micrograms/ml sex change.

Polynucleotide of the present invention and polypeptide can be used as research reagent and material, to find animal and human's class treatment of diseases and diagnostic method.Carrier, host cell, expression

The present invention also relates to carrier, it comprises a kind of polynucleotide of the present invention or multiple polynucleotide, also relates to carrier of the present invention through genetic engineering modified host cell, and relates to recombinant technology and produce polypeptide of the present invention.By the RNA that DNA construct of the present invention obtains, available cell free translation system produces this proteinoid.

In order to produce recombinant chou, can pass through genetic engineering modified host cell, make it to have expression system or its part of Nucleotide more than the present invention.The method of describing in the laboratory manual of many standards can realize that polynucleotide import host cell, people such as Davis for example, people such as molecular biology basic methods (BASIC METHODS IN MOLECULAR BIOLOGY) (1986) and Sambrook, molecular cloning: laboratory manual, second edition (MOLECULARCLONING:A LABORATORY MANUAL, 2nd Ed.), press of cold spring harbor laboratory, the for example calcium phosphate transfection of describing in the cold spring port, New York (1989), the transfection of DEAE-dextran mediation, transvection, microinjection, cationic-liposome-mediated transfection, electroporation, transduction, scrape loading, particle bombardment imports or injection.

The representative example of suitable host comprises bacterial cell, for example streptococcus (Streptococci), Staphylococcus (Staphylococci), intestinal bacteria (E.coli), streptomyces (Streptomyces) and Bacillus subtillis (Bacillus subtilis) cell; Fungal cell, for example yeast cell and Aspergillus (Aspergillus) cell; Insect cell is Drosophila S2 (Drosophila S2) and Spodoptera Sf9 (Spodoptera Sf9) cell for example; Zooblast is CHO, COS, Hela, C127,3T3, BHK, HEK293 and Bao Si melanoma cells (Bowes melanoma cells) for example; And vegetable cell.

Can use many expression systems.This type systematic especially comprise be derived from chromosomal, system episome and virus, for example, be derived from bacterial plasmid, phage, transposon, the yeast episome, parenthesis, the yeast chromosomal composition, virus is (as baculovirus (baculoviruses), papovavirus (papova viruses) is as SV40, vaccinia virus, adenovirus, fowlpox virus (fowl pox viruses), pseudorabies virus (pseudorabies viruses) and retroviral (retroviruses)) plasmid, and the plasmid that is derived from its combination, be derived from the plasmid of plasmid and phage genetic constitution as those, as clay and phagemid.Expression system can comprise the control region, and its adjusting also causes expression.Usually can use arbitrary system or carrier, it is suitable for keeping, breeds or expresses polynucleotide to produce polypeptide in the host.One of available many known and routine techniquess will be suitable nucleotide sequence insertion expression system, people such as Sambrook for example, molecular cloning: laboratory manual (source is the same).

Suitable secretion signal can be added target polypeptides, make to be translated protein secreting and to go into endoplasmic, periplasmic space or secrete external environment to born of the same parents.These signals can be endogenous for polypeptide or their allos signals.

If the IGS1 polypeptide expression is used for screening assay, preferably produce polypeptide usually at cell surface.In this case, can be before using screening to analyze harvested cell.When the affinity of IGS1 polypeptide or functionally active are modified by receptor active modifying protein (RAMP), preferably and normally need at the relevant RAMP of cell surface coexpression as much as possible.In this case, need before analyzing, screening gather in the crops the cell of expressing IGS1 polypeptide and relevant RAMP equally.If the IGS1 polypeptide is secreted to substratum, recyclable substratum is to reclaim and purified polypeptide; If be created in the born of the same parents, lysing cell at first before reclaiming polypeptide.

Can adopt known method from the reconstitution cell culture, to reclaim and purifying IGS1 polypeptide, comprising ammonium sulfate or ethanol sedimentation, acid extraction, negatively charged ion or cation-exchange chromatography, phosphorylated cotton chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxyapatite chromatography and lectin chromatography.Most preferably, use the high performance liquid chromatography purifying.Can use known techniques to carry out proteinic refolding, make the polypeptide of sex change in separation and/or purge process be regenerated as activity conformation.Diagnostic assay

The present invention also relates to the purposes of IGS1 polynucleotide as diagnostic reagent.Detection to the mutant form of the IGS1 gene relevant with dysfunction can provide a kind of diagnostic tool, and it can be added into or define a kind of disease or disease susceptibility, its come from IGS1 low expression, cross to express or express and change.Equally in this case, need the coexpression of relevant receptor active modifying protein, to obtain the diagnostic assay of desired quality.Can detect in dna level by a series of technology and carry the individuality of IGS1 transgenation.

Diagnostic nucleic acid can be from experimenter's cell.For example come autoblood, urine, saliva, examination of living tissue or necrotomy material.Genomic dna can be directly used in detection or by PCR or other amplification technique it is carried out enzymatic amplification before analysis.RNA or cDNA also can use in a similar manner.Can detect deletion and insertion by size with normal genotype comparison amplified production.The DNA of amplification and the IGS1 nucleotide sequence hybridization of mark can be identified point mutation.The sequence of mating fully can be distinguished with the two strands of mispairing by RNase digestion or by the difference of denaturation temperature and to be come.By the change of dna fragmentation electrophoretic mobility in being with or without the gel of denaturing agent, or also can detect the difference of dna sequence dna by direct dna sequencing.See for example people such as Myers, science (1985) 230:1242.Also can measure the change that discloses specific site place sequence by the nuclease protection, for example RNase and S1 protection or chemical chop method are measured in wherein said nuclease protection.See people such as Cotton, newspaper (1985) 85:4397-4401 of institute of NAS.In another embodiment, can be by making up the effective scanning that the Oligonucleolide primers array carries out for example transgenation, wherein said primer comprises IGS1 nucleotide sequence or its fragment.The array technique method is known, and the tool general applicability, can be used for solving the many problems in the molecular genetics, comprises genetic expression, genetic linkage and hereditary variability.(for example seeing: people such as M.Chee, science, 274 volumes, 610-613 page or leaf (1996)).

Diagnostic assay provides the diagnosis of especially above-mentioned disease susceptibility or measuring method, and method is by the sudden change in the described method detection IGS1 gene.Diagnostic assay provides especially to diagnosis or measuring method psychiatric and the disorderly susceptibility of CNS, especially motion function obstacle, ataxia or disease, for example twitch, tremble, Tourette's syndrome, parkinsons disease, huntington disease, dyskinesia, dystonia and spasm, method is to detect sudden change in the IGS1 gene by described method.

In addition, especially above-mentioned disease can be diagnosed by comprising the method for measuring sample, and wherein sample comes from the experimenter of IGS1 polypeptide or decline of IGS1 mRNA horizontal abnormality or rising.Can diagnose by comprising the method for measuring sample, particularly psychiatric and CNS disorder, especially motion function obstacle, ataxia or disease, for example twitch, tremble, Tourette's syndrome, parkinsons disease, huntington disease, dyskinesia, dystonia and spasm, wherein sample comes from the experimenter that IGS1 polypeptide or IGS1 mRNA horizontal abnormality descend or raise.

Can use arbitrary well known in the art, be used for measuring decline or the rising of expressing for example PCR, RT-PCR, RNase protection, Northern trace and other hybridizing method in the method for the quantitative polynucleotide of rna level.The determination techniques that is used for measuring from host's sample protein level (as IGS1) is as well known to those skilled in the art.This type of measuring method comprises that radioimmunoassay, competition are in conjunction with mensuration, Western engram analysis and ELISA mensuration.

The present invention relates to diagnostic kit on the other hand, is used to diagnose especially above-mentioned disease or to the diagnosis of susceptibility of one of above-mentioned disease.Especially, the present invention relates to psychiatric and the diagnostic kit CNS disorder, especially motion function obstacle, ataxia or disease are for example twitched, are trembled, Tourette's syndrome, parkinsons disease, huntington disease, dyskinesia, dystonia and spasm.

Test kit can comprise:

(a) IGS1 polynucleotide, the preferably nucleotide sequence of SEQ ID NO:1 or its fragment; And/or

(b) with (a) complementary nucleotide sequence; And/or

(c) IGS1 polypeptide, the preferably polypeptide of SEQ ID NO:2 or its fragment; And/or

(d) at the antibody of IGS1 polypeptide, preferably at the antibody of the polypeptide of SEQ ID NO:2; And/or

(e) RAMP polypeptide, it is that the associated biomolecule characteristic or the antigenic characteristic of IGS1 polypeptide is required.

Be to be understood that in arbitrary this type of test kit (a) and (b), (c), (d) and (e) can comprise its basal component.Karyomit(e) is measured

Nucleotide sequence of the present invention identifies it also is valuable to karyomit(e).This sequence specific ground target and can with the specific position hybridization on the human chromosome of individuality.Is the first step important step that those sequences and gene-correlation disease are connected according to the present invention to the mapping of karyomit(e) correlated series.In case certain sequence is mapped to accurate chromosome position, physical location and the genetic map data of this sequence on karyomit(e) are interrelated.These type of data can be at, V.McKusick for example, human Mendelian inheritance (Mendelian Inheritance in Man) (by with online acquisition of Johns Hopkins University's Welch medical library) in find.Identify that by linkage analysis (physics adjoins the common succession of gene) mapping is to the gene of same chromosomal region and the relation of disease then.

Also can measure the difference of affected and unaffected individual cDNA or genome sequence.If observed certain sudden change at some or all affected individualities, and do not observed this sudden change at arbitrary normal individual, then this sudden change may be the factor that causes disease.Antibody

Polynucleotide of the present invention or its fragment or its analogue, or the cell of expressing their (if necessary, expressing together with relevant RAMP) also can be used as immunogen, to produce to the specific antibody of IGS1 polypeptide immune.Term " immunologic opsonin " refers to that antibody compares to the affinity of other related polypeptide in the prior art with them to the affinity of peptide more than the present invention, and antibody has obviously bigger affinity to the former.

Can use ordinary method, preferably inhuman by polypeptide or fragment, analogue or cell with epi-position are granted animal, to obtain to produce antibody at the IGS1 polypeptide.For MONOCLONAL ANTIBODIES SPECIFIC FOR, can use arbitrary providing to cultivate the technology that produces antibody by successive clone.Example comprises hybridoma technology (Kohler, G. and Milstein, C., nature (1975) 256:495-497), three knurl trioma technology, human B cell hybridoma technology (people such as Kozbor, immunology today (Immunology Today) (1983) 4:72) and EBV-hybridoma technology (people such as Cole, monoclonal antibody and oncotherapy (MONOCLONAL ANTIBODIES ANDCANCER THERAPY), 77-96 page or leaf, Alan R.Liss.Inc., 1985).

Above-mentioned antibody can be used for separating or identifying the clone of express polypeptide, or by the affinity chromatography purified polypeptide.

This type of anti-IGS1 polypeptide antibody or anti-IGS1 polypeptide-RAMP mixture antibody also can be used for the especially above-mentioned disease of treatment.Especially, this type of anti-IGS1 polypeptide antibody or anti-IGS1 polypeptide-RAMP mixture antibody can be used for treating psychiatric and CNS is disorderly, especially motion function obstacle, ataxia or disease are for example twitched, are trembled, Tourette's syndrome, parkinsons disease, huntington disease, dyskinesia, dystonia and spasm.Animal

Another aspect of the present invention relates to the system based on the non-human animal, and it is as expressing because of IGS1 is unusual or the active disorderly model that causes unusually.The activity that also can be used for further characterizing the IGS1 gene based on non-human animal's model system.This system can be used as authenticating compound and the part of the screening strategy that designs, and wherein said compound can be treated the disorder based on IGS1, especially above-mentioned disease.Especially, this system can be used as authenticating compound and the part of the screening strategy that designs, wherein said compound can be treated based on the psychiatric of IGS1 and CNS disorder, especially motion function obstacle, ataxia or disease are for example twitched, are trembled, Tourette's syndrome, parkinsons disease, huntington disease, dyskinesia, dystonia and spasm.

By this way, can be used for identifying medical compounds, therapy and intervention method based on the model of animal, they can treat effectively that unusual IGS1 expresses or the active unusual and disorder that causes.In addition, this type of animal model can be used for determining the LD50 and the ED50 of animal subject.These data can be used for determining the interior validity of body of potential IGS1 disorder treatment.

Can comprise that the transgenic animal of non-recombinant animal and recombination engineering, wherein said disorder are based on that unusual IGS1 expresses or active unusual based on the IGS1 disorder based on the model system of animal.

The model of the animal of IGS1 disorder comprises for example genetic model.Demonstration can be by for example using based on the animal model of the disorderly sample symptom of IGS1, and the technology of the generation transgenic animal that aforesaid those IGS1 sequences and those skilled in the art are known combines, and carries out genetically engineered design.For example, the IGS1 sequence can be imported the genome of target animal, and make it to express and/or wrong the expression, perhaps, if when having endogenous IGS1 sequence, they can be crossed and be expressed, wrong express or additionally they can be destroyed, so that IGS1 expression of gene deficiency or inactivation.

To express or the wrong IGS1 of expression gene order in order crossing, the encoding part of IGS1 gene order can be connected with the adjusting sequence, the latter can drive high-caliber genetic expression, or expresses this gene in the cell type of the target animal type of not expressing this gene usually.This type of regulatory region will be known for those skilled in the art, and need not undo experimentation and can utilize.

For the low expression of endogenous IGS1 gene order, the sequence that the design of separable and genetically engineered is such, make its genome that imports target animal again after, the allelotrope of deactivatable or " knocking out " endogenous IGS1 gene.Preferably, engineered IGS1 gene order imports by gene targeting, makes endogenous IGS1 sequence destroyed after engineered IGS1 gene order is integrated into the animal gene group.

The arbitrary class animal that can be used to produce the animal model of IGS1 associated disorders includes, but not limited to the animal of mouse, rat, rabbit, squirrel, cavy, pig, piggy, goat and non-human primates, for example baboon, monkey and chimpanzee.

Available arbitrary technology well known in the art imports animal with the IGS1 transgenosis, to produce the initial system of transgenic animal.This type of technology includes, but not limited to protokaryon microinjection (Hoppe, P.C. and Wagner, T.E., 1989, U.S. Patent number 4,873,191); The germline gene of retroviral mediation shifts (people such as van der Putten, institute of NAS reports 82:6148-6152,1985); The gene targeting of embryonic stem cell (people such as Thompson, cell (Cell) 56:313-321,1989); Embryo's electroporation (Lo, molecular cytobiology (Mol.Cell.Biol.) 3:1803-1814,1983); And the transgenosis of sperm mediation people such as (, cell 57:717-723,1989) Lavitrano etc.The summary of this type of technology is seen Gordon, transgenic animal, international cytology comment (Intl.Rev.Cytol.) 115:171-229,1989.

The invention provides and all carry the genetically modified transgenic animal of IGS1 in all cells, and carry this genetically modified animal in some cells (but not all cells), promptly inlay animal.(for example see the technology that Jakobovits describes, current biology (Curr.Biol.) 4; 761-763,1994).Transgenosis can be used as single transgenosis and integrates or integrate as concatermer, for example head-head series connection or head-tail series connection.By people's such as for example following Lasko method (institute of NAS reports 89:6232-6236 for Lasko, people such as M., 1992), this transgenosis can be imported specific cell type and activation by selectivity.

This type of cell type specificity activates required adjusting sequence and depends on specific target cell type, and this point is obvious to those skilled in the art.

When wishing that the IGS1 transgenosis is integrated in the chromosomal foci of endogenous IGS1 gene, preferred gene is practiced shooting.In brief, when desire is used this technology, design is as the carrier of integrating purpose, wherein said carrier contains some nucleotide sequences (for example nucleotide sequence of mouse IGS1 gene) with the endogenous IGS1 dna homolog of target, by with the homologous recombination of chromosome sequence, import and destroy the function of the allelic nucleotide sequence of endogenous IGS1 gene or IGS1 gene.By people's (Gu, people such as H., science 265:103-106,1994) such as for example following Gu method, this transgenosis also can be imported specific cell type by selectivity, so only inactivation target native gene in this cell type.The required adjusting sequence of this type of cell type specificity inactivation depends on specific target cell type, and is obvious to those skilled in the art.

In case produced transgenic animal, available standards technical measurement reorganization IGS1 gene and protein expression.Initial screening can come analyze animal tissue by Southern engram analysis or round pcr, and whether genetically modified integration takes place to measure.Also can use the genetically modified mRNA expression level of IGS1 in the following technical evaluation transgenic animal tissue, wherein technology includes, but not limited to Northern engram analysis, in situ hybridization analysis and RT-PCR from animal tissues's sample.The tissue sample of expressing target gene also can use the assessment of antibody mediated immunity cytochemistry, and wherein antibody is special to the transgenosis target product of target gene.Then to further assessing, to identify indicating characteristic those animals based on the disorderly symptom of IGS1 with the IGS1 transgenic animal that are easy to detected horizontal expression IGS1 gene mRNA or IGS1 transgenosis peptide (antibody with direct anti-target gene product epi-position detects by immunocytochemistry).

In case producing the genetically modified initial animal of IGS1 (is that those express the proteinic animal of IGS1 in target cell or tissue, and preferably, demonstration is based on those animals of the disorderly symptom of IGS1), can breed them, close breeding, outbreeding or cross-breeding, to produce particular animals colony.The example of this type of breeding strategy includes, but are not limited to: will have the initial animal outbreeding more than an integration site, and separate strain to set up; To separate the strain close breeding, with generation compound IGS1 transgenics, because the genetically modified adduction expressional function of each IGS1, but its high level expression target IGS1 transgenosis; The transgenic animal hybridization of heterozygote, to produce the animal of isozygotying in the specific integration site, purpose is to improve expression and need not to screen animal by DNA analysis; With isolating homozygote incross, to produce compound heterozygote or homozygote strain; The animal that breeding has different close breeding genetic backgrounds changes allelotrope to the genetically modified expression of IGS1 and to producing the influence of IGS1 sample symptom with check.A kind of method is with genetically modified initial animal of IGS1 and wild-type incross, to produce the F1 generation of the disorderly sample symptom that shows that aforesaid those IGS1 are relevant.Then with F1 for inbreeding, to develop a kind of homozygote strain, can survive if find homozygous target gene transgenic animal.Vaccine

Another aspect of the present invention relates to the method for inducing the mammalian immune reaction; it comprises grants (for example by inoculation) Mammals IGS1 polypeptide or its fragment; if necessary; together grant with the RAMP polypeptide; be enough to produce antibody and/or t cell immune response, avoid one of especially above-mentioned disease to protect described animal.

Another aspect of the present invention relates to the method for inducing the mammalian immune reaction, and it comprises by carrier sends the IGS1 polypeptide, and wherein carrier can with the induction of immunity reaction, protect described animal to avoid disease in expression in vivo IGS1 polynucleotide.

The further aspect of the present invention relates to immunity/vaccine preparation (composition), and it can induce the immune response to the IGS1 polypeptide after importing mammalian hosts in Mammals, and wherein composition comprises IGS1 polypeptide or IGS1 gene.This para-immunity/vaccine preparation (composition) can be therapeutic immunization/vaccine preparation or preventative immunity/vaccine preparation.Vaccine preparation can further comprise suitable carriers.Because the IGS1 polypeptide can be degraded under one's belt, so parenteral administration (comprising subcutaneous injection, intramuscularly, intravenous injection, intradermal injection etc.) preferably.The preparation that is suitable for parenteral administration comprises water-based and non-aqueous aseptic injectable solution, the isoosmotic solute of blood that it can comprise antioxidant, damping fluid, fungistat and can make preparation and receptor; And water-based and non-aqueous sterile suspension, it can comprise the suspension factor or the thickening factor.Preparation can unitary dose or multiple doses be present in the container, for example sealed ampoule and phial, and can store under the lyophilize condition only need to add sterile liquid carrier before use.Vaccine preparation also can comprise adjuvant system, to promote the immunogenicity of preparation, oil-in-water system for example well known in the art and other system.Dosage depends on the specific activity of vaccine, and is easy to measure by normal experiment.Screening assay

IGS1 polypeptide of the present invention can be used as the screening method of compound, and wherein compound combines with this receptor, and the activation of activation (agonist) or inhibition (antagonist) the present invention's receptor polypeptides.Like this, polypeptide of the present invention also can be used for for example being evaluated at, and in cell, acellular prepared product, chemical library and the natural product mixture, the small molecules substrate combines with part.These substrates and part can be natural substrate and parts, or the stand-in of structure or function.

The IGS1 polypeptide is responsible for biological function, comprises the pathology aspect.Wish in view of the above to find compound and medicine, it stimulates IGS1 on the one hand, and it can suppress the function of IGS1 on the other hand.Usually, agonist is used for treatment and the prevention purpose to especially above-mentioned disease situation.Especially, agonist is used for the treatment of psychiatric and CNS disorder and prevention purpose, especially motion function obstacle, ataxia or disease are for example twitched, are trembled, Tourette's syndrome, parkinsons disease, huntington disease, dyskinesia, dystonia and spasm.

Antagonist can be used for a series of treatments of especially above-mentioned disease situation and prevention purpose.Especially, antagonist is used for the treatment of psychiatric and CNS disorder and prevention purpose, especially motion function obstacle, ataxia or disease are for example twitched, are trembled, Tourette's syndrome, parkinsons disease, huntington disease, dyskinesia, dystonia and spasm.

Usually, this screening step comprises and produces suitable cell, and it is at surface expression receptor polypeptides of the present invention, and if necessary, with RAMP at its surperficial coexpression.This type of cell comprises from Mammals, yeast, Drosophila or colibacillary cell.Then, the cell cytolemma of expressed receptor (or contain) of expressing this receptor is contacted with test compound, with the observation combination, or the stimulation of functional response or inhibition.

A kind of triage techniques is included in the system and uses the cell (for example, the Chinese hamster ovary celI of transfection) of expressing acceptor of the present invention, and wherein system can measure the cellular calcium that pH in the outer pH of born of the same parents, the born of the same parents or receptor activation cause and changes.Whether in this technology, compound and the cells contacting of expressing receptor polypeptides of the present invention are measured second messenger's reaction then, and for example signal transduction, pH change or the change of calcium level, activate or suppressed this receptor to determine the potential compound.

Another kind method relates to the screening of acceptor inhibitor, and it is undertaken by measuring receptor-mediated Signal Regulation, for example cAMP accumulation and/or adenylate cyclase activity.This method comprises with acceptor transfecting eukaryotic cells of the present invention, with at the cell surface expression this receptor.Then when having the potential antagonist, with the agonist of cellular exposure in acceptor of the present invention.If potential antagonist and receptors bind can suppress the combination of acceptor like this, regulate the signal of agonist mediation.

Another kind of detect the agonist of acceptor of the present invention or the method for antagonist is at U.S. Patent number 5,482, describe in 835 based on the zymic technology.

Mensuration can only need the combination of test candidate compound, and wherein the adhesion with the cell that has acceptor can relate to the competition thing competition with mark by detecting with the direct or indirect bonded mark of candidate compound in perhaps measuring.Further, use the detection system that is suitable for the surface is carried the cell of acceptor, whether these mensuration can test candidate compound and cause signal to produce by activated receptor.Usually when known agonist exists, measure the inhibitor of activation, and when candidate compound exists, observe the influence of agonist activation.

Further, mensuration can only need comprise following steps: candidate compound is mixed with the solution that contains the IGS1 polypeptide to form mixture, measure IGS1 activity in the mixture, and active and standard compares with the IGS1 of mixture.

IGS1 cDNA, protein and this proteinic antibody also can be used for configuration to be measured, and is used to detect the interior IGS1 mRNA of compound pair cell of interpolation and the influence that protein produces.For example by standard method well known in the art, make up ELISA with mono-clonal and polyclonal antibody, to measure proteinic secretion level of IGS1 or cell related levels, and it can be used for from find such medicament through the cell or tissue of proper handling, and it can suppress or strengthen the generation (also being called antagonist and agonist) of IGS1.The standard method of carrying out screening assay is that this area is known.

The example of potential IGS1 antagonist comprise antibody or, sometimes, for oligonucleotide or with the closely-related protein of the part of IGS1, for example fragment of part or small molecules, itself and receptors bind but do not induce reaction cause receptor active to be suppressed.

So on the other hand, the present invention relates to the screening reagent box, it is used to identify agonist, antagonist, part, acceptor, substrate, enzyme of IGS1 polypeptide etc.; Perhaps be used to identify the compound that reduces or strengthen the generation of IGS1 polypeptide, it comprises:

(a) IGS1 polypeptide, the preferably polypeptide of SEQ ID NO:2;

(b) express the reconstitution cell of IGS1 polypeptide, preferably express the reconstitution cell of SEQ ID NO:2;

(c) express the cytolemma of IGS1 polypeptide, preferably express the cytolemma of SEQ ID NO:2; Or

(d) at the antibody of IGS1 polypeptide, the antibody of SEQ ID NO:2 preferably.

Be to be understood that in arbitrary this type of test kit (a) and (b), (c) or (d) can comprise its basal component.Prevention and methods of treatment

The invention provides the method for the excessive or not enough relevant abnormal condition of treatment and IGS1 activity.

If IGS1 is excessively active, can adopt certain methods.A kind of method comprises grants experimenter with pharmaceutically acceptable carrier with significant quantity with aforesaid inhibitor compound (antagonist), by combining of block ligand and IGS1, or by the interaction of inhibition with RAMP polypeptide or second signal, thereby the inhibition activation is alleviated abnormal condition thus.

In another approach, can use the soluble form of IGS1 polypeptide, it still can compete the ground binding partner with endogenous IGS1.The general embodiment of this type of competition thing comprises the IGS1 polypeptide fragment.

Also have another kind of method, the available expression of the expression of the gene of the endogenous IGS1 that encodes-interrupter technique suppresses.Known this type of technology relates to the use of antisense sequences, this antisense sequences or produce in inside, or use separately.For example see, O ' Connor, neurochemistry magazine (J Neurochem) (1991) 56:560 oligodeoxynucleotide is as the antisense inhibitor (Oligodeoxynucleotidesas Antisense Inhibitors of Gene Expression) of genetic expression, CRC Press, Boca Raton, U.S. Florida (1988).In addition, can provide the oligonucleotide that can form triple helix with gene.For example see people such as Lee, nucleic acids research (1979) 6:3073; People such as Cooney, science (1988) 241:456; People such as Derven, science, (1991) 251:1360.Itself can use these oligomers, perhaps can express relevant oligomer in vivo.Synthetic antisense or three chain oligonucleotide can contain modified base or modified main chain.The latter's example comprises methyl phosphorodithioate, thiophosphatephosphorothioate or peptide nucleic acid(PNA) main chain.In antisense or three chain oligonucleotide, mix this type of main chain, be not subjected to the degraded of nuclease with protection, and be that this area is known.Antisense or three chain molecules that synthetic has these or other modified main chain have also formed a part of the present invention.

In addition, can use polypeptide expression to the sequence-specific ribozyme prevention of IGS1 mRNA IGS1.Ribozyme is the RNA with enzymic activity, it can be natural or synthetic (see for example Usman, people such as N., the up-to-date viewpoint of structure biology (Curr.Opin.Struct.Biol.) (1996) 6 (4), 527-33).The synthetic ribozyme can be designed to cut IGS1 mRNA specifically in selected site, stops IGS1mRNA to be translated as functional polypeptide thus.Available natural ribose phosphate backbone and natural base synthetic kernel enzyme are as usually seen in the RNA molecule.In addition, available non-natural main chain synthesizes ribozyme, so that the protection of avoiding nuclease degradation to be provided, and 2 '-O-methyl RNA for example, and can contain modified base.

For treatment and IGS1 and its active low relevant abnormal condition of expressing, also can adopt certain methods.A kind of method comprises grants experimenter with the pharmaceutically acceptable carrier combination with the treatment significant quantity with the compound (being above-mentioned agonist) of activation IGS1, therefore to alleviate abnormal condition.In addition, can use gene therapy, produce endogenous IGS1 to realize the intravital relevant cell of experimenter.For example, as discussed above, polynucleotide of the present invention can be designed by genetically engineered, so that express in the retroviral vector of replication defective.Separable then this retroviral expression construct also imports packing cell with it, and wherein packing cell makes packing cell can produce the infectious virus particle that contains target gene now with the retroviral plasmid vector transduction of the RNA that contains code book invention polypeptide.These producer's cells can be granted the experimenter and are used for the construct in vitro cell, and express polypeptide in vivo.The summary of gene therapy is seen the 20th chapter in people's molecular genetics (Human Molecular Genetics) book, gene therapy and other methods of treatment (Chapter 20 based on molecular genetic, Gene Therapy andother Molecular Genetic-based Therapeutic Approaches) (and quoting as a reference) herein, Strachan T. and Read A.P., BIOS Scientific Publishers Ltd (1996).

Above-mentioned arbitrary methods of treatment can be used for the experimenter of arbitrary this type of treatment of needs, for example comprises Mammals such as dog, cat, ox, horse, rabbit, monkey and most preferably, people.

Preparation and using

Peptide, for example soluble form of IGS1 polypeptide, and agonist and antagonist peptide or small molecules can form preparation with the suitable pharmaceutical carrier combination.This type of preparation comprises the polypeptide or the compound for the treatment of significant quantity, and pharmaceutically acceptable carrier or vehicle.Preparation should be the pattern that is suitable for using, and in the technical scope of this area.The invention further relates to pharmacy packing and test kit, they contain one or more containers of the composition filling of useful one or more above-mentioned compositions of the present invention.

Polypeptide of the present invention and other compound can use separately, or with other compound, as the combination of treatment compound.

The preferred form that pharmaceutical composition is carried out systemic application comprises injection, generally by intravenous injection.Also available other injecting pathway, for example subcutaneous injection, intramuscularly or intraperitoneal injection.Other approach that is used for systemic application comprises that use permeate agent (as biliary salts or fusidic acid or other stain remover) is through mucous membrane with through dermal administration.In addition, if having suitable Enteral formulations or capsule preparations, also can be Orally administered.

Required dosage range depends on the character of the character of the selection of peptide or compound, route of administration, preparation, experimenter's situation and cures mainly practitioner's decision.Suitable dosage ranges is 0.1-100 μ g/kg experimenter.But in view of chemical compound lot is available, and different route of administration has different validity, can estimate that there is big difference in required dosage.For example, can estimate Orally administered and the intravenous injection comparison, the former needs higher dosage.The daily experience adjustment of the difference available standards between these dosage levels is optimized, and this point is that this area is known.

The polypeptide that is used for the treatment of also can produce in subject endogenously, is commonly referred to aforesaid " gene therapy " on form of therapy.So for example, can use polynucleotide (as DNA or RNA) genetic engineering modified from experimenter's cell, with the coded polypeptide that exsomatizes, and for example, by the use of retroviral plasmid vector.Then cell is imported the experimenter.

The following example only is used for further illustrating in more detail the present invention, in any case so these embodiment be not used in and limit the scope of the invention.The homology PCR clone of the cDNA clone embodiment 1a. genomic fragment of the g protein coupled receptor that embodiment 1. codings are new, the wherein new g protein coupled receptor (GPCR) of genomic fragment coding.

The homologous clone strategy of PCR-based is used for the separate part genomic dna sequence, the new g protein coupled receptor of wherein said genomic dna sequence coding.Ring n02 (TM3/12) intersection designs following forward (F11) and reverse (R13) degenerate pcr primer in the conserved regions of neurotensin acceptor gene family membrane spaning domain 1 (TM1) and in membrane spaning domain 3 and the born of the same parents respectively:

F11(TM1)：

5’-CATCTTCGTCGTCGGCAC(A，C，G?orT)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’-GGGTGGCAGATGGCCA(A?or?G)(A?or?G)(C?or?T)A(A，C，G?orT)c(G?or?T)(C?or?T)TC-3’

(SEQ?ID?NO：4)

In addition, designed the few primer (HNTR1F1STOP) of one 3 ' sealing:

HNTR1F1STOP：

5’-ACGGTGGGCAACACGGTGACGGCGTT-3’-3’-dA

(SEQ?ID?NO：5)

3 ' sealing primer is to being special in people's neurotensin acceptor (NTR1) cDNA TM1 coding region, and overlap with the degeneracy forward primer (and competition).Its 3 '-end is with the sealing of 3 '-Desoxyadenosine group, to stop the catalytic extension of polysaccharase (Eurogentec, Belgium catalogueOL-0401-0302).

In 60 μ l volumes, carry out the PCR reaction, wherein contain 100ng human gene group DNA (Clontech), 6 μ l, 10 * PCR damping fluid II (100mM Tris-HCl pH8.3; 500mMKCl, Perkin Elmer), 3. μ l 25mM MgCl ₂, 0.36 μ l dNTP (every kind of dNTP25mM), 1.5 AmpliTaq of unit ^TMThe forward of polysaccharase (Perkin Elmer), every degeneracy and each 30pmole of reverse primer, and 3 ' sealing primer 100pmole.Reaction tubes was in 94 ℃ of heating 2 minutes, carry out (94 ℃ of 20 round-robin sex change then, 30 seconds), annealing (55 ℃, 1 minute ,-0.25 ℃/circulation of each minimizing) and extension (72 ℃, 1 minute), next carry out another and take turns (94 ℃ of 20 round-robin sex change, 30 seconds), annealing (50 ℃, 1 minute) and the extension (72 ℃, 1 minute).At last in 72 ℃ of 5 minutes reacting by heating pipes.2% sepharose separates the size of PCR reaction product and uses ethidium bromide staining.Use Qiaex-II ^TMExpection size on purification kit (QiagenInc.) the purifying gel (± 300bp) fragment, and be connected to the pGEM-T plasmid according to supplier (pGEM-T test kit Promega) recommendation step.The recombinant plasmid of Chan Shenging is used for transformed competence colibacillus intestinal bacteria SURE like this ^TM2 bacteriums (Stratagene).

Cell transformed is laid on the LB agar plate that contains penbritin (100 μ g/ml), IPTG (0.5mM) and X-gal (50 μ g/ml).Colony lift to Hybond N+ film (Amersham) is gone up and according to people's such as Buluwela microwave oven step (17,452 pages of nucleic acids research; 1989) sex change and fixed dna.Shift colony with 65 ℃ of prehybridizations of improvement Church damping fluid (0.5M phosphoric acid salt, 7%SDS, 10mM EDTA) 2 hours, wait mole containing then ³²2 * 10 of people's neurotensin acceptor 1 of P-mark and 2 cDNA probes (NTR1/2) ⁶65 ℃ of hybridization of spending the night in the same damping fluid of cpm/ml.According to the guidance that supplier provides, use Prime-It II kit ^TM(Stratagene) by random priming will [α- ³²P] dCTP mixes, and carries out the radio-labeling of cDNA probe, makes its specific activity＞10 ⁹Cpm/ μ g, wherein said cDNA probe contains whole encoding sequences of people NTR1 and NTR2.Washing hybridization filter membrane (washed under with 2 * SSC/0.1%SDS room temperature 2 * 30 minutes, and used 0.1 * SSC, 0.1%SDS to wash each 40 minutes 2 times then) under the high stringent condition, and radioautograph is spent the night in 65 ℃.Select some high tightly to show that the white at random colony of amixia signal is used for dna sequence analysis after washing film.

Stop terminal cycle sequencing reaction kit (PE-ABI) with ABI Prism BigDye and carry out dna sequencing reaction.Precipitate purification cycle sequencing reaction product and be splined on ABI 373 automatic sequencers by EtOH/NaOAc.Two clones about the same (HNT642 and HNT768) have been identified, a newcomer's part in its GPCR family of as if encoding.We claim that this new GPCR is IGS1.

Table 3: used few primer overview

SEQ?ID?NO：3	?F11：5’-CATCTTCGTCGTCGGCAC(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		SEQ?ID?NO：4	?R13：5’-GGGTGGCAGATGGCCA(A?or?G)(A?or?G)(C?or?T)A(A，C，G?or?T)C(G ?or?T)(C?or?T)TC-3’
SEQ?ID?NO：5	?HNTR1F1Stop：5’-ACGGTGGGCAACACGGTGACGGCGTT-3’-3’-dA	SEQ?ID?NO：4
SEQ?ID?NO：5	?HNTR1F1Stop：5’-ACGGTGGGCAACACGGTGACGGCGTT-3’-3’-dA	SEQ?ID?NO：6	?AP1：5’-CCATCCTAATACGACTCACTATAGGGC-3’
SEQ?ID?NO：7	?AP2：5’-ACTCACTATAGGGCTCGAGCGGC-3’	SEQ?ID?NO：6	?AP1：5’-CCATCCTAATACGACTCACTATAGGGC-3’
SEQ?ID?NO：7	?AP2：5’-ACTCACTATAGGGCTCGAGCGGC-3’	SEQ?ID?NO：8	?IP11260：5’-TTTATCTTTAACCTCCTCGTCACCGACC-3’
SEQ?ID?NO：9	?IP11261：5’-TAGTGTTGCAGCGCAAGCCG-3’	SEQ?ID?NO：8	?IP11260：5’-TTTATCTTTAACCTCCTCGTCACCGACC-3’
SEQ?ID?NO：9	?IP11261：5’-TAGTGTTGCAGCGCAAGCCG-3’	SEQ?ID?NO：10	?IP11262：5’-GGGAGCGTTCCACTGACACCAAGACAATGG-3’
SEQ?ID?NO：11	?IP11263：5’-CAGCGTTCCACTGACACCAAGACAATGG-3’	SEQ?ID?NO：10	?IP11262：5’-GGGAGCGTTCCACTGACACCAAGACAATGG-3’
SEQ?ID?NO：11	?IP11263：5’-CAGCGTTCCACTGACACCAAGACAATGG-3’	SEQ?ID?NO：12	?IP11264：5’-AAGGCGAACAGGTGGGTGAGGCTAACC-3’
SEQ?ID?NO：13	?IP11515：5’-TGGCGAAGGCGAACAGGTGG-3’	SEQ?ID?NO：12	?IP11264：5’-AAGGCGAACAGGTGGGTGAGGCTAACC-3’
SEQ?ID?NO：13	?IP11515：5’-TGGCGAAGGCGAACAGGTGG-3’	SEQ?ID?NO：14	?IP11516：5’-GCGAAGGCGAACAGGTGGGTGAGG-3’
SEQ?ID?NO：15	?IP11684：5’-CTAGTGTTGCAGCGCAAGCCGCAG-3’	SEQ?ID?NO：14	?IP11516：5’-GCGAAGGCGAACAGGTGGGTGAGG-3’
SEQ?ID?NO：15	?IP11684：5’-CTAGTGTTGCAGCGCAAGCCGCAG-3’	SEQ?ID?NO：16	?IP12261：5’-CACAGAAAGCATAACCAGTGATTGAACC-3’
SEQ?ID?NO：17	?IP12262：5’-GCTTTAGGTTCCTGGAATCCCATTTGG-3’	SEQ?ID?NO：16	?IP12261：5’-CACAGAAAGCATAACCAGTGATTGAACC-3’
SEQ?ID?NO：17	?IP12262：5’-GCTTTAGGTTCCTGGAATCCCATTTGG-3’	SEQ?ID?NO：18	?IP12264：5’-TTGTCACCAGCATAGGCACTGAGTG-3’

Embodiment 1b. contains the clone of the cDNA fragment of complete IGS1 encoding sequence

Obtain the complete encoding sequence of IGS1 cDNA by the rapid amplifying method (RACE analysis) of cDNA end.Use Marathon-Ready ^TMHuman brain cDNA (Clontech n ⁰7400-1) carry out 5 ' and 3 ' RACE PCR, the primer is Marathon ^TMThe joint primer 1 that cDNA amplification kit (Clontech K1802-1) provides (AP1:SEQ ID NO:6) and based on the IGS1 Auele Specific Primer IP11261 (3 ' RACE of the dna sequence dna (Fig. 1) of clone HNT642 and HNT768; And IP11262 and IP11263 (5 ' RACE SEQ ID NO:9); Be respectively SEQ ID NO:10 and 11).Next use joint primer 2 (AP2; SEQ ID NO:7) and IGS1 specificity nested primer IP11260 (3 ' RACE; And IP11515 and IP11516 (5 ' RACE SEQ ID NO:8); Be respectively SEQ ID NO:13 and 14) carry out nido RACE PCR.According to the Marathon-Ready that Clontech provided ^TMGuidance in the cDNA user manual is carried out first and nest-type PRC RACE reaction.Dye with 1% sepharose separation nest-type PRC RACE product and with EtBr.With the gel trace to Hybond N+ film, and with clone HNT642 it ³²The insertion fragment of P-mark is in the hybridization of spending the night of 65 ℃ of Church hybridization buffers.

The Southern engram analysis of AP2/IP11515 and AP2/IP11516 5 ' RACE nest-type PRC reaction all show some positive bands (± 200bp, ± 250bp, ± 330bp, ± 360bp, ± 400bp and ± 700bp).Each band from gel these bands of purifying also is cloned into pGEM-T plasmid vector (the independent PCR fragment of hybrid IP 11515 and IP11516 nido 5 ' RACE reaction before the clone).To dropping into row order-checking (=clone HNT1393-1412) from each segmental 3-4 random set (Fig. 1).

Some bands of nido AP2/IP11260 3 '-RACE PCR reaction and display.From the gel purifying can with the maximum segment of 3 ' nido RACE PCR of IGS1 probe hybridization (± 1,550bp), and be connected to pGEM-T (Promega), be used for transformed competence colibacillus intestinal bacteria SURE II cell.With clone HNT642 it ³²The insertion fragment of p-mark is carried out colony hybridization, identifies from the special transformant of the IGS1 of this ligation.Carry out the colony blot hybridization with probe as previously mentioned, and under high stringent condition, wash (0.1 * SSC 0.1%SDS washed 30 minutes for 65 ℃).Screening by hybridization to 3 ' RACE nest-type PRC library has produced 3 positive colonies.To two in them check order (HNT1413-1414).

In two additional experiments, provide step in the handbook according to supplier (Clontech PT1156-1), from Marathon-Ready ^TMHuman brain cDNA (Clontech) obtains other three 3 ' RACE cDNA clones (HB4686, HB4687 and HB4688).In an experiment, the product (obtaining with IGS1 Auele Specific Primer IIP11261 and joint primer AP1) from elementary 3 ' RACE reaction is increased to IP11260/IP11261 (being respectively SEQ ID NO:8 and 16) with the half-nest type primer again.Produced like this anticipation ± the 1400bp fragment, with its purifying and be cloned into the pGEM-T plasmid vector from the gel, produce clone HB4686 and HB4687.In another experiment, elementary 3 ' RACE PCR reaction product (obtaining with IGS1 Auele Specific Primer IP11684 (SEQ ID NO:15) and joint primer AP1) is increased to IP11260/IP11261 with nested primer again.From this reaction, produce one ± 1400bp fragment, be purified and be cloned into the pGEM-T plasmid vector, produced clone HB4688.

To the order-checking of the separative IGS1 cDNA clone of institute total length, and can be assembled into single contig (Fig. 1).This sentences the cDNA sequence part that IGS1 DNA (SEQ ID NO:1) provides adjacency, wherein the cDNA sequence of adjacency be at least four independently cDNA clone measure determined.Translation to this contig demonstrates a long opening code-reading frame, and it is measurable for containing 508 amino acid whose protein, and this protein demonstrates and gpcr protein matter (IGS1PROT; SEQ ID NO:2) has good homology.The dna sequence dna and the expressed sequence tag database (dbest) of IGS1 contig are carried out computer assisted homology research (Blastn; People such as Altschul S.F., [1997], nucleic acids research, 25:3389-3402), show to have EST20889 (registration number AA318717) and EST registration number AI672141 that they are all overlapping with 3 ' end of IGS1 contig, but are positioned at IGS1 opening code-reading frame outer (Fig. 1).Embodiment 1c. separates the cDNA fragment of the adjacency that contains total length IGS1 encoding sequence

By clone HNT1398 and HNT1413 template are carried out the IGS1 cDNA clone that overlapping-PCR produces adjacency.(50 μ l) uses primer I P12264/IP11264 (being respectively SEQ ID NO:18 and 12) and IP11260/IP11262 (being respectively SEQ ID NO:8 and 17) that 100ng HNT1398 plasmid DNA and 100ng HNT1413 plasmid are carried out pcr amplification respectively in independently reacting, and (uses Expand ^TMHigh Fidelity PCR system [Boehringer] sex change [94 ℃, 30 seconds], annealing [60 ℃, 30 seconds] and 30 circulations of extension [72 ℃, 1 minute] reaction).Each PCR reaction product is got 1 μ l merge, and IP12264/IP12261 is increased under similarity condition again with primer.This overlapping-PCR has produced the band of one ± 1730bp, with its purifying and connect into the pGEM-T plasmid vector from the gel.Recombinant plasmid is used for transformed competence colibacillus bacillus coli DH 5 alpha F ' bacterium.Cell transformed is laid on the LB agar plate that contains penbritin (100 μ g/ml).Prepare plasmid DNA the colony at random from some, and determine to insert segmental size by restrictive diges-tion.To contain ± 1730bp inserts segmental three cloning and sequencings.The sequence of clone HB4693 and the total identical (see figure 1) of IGS1 cDNA sequence.The bacterial strain that has plasmid HB4693 is after being laid on the LB agar plate that contains penbritin (100 μ g/ml) once more, clone and be preserved in Innogenetics bacterial classification catalogue (ICCG#4297) again and be positioned at the fungi strain preservation center (CBS) of Baarn, Holland (preserving number CBS102049).Plasmid DNA from clone's strain isolated preparation is again checked order once more, and find identical with the consensus sequence that recorded in the past.

Attention: we found afterwards that primer I P12262 sequence was not included in the insertion sequence of clone HNT1413, and therefore can not produce amplicon from the HNT1413 template.We infer and directly overlapping by between the amplicon of HNT1413 plasmid DNA (being present in overlapping PCR reaction tubes as carrying to pollute) and the generation of HNT1398 template to have caused the successful amplification of overlapping fragments thus.The NORTHERN of embodiment 2.IGS1 and " MTE array " are analyzed the structure of embodiment 2a.pcDNA3.1 (+) hu IGS1 expression vector

5 μ g pcDNA3.1 (+) (Invitrogen) cut (37 ℃, 3 hours) with Hind III enzyme, use T when having dNTP (0.25mM f.c.) ₄Polysaccharase is mended flat, and carries out gel analysis.With linearizing DNA wash-out (using the Qiaex II of Qiagen to extract test kit) and be dissolved in 40 μ lH from the gel ₂O.This DNA digests with Not I, and carries out gel analysis once more.With the Qiaex II gel extraction kit 5364bp carrier segments that wash-out obtained from the gel, and be dissolved in 40 μ lH ₂O.Get 5 μ l and on gel, analyze, to check size, amount and purity.

5 μ g pGEM-T hu IGS1 plasmids (ICCG#4297) can obtain people IGS1 encoding sequence after digesting (37 ℃, 3 hours) with Nae I/Not I.Agarose gel electrophoresis shows that digestion has produced three fragments of 400bp, 1629bp and 2702bp.The fragment (Qiaex II) of wash-out 1629bp from the gel, and be dissolved in 40 μ l H again ₂O.Getting 5 μ l analyzes on gel.

To Ready-To-Go ligase enzyme pipe (T ₄Dna ligase, Amersham PharmaciaBiotech) pcDNA3.1 (+) carrier, the 3 μ l that add 1 μ lHind III digestion insert fragment and 16 μ lH ₂O was in room temperature incubation 1 hour.Ligation mixture with 2 μ l transforms through chemically treated competence DH5 α F ' bacterium.The bacterium that 200 μ l are transformed is laid on the LB flat board, and (100 μ g penbritins/ml) are in 37 ℃ of overnight growth.Select 16 clones at random, and be incubated in the LB substratum that 3ml contains penbritin.Use BioRobot ^TM9600 nucleic acid purification systems (Qiagen) prepare plasmid DNA, and carry out restriction analysis by Not I, Pst I and Sph I Restriction Enzyme.The DNA that has the colony of correct restriction enzyme digestion pattern from one is partly checked order, to confirm that inserting the site also finds to have the sequence of expectation.The clone of part order-checking is preserved in Innogenetics bacterial classification catalogue (ICCG#4350), and prepares a large amount of DNA (MegaPrep, Qiagen500 test kit) from preservation strain.The sequential analysis of this large-scale DNA prepared product (3 μ g/ μ l, totally 500 μ l) has confirmed the sequence of estimating.Embodiment 2b.MTE (many tissue expressions) array analysis

25ng people IGS1 DNA (from peDNA3.1 huIGS1[ICCG#4350] 1093bpAat II insert fragment) with (α- ³²P)-the dCTP mark.With this label probe of Micro Bio-Spin P-30 post (BioRad) purifying.16 * 10 ⁶The huIGS1 cDNA probe of cpm mark and the C of 30 μ g ₀The smart DNA of the cut-out Pacific herring of t-1,150 μ g and 50 μ l, 20 * SSC are mixed into cumulative volume 200 μ l, in 95 ℃ of heating 5 minutes, then in 68 ℃ of incubations 30 minutes.This mixture is added 5ml Express Hyb solution, and be evenly distributed on the many tissue expressions of people (MTE) arrays (Clontech#7775-1).This array is in 68 ℃ of hybridization of spending the night.With 65 ℃ of 2 * SSC/1%SDS developing and printing mark 4 times 20 minutes, use 55 ℃ of 0.1 * SSC/0.5%SDS to wash then 2 times 20 minutes.The X line film radioautograph of this trace.

The hybridization of IGS1 probe on the MTE array shows to have only caudatum and the strong signal of lenticular nucleus tool (Fig. 2).Embodiment 2c.Northern engram analysis

25ng people IGS1 DNA (from peDNA3.1 huIGS1[ICCG#4350] 1093bpAat II insert fragment) with (α- ³²P)-the dCTP mark.With this label probe of Micro Bio-Spin P-30 post (BioRad) purifying.8 * 10 ⁶The huIGS1 cDNA probe of cpm mark is in 95 ℃ of sex change 5 minutes, and it is added 5ml Express Hyb solution, and is evenly distributed on human brain MTN Blots II or the IV (being respectively Clontech#7755-1 and #7769-1).This trace is in 68 ℃ of hybridization of spending the night.With 2 * SSC/0.05%SDS room temperature developing and printing mark 4 times 10 minutes, use 50 ℃ of 0.1 * SSC/0.1%SDS to wash then 2 times 40 minutes.The X line film radioautograph of this trace.

The IGS1 probe is positioned at two treaties 4,400 of lenticular nucleus and caudatum and the strong band (Fig. 3) of 9,000 Nucleotide (nt) with showing from the Northern blot hybridization of different human brains district RNA.Lower band is a caudatum, and is intensive slightly, and another is lenticular situation.Also can see 4,400 and 9 in dorsal thalamus, the band of 000nt, but both are all very weak.In addition in black substance can detect one very weak 9,000nt transcript, but do not have 4, the band of 400nt.Observe at last in cerebellum, medullary substance and amygdaloid body extremely weak 9, the 000nt band.Can not observe 4 in dorsal thalamus and black substance, the band of 400nt.These results and MTE analytical results match, and it is according to being: the strongly expressed of observing IGS1 in caudatum and lenticular nucleus.But do not reckon with and to have 2 transcripts.Though 4,400nt band most probable is corresponding to IGS1 mRNA, to 9, the source of 000nt band is not clear.Because the IGS1 gene does not comprise intron (not having in the coding region at least), so 9, the 000nt transcript may not be the transcript of not montage or other montage.It may be the IGS1 transcript with other poly-adenosine action site, and perhaps it is a kind of class of cross hybridization.Our supposition: detect only one very weak 9,000nt transcript and do not detect 4 is during the situation of 400nt transcript, this is because 9, the 000nt transcript is than 4, and the 400nt transcript is strong slightly, and this is than low strap so just under the detectability that Northern measures.

IGS1 in situ hybridization by rat brain is analyzed and has further been confirmed these results, its with above-mentioned same anatomical area in detected the expression of IGS1.The IGS1 part embodiment 1a. that embodiment 3. screenings are inferred makes up the CHOG 16-cell of IGS1 transfection

For identifying the part of IGS1, with IGS1 stable transfection Chinese hamster ovary (CHO) cell.Because the G protein coupling of IGS1 mechanism is unknown, so used specific Chinese hamster ovary celI strain, it expresses G-Protein G 16, and (CHOG 16, Molecular Devices), be GPCR known " ubiquity conjugant " (Milligan G. waits people (1996) pharmaceutical science trend (TrendsPharmacol.Sci.) 17:235-7).

Material therefor comprises: the IGS1-pREP9 carrier; SuperFect Transfection Reagent (Qiagen); Growth medium: CHO-S-SFM II (Gibco BRL) replenishes 10%FCS, 2mM L-glutaminate, hygromycin B 400 μ g/ml; Select substratum: CHO-S-SFM II (Gibco BRL), replenish 10%FCS, 2mM L-glutaminate, hygromycin B 400 μ g/ml and Geneticin 500 μ g/ml; The little test kit of RNeasy (Qiagen), DNase I (Ambion, 2U/ μ l), SuperScript II (Gibco BRL), SuperScript II200U (Gibco BRL), AmpliTaq (PerkinElmer)

By Xho I/Nhe I site with the IGS1 encoding sequence from pcDNA3.1huIGS1[ICCG#4350] be cloned into pREP9 (Invitrogen).Described by supplier with SuperFect (Qiagen) transfection CHOG α 16 cells.In the T25 flask, carry out transfection., discard substratum and change the selection substratum after 24 hours with the growth medium cultivation.After growth converges sheet in selecting substratum, polyclone is gone down to posterity twice in the T25 flask.Cell is inoculated by limiting dilution, to obtain mono-clonal.

Select mono-clonal by RT-PCR.14 mono-clonals are tested.According to the method that is provided, use the little test kit of RNeasy (Qiagen) from mono-clonal (from 1 remittance film perforation of 24 orifice plates) isolation of RNA.DNase I (Ambion, 2U/ μ l) handles RNA, each sample 1U.With SuperScript II (Gibco BRL) half RNA sample is carried out RT-PCR.RNA and oligo-dT16 (0.6 μ M) carried out primer annealing in 10 minutes in 65 ℃, placed 15 ℃ then.Add the first chain damping fluid (Gibco BRL), and each 0.43mM of dNTP, DTT10mM, 20U RNasin (Promega, 40U/ μ l) and SuperScript II200U (GibcoBRL, 200U/ μ l) be to final volume 30 μ l, then in 42 ℃ of incubations 1 hour.

Carry out the PCR of 25 μ l with IGS1 specificity inner primer, AmpliTaq (PerkinElmer).At first carry out 35 round-robin PCR.In order to confirm positive monoclonal and to obtain a best mono-clonal, carry out another with less circulation and higher annealing temperature and take turns PCR.Each PCR reacts with 2 μ l, the first chain cDNA (from 30 μ l).

6 best mono-clonals are grown in the T25 flask and are converged sheet and freezing in the growth medium that contains 10%DMSO.Embodiment 3b. measures intracellular Ca2+

Measure the caused intracellular Ca2+ transfer of the reaction of inferring part with Fluorometric Imaging Plate Reader (FLIPR), thereby CHOG 16-IGS1 cell is carried out functional screening.

The preparation of pair cell has been used following material: clean, flat, black hole 96 orifice plates (Costar); Growth medium: the Nut-Mix F-12 (HAM) that contains Glutamax (Gibco) that replenishes 10% foetal calf serum (Gibco); Incubator: 5%CO2,37 ℃ (Nuaire).

At preceding 24 hours of experiment or 48 hours inoculating cells to black wall microwell plate.The cell density that desire was cultivated 48 hours is 0.8 * 10 ^-4The cell density that individual cells/well and desire were cultivated 24 hours is 2.2 * 10 ^-4Individual cells/well.Institute carries out under aseptic condition in steps.

To sample on the dyestuff, used following material: 2mM dyestuff stock solution: 1mg Fluo-4 (Molecular Probes) is dissolved in low water (low-water) DMSO (Sigma) (fluid preservation is at-20 ℃) of 443 μ l; 20% general sieve nicotinic acid (pluronic acid) solution:, the general sieve nicotinic acid of 400mg (Sigma) is dissolved in the low water DMSO (Sigma) (room temperature preservation) of 2ml in 37 ℃; Dyestuff/general sieve nicotinic acid mixed solution: before the use, isopyknic dyestuff stock solution is mixed (final concentration of dyestuff and general sieve nicotinic acid is respectively 1mM and 10%) with 20% general sieve nicotinic acid; 4-(dipropyl sulfamyl) phenylformic acid, the 250mM stock solution: 710mg 4-(dipropyl sulfamyl) phenylformic acid (Sigma) is dissolved in 5ml 1N NaOH, and mixes with no phenol red Hank ' s BSS (Gibco) that 5ml has replenished 20mM HEPES; Sample-loading buffer: 10.5ml has replenished no phenol red Hank ' the s BSS (Gibco) of 20mM HEPES, 105 μ l 4-(dipropyl sulfamyl) phenylformic acid, 210 μ l1M HEPES; Lavation buffer solution: no phenol red Hank ' the s BSS (Gibco) and 2.5mM 4-(dipropyl sulfamyl) phenylformic acid that have replenished 20mM HEPES.

2mM dyestuff stock solution adds in the sample-loading buffer immediately with after the general sieve nicotinic acid of isopyknic 20% (w/v) mixes.Under the situation of not destroying remittance sheet cellular layer, the sucking-off growth medium.Every hole is divided with Multidrop (Labsystems) and is added 100 μ l sample-loading buffers.Cell is in 5%CO ₂, 37 ℃ of incubators were cultivated 30 minutes.For calculating background fluorescence, sample on the dyestuff is not carried out in some holes.On the dyestuff behind the sample, wash cell three times in (Denley cell washing instrument automatically), so that basic fluorescence reduces on the background 20,000-50,000 counting with lavation buffer solution.Add 100 μ l lavation buffer solutions, and in 37 ℃ be cultured to begin the experiment.

Dilute screened compound with no phenol red Hank ' the s BSS (Gibco) that has replenished 20mM HEPES (Gibco) and 0.1%BSA (Sigma).Detect intracellular Ca2+ by manufacturer (Molecular Devices) is described with FLIPR.The FLIPR parameter of setting up adds for exposure 0.4 second, wave filter 1,50 μ l liquid, pipettor height 125 μ l, and velocity of diffusion 40 μ l/ do not have mixing second.

Sequence table＜110〉SOLVAY PHARMACEUTICALS B.V.＜120〉new human G-protein coupled receptor＜130〉SPW99.04＜140〉＜141＜160〉18＜170〉PatentIn Ver.2.1＜210〉1＜211〉1659＜212〉DNA＜213〉people (Homo sapiens)＜220〉＜221〉CDS＜222〉(36) .. (1559)＜400〉1gcctgcaacc tgtcycacgc cctctggctg ttgcc atg acg tcc acc tgc acc 53

Met?Thr?Ser?Thr?Cys?Thr

1???????????????5aac?agc?acg?cgc?gag?agt?aac?agc?agc?cac?acg?tgc?atg?ccc?ctc?tcc????101Asn?Ser?Thr?Arg?Glu?Ser?Asn?Ser?Ser?His?Thr?Cys?Met?Pro?Leu?Ser

10??????????????????15??????????????????20aaa?atg?ccc?atc?agc?ctg?gcc?cac?ggc?atc?atc?cgc?tca?acc?gtg?ctg????149Lys?Met?Pro?Ile?Ser?Leu?Ala?His?Gly?Ile?Ile?Arg?Ser?Thr?Val?Leu

25??????????????????30??????????????????35gtt?atc?ttc?ctc?gcc?gcc?tct?ttc?gtc?ggc?aac?ata?gtg?ctg?gcg?cta????197Val?Ile?Phe?Leu?Ala?Ala?Ser?Phe?Val?Gly?Asn?Ile?Val?Leu?Ala?Leu

40??????????????????45??????????????????50gtg?ttg?cag?cgc?aag?ccg?cag?ctg?ctg?cag?gtg?acc?aac?cgt?ttt?atc????245Val?Leu?Gln?Arg?Lys?Pro?Gln?Leu?Leu?Gln?Val?Thr?Asn?Arg?Phe?Ile?55??????????????????60??????????????????65??????????????????70ttt?aac?ctc?ctc?gtc?acc?gac?ctg?ctg?cag?att?tcg?ctc?gtg?gcc?ccc????293Phe?Asn?Leu?Leu?Val?Thr?Asp?Leu?Leu?Gln?Ile?Ser?Leu?Val?Ala?Pro

75??????????????????80??????????????????85tgg?gtg?gtg?gcc?acc?tct?gtg?cct?ctc?ttc?tgg?ccc?ctc?aac?agc?cac????341Trp?Val?Val?Ala?Thr?Ser?Val?Pro?Leu?Phe?Trp?Pro?Leu?Asn?Ser?His

90??????????????????95?????????????????100ttc?tgc?acg?gcc?ctg?gtt?agc?ctc?acc?cac?ctg?ttc?gcc?ttc?gcc?agc????389Phe?Cys?Thr?Ala?Leu?Val?Ser?Leu?Thr?His?Leu?Phe?Ala?Phe?Ala?Ser

105?????????????????110?????????????????115gtc?aac?acc?att?gtc?ttg?gtg?tca?gtg?gat?cgc?tac?ttg?tcc?atc?atc????437Val?Asn?Thr?Ile?Val?Leu?Val?Ser?Val?Asp?Arg?Tyr?Leu?Ser?Ile?Ile

120?????????????????125?????????????????130cac?cct?ctc?tcc?tac?ccg?tcc?aag?atg?acc?cag?cgc?cgc?ggt?tac?ctg????485His?Pro?Leu?Ser?Tyr?Pro?Ser?Lys?Met?Thr?Gln?Arg?Arg?Gly?Tyr?Leu135?????????????????140?????????????????145?????????????????150ctc?ctc?tat?ggc?acc?tgg?att?gtg?gcc?atc?ctg?cag?agc?act?cct?cca????533Leu?Leu?Tyr?Gly?Thr?Trp?Ile?Val?Ala?Ile?Leu?Gln?Ser?Thr?Pro?Pro

155?????????????????160?????????????????165ctc?tac?ggc?tgg?ggc?cag?gct?gcc?ttt?gat?gag?cgc?aat?gct?ctc?tgc????581Leu?Tyr?Gly?Trp?Gly?Gln?Ala?Ala?Phe?Asp?Glu?Arg?Asn?Ala?Leu?Cys

170?????????????????175?????????????????180tcc?atg?atc?tgg?ggg?gcc?agc?ccc?agc?tac?act?att?ctc?agc?gtg?gtg????629Ser?Met?Ile?Trp?Gly?Ala?Ser?Pro?Ser?Tyr?Thr?Ile?Leu?Ser?Val?Val

185?????????????????190?????????????????195tcc?ttc?atc?gtc?att?cca?ctg?att?gtc?atg?att?gcc?tgc?tac?tcc?gtg????677Ser?Phe?Ile?Val?Ile?Pro?Leu?Ile?Val?Met?Ile?Ala?Cys?Tyr?Ser?Val

200?????????????????205?????????????????210gtg?ttc?tgt?gca?gcc?cgg?agg?cag?cat?gct?ctg?ctg?tac?aat?gtc?aag????725Val?Phe?Cys?Ala?Ala?Arg?Arg?Gln?His?Ala?Leu?Leu?Tyr?Asn?Val?Lys215?????????????????220?????????????????225?????????????????230aga?cac?agc?ttg?gaa?gtg?cga?gtc?aag?gac?tgt?gtg?gag?aat?gag?gat????773Arg?His?Ser?Leu?Glu?Val?Arg?Val?Lys?Asp?Cys?Val?Glu?Asn?Glu?Asp

235?????????????????240?????????????????245gaa?gag?gga?gca?gag?aag?aag?gag?gag?ttc?cag?gat?gag?agt?gag?ttt????821Glu?Glu?Gly?Ala?Glu?Lys?Lys?Glu?Glu?Phe?Gln?Asp?Glu?Ser?Glu?Phe

250?????????????????255?????????????????260cgc?cgc?cag?cat?gaa?ggt?gag?gtc?aag?gcc?aag?gag?ggc?aga?atg?gaa????869Arg?Arg?Gln?His?Glu?Gly?Glu?Val?Lys?Ala?Lys?Glu?Gly?Arg?Met?Glu

265?????????????????270?????????????????275gcc?aag?gac?ggc?agc?ctg?aag?gcc?aag?gaa?gga?agc?acg?ggg?acc?agt????917Ala?Lys?Asp?Gly?Ser?Leu?Lys?Ala?Lys?Glu?Gly?Ser?Thr?Gly?Thr?Ser

280?????????????????285?????????????????290gag?agt?agt?gta?gag?gcc?agg?ggc?agc?gag?gag?gtc?aga?gag?agc?agc????965Glu?Ser?Ser?Val?Glu?Ala?Arg?Gly?Ser?Glu?Glu?Val?Arg?Glu?Ser?Ser295?????????????????300?????????????????305?????????????????310acg?gtg?gcc?agc?gac?ggc?agc?atg?gag?ggt?aag?gaa?ggc?agc?acc?aaa????1013Thr?Val?Ala?Ser?Asp?Gly?Ser?Met?Glu?Gly?Lys?Glu?Gly?Ser?Thr?Lys

315?????????????????320?????????????????325gtt?gag?gag?aac?agc?atg?aag?gca?gac?aag?ggt?cgc?aca?gag?gtc?aac????1061Val?Glu?Glu?Asn?Ser?Met?Lys?Ala?Asp?Lys?Gly?Arg?Thr?Glu?Val?Asn

330?????????????????335?????????????????340cag?tgc?agc?att?gac?ttg?ggt?gaa?gat?ggc?atg?gag?ttt?ggt?gaa?gac????1109Gln?Cys?Ser?Ile?Asp?Leu?Gly?Glu?Asp?Gly?Met?Glu?Phe?Gly?Glu?Asp

345?????????????????350?????????????????355gac?atc?aat?ttc?agt?gag?gat?gac?gtc?gag?gca?gtg?aac?atc?ccg?gag????1157Asp?Ile?Asn?Phe?Ser?Glu?Asp?Asp?Val?Glu?Ala?Val?Asn?Ile?Pro?Glu

360?????????????????365?????????????????370agc?ctc?cca?ccc?agt?cgt?cgt?aac?agc?aac?agc?aac?cct?cct?ctg?ccc????1205Ser?Leu?Pro?Pro?Ser?Arg?Arg?Asn?Ser?Asn?Ser?Asn?Pro?Pro?Leu?Pro375?????????????????380?????????????????385?????????????????390agg?tgc?tac?cag?tgc?aaa?gct?gct?aaa?gtg?atc?ttc?atc?atc?att?ttc????1253Arg?Cys?Tyr?Gln?Cys?Lys?Ala?Ala?Lys?Val?Ile?Phe?Ile?Ile?Ile?Phe

395?????????????????400?????????????????405tcc?tat?gtg?cta?tcc?ctg?ggg?ccc?tac?tgc?ttt?tta?gca?gtc?ctg?gcc????1301Ser?Tyr?Val?Leu?Ser?Leu?Gly?Pro?Tyr?Cys?Phe?Leu?Ala?Val?Leu?Ala

410?????????????????415?????????????????420gtg?tgg?gtg?gat?gtc?gaa?acc?cag?gta?ccc?cag?tgg?gtg?atc?acc?ata????1349Val?Trp?Val?Asp?Val?Glu?Thr?Gln?Val?Pro?Gln?Trp?Val?Ile?Thr?Ile

425?????????????????430?????????????????435atc?atc?tgg?ctt?ttc?ttc?ctg?cag?tgc?tgc?atc?cac?ccc?tat?gtc?tat????1397Ile?Ile?Trp?Leu?Phe?Phe?Leu?Gln?Cys?Cys?Ile?His?Pro?Tyr?Val?Tyr

440?????????????????445?????????????????450ggc?tac?atg?cac?aag?acc?att?aag?aag?gaa?atc?cag?gac?atg?ctg?aag????1445Gly?Tyr?Met?His?Lys?Thr?Ile?Lys?Lys?Glu?Ile?Gln?Asp?Met?Leu?Lys455?????????????????460?????????????????465?????????????????470aag?ttc?ttc?tgc?aag?gaa?aag?ccc?ccg?aaa?gaa?gat?agc?cac?cca?gac????1493Lys?Phe?Phe?Cys?Lys?Glu?Lys?Pro?Pro?Lys?Glu?Asp?Ser?His?Pro?Asp

475?????????????????480?????????????????485ctg?ccc?gga?aca?gag?ggt?ggg?act?gaa?ggc?aag?att?gtc?cct?tcc?tac????1541Leu?Pro?Gly?Thr?Glu?Gly?Gly?Thr?Glu?Gly?Lys?Ile?Val?Pro?Ser?Tyr

490?????????????????495?????????????????500gat?tct?gct?act?ttt?cct?tgaagttagt?tctaaggcaa?accttgaaaa???????????1589Asp?Ser?Ala?Thr?Phe?Pro

505tcagtccttc agccacagct atttagagct ttaaaactac caggttcaat cactggttat 1649gctttctgtg 1659＜210〉2＜211〉508＜212〉PRT＜213〉people＜400〉2Met Thr Ser Thr Cys Thr Asn Ser Thr Arg Glu Ser Asn Ser Ser His 15 10 15Thr Cys Met Pro Leu Ser Lys Met Pro Ile Ser Leu Ala His Gly Ile

20??????????????????25??????????????????30Ile?Arg?Ser?Thr?Val?Leu?Val?Ile?Phe?Leu?Ala?Ala?Ser?Phe?Val?Gly

35??????????????????40??????????????????45Asn?Ile?Val?Leu?Ala?Leu?Val?Leu?Gln?Arg?Lys?Pro?Gln?Leu?Leu?Gln

50??????????????????55??????????????????60Val?Thr?Asn?Arg?Phe?Ile?Phe?Asn?Leu?Leu?Val?Thr?Asp?Leu?Leu?Gln?65??????????????????70??????????????????75??????????????????80Ile?Ser?Leu?Val?Ala?Pro?Trp?Val?Val?Ala?Thr?Ser?Val?Pro?Leu?Phe

85??????????????????90??????????????????95Trp?Pro?Leu?Asn?Ser?His?Phe?Cys?Thr?Ala?Leu?val?Ser?Leu?Thr?His

100?????????????????105?????????????????110Leu?Phe?Ala?Phe?Ala?Ser?Val?Asn?Thr?Ile?Val?Leu?Val?Ser?Val?Asp

115?????????????????120?????????????????125Arg?Tyr?Leu?Ser?Ile?Ile?His?Pro?Leu?Ser?Tyr?Pro?Ser?Lys?Met?Thr

130?????????????????135?????????????????140Gln?Arg?Arg?Gly?Tyr?Leu?Leu?Leu?Tyr?Gly?Thr?Trp?Ile?Val?Ala?Ile145?????????????????150?????????????????155?????????????????160Leu?Gln?Ser?Thr?Pro?Pro?Leu?Tyr?Gly?Trp?Gly?Gln?Ala?Ala?Phe?Asp

165?????????????????170?????????????????175Glu?Arg?Asn?Ala?Leu?Cys?Ser?Met?Ile?Trp?Gly?Ala?Ser?Pro?Ser?Tyr

180?????????????????185?????????????????190Thr?Ile?Leu?Ser?Val?Val?Ser?Phe?Ile?Val?Ile?Pro?Leu?Ile?Val?Met

195?????????????????200?????????????????205Ile?Ala?Cys?Tyr?Ser?Val?Val?Phe?Cys?Ala?Ala?Arg?Arg?Gln?His?Ala

210?????????????????215?????????????????220Leu?Leu?Tyr?Asn?Val?Lys?Arg?His?Ser?Leu?Glu?Val?Arg?Val?Lys?Asp225?????????????????230?????????????????235?????????????????240Cys?Val?Glu?Asn?Glu?Asp?Glu?Glu?Gly?Ala?Glu?Lys?Lys?Glu?Glu?Phe

245?????????????????250?????????????????255Gln?Asp?Glu?Ser?Glu?Phe?Arg?Arg?Gln?His?Glu?Gly?Glu?Val?Lys?Ala

260?????????????????265?????????????????270Lys?Glu?Gly?Arg?Met?Glu?Ala?Lys?Asp?Gly?Ser?Leu?Lys?Ala?Lys?Glu

275?????????????????280?????????????????285Gly?Ser?Thr?Gly?Thr?Ser?Glu?Ser?Ser?Val?Glu?Ala?Arg?Gly?Ser?Glu

290?????????????????295?????????????????300Glu?Val?Arg?Glu?Ser?Ser?Thr?Val?Ala?Ser?Asp?Gly?Ser?Met?Glu?Gly305?????????????????310?????????????????315?????????????????320Lys?Glu?Gly?Ser?Thr?Lys?Val?Glu?Glu?Asn?Ser?Met?Lys?Ala?Asp?Lys

325?????????????????330?????????????????335Gly?Arg?Thr?Glu?Val?Asn?Gln?Cys?Ser?Ile?Asp?Leu?Gly?Glu?Asp?Gly

340?????????????????345?????????????????350Met?Glu?Phe?Gly?Glu?Asp?Asp?Ile?Asn?Phe?Ser?Glu?Asp?Asp?Val?Glu

355?????????????????360?????????????????365Ala?Val?Asn?Ile?Pro?Glu?Ser?Leu?Pro?Pro?Ser?Arg?Arg?Asn?Ser?Asn

370?????????????????375?????????????????380Ser?Asn?Pro?Pro?Leu?Pro?Arg?Cys?Tyr?Gln?Cys?Lys?Ala?Ala?Lys?Val385?????????????????390?????????????????395?????????????????400Ile?Phe?Ile?Ile?Ile?Phe?Ser?Tyr?Val?Leu?Ser?Leu?Gly?Pro?Tyr?Cys

405?????????????????410?????????????????415Phe?Leu?Ala?Val?Leu?Ala?Val?Trp?Val?Asp?Val?Glu?Thr?Gln?Val?Pro

420?????????????????425?????????????????430Gln?Trp?Val?Ile?Thr?Ile?Ile?Ile?Trp?Leu?Phe?Phe?Leu?Gln?Cys?Cys

435?????????????????440?????????????????445Ile?His?Pro?Tyr?val?Tyr?Gly?Tyr?Met?His?Lys?Thr?Ile?Lys?Lys?Glu

450?????????????????455?????????????????460Ile?Gln?Asp?Met?Leu?Lys?Lys?Phe?Phe?Cys?Lys?Glu?Lys?Pro?Pro?Lys465?????????????????470??????????????????475????????????????480Glu?Asp?Ser?His?Pro?Asp?Leu?Pro?Gly?Thr?Glu?Gly?Gly?Thr?Glu?Gly

485?????????????????490?????????????????495Lys?Ile?val?Pro?Ser?Tyr?Asp?Ser?Ala?Thr?Phe?Pro

500 505＜210〉3＜211〉27＜212〉DNA＜213〉＜220〉＜223〉：＜220〉＜221〉＜222〉 ( 19 )＜223〉＜220〉＜221〉＜222〉 ( 22 )＜223〉＜220〉＜221〉＜222〉 ( 25 )＜223〉＜400〉3catcttcgtc gtcggcacng ynggnaa 27＜210〉4＜211〉26＜212〉DNA＜213〉＜220〉＜223〉：＜220〉＜221〉＜222〉 ( 21 )＜223〉＜400〉4gggtggcaga tggccarrya nckytc 26＜210〉5＜211〉27＜212〉DNA＜213〉＜220〉＜223〉：＜220〉＜221〉misc_feature＜222〉 ( 27 )＜223〉：3’-＜400〉5acggtgggca acacggtgac ggcgtta 27＜210〉6＜211〉27＜212〉DNA＜213〉＜220〉＜223〉：＜400〉6ccatcctaat acgactcact atagggc 27＜210〉7＜211〉23＜212〉DNA＜213〉＜220〉＜223〉：＜400〉7actcactata gggctcgagc ggc 23＜210〉8＜211〉28＜212〉DNA＜213〉＜220〉＜223〉：＜400〉8tttatcttta acctcctcgt caccgacc 28＜210〉9＜211〉20＜212〉DNA＜213〉＜220〉＜223〉：＜400〉9tagtgttgca gcgcaagccg 20＜210〉10＜211〉30＜212〉DNA＜213〉＜220〉＜223〉：＜400〉10ggcagcgttc cactgacacc aagacaatgg 30＜210〉11＜211〉28＜212〉DNA＜213〉＜220〉＜223〉：＜400〉11cagcgttcca ctgacaccaa gacaatgg 28＜210〉12＜211〉27＜212〉DNA＜213〉＜220〉＜223〉：＜400〉12aaggcgaaca ggtgggtgag gctaacc 27＜210〉13＜211〉20＜212〉DNA＜213〉＜220〉＜223〉：＜400〉13tggcgaaggc gaacaggtgg 20＜210〉14＜211〉24＜212〉DNA＜213〉＜220〉＜223〉：＜400〉14gcgaaggcga acaggtgggt gagg 24＜210〉15＜211〉24＜212〉DNA＜213〉＜220〉＜223〉：＜400〉15ctagtgttgc agcgcaagcc gcag 24＜210〉16＜211〉28＜212〉DNA＜213〉＜220〉＜223〉：＜400〉16cacagaaagc ataaccagtg attgaacc 28＜210〉17＜211〉27＜212〉DNA＜213〉＜220〉＜223〉：＜400〉17gctttaggtt cctggaatcc catttgg 27＜210〉18＜211〉25＜212〉DNA＜213〉＜220〉＜223〉：＜400〉18ttgtcaccag cataggcact gagtg 25

Claims

1. isolating polynucleotide, it comprises and is selected from following nucleotide sequence:

A) nucleotide sequence, its coding is according to the IGS1 polypeptide of SEQ ID NO:2;

B) nucleotide sequence of coded polypeptide, wherein said polypeptide inserts fragment coding by DNA, described insertion fragment is included in preserving number CBS 102049, is preserved in the preservation thing at fungi strain preservation center of Holland, particularly corresponding to the nucleotide sequence of SEQ ID NO:1;

C) nucleotide sequence, its total length have 80% at least, and the sequence of (preferably at least 90%) is identical with (a) or nucleotide sequence (b);

D) nucleotide sequence, its with (a) or (b) or nucleotide sequence complementation (c).

2. the polynucleotide of claim 1, wherein said polynucleotide contain the nucleotide sequence that is contained in SEQ IDNO:1, and the IGS1 polypeptide of SEQ ID NO:1 coding SEQ ID NO:2.

3. the polynucleotide of claim 1, wherein said polynucleotide total length comprise at least 80% with the identical nucleotide sequence of nucleotide sequence of SEQ ID NO:1.

4. the polynucleotide of claim 3, it is the polynucleotide of SEQ ID NO:1.

5. the polynucleotide of claim 1-4, it is DNA or RNA.

6. hybridization probe, it comprises the polynucleotide of claim 1 or it has 5 Nucleotide at least, and the fragment between 30 to 50 Nucleotide preferably.

7.DNA or the RNA molecule, it contains expression system, wherein when described expression system was arranged in proper host cell, this expression system can produce the IGS1 polypeptide that comprises aminoacid sequence, peptide has at least 80% identity more than itself and the SEQ ID NO:2.

8. host cell, it comprises the expression system of claim 7.

9. host cell according to Claim 8, it is a yeast cell.

10. host cell according to Claim 8, it is a zooblast.

11.IGS1 the membrane prepare thing of acceptor, it comes from according to Claim 8-10 cell.

12. a method that produces the IGS1 polypeptide, it is included in the host who cultivates claim 8 under the condition that is enough to produce this polypeptide, and reclaims polypeptide from culture.

13. a generation can produce the method for the cell of IGS1 polypeptide, comprises that the expression system with claim 7 transforms or transfectional cell, makes cell can produce the IGS1 polypeptide under suitable culture condition.

14.IGS1 polypeptide, the aminoacid sequence total length at least 80% that it comprises is identical with the aminoacid sequence of SEQ ID NO:2.

15. the polypeptide of claim 14, it comprises the aminoacid sequence of SEQ ID NO:2.

16. an antibody, its IGS1 polypeptide to claim 14 has immunologic opsonin.

17. a method for the treatment of the experimenter, wherein the experimenter need strengthen the active or expression of the IGS1 polypeptide receptor of claim 14, comprising:

(a) grant the agonist that the experimenter treats this receptor of significant quantity; And/or

(b) provide the experimenter isolating polynucleotide, wherein comprise the identical nucleotide sequence of nucleotide sequence of total length at least 80% and the IGS1 polypeptide of coding SEQID NO:2; Or comprise with described nucleotide sequence complementary, be can body in the nucleotide sequence of the form that produces of the described receptor active of influence.

18. a method for the treatment of the experimenter, wherein the experimenter need suppress the active or expression of the IGS1 polypeptide receptor of claim 14, comprising:

(a) grant the antagonist that the experimenter treats this receptor of significant quantity; And/or

(b) use polynucleotide to the experimenter, described polynucleotide suppress the expression of the nucleotide sequence of coding this receptor; And/or

(c) grant the polypeptide that the experimenter treats significant quantity, this polypeptide and its part of described receptor competition.

19. a method of diagnosing experimenter's disease or disease susceptibility, described disease relate to the IGS1 polypeptide expression or the activity of claim 14 among the experimenter, comprising:

(a) in described experimenter's genome, exist in the nucleotide sequence of the described IGS1 polypeptide of definite coding and whether suddenly change; And/or

(b), analyze whether the IGS1 polypeptide expresses or the amount of its expression to sample from described experimenter.

20. the method for the agonist of an IGS1 polypeptide of identifying claim 14 comprises:

(a) with test compound and the cells contacting that produces the IGS1 polypeptide; And

(b) whether the determination test compound influences the signal that produces by the activation of IGS1 polypeptide.

21. agonist of identifying by the method for claim 20.

22. the method for the antagonist of the IGS1 polypeptide of evaluation claim 14 comprises:

(a) with agonist and the cells contacting that produces the IGS1 polypeptide; And

(b) when candidate compound exists, measure the signal that produces by described agonist and whether disappear.

23. antagonist of identifying by the method for claim 22.

24. recombinant host cell or its film of expressing the IGS1 polypeptide, its method by claim 13 produces.

25. a method of setting up the non-human animal of genetic modification, it may further comprise the steps:

A) encoding part with polynucleotide is connected with the adjusting sequence, wherein said polynucleotide have the protein of aminoacid sequence of SEQ ID NO:2 by coding basically or the nucleotide sequence of its bioactive fragment is formed, and wherein said adjusting sequence can drive high-caliber genetic expression or drive the gene of not expressing under the normal circumstances in described animal and express in a kind of cell type.

B) encoding part of genetically engineered design polynucleotide, and described sequence is imported again in the genome of described animal, the allelotrope that makes native gene by this way is inactivation wholly or in part, wherein said polynucleotide have the protein of aminoacid sequence of SEQ ID NO:2 by coding basically or the nucleotide sequence of its bioactive fragment is formed, protein or its bioactive fragment of the aminoacid sequence of the allelotrope coding tool SEQ ID NO:2 of described native gene.