WO2001048189A1 - Novel guanosine triphosphate-binding protein-coupled receptors, genes thereof and production and use of the same - Google Patents

Abstract

Fifteen novel genes sustaining hydrophobic domains, which are seemingly 7 transmembrane domains characteristic to G protein-coupled receptors, are successfully isolated by human tissue cDNA screening. These genes and proteins which are the expression products thereof are usable in screening ligands, screening agonists or antagonists which are useful as drugs, diagnosing diseases in which these gene participate, etc.

Description

 Description Novel guanosine triphosphate-binding protein-coupled receptor, their genes, and their production and use

 The present invention relates to novel G protein-coupled receptors and their genes, and their production and use. Background art

G protein-coupled receptors are a general term for a group of cell membrane receptors that transmit signals into cells through the activation of trimeric GTP-binding proteins. G protein-coupled receptors are also called “seven-transmembrane receptors” because of their structural characteristics of having seven transmembrane regions in the molecule. G protein-coupled receptors transmit information on various physiologically active substances from the cell membrane into cells through the activation of trimeric GTP-binding proteins and the resulting changes in intracellular second messengers. Intracellular second messengers which are controlled by trimeric GTP-binding proteins, cAMP which via adenylyl two cyclase, although such Ca ² + which via a phospholipase C is well known, via trimeric GTP-binding protein Recently, it has been revealed that many cellular proteins are targeted, such as the control of channels and the activation of phosphorylase (Annu. Rev. Neurosci, (97) 20: 399). Substrates (ligands) for G protein-coupled receptors are very diverse, including proteinaceous hormones, chemokines, peptides, amines, lipid-derived substances, and proteases such as thrombin. At present, the number of G protein-coupled receptors whose genes have been identified is less than 300 in humans excluding sensory organ receptors, but the number of G protein-coupled receptors whose ligands have been identified is about There are only 140 types and the ligand is unknown. —There are more than 100 types of “fan G protein-coupled receptors”. However, it is assumed that there are at least 400, and sometimes as many as 1000, G-protein coupled receptors in the actual human genome (Trends Pharmacol. Sci. (97) 18: 430). This means that the number of orphan G protein-coupled receptors of unknown function will explode with the rapid progress of genome analysis in the future. Over 90% of drugs created by global pharmaceutical companies so far target interactions in the extracellular space, of which small molecule drugs related to G protein-coupled receptors are mostly Occupy. The basis for this is that diseases associated with G protein-coupled receptors are very rare, including genetic diseases, cerebral nervous system, circulatory system, digestive system, immune system, exercise system, urogenital system, etc. Therefore, many pharmaceutical companies have recently possessed an orphan G protein-coupled receptor that was revealed by genomic analysis, and have spent much of their time searching for ligands and elucidating physiological functions. Against this background, there have recently been reports of successful searches for physiological ligands for novel G protein-coupled receptors. For example, calcitonin gene-related peptide receptor (J. Biol. Chem. (96) 271: 11325), orexin (Cell (98) 92: 573) and prolactin-releasing peptide (Nature (98) 393: 272) This case had a great impact on basic research in the life science field.

In particular, human-fan G protein-coupled receptors have received a great deal of attention as targets that are likely to lead to new drug development. In general, since there is no specific ligand for orphan'G protein-coupled receptor, it has been difficult to develop its agonist and angyu gonist. However, in recent years, it has been proposed to create a drug targeting an orphan G protein-coupled receptor by combining an extensive compound library with high-throughput screening (Trends Pharmacol. Sc i. (97) 18: 430, Br. J. Pharm. (98) 125: 1387). In other words, the orphan G protein-coupled receptor identified by genetic manipulation is used to screen physiological agonism by function screening using changes in intracellular second messenger cAMP and Ca ²⁺ as indices. This is to discover strikes and perform in vivo functional analysis. At this time, by using a compound library to increase the throughput of the screening, it was possible to discover specific surrogate agonists for orphan G protein-coupled receptors and angel gonists, and eventually to identify specific Theoretically, the development of therapeutic drugs for diseases is possible. Disclosure of the invention

 The present invention has been made in view of the current situation surrounding such a G protein-coupled receptor, and an object thereof is to provide a novel G protein-coupled receptor and a gene thereof, as well as methods for producing and using the same. It is in. The aim is to provide these molecules as targets for drug development research.

 The present inventors have conducted intensive studies in order to solve the above-mentioned problems, and as a result, by carrying out the polymerase chain reaction using human tissue cDNA as a 鎵 type, the characteristic of G protein-coupled receptor We have succeeded in isolating 15 novel genes that have a hydrophobic region that is considered to be a transmembrane domain. These genes and the proteins that are their translation products can be used for screening for ligands and for screening agonists that are useful as pharmaceuticals.

 That is, the present invention relates to novel G protein-coupled receptors and their genes, and their production and use, and more specifically,

 (1) the DNA according to any one of (a) to (d) below, which encodes a guanosine triphosphate binding protein-coupled receptor;

 (a) SEQ ID NO: 1 to 8, 33 to 34, or 41 to 45, a DNA encoding a protein comprising the amino acid sequence described in any one of:

(b) SEQ ID NO: 9 to 16; 35 to 36; 46 to 50 (c) SEQ ID NO: consisting of an amino acid sequence in which one or more amino acids are substituted, deleted, added and / or inserted in the amino acid sequence described in any one of 1 to 8, 33 to 34, 41 to 45 DNA encoding proteins,

 (d) a DNA that hybridizes under stringent conditions to a DNA consisting of the nucleotide sequence of any one of SEQ ID NOs: 9 to 16, 35 to 36, and 46 to 50,

(2) a DNA encoding a partial peptide of a protein consisting of the amino acid sequence of any one of SEQ ID NOs: 1 to 8, 33 to 34, and 41 to 45;

 (3) a vector containing the DNA of (1) or (2),

 (4) a transformant carrying the DNA of (1) or (2) or the vector of (3),

 (5) a protein or peptide encoded by the DNA of (1) or (2),

 (6) the step of culturing the transformant according to (4), and recovering the protein or peptide expressed from the transformant or the culture supernatant thereof; Production method,

 (7) A method for screening a ligand that binds to a protein according to (5),

 (a) contacting a test sample with the protein or peptide according to (5),

(b) selecting a compound that binds to the protein or peptide;

(8) A method for screening a compound having an activity of inhibiting the binding of the protein according to (1) or (2) to a ligand thereof,

 (a) contacting a ligand with the protein or the partial peptide thereof according to (1) or (2) in the presence of the test sample, and detecting the binding activity between the protein or the partial peptide and the ligand;

(b) selecting a compound that reduces the binding activity detected in step (a) as compared to the binding activity in the absence of the test sample; (9) A method for screening for a compound that inhibits or promotes the activity of the protein according to (1) or (2),

 (a) contacting a cell expressing the protein with a ligand for the protein in the presence of a test sample;

 (b) detecting a change in a cell due to binding of the ligand to the protein,

(c) selecting a compound that suppresses or enhances the change in the cells detected in step (b) as compared with the change in the cells in the absence of the test sample;

 (10) The method according to (8) or (9), wherein the change in the cell is a change in cAMP concentration or a change in calcium concentration.

 (1 1) an antibody that binds to the protein of (1) or (2),

 (12) a compound isolated by the screening according to any of (7) to (10), and

 (13) A pharmaceutical composition comprising the compound according to (12) as an active ingredient, and

(14) SEQ ID NO: 9 to 16, 35 to 36, 46 to 50 or a nucleotide having a nucleotide length of at least 15 nucleotides, which is complementary to a complementary strand thereof. Is what you do.

 In the present invention, the “G protein-coupled receptor” refers to a cell membrane receptor that transmits a signal into a cell through activation of a GTP-binding protein.

 In the present invention, “ligand” refers to a physiological substance that binds to a G protein-coupled receptor and transmits a signal into a cell. Here, “physiological substance” refers to a compound that binds to a G protein-coupled receptor in a living body.

In the present invention, “agonist” refers to a compound capable of transmitting a signal into cells by binding to a G protein-coupled receptor, and includes physiological substances, artificially synthesized compounds, and naturally occurring compounds. Including. In the present invention, the term “angigonist” refers to a compound that inhibits the binding of a ligand to a G protein-coupled receptor or the transmission of a signal into a cell, and is a physiological substance or an artificially synthesized substance. Compounds, including naturally occurring compounds.

 The present invention provides a novel G protein-coupled receptor and a DNA encoding the protein. Included in the present invention, 15 human-derived cDNA clones isolated by the present inventors, `` GPRv4 '', `` GPRvll '', `` GPRvl3 '', `` GPRvl4 '', `` GPRvl5 '', `` GPRvl9 '', Named "GPRv20", "GPRv31", "GPRv38", "GPRv39", "GPRv68", "GPRv77", "GPRv78", "GPRv79", "GPRv81" (If necessary, collect these clones together. GPRv ”). The nucleotide sequences of these cDNAs are shown in SEQ ID NOs: 9 to 16, 35 to 36, and 46 to 50, and the amino acid sequences of the proteins encoded by the cDNAs are shown in SEQ ID NOs: 1 to 8, 33 to 3 4, 4 1 to 45 are shown.

 As a result of BLAST search, all proteins encoded by the GPRv cDNA showed significant amino acid sequence homology with known G protein-coupled receptors. Specifically, `` GPRv 4 '' has 31% homology to ORYLA PROBABLE G PROTEIN-COUPLED RECEPTOR (Q91178, 428aa), and `` GPRvll '' has HUMAN NEUROPEPTIDE Y RECEPTOR TYPE 2 (P49146,

381aa) to 31% homology, `` GPRvl3 '' to 39% homology to PONPY C5A ANAPHYLATOXIN CHEM0 TACTIC RECEPTOR (P79234, 340aa), `` GPRvl4 '' to CHICK

40% homology to P2Y PURIN0CEPT0R 5 (P32250, 308aa), `` GPRvl5 '' 26% homology to H UMAN 5-HYDR0XYTRYPTAMINE IE RECEPTOR (P28566, 365aa), `` GPRvl9 '' to APIME OPSIN , BLUE-SENSITIVE (P90680, 377aa) has 25% homology, `` GPRv20 '' has 38% homology to RAT MAS P-ONCOGENE (P12526, 324aa), and `` GPRv31 '' has SHEEP 29% homology to THYROTROPIN- RELEASING HORMONE RECEPT OR (Q28596, 398aa), `` GPRv38 '' is P2Y PURIN0CEPT0R 7

(Q15722, 352aa) has 46% homology, `` GPRv39 '' has 35% homology to RAT NEUROTENSIN RECE PTOR TYPE 1 (P20789, 424aa), and `` Factory GPRv68 '' has TYPE-IB A 39% homology to NGIOTENSIN II RECEPTOR (Q13725, 359aa), `` GPRv77 '' shows 29% homology to HUMAN PUTATIVE G PROTEIN-COUPLED RECEPTOR GPR17 (R12) (Q13304, 339aa), `` GPRv78 "Has 39% homology to HUMAN GALA IN RECEPTOR TYPE 2 (04360 3, 387aa)," GPRv79 "has 39% homology to RAT MAS PR0T0-ONCOGENE (P12 526, 324aa)," GPRv81 ”is HUMAN 5-HYDR0XYTRYPTAMINE

Each showed 25% homology to IB RECEPTOR (P28222, 390aa).

 In addition, the protein encoded by the GPRv cDNA isolated by the present inventors (hereinafter sometimes referred to as “GPRv protein”) has seven transmembrane domains characteristic of a G protein-coupled receptor. It retained possible hydrophobic regions. From these facts, it is considered that all GPRv cDNAs encode proteins belonging to the G protein-coupled receptor family. G protein-coupled receptors have the activity of transmitting signals into cells through the activation of G proteins by the action of their ligands, and as described above, include genetic diseases, brain nervous system, circulatory It has been implicated in numerous areas of disease, including system, digestive, immune, motor, and genitourinary. Therefore, the GPRv protein can be used for screening of agonists and angiogonists that regulate the function of the GPRv protein, and is an important target for the development of drugs for the above diseases.

 The present invention also provides a protein functionally equivalent to the GPRv protein. here

“Functionally equivalent” means that the protein of interest has biological properties equivalent to the GPRv protein. The biological properties of GPRv proteins include the activity of transducing signals into cells through the activation of trimeric GTP-binding proteins. Trimeric GTP-binding proteins fall into one of three categories, depending on the type of intracellular signaling system that activates, Gq type that increases Ca ²⁺ , Gs type that increases cAMP, and Gi type that suppresses cAMP. (Trends Pharmacol. Sci. (99) 20: 118). Therefore, whether the target protein has the same biological properties as the GPRv protein This can be evaluated, for example, by detecting a change in intracellular cAMP concentration or calcium concentration due to the activation.

 One embodiment of a method for preparing a protein functionally equivalent to the GPRv protein includes a method of introducing a mutation into an amino acid sequence in a protein. Such methods include, for example, site-directed mutagenesis (Current Protocols in Molecular Biology edit. Ausubel et al. (1987) Publish. John Wiley & Sons Section 8.1-8.5)). Amino acid mutations in proteins may also occur in nature. In the present invention, the amino acid sequence of the GPRv protein (SEQ ID NO: 1 from 8, 33 to 34, 41 to 45) irrespective of whether it is artificial or naturally occurring is 1 or It includes proteins in which a plurality of amino acids are mutated by substitution, deletion, insertion, Z or addition, and the like, and includes proteins functionally equivalent to the GPRv protein. The number and location of amino acid mutations in these proteins are not limited as long as the function of the GPRv protein is maintained. The number of mutations will typically be within 10% of all amino acids, preferably within 5% of all amino acids, and more preferably within 1% of all amino acids.

Another embodiment of a method for preparing a protein functionally equivalent to the GPRv protein includes a method utilizing a hybridization technique or a gene amplification technique. That is, those skilled in the art can use a hybridization technique (Current Protocols in Molecular Biology edit. Ausubel et al. (1987) Publish. John Wiley & Sons Section 6.3-6.4) to make a DNA sequence encoding a GPRv protein. (SEQ ID NOS: 9 to 16, 35 to 36, 46 to 50) or a portion thereof, from which a highly homologous DNA is isolated from a DNA sample derived from a homologous or heterologous organism. Obtaining a protein functionally equivalent to the GPRv protein from the DNA can be usually performed. Thus, a protein encoded by a DNA that hybridizes with a DNA encoding a GPRv protein and that is functionally equivalent to the GPRv protein is also included in the protein of the present invention. Examples of organisms for isolating such proteins include, but are not limited to, rats, mice, egrets, chicks, birds, and sea lions, in addition to humans.

 Stringent hybridization conditions for isolating DNA that encodes a protein functionally equivalent to the GPRv protein are usually of the order of `` lxSSC, 0.1% SDS, 37 ° C ''. The more strict conditions are about 0.5xSSC, 0.1¾SDS, 42 ° C, and the more severe conditions are about 0.2xSSC, 0.1% SDS, 65 ° C. Is the condition. Thus, the isolation of DNA having higher homology to the probe sequence can be expected as the hybridization conditions become more severe. However, the combination of the above SSC, SDS and temperature conditions is merely an example, and those skilled in the art will recognize the above or other factors (eg, probe concentration, probe length, etc.) that determine the stringency of a high predication solution. , Hybridization reaction time, etc.) as appropriate, it is possible to realize the same stringency as described above.

 The protein encoded by DNA isolated using such a hybridization technique usually has high homology in amino acid sequence with the GPRv protein. High homology refers to sequence homology of at least 40% or more, preferably 60% or more, more preferably 80% or more (eg, 90% or more and 95% or more).

The identity of the amino acid sequence or nucleotide sequence can be determined by the algorithm BLAST by Karl in and Altschul (Proc. Natl. Acad. Sei. USA 90: 5873-5877, 1993). Based on this algorithm, programs called BLASTN and BLASTX have been developed (Altschul et al. J. ol. Biol. 215: 403-410, 1990). When analyzing a nucleotide sequence by BLASTN based on BLAST, the parameters are, for example, score = 100 and word length = 12. Also, when analyzing an amino acid sequence by BLA STX based on BLAST, the parameters are, for example, score = 50 and word length = 3. When using BLAST and Gapped BLAST programs Use the default parameters of each program. The specific methods of these analysis methods are known (http: @ www.ncbi.nlm.nih.gov.).

 Also, using a gene amplification technique (PCR) (Current protocols in Molecular Biology edit. Ausubel et al. (1987) Publish. John Wiley & Sons Section 6.1-6.4), a DNA sequence encoding a GPRv protein (SEQ ID NO: 9). From 16 to 35, 36 to 36, and 46 to 50), a primer was designed, a DNA fragment having high homology to the DNA sequence encoding the GPRv protein was isolated, and the DNA was isolated. Based on this, it is also possible to obtain a protein functionally equivalent to the GPRv protein.

 The present invention also includes a partial peptide of the protein of the present invention. This partial peptide includes a peptide that binds to a ligand but does not transmit a signal. An affinity column prepared based on such a peptide can be suitably used for screening of a ligand. Further, the partial peptide of the protein of the present invention can also be used for preparing an antibody. The partial peptide of the present invention can be produced, for example, by a genetic technique, a known peptide synthesis method, or by cleaving the protein of the present invention with an appropriate peptidase. The partial peptide of the present invention usually has 8 amino acid residues or more, preferably 12 amino acid residues or more (for example, 15 amino acid residues or more).

The protein of the present invention can be prepared as a recombinant protein or as a natural protein. The recombinant protein can be prepared, for example, by introducing a vector into which a DNA encoding the protein of the present invention is inserted into an appropriate host cell as described below, and purifying the protein expressed in the transformant. . On the other hand, a natural protein can be prepared using, for example, an affinity column to which an antibody against the protein of the present invention described below is bound (Current Protocols in Molecular Biology edit. Ausubel et al. (1987)). Publish. John Wiley & Sons Section 16. 16.16). The antibody used for affinity purification may be a polyclonal antibody or a monoclonal antibody. In addition, in vitro translation (For example, see “0n the fidelity of mRNA translation in the nuclease-treate d rabbit reticulocyte lysate system. Dasso, MC, Jackson, RJ (1989) NAR 17: 3129-3144”) and the like. It is also possible. The present invention also provides a DNA encoding the protein of the present invention. The form of the DNA of the present invention is not particularly limited as long as it can encode the protein of the present invention, and includes genomic DNA, chemically synthesized DNA, and the like in addition to cDNA. In addition, DNAs having any base sequence based on the degeneracy of the genetic code are included as long as they can encode the protein of the present invention. As described above, the DNA of the present invention comprises a DNA sequence encoding a GPRv protein (SEQ ID NOS: 9 to 16, 35 to 36, 46 to 50) or a part thereof as a probe. It can be isolated by a conventional method such as the PCR method using a primer synthesized based on these DNA sequences.

The present invention also provides a vector into which the DNA of the present invention has been inserted. The vector of the present invention is not particularly limited as long as it stably retains the inserted MA. For example, if Escherichia coli is used as a host, pBluescript vector-1 (Stratagene Inc.) may be used as a vector for cloning. Is preferred. When a vector is used for the purpose of producing the protein of the present invention, an expression vector is particularly useful. The expression vector is not particularly limited as long as it is a vector that expresses a protein in a test tube, in E. coli, in cultured cells, or in an individual organism. For example, pBEST vector (Promega) for expression in a test tube PET vector (manufactured by Invitrogen) for Escherichia coli, PME18S-FL3 vector (GenBank Accession No. AB009864) for cultured cells, pME18S vector (Mol Cell Biol. 8: 466-472 (Mol Cell Biol. 8: 466-472) for living organisms. 1988)). Insertion of the DNA of the present invention into a vector can be performed by a conventional method, for example, by a ligase reaction using a restriction enzyme site (Current protocols in Molecular Biology edit. Ausubel et al. (1987) Publish. John Wiley & Sons. Section 11.4-11. 11). The present invention also provides a transformant carrying the DNA of the present invention or the vector of the present invention. The host cell into which the vector of the present invention is introduced is not particularly limited, and various host cells can be used depending on the purpose. Examples of eukaryotic cells for highly expressing a protein include COS cells and CH0 cells. Vector introduction into host cells includes, for example, calcium phosphate precipitation, electropulse perforation (Current protocols in Molecular Biology edit. Ausubel et al. (1987) Publish. John Wiley & Sons.Section 9.1-9.9), It can be performed by a known method such as a lipofectamine method (manufactured by GIBCO-BRL) or a microinjection method.

The present invention also relates to a DNA encoding the protein of the present invention (a DNA comprising the nucleotide sequence of any one of SEQ ID NOs: 9 to 16, 35 to 36, and 46 to 50, or a complementary strand thereof). A) a nucleotide having a chain length of at least 15 nucleotides that is complementary to Here, the “complementary strand” refers to one strand of a double-stranded nucleic acid consisting of A: T (U for RNA) and G: C base pairs, with respect to the other strand. The term "complementary" is not limited to a sequence completely complementary to at least 15 contiguous nucleotide regions, but is at least 70%, preferably at least 80%, more preferably 90%, and still more preferably 95%. What is necessary is that they have homology on the base sequence of at least%. The algorithm described in this specification may be used as an algorithm for determining homology. Such nucleotides can be used as a probe for detecting and isolating the MA of the present invention, and as a primer for amplifying the DNA of the present invention. When used as a primer, it usually has a chain length of 15 bp to 100 bp, preferably 15 bp to 35 bp. When used as a probe, a nucleotide having a chain length of at least 15 bp containing at least a part or the entire sequence of the MA of the present invention is used. Such nucleotides preferably specifically hybridize to DNA encoding the protein of the present invention. "Specifically hybridizes" means under normal hybridization conditions, preferably under stringency. The DNA that encodes the protein of the present invention (SEQ ID NOs: 9 to 16, 35 to 36, 46 to 50) hybridizes with the DNA encoding the protein of the present invention under a simple condition, and encodes another protein. NA means do not hybridize.

 These nucleotides can be used for testing and diagnosing abnormalities of the protein of the present invention. For example, abnormal expression of the DNA encoding the protein of the present invention can be examined by Northern hybridization or RT-PCR using these nucleotides as probes or primers. In addition, the DNA encoding the protein of the present invention and its expression control region are amplified by polymerase chain reaction (PCR) using these nucleotides as primers, and the DNA is amplified by methods such as RFLP analysis, SSCP, and sequencing. Inspection and diagnosis of sequence abnormalities.

 In addition, these nucleotides include antisense DNA for suppressing the expression of the protein of the present invention. The antisense MA has a chain length of at least 15 bp or more, preferably 100 bp, more preferably 500 bp or more, and usually has a chain length of 3000 bp or less, preferably 2000 bp or less in order to cause an antisense effect. Such antisense MA may also be applied to gene therapy for diseases caused by abnormalities (functional abnormalities or current abnormalities) of the protein of the present invention. The antisense DNA is, for example, a phosphorothionate based on the sequence information of a DNA encoding the protein of the present invention (eg, SEQ ID NOS: 9 to 16, 35 to 36, 46 to 50). Nucleic Acids Res 16, 3209-21 (1988)), etc. can be prepared by the method (Stein, 1988 Physicochemical properties oi phosphorotnioate oligodeoxynucle otides. Nucleic Acids Res 16, 3209-21 (1988)).

When used for gene therapy, the nucleotides of the present invention can be used, for example, in ex vivo by using a viral vector such as a retrovirus vector, an adenovirus vector, an adeno-associated virus vector, or a non-viral vector such as a ribosome. o It may be possible to administer to patients by the method or in vivo method. The present invention also provides an antibody that binds to the protein of the present invention. The form of the antibody of the present invention is not particularly limited, and includes a polyclonal antibody, a monoclonal antibody, and a part thereof having antigen-binding properties. In addition, antibodies of all classes are included. Furthermore, the antibodies of the present invention also include special antibodies such as humanized antibodies.

 In the case of a polyclonal antibody, the antibody of the present invention can be obtained by synthesizing an oligonucleotide corresponding to the amino acid sequence of the protein of the present invention according to a conventional method, and immunizing a rabbit (Current protocols in Molecular Biology edit. Ausubel et al. (1987) Publish. John Wiley & Sons. Section 11.12-11.13). In the case of a monoclonal antibody, a mouse is immunized with a protein expressed and purified in Escherichia coli according to a conventional method, and a hybridoma cell obtained by fusing the spleen cell and myeloma cell thereof is prepared. It can be obtained from dorma cells (Current protocols in Molecular Biology edit. Ausubel et al. (1987) Publish. John Wiley & Sons. Section 11.4- 11.11).

 Antibodies that bind to the protein of the present invention may be used, for example, for the examination and diagnosis of abnormal expression or structural abnormality of the protein of the present invention, in addition to purification of the protein of the present invention. Specifically, for example, proteins are extracted from tissues, blood, or cells, and abnormalities in expression and structure are detected through detection of the proteins of the present invention by methods such as Western blotting, immunoprecipitation, and ELISA. Inspection / presence can be diagnosed.

It is also conceivable to use an antibody that binds to the protein of the present invention for the purpose of treating a disease associated with the protein of the present invention. The antibody of the present invention can act as an agonist of the protein of the present invention. When the antibody is used for the purpose of treating a patient, a human antibody or a humanized antibody is preferred because of its low immunogenicity. Human antibodies include mice in which the immune system has been replaced with that of a human (eg, "" Functional transpant of megabase human immunoglobulin loci recapitulates human antibody responses in mice, Mendez, MJ et al. (1997) Nat. Genet.15 : 146-156 "). In addition, humanized antibodies are It can be prepared by genetic recombination using the hypervariable region of a null antibody (Methods in Enzymology 203, 99-121 (1991)).

 The present invention also provides a method for screening for a ligand that binds to the protein of the present invention, using the protein of the present invention. This screening method includes (a) a step of bringing a test sample into contact with a protein of the present invention or a partial peptide thereof, and (b) a step of selecting a compound that binds to the protein or a partial peptide thereof.

 The test sample is not particularly limited. For example, known compounds or peptides whose ligand activities of various G protein-coupled receptors are unknown (for example, those registered in a chemical file) or phage A random 'peptide group created by applying a display method (J. Mol. Biol. (1991) 222, 301-310) can be used. In addition, culture supernatants of microorganisms and natural components derived from plants and marine organisms are also targets for screening. Other examples include, but are not limited to, brain and other biological tissue extracts, cell extracts, and expression products of gene libraries.

 The protein of the present invention used for screening may be, for example, a form expressed on a cell surface, a form as a cell membrane fraction of the cell, or a form bound to an affinity column.

Specific screening techniques include, for example, a method of contacting a test sample with an affinity column for the protein of the present invention to purify a compound that binds to the protein of the present invention, and a number of known methods such as a West Western plotting method. Methods are available. When these methods are used, the test sample is appropriately labeled, and the binding to the protein of the present invention can be detected using the label. In addition to these methods, a cell membrane that expresses the protein of the present invention is prepared and immobilized on a chip, and the dissociation of the trimeric GTP-binding protein upon ligand binding is determined by surface plasmon resonance (surface plasmon resonance). It is also possible to use a method of detecting by resonance (Nature Biotechnology (99) 17: 1105). Further, the binding activity between the test sample and the protein of the present invention can be detected by using, as an index, a change in cells caused by binding of the test sample to the protein of the present invention expressed on the cell surface. Such changes include, but are not limited to, changes in intracellular Ca ²⁺ levels and changes in cAMP levels. Specifically, agonist activity for G protein-coupled receptors can be measured by the GTP-S binding method.

As one example of this method, 20 mM HEPES cell membranes was expressed G protein-coupled receptors (pH7.4), lOOmM NaCl, lOmM gCl 2, 50 in M GDP solution, labeled with ³⁵ S GTP After mixing with 400 pM of αS and incubating in the presence and absence of the test sample, filtration is performed, and a method of comparing the radioactivity of the bound GTPαS can be used.

In addition, G protein-coupled receptors share a system that transmits signals into cells via activation of trimeric GTP-binding proteins. Trimeric GTP-binding proteins are classified into three types, depending on the type of intracellular signaling system that activates, Gq type that increases Ca ²⁺ , Gs type that increases cAMP, and Gi type that suppresses cAMP. This chimerized was Sabuyuni' Bok and non Gq protein shed Sabuyunitto and other G proteins by applying the positive signals upon ligand subscription-learning intracellular pathway of Gq, thereby resulting in Ca ^{2 +} increase It is possible. The elevated Ca ²⁺ level can be detected using the repo overnight gene system having TRE (TP A responsive element) upstream, a staining indicator such as Fluor-3, and a change in the fluorescent protein aequorin as an index. Similarly, Gs protein subunits are chimerized with other G protein subunits, and a positive signal is consequently increased to cAMP, a Gs intracellular transduction pathway, and CRE (cAMP-responsive element) is upstream. It is also possible to use as an index the change in the repo overnight gene system in (Trends Pharmacol. Sci. (99) 20: 118).

There are no particular restrictions on the host cell that expresses the protein of the present invention in this screening system, and various host cells may be used depending on the purpose. Cells, CHO cells, HEK293 cells, and the like. Examples of the vector for expressing the protein of the present invention in vertebrate cells include a promoter located upstream of a gene encoding the protein of the present invention, an RNA splice site, a polyadenylation site, a transcription termination sequence, and an origin of replication. Those having the above can be preferably used. For example, pSV2dhfr (ol.Cell.Biol. (1981) 1, 854-864) having the early promoter of SV40, pEF-BOS (Nucleic Acids Res. (1990) 18, 5322), pCDM8 (Nature (1987) ) 329, 840-842), pCEP4 (Invitrogen) and the like are useful vectors for expressing G protein-coupled receptors. The DNA of the present invention can be inserted into a vector by a ligase reaction using a restriction enzyme site according to a conventional method (Current protocol in Molecular Biology eait. Ausubel et al. (1987) Publish. John Wiley). & Sons. Section 11.4 ~; 11.11). In addition, the introduction of a vector into a host cell can be performed, for example, by the calcium phosphate precipitation method or the electric pulse perforation method (Current protocols in Molecular Biology edit. Ausubel et al. (1987) Publish. John Wiley & Sons. Section 9.1-9.9). ), The ribofectamine method (GIBC0-BRL), the FuGENE6 reagent (Beringermannheim), the microinjection method, and the like.

If the ligand is isolated by the above-described method for screening a ligand that binds to the protein of the present invention, a compound that inhibits the interaction between the protein and the ligand of the present invention can be screened. Therefore, the present invention also provides a method for screening a compound having an activity of inhibiting the binding of the protein of the present invention to its ligand. This screening method comprises the steps of: (a) contacting a ligand of the protein or its partial peptide in the presence of a test sample with a ligand, and detecting the binding activity between the protein or its partial peptide and the ligand; b) selecting a compound that reduces the binding activity detected in step (a) as compared to the binding activity in the absence of the test sample. The test sample is not particularly limited. For example, a compound group obtained by combinatorial chemistry—technology (Tetrahedron (1995) 51, 8135-8137), or a phage display method (J. Mol. Biol. (1991) 222, 301-310) can be used. Screening also includes culture supernatants of microorganisms and natural components derived from plants and marine organisms. Other examples include, but are not limited to, brain and other biological tissue extracts, cell extracts, expression products of gene libraries, synthetic low molecular compounds, synthetic peptides, natural compounds, and the like.

 The protein of the present invention used in the screening may be, for example, in a form expressed on the cell surface, as a cell membrane fraction of the cell, or in a form bound to an affinity column.

 As a specific screening method, for example, a method in which a ligand is labeled with a radioisotope or the like, and the ligand is contacted with the protein of the present invention in the presence of a test sample, and then compared with the case where detection is performed in the absence of a test sample Then, a method of detecting a compound that reduces the binding activity between the protein and the ligand of the present invention based on the label attached to the ligand can be used. In addition, as in the case of the screening of the ligand binding to the protein of the present invention, it is also possible to perform screening using changes in cells as an index. That is, a compound expressing the protein of the present invention is brought into contact with a ligand in the presence of a test sample, and a compound that reduces a change in the cell compared to the case where the ligand is detected in the absence of the test sample is selected. Thus, it is possible to screen for a compound that inhibits the binding between the protein of the present invention and a ligand. Cells expressing the protein of the present invention can be prepared in the same manner as in the above-described screening for a ligand that binds to the protein of the present invention. The compound isolated by this screening is a candidate for the agonist of the protein of the present invention.

The present invention also provides a method for screening a compound that inhibits or promotes the activity of the protein of the present invention. This screening method is based on (a) the existence of a test sample. Contacting a cell that expresses the protein of the present invention with a ligand of the protein in the presence of the protein, (b) detecting a change in the cell due to binding of the ligand to the protein of the present invention, (c) absence of a test sample Selecting a compound that suppresses or enhances the change in the cell detected in step (b) as compared to the change in the cell below.

As a test sample, a compound group obtained by a combinatorial chemistry technique, a phage display method, etc. are applied in the same manner as the above-mentioned screening method of a compound that inhibits the binding between the protein and the ligand of the present invention. Random peptides, culture supernatants of microorganisms, natural components derived from plants and marine organisms, biological tissue extracts, cell extracts, expression products of gene libraries, synthetic low molecular compounds, synthetic peptides And natural compounds. In addition, a compound isolated by screening a compound that inhibits the binding between the protein of the present invention and a ligand can be used as a test sample. Cells expressing the protein of the present invention can be prepared in the same manner as in the above-described screening for a ligand that binds to the protein of the present invention. Changes in the cells after contact with the test sample can be detected by using changes in intracellular Ca ²⁺ levels and cAMP levels as indices, as in the above-described screening method. When detecting intracellular signal transduction, it is also possible to detect using a measurement system such as a repo overnight system using luciferase or the like as a reporter gene.

As a result of this detection, if the change in cells when the test sample is contacted is suppressed compared to the change in cells when the ligand is contacted in the absence of the test sample, The sample is determined to be a compound that inhibits the activity of the protein of the present invention. Conversely, if the test sample enhances the change in the cells, the compound is determined to be a compound that promotes the activity of the protein of the present invention. Here, “promoting or inhibiting the activity of the protein of the present invention” means whether the action is direct or indirect on the protein of the present invention. It means that the activity of the protein of the present invention is promoted or inhibited. Accordingly, compounds isolated by this screening include compounds that act on the protein or ligand of the present invention to inhibit or promote their binding and thereby inhibit or promote the activity of the protein of the present invention. However, compounds that do not inhibit or promote these bindings per se but result in inhibiting or promoting the activity of the protein of the present invention are also included. Such compounds include, for example, compounds that do not inhibit the binding of the protein of the present invention to the ligand, but inhibit or promote intracellular signaling pathways.

 When a compound isolated by the screening method of the present invention is used as a pharmaceutical, the isolated compound itself is administered directly to a patient or administered as a pharmaceutical composition formulated by a known pharmaceutical method. It is also possible to do. For example, it is conceivable to administer the composition by appropriately combining it with a pharmacologically acceptable carrier or vehicle, specifically, sterile water, physiological saline, vegetable oil, emulsifier, suspension, and the like. Administration to a patient can be generally performed by methods known to those skilled in the art, such as, for example, intraarterial injection, intravenous injection, and subcutaneous injection. The dose varies depending on the weight and age of the patient, the administration method, and the like, but those skilled in the art can appropriately select an appropriate dose. In addition, if the compound can be encoded by DNA, the DNA may be incorporated into a gene therapy vector to perform gene therapy. BRIEF DESCRIPTION OF THE FIGURES

 FIG. 1 is a diagram showing the result of performing a BLAST search on the entire sequence of SWISS-PR0T using the amino acid sequence of “GPRv4” as rQueryj. It showed 31% homology to ORYLA PROBABLE G PR0TEIN-C0UPL ED RECEPTOR.

FIG. 2 is a diagram showing the results of performing a BLAST search on the entire sequence of SWISS-PR0T using the rcPRvllj amino acid sequence as Queryj. It showed 31% homology to HUMAN NEUROPEPTIDE Y RECEPTOR TYPE 2. FIG. 3 is a diagram showing the results of performing a BLAST search on all SWISS-PROT sequences using the “GPRvl3” amino acid sequence as rQueryj. PONPY C5A showed 39% homology to ANAPHYLATOXIN CHEM0T ACTIC RECEPTOR.

 FIG. 4 is a diagram showing the results of performing a BLAST search on the entire SWISS-PR0T sequence using the rGPRvHj amino acid sequence as rQueryj. It showed 40% homology to CHICK P2Y PURIN0CEPT0R5.

 FIG. 5 is a diagram showing the results of performing a BLAST search on the entire SWISS-PROT sequence using the amino acid sequence of “GPRvl5” as Queryj. It showed 26% homology to HUMAN 5-HYDR0XYTRYPTAMINE IE.

 FIG. 6 is a diagram showing the results of performing a BLAST search on the entire sequence of SWISS-PROT using the amino acid sequence of “GPRvl9” as rQueryj. It showed 25% homology to APIME OPSIN and BLUE-SENSITIVE.

 FIG. 7 is a diagram showing the results of performing a BLAST search on all SWISS-PROT sequences using the amino acid sequence of “GPRv20” as rQueryj. It showed 38% homology to RAT MAS PR0T0-ONCOGENE.

 Figure 8 shows the results of a BLAST search of all SWISS-PROT sequences using the amino acid sequence of “GPRv31” as “Queryj.” 29% homology to SHEEP THYROTROPIN- RELEASING HORMONE RECEPTOR showed that.

 FIG. 9 is a diagram showing the result of performing a BLAST search on the entire SWISS-PROT sequence with the amino acid sequence of “GPRv38” set to rQuer j. It showed 46% homology to P2Y PURIN0CEPT0R 7 (Q15722).

FIG. 10 is a diagram showing the result of performing a BLAST search on the entire sequence of SWISS-PR0T using the amino acid sequence of “GPRv39” as rQueryj. It showed 35% homology to NEUROTENSIN RECEPTOR TYPE 1 (P20789). FIG. 11 is a diagram showing the results of performing a BLAST search on the entire SWISS-PROT sequence using the amino acid sequence of “GPRv68” as rQueryj. 39% showed the highest homology to TYPE-IB ANGIOTENSIN II RECEPTOR (Q13725).

 FIG. 12 is a diagram showing the result of performing a BLAST search on the entire SWISS-PR0T sequence using the amino acid sequence of “GPRv77” as rQueryj. It showed the highest homology at 29 ° to HUMAN PUTATIVE G PROTEIN-COU PLED RECEPTOR GPR17 (R12) (Q13304). FIG. 13 is a diagram showing the results of performing a BLAST search on the entire SWISS-PROT sequence using the amino acid sequence of “GPRv78” as rQueryj. 39% showed the highest homology with HUMAN GALANIN RECEPTOR TYPE 2 (043603).

 FIG. 14 is a diagram showing the results of performing a BLAST search on the entire SWISS-PROT sequence using the amino acid sequence of “GPRv79” as rQueryj. 39% showed the highest homology to RAT MAS PR0T0-0NC0GENE (P125 26).

 FIG. 15 is a diagram showing the results of performing a BLAST search on the entire sequence of SWISS-PR0T using the amino acid sequence of “GPRv81” as rQueryj. 25% showed the highest homology to HUMAN 5-HYDR0XYTRYPTAMINE IB RECEPTOR (P28222). BEST MODE FOR CARRYING OUT THE INVENTION

 Next, the present invention will be described more specifically with reference to examples, but the present invention is not limited to the following examples. Unless otherwise specified, it can be performed according to a known method (Maniat is, T. at al. (1982): "Molecular Cloning-A Laboratory Manual" Cold Spring Harbor Laboratory, NY).

 [Example 1] Isolation of a gene encoding a novel G protein-coupled receptor

The full length cDNA encoding the novel G protein-coupled receptor of the present invention (GPRv4, GPRvll, GPRvl3, GPRvH, GPRvl 5, GPRvl9, GPRv20, GPRv31, GPRv38, GPRv39, GPRv68, GPRv77, GPRv78, GPRv7 9, GPRv81) Obtained by PCR. To amplify the novel G protein-coupled receptor GPRv4, Marathon Ready cDNA derived from human fetal brain (Clontech) is used as type II cDNA, and as a forward primer, 5,-ATGGCCA ACTCCACAGGGCTGAACGCCT-3 '(SEQ ID NO: 17) 5, -TCAGGAGAGAGAACTCTCAGGTGGCCCC-3 '(SEQ ID NO: 18) was used as a reverse primer. PCR was carried out using Pyrobes tDNA polymerase (Takara Shuzo) in the presence of 5% formamide, after 94 ° C (2 minutes), 98 ° C (30 seconds) / 65 ° C (30 seconds) / 75 ° C (2 Min) cycle was repeated 30 times. As a result, a DNA fragment of about 1.1 kbp was amplified. This fragment was cloned using pCR2.1 plasmid (Invitrogen). The nucleotide sequence of the obtained clone was analyzed using the ABI377 DNA Sequencer (Applied Biosystems) according to the didoxy-one-mine method. The elucidated sequence is shown in SEQ ID NO: 9.

 This sequence has an open reading frame of 1107 bases (from 1st to 1107th of SEQ ID NO: 9). The amino acid sequence (368 amino acids) predicted from the open reading frame is shown in SEQ ID NO: 1. The predicted amino acid sequence has a hydrophobic region that is thought to be seven transmembrane domains that are characteristic of G protein-coupled receptors, indicating that this gene encodes a G protein-coupled receptor. Revealed.

To amplify the novel G protein-coupled receptor GPRvll, Marathon Ready cDNA (Clontech) derived from human fetus is used as type II cDNA, and as a forward primer, 5,-ATGCAGGC GCTTAACATTACCCCGGAGC-3 '(SEQ ID NO: 19), reverse primer 5, -T TAATGCCCACTGTCTAAAGGAGAATTC-3 '(SEQ ID NO: 20) was used. PCR was performed using Pyrobest DNA polymerase (Takara Shuzo) in the presence of 5% formamide, followed by five cycles of 94 ° C (2.5 seconds), followed by 94 ° C (5 seconds) / 72 ° C (2 minutes). The cycle of 94 ° C (5 seconds) / 70 ° C (2 minutes) was repeated 5 times, and the cycle of 94 ° C (5 seconds) / 68 ° C (2 minutes) was repeated 25 times. As a result, a DNA fragment of about 1.2 kbp was amplified. This fragment was cloned using pCR2.1 plasmid (Invitrogen). The nucleotide sequence of the clone obtained is Analysis was performed using the ABI377 DNA Sequencer (Applied Biosystems) by the dideoxy terminator method. The elucidated sequence is shown in SEQ ID NO: 10.

 This sequence has an open reading frame of 1296 bases (1st to 1296th of SEQ ID NO: 10). The amino acid sequence (431 amino acids) predicted from the open reading frame is shown in SEQ ID NO: 2. The predicted amino acid sequence has a hydrophobic region that seems to be seven transmembrane domains characteristic of G protein-coupled receptors, indicating that this gene encodes a G protein-coupled receptor did.

 To amplify the novel G protein-coupled receptor GPRvl3, Marathon Ready cDNA (Clontech) derived from human placenta was used as type I cDNA, and as a forward primer, 5,-ATGGGGM CGATTCTGTCAGCTACGAGT-3 '(SEQ ID NO: 21), reverse 5, -C TACACCTCCATCTCCGAGACCAGGTCA-3 '(SEQ ID NO: 22) was used as a primer. PCR was performed using Pyrobest DNA polymerase (Takara Shuzo Co., Ltd.) in the presence of 5 formamide, followed by five cycles of 94 ° C (2.5 minutes) followed by 94 ° C (5 seconds) / 72 ° C (2 minutes). The cycle of 5 ° C (5 seconds) / 70 ° C (2 minutes) was repeated 5 times, and the cycle of 94 ° C (5 seconds) / 68 ° C (2 minutes) was repeated 25 times. As a result, a DNA fragment of about 1.1 kbp was amplified. This fragment was cloned using pCR2.1 plasmid (Invitrogen). The nucleotide sequence of the obtained clone was analyzed using the ABI377 DNA Sequencer (Applied Biosystems) by the dideoxy-mine-one-one method. The elucidated sequence is shown in SEQ ID NO: 11.

The sequence has an open reading frame of 1014 bases (1st to 1014th of SEQ ID NO: 11). The amino acid sequence (337 amino acids) predicted from the open reading frame is shown in SEQ ID NO: 3. The predicted amino acid sequence has a hydrophobic region that seems to be seven transmembrane domains characteristic of G protein-coupled receptors, indicating that this gene encodes a G protein-coupled receptor did. To amplify the novel G protein-coupled receptor GPRvl4, Marathon Ready cDNA (Clontech) derived from human fetal brain was used as type I cDNA, and as a forward primer 5, -ATGTTA GCCAACAGCTCCTCAACCAACA-3 '(SEQ ID NO: 23) ), 5, -TCAGAGGGCGGAATCCTGGGGACACTGT-3 ′ (SEQ ID NO: 24) was used as the reverse primer. PCR was performed using Pyrobe st DNA polymerase (Takara Shuzo) in the presence of 5% formamide at 94 ° C (2.5 minutes), followed by 5 cycles of 94 ° C (5 seconds) / 72 ° C (2 minutes). The cycle of 94 ° C (5 seconds) / 70 ° C (2 minutes) was repeated 5 times, and the cycle of 94 ° C (5 seconds) / 68 ° C (2 minutes) was repeated 25 times. As a result, a DNA fragment of about 1.1 kbp was amplified. This fragment was cloned using pCR2.1 plasmid (Invitrogen). The nucleotide sequence of the obtained clone was analyzed using the ABI377 DNA Sequencer (Applied Biosystems) by the dideoxy-mine-one-one method. The elucidated sequence is shown in SEQ ID NO: 12.

 This sequence has an open reading frame of 1119 bases (1st to 1119th of SEQ ID NO: 12). The amino acid sequence (372 amino acids) predicted from the open reading frame is shown in SEQ ID NO: 4. The predicted amino acid sequence has a hydrophobic region that seems to be seven transmembrane domains characteristic of G protein-coupled receptors, indicating that this gene encodes a G protein-coupled receptor did.

To amplify the novel G protein-coupled receptor GPRvl5, Marathon Ready cDNA (Clontech) derived from human fetal brain was used as type I cDNA, and as a forward primer 5, -ATGAGT GATGAGCGGCGGCTGCCTGGCAG-3 '(SEQ ID NO: 25) ), 5′-CTAGGACGCGGAGCCCAGCGAGTCCGAG-3 ′ (SEQ ID NO: 26) was used as the reverse primer. PCR was performed using Pyrobest DNA polymerase (Takara Shuzo) in the presence of 5% formamide, followed by five cycles of 98 ° C (5 seconds) / 72 ° C (4 minutes) after 94 ° C (2.5 minutes). The cycle of 98 ° C (5 seconds) / 70 ° C (4 minutes) was repeated 5 times, and the cycle of 98 ° C (5 seconds) / 68 ° C (4 minutes) was repeated 25 times. As a result, a DNA fragment of about 1.8 kbp was amplified. This fragment was cloned using pCR2.1 pi asmid (Invitrogen). Base sequence of the clone obtained The columns were analyzed using the ABI377 DNA Sequencer (Applied Biosystems) according to the dideoxy and mineral method. The elucidated sequence is shown in SEQ ID NO: 13. This sequence has an open reading frame of 1830 bases (SEQ ID NO: 13). The amino acid sequence (609 amino acid) predicted from the open reading frame is shown in SEQ ID NO: 5. The predicted amino acid sequence has a hydrophobic region that is thought to be the seven transmembrane domains characteristic of a G protein-coupled receptor, indicating that this gene encodes a G protein-coupled receptor did.

 To amplify the novel G protein-coupled receptor GPRvl9, Marathon Ready cDNA (Clontech) derived from human placenta was used for type III cDM, and as a forward primer, 5,-ATGATGGG ACTCACCGAGGGGGTGTTCC-3 '(SEQ ID NO: 27), 5, -C TAAGAGAAAATGGGTCCCTTGGATCCAG-3 '(SEQ ID NO: 28) was used as the reverse primer. PCR using Pyrobes t DNA polymerase (Takara Shuzo), 94 ° C (2 minutes), 94 ° C (30 seconds) / 55 ° C

(30 seconds) / 72 ° C (2 minutes) cycle was repeated 30 times. As a result, a DNA fragment of about 1.0 kbp was amplified. This fragment was cloned using pCR2.1 plasmid (Invitrogen). The nucleotide sequence of the resulting clone was analyzed using the ABI377 DNA Sequencer (Applied Biosystems) according to the dideoxy and mineral method. The sequence identified is shown in SEQ ID NO: 14.

 This sequence has an open reading frame of 951 bases (SEQ ID NO: 14). The amino acid sequence (316 amino acid) predicted from the open reading frame is shown in SEQ ID NO: 6. Since the predicted amino acid sequence has seven transmembrane domains that are likely to be characteristic of G protein-coupled receptors, the gene may encode a G protein-coupled receptor. found.

To amplify the novel G protein-coupled receptor GPRv20, Marathon Ready cDNA (Clontech) derived from human fetus is used as type I cDNA, and as a forward primer, 5,-ATGGATCC AACCATCTCAACCTTGGACAC-3 '(SEQ ID NO: 29), reverse 5, -TCAGGTTAGATAAACATCTATTTGAAGAC-3 '(SEQ ID NO: 30) was used as a primer. PCR Pyrobe Using st DNA polymerase (Takara Shuzo), 5 cycles of 94 ° C (5 seconds) / 72 ° C (4 minutes) after 94 ° C (2.5 minutes) in the presence of 5% formamide, 94 ° C C (5 seconds) / 70 ° C

(4 min) cycle was repeated 5 times, and 94 ° C (5 sec) / 68 ° C (4 min) cycle was repeated 25 times. As a result, a MA fragment of about 1.1 kbp was amplified. This fragment was cloned using pCR2.1 pi asmid (Invitrogen). The nucleotide sequence of the obtained clone was analyzed using the ABI377 DNA Sequencer (Applied Biosystems) by the dideoxy-one-mine method. The elucidated sequence is shown in SEQ ID NO: 15. This sequence has an open reading frame of 1116 bases (SEQ ID NO: 15). The amino acid sequence (322 amino acids) predicted from the open reading frame is shown in SEQ ID NO: 7. The predicted amino acid sequence has a hydrophobic region that is thought to be the seven transmembrane domains characteristic of a G protein-coupled receptor, indicating that this gene encodes a G protein-coupled receptor did.

 To amplify the novel G protein-coupled receptor GPRv31, Marathon Ready cDNA (Clontech) derived from human fetus is used as type I cDNA, 5, -ATGGTTGG AGACACATTAAAACTTCTG-3 '(SEQ ID NO: 31) as the forward primer, reverse primer 5, -TC ATGGCATGACAACCAGATTAGGAAAG-3 '(SEQ ID NO: 32) was used. PCR was performed using Pyrobest DNA polymerase (Takara Shuzo), followed by a cycle of 94 ° C (30 seconds) / 50 ° C (30 seconds) / 72 ° C (2 minutes) after 94 ° C (2 minutes). Repeated times. As a result, a DNA fragment of about 1.1 kbp was amplified. This fragment was cloned using pCR2.1 plasmid (Invitrogen). The nucleotide sequence of the obtained clone was analyzed using the ABI377 DNA Sequencer (Applied Biosystems) by the dideoxy-one-mine-one-one method. The clearly identified sequence is shown in SEQ ID NO: 16.

This sequence has an open reading frame of 1062 bases (SEQ ID NO: 16). The amino acid sequence (353 amino acid) predicted from the open reading frame is shown in SEQ ID NO: 8. The predicted amino acid sequence is the G protein-coupled receptor The presence of the characteristic seven transmembrane hydrophobic regions suggests that this gene encodes a G protein-coupled receptor.

 To amplify the novel G protein-coupled receptor GPRv38, a human brain-derived Marathon Ready cDNA (Clontech) was used as type I cMA, and as a forward primer, 5, -ATGTCGGTCT GCTACCGTCCCCCAGGGA-3 '(SEQ ID NO: 37) and reverse 5, -TCA AAGGTCCCATTCCGGACCGTCCTTC-3 ′ (SEQ ID NO: 38) was used as a primer. PCR was performed using Pyrobest DNA polymerase (Takara Shuzo) in the presence of 5% formamide, followed by 5 cycles of 98 ° C (2.5 minutes) followed by 98 ° C (5 seconds) / 72 ° C (4 minutes). The cycle of 98 ° C (5 seconds) / 70 ° C (4 minutes) was repeated 5 times, and the cycle of 98 ° C (5 seconds) / 68 ° C (4 minutes) was repeated 25 times. As a result, a DNA fragment of about 1.1 kbp was amplified. This fragment was cloned using pCR2.1 plasmid (Invitrogen). The nucleotide sequence of the obtained clone was analyzed using the ABI377 DNA Sequencer (Applied Biosystems) according to the dideoxy-one-one-one method. The elucidated sequence is shown in SEQ ID NO: 35. The sequence has an open reading frame of 1077 bases (SEQ ID NO: 35). The amino acid sequence (358 amino acids) predicted from the open reading frame is shown in SEQ ID NO: 33. Since the predicted amino acid sequence has a hydrophobic region that seems to be seven transmembrane domains that are characteristic of G protein-coupled receptors, it was determined that this gene encodes a G protein-coupled receptor did.

To amplify the novel G protein-coupled receptor GPRv39, Marathon Ready cDNA (Clontech) derived from human fetal brain was used as type II cDNA, and 5, -ATGTCA GGGATGGAAAAACTTCAGAATG-3 'as the forward primer (SEQ ID NO: 39) 5, -TCAGGTTTTGTTAAAGTGGAAGCTTTGATAG-3 '(SEQ ID NO: 40) was used as a reverse primer. PCR was performed using Pyr obest DNA polymerase (Takara Shuzo) in the presence of 5% formamide, after 94 ° C (2 minutes), 94 ° C (30 seconds) / 50 ° C (30 seconds) / 72 ° C ( (1.5 minutes) was repeated 35 times. As a result, a DNA fragment of about 1.2 kbp was amplified. This fragment was cloned using pCR2.1 lasmid (Invitrogen). Of the resulting clone The nucleotide sequence was analyzed using the ABI377 DNA Sequencer (Applied Biosystems) by the dideoxy-mine-one-one method. The elucidated sequence is shown in SEQ ID NO: 36.

 This sequence has an open reading frame of 1248 bases (SEQ ID NO: 36). The amino acid sequence (415 amino acid) predicted from the open reading frame is shown in SEQ ID NO: 34. Since the predicted amino acid sequence has a hydrophobic region that seems to be seven transmembrane domains that are characteristic of G protein-coupled receptors, it was determined that this gene encodes a G protein-coupled receptor did.

 To amplify the novel G protein-coupled receptor GPRv68, human genomic DNA (Clontech) was converted to type I cDNA, 5, -ATGCAGATGGCCGATGCAGCCACGATA G-3 '(SEQ ID NO: 51) as a forward primer, and 5, -TCAGTAGGCAGAGCTGCTGG as a reverse primer. GCAGCAGG-3 ′ (SEQ ID NO: 52) was used. For PCR, use Pyrobest DNA polymerase (Takara Shuzo), and cycle 98 ° C (30 seconds) / 55 ° C (30 seconds) / 72 ° C (4 minutes) after 98 ° C (2.5 minutes). Repeated 35 times. As a result, a DNA fragment of about 1.4 kbp was amplified. This fragment was cloned using pCR2.1 plasmid (Invitrogen). The nucleotide sequence of the obtained clone was analyzed using the ABI377 DNA Sequencer (Applied Biosystems) by the dideoxy-one-mine-one-one method. The determined sequence is shown in SEQ ID NO: 46.

 This sequence has an open reading frame of 1410 bases (SEQ ID NO: 46). The amino acid sequence (469 amino acid) predicted from the open reading frame is shown in SEQ ID NO: 41. Since the predicted amino acid sequence has a hydrophobic region that seems to be seven transmembrane domains that are characteristic of G protein-coupled receptors, it was determined that this gene encodes a G protein-coupled receptor did.

To amplify the novel G protein-coupled receptor GPRv77, Marathon Ready cDNA (Clontech) derived from human fetal brain was converted into a gun-shaped cDNA, and the forward primer was 5, -atgaac aacaatacaacatgtattcaac-3 '(Nishiki Numbers: 5 3), Reno, 1 Sufu. 5, as a limer -tcaaccatatgattgcatatgtgctgaa-3 ′ (SEQ ID NO: 54) was used. PCR was performed using Pyrobe st DNA polymerase (Takara Shuzo), followed by 30 cycles of 94 ° C (30 seconds) / 55 ° C (30 seconds) / 72 ° C (3 minutes) after 94 ° C (2.5 minutes). Repeated times. As a result, a DNA fragment of about 1.0 kbp was amplified. This fragment was cloned using pCR2.1 plasmid (Invitrogen). The nucleotide sequence of the obtained clone was analyzed using the ABI377 DNA Sequencer (Applied Biosystems) by the dideoxy-mine-one-time method. The elucidated sequence is shown in SEQ ID NO: 47.

 This sequence has an open reading frame of 1011 bases (SEQ ID NO: 47). The amino acid sequence (336 amino acid) predicted from the open reading frame is shown in SEQ ID NO: 42. Since the predicted amino acid sequence has a hydrophobic region that seems to be seven transmembrane domains that are characteristic of G protein-coupled receptors, it was determined that this gene encodes a G protein-coupled receptor did.

 For amplification of the novel G protein-coupled receptor GPRv78, Marathon Ready cDNA (Clontech) derived from human fetal brain was used as type I cDNA, and as a forward primer, 5, -ATGCAC ACCGTGGCTACGTCCGGACCCA-3 '(SEQ ID NO: 55) As a reverse primer, 5'-TCAGAGAGGGGCGTTGTCCTCCCCCAGG-3 '(SEQ ID NO: 56) was used. PCR was performed using Pyrobe st DNA polymerase (Takara Shuzo), 5 cycles of 98 ° C (5 seconds) / 98 ° C (5 seconds) / 72 ° C (4 minutes) in the presence of 5% formamide. The cycle of 98 ° C. (5 seconds) / 70 ° C. (4 minutes) was repeated 5 times, and the cycle of 98 ° C. (5 seconds) / 68 ° C. (4 minutes) was repeated 25 times. As a result, a DNA fragment of about 1.2 kbp was amplified. This fragment was cloned using pCR2.1 plasmid (Invitrogen). The nucleotide sequence of the clone obtained was determined by the ABI377 DNA Sequencer (Applied

(Biosystems). The elucidated sequence is shown in SEQ ID NO: 48.

This sequence has an open reading frame of 1197 bases (SEQ ID NO: 48). Amino acid sequence predicted from open reading frame (398 Amino acid) is shown in SEQ ID NO: 43. Since the predicted amino acid sequence has a hydrophobic region that seems to be seven transmembrane domains that are characteristic of G protein-coupled receptors, it was determined that this gene encodes a G protein-coupled receptor did.

 To amplify the novel G protein-coupled receptor GPRv79, human genomic DNA (Clontech) was converted to type I cDNA, 5'-atggatccaaccaccccggcctgggga a-3 (SEQ ID NO: 57) as the primary primer, and 5,5 as the reverse primer. -ctacaccagactgcttctcg acatctcc-3, (SEQ ID NO: 58) was used. For PCR, Pyrobest DNA polymerase (Takara Shuzo) was used, and after 94 ° C (2 minutes), a cycle of 94 ° C (30 seconds) / 55 ° C (30 seconds) / 72 ° C (2.5 minutes) was performed. Repeated 30 times. As a result, a DNA fragment of about 1.0 kbp was amplified. This fragment was cloned using pCR2.1 plasmid (Invitrogen). The nucleotide sequence of the resulting clone was analyzed by the dideoxy terminator method using an ABI377 DNA Sequencer (Applied Biosystems). The determined sequence is shown in SEQ ID NO: 49.

 The sequence has an open reading frame of 993 bases (SEQ ID NO: 49). The amino acid sequence (330 amino acids) predicted from the open reading frame is shown in SEQ ID NO: 44. The predicted amino acid sequence has a hydrophobic region that is thought to be the seven transmembrane domains characteristic of a G protein-coupled receptor, indicating that this gene encodes a G protein-coupled receptor did.

To amplify the novel G protein-coupled receptor GPRv81, human genomic DNA (Clontech) is converted to type II cDNA, 5'-ATGGGGGATGAGCTGGCACCTTGCCCT G-3, (SEQ ID NO: 59) as a forward primer, and 5 CTAGGAAATGGTAAAGATGG CCTGGTGC- as a reverse primer. 3 ′ (SEQ ID NO: 60) was used. For PCR, Pyrobest DNA polymerase (Takara Shuzo) was used, and after a cycle of 94 ° C (2 minutes), a cycle of 94 ° C (30 seconds) / 55 ° C (30 seconds) / 72 ° C (2.5 minutes) was performed. Repeated 30 times. As a result, a DNA fragment of about 1.0 kbp was amplified. This fragment was cloned using pCR2.1 plasmid (Invitrogen). The nucleotide sequence of the obtained clone was determined by ABI377 Analysis was performed using a DNA Sequencer (Applied Biosystems). The elucidated sequence is shown in SEQ ID NO: 50.

 This sequence has an open reading frame (SEQ ID NO: 50) of 1044 bases. The amino acid sequence (347 amino acids) predicted from the open reading frame is shown in SEQ ID NO: 45. Since the predicted amino acid sequence has a hydrophobic region that seems to be seven transmembrane domains that are characteristic of G protein-coupled receptors, it was determined that this gene encodes a G protein-coupled receptor did.

 [Example 2] BLAST search for SWISS-PR0T using the amino acid sequence of a novel G protein-coupled receptor

 BLAST (Basic local alignment search tool) for SWISS-PR0T in the amino acid sequence of “GPRv4” [SF Altschul et al., J. Mol. Biol., 215: 403-410 (1990)] Shown in 1. “GPRv4” showed the highest homology at 31 ° to ORYLA PROBABLE G PROTEIN-COUPLED RECEPTOR (Q91178, 428aa) among known G protein-coupled receptors. This proved that "GPRv4" was a novel G protein-coupled receptor.

 The BLAST search results for SWISS-PR0T with the amino acid sequence of “GPRvll” are shown in FIG. “GPRvll” showed the highest homology at 31 ° to HUMAN NEUROPEPTIDE Y RECEPTOR TYPE 2 (P49146, 381aa) among known G protein-coupled receptors. This proved that "GPRvll" is a novel G protein-coupled receptor.

 The BLAST search results for SWISS-PR0T with the amino acid sequence of “GPRvl3” are shown in FIG. “GPRvl3” showed the highest homology at 39% to P0NPY C5A A APHYLAT OXIN CHEMOTACTIC RECEPTOR (P79234, 340aa) among known G protein-coupled receptors. This proved that “GPRvl3” is a novel G protein-coupled receptor.

BLAST search results for SWISS-PR0T with the amino acid sequence of “GPRvl4” are shown in FIG. "GPRvl4 I is a known G protein-coupled receptor that is CHICK P2Y PURIN0CEP 40% showed the highest homology to TOR 5 (P32250, 308aa). This proved that "GPRvl4" was a novel G protein-coupled receptor.

 BLAST search results for SWISS-PR0T with the amino acid sequence of “GPRvl5” are shown in FIG. “GPRvl5” showed the highest homology at 26% to HUMAN 5-HYDR0XYTRYP TAMINE IE RECEPTOR (P28566, 365aa) among known G protein-coupled receptors. This proved that “GPRvl5” is a novel G protein-coupled receptor.

 The results of a BLAST search for SWISS-PR0T with the amino acid sequence of “GPRvl9” are shown in FIG. "GPRvl9" showed the highest homology at 25% among APIME OPSIN and BLUE-S ENSITIVE (P90680, 377aa) among known G protein-coupled receptors. This indicated that "GPRvl9" was a novel G protein-coupled receptor.

 BLAST search results for SWISS-PR0T with the amino acid sequence of “GPRv20” are shown in FIG. “GPRv20” showed the highest homology of 38% to RAT MAS PR0T0-0NC0G ENE (P12526, 324aa) among known G protein-coupled receptors. This indicated that “GPRv20” was a novel G protein-coupled receptor.

 The results of a BLAST search for SWISS-PR0T with the amino acid sequence of “GPRv31” are shown in FIG. “GPRv31” showed the highest homology at 29% to SHEEP THYR0TR0PIN-R ELEASING HORMONE RECEPTOR (Q28596, 398aa) among known G protein-coupled receptors. This indicated that “GPRv31” is a novel G protein-coupled receptor.

 FIG. 9 shows the results of a BLAST search for SWISS-PR0T using the amino acid sequence of “GPRv38”. “GPRv38” was not the same among known G protein-coupled receptors and showed the highest homology at 46% to P2Y PURINOCEPTOR 7 (Q15722, 352aa). This indicated that “GPRv38” is a novel G protein-coupled receptor.

FIG. 10 shows the results of a BLAST search for SWISS-PR0T using the amino acid sequence of “GPRv39”. “GPRv39” is the same among known G protein-coupled receptors, and is the highest at 35% of RAT NEUROTENSIN RECEPTOR TYPE 1 (P20789, 424aa) Showed high homology. This indicated that “GPRv39” is a novel G protein-coupled receptor.

 BLAST search results for SWISS-PROT with the amino acid sequence of “GPRv68” are shown in FIG. “GPRv68” was not the same among known G protein-coupled receptors, and showed the highest homology at 39 ° to TYPE-IB ANGIOTENSIN II RECEPTOR (Q13725, 359aa). This indicated that “GPRv68” is a novel G protein-coupled receptor.

 BLAST search results for SWISS-PR0T with the amino acid sequence of “GPRv77” are shown in FIG. `` GPRv77 '' has no known G protein-coupled receptor, and has the highest homology at 29% with HUMAN PUTATIVE G PROTEIN-COUPLED RECEPTOR GPR17 (R12) (Q13304, 339a a) showed that. This proved that “GPRv77” is a novel G protein-coupled receptor.

 BLAST search results for SWISS-PROT with the amino acid sequence of “GPRv78” are shown in FIG. “GPRv78” was not the same among known G protein-coupled receptors, and showed the highest homology at 39% with HUMAN GAL IN IN RECEPTOR TYPE 2 (043603, 387aa). This proved that "GPRv78" is a novel G protein-coupled receptor.

 The BLAST search result for SWISS-PR0T with the amino acid sequence of “GPRv79” is shown in FIG. “GPRv79” was not the same as any known G protein-coupled receptor, and showed the highest homology at 39% to RAT MAS PROTO-ONCOGENE (P12526, 324aa). From this, it was determined that “GPRv79” is a novel G protein-coupled receptor.

The BLAST search results for SWISS-PR0T for the amino acid sequence of “GPRv81” are shown in FIG. `` GPRv81 '' is not the same among known G protein-coupled receptors, and is 25% of HUMAN 5-HYDROXYTRYPTAMINE IB RECEPTOR (P28222, 390aa). It showed the highest homology. This proved that “GPRv81” is a novel G protein-coupled receptor. Industrial applicability

 According to the present invention, a novel G protein-coupled receptor (GPRv4, GPRvll, GPRvl3, GPRv, GPRvl5, GPRvl9, GPRv20, GPRv31, GPRv38, GPRv39, GPRv68, GPRv77, GPRv78, GPRv79, GPRv81) A vector containing the gene, a host cell containing the vector, and a method for producing the protein are provided. Furthermore, a method for screening a compound that modifies the activity of the protein was provided. The protein of the present invention, its gene, or a compound that modulates the activity of the protein of the present invention is expected to be used for the development of new preventive or therapeutic agents for diseases involving the G protein-coupled receptor protein of the present invention. Is done.

Claims

The scope of the claims 1. The DNA according to any one of the following (a) to (d), which encodes a guanosine triphosphate binding protein-coupled receptor.  (a) DNA encoding a protein consisting of the amino acid sequence described in any one of SEQ ID NOs: 1 to 8, 33 to 34, and 41 to 45.  (b) a DNA comprising the code region of the nucleotide sequence of any one of SEQ ID NOs: 9 to 16, 35 to 36, and 46 to 50;  (c) SEQ ID NO: 1 amino acid sequence in which one or more amino acids have been substituted, deleted, added and / or inserted in the amino acid sequence described in any one of 8, 33 to 34, and 41 to 45 DNA encoding a protein consisting of  (d) a DNA that hybridizes under stringent conditions to a DNA consisting of the nucleotide sequence of any one of SEQ ID NOS: 9 to 16, 35 to 36, and 46 to 50.  2. A DNA encoding a partial peptide of a protein consisting of the amino acid sequence of any one of SEQ ID NOs: 1 to 8, 33 to 34, and 41 to 45.  3. A vector containing the MA according to claim 1 or 2.  4. A transformant carrying the DNA according to claim 1 or 2 or the vector according to claim 3.  5. A protein or peptide c encoded by the DNA of claim 1 or 2 c. 6. culturing the transformant according to claim 4, and recovering the expressed protein or peptide from the transformant or a culture supernatant thereof, the method according to claim 5, Production method.  7. A method for screening a ligand that binds to the protein according to claim 5,  (a) contacting a test sample with the protein or peptide according to claim 5, (b) selecting a compound that binds to the protein or peptide. 8. A method for screening a compound having an activity of inhibiting the binding between the protein of claim 1 or 2 and a ligand thereof,  (a) contacting a ligand with the protein or a partial peptide thereof according to claim 1 or 2 in the presence of a test sample, and detecting a binding activity between the protein or a partial peptide thereof and the ligand;  (b) selecting a compound that reduces the binding activity detected in step (a) as compared to the binding activity in the absence of the test sample.  9. A method for screening a compound that inhibits or promotes the activity of the protein according to claim 1 or 2,  (a) contacting a cell expressing the protein with a ligand for the protein in the presence of a test sample;  (b) detecting a change in a cell due to binding of the ligand to the protein, (c) selecting a compound that suppresses or enhances the change in the cell detected in step (b) as compared to the change in the cell in the absence of the test sample.  10. The method according to claim 8 or 9, wherein the change in the cell is a change in cAMP concentration or a change in calcium concentration.  1 1. An antibody that binds to the protein according to claim 1 or 2.  12. A compound isolated by the screening according to any of claims 7 to 10.  13. A pharmaceutical composition comprising the compound according to claim 12 as an active ingredient.  14. SEQ ID NO: A nucleotide having a length of at least 15 nucleotides, which is complementary to a DNA consisting of the nucleotide sequence of any of 9 to 16, 35 to 36, and 46 to 50 or a complementary strand thereof.