JP2001029083A - Novel g-protein conjugated type receptor - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a novel G-protein conjugated type receptor which comprises a G-protein conjugated type receptor containing a specific amino acid sequence, possesses high possibility as a target molecule for creating medicines and is used for the screening, or the like, of prophylactic or therapeutic drugs against central nervous system diseases, e.g. schizophreia, or the like. SOLUTION: This G-protein conjugated type receptor is a novel G-protein conjugated type receptor containing the amino acid sequence represented by the formula or a G-protein conjugated type receptor exerting the same effect as this receptor, possesses high possibility as a target molecule for creating medicines and is useful in the method, or the like, of screening out an agonist or antagonist for the development, or the like, of prophylactic or therapeutic drugs against central nervous system diseases, e.g. schizophrenia, Parkinson's disease, ache. This receptor is obtained by extracting mRNA from human cells or tissues by a conventional method, carrying out reverse transcriptase- polymerase chain reaction using this mRNA as a template by the use of a primer intercalating this receptor mRNA or a part of mRNA domain and integrating the resultant gene into an expression vector to express it in a host cell.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention belongs to the field of genetic engineering, and relates to a novel G protein-coupled receptor, a gene encoding the G protein-coupled receptor, a method for producing the G protein-coupled receptor, and a G protein-coupled receptor. The present invention relates to an antibody against a type receptor and a screening method using the G protein-coupled receptor.

[0002]

BACKGROUND OF THE INVENTION It is well known that many medically important biological processes are mediated by proteins and / or second messengers involved in signal transduction pathways, including G proteins (Lefkowitz). , Nature, 1
991, 351: 353-354). Among them, a group of cell membrane receptors that transmit a signal into a cell through activation of a trimeric GTP-binding protein is generally referred to as “G protein-coupled receptor”. Examples of such proteins include adrenaline and dopamine receptors (Kobilka, BK et al., Proc. Natl. Ac
ad. Sci. USA, 1987, 84: 46-50; Kobilka, BK et al., Sci.
ence, 1987, 238: 650-656; Bunzow, JR et al., Nature, 1
988, 336: 783-787). All known G protein-coupled receptors have an extracellular amino terminus and an intracellular carboxyl terminus, and form a superfamily that shares a structure that penetrates the cell membrane seven times. It may be collectively referred to as a "penetrating receptor." The G protein-coupled receptor transmits information of various physiologically active substances from the cell membrane to the cell via the activation of the trimeric GTP-binding protein and the resulting change in the intracellular second messenger. Trimeric GT
Intracellular second messengers controlled by P-binding proteins are well known, such as cAMP via adenylate cyclase and Ca ⁺⁺ via phospholipase C. control,
It has recently been revealed that many cellular proteins, such as the activation of kinases, are targets (Gudermann,
T. et al. (1997) Annu. Rev. Neurosci., 20, 399-42
7). Many of the bioactive substances that transmit information through G protein-coupled receptors include neurotransmitters, hormones, chemokines, lipid-derived signal transmitters, divalent ions, and proteases. It is. Each of these physiologically active substances has a specific G protein-coupled receptor, and transmits that information into cells.

In many cases, G protein-coupled receptors are preceded by gene cloning due to structural similarity within their superfamily. Receptors that do not correspond to endogenous ligands are called orphan G protein-coupled receptors. Have been. Generally, it has been difficult to develop agonists and antagonists of the orphan G protein-coupled receptor since no specific ligand has been discovered. However, in recent years, it has been proposed to create a drug targeting an orphan G protein-coupled receptor by combining an enhanced compound library with high-performance high-throughput screening (Stad
el, J. et al. (1997) Trends Pharmacol. Sci., 18, 4
30-437). That is, measurement of cAMP and Ca ⁺⁺ , which are second messengers of many G protein-coupled receptors, GTPase activity which is an index of activation of trimeric GTP binding protein, G protein binding measurement of GTPγS, etc. By increasing the throughput, it is possible to screen an agonist for an orphan G protein-coupled receptor from a compound library, and discover specific agonists and antagonists using the compound,
This means that it is possible to develop therapeutics for specific diseases. Under such circumstances, the discovery of a novel G protein-coupled receptor that can be a therapeutic target for a new disease can be regarded as the most important step in creating a drug that acts on a G protein-coupled receptor.

[0004] To date, hundreds of G protein-coupled receptors have been cloned from eukaryotes. For humans, G protein-coupled receptors that have been associated with more than one hundred endogenous ligands have been cloned.
To date, hundreds of therapeutic agents targeting these receptors have been utilized (Wilson, J. et al.
(1998) British J. of Pharmacol. 125, 1387-1392).
G protein-coupled receptors target a wide variety of diseases, and act on G protein-coupled receptors in the fields of the central nervous system, cardiovascular system, inflammatory immune system, digestive system, motor system, and urogenital system. (Stadel, J. et al. (1997) Trends Pharmacol. Sci.,
18, 430-437). This suggests that G protein-coupled receptor agonists or antagonists are highly likely to be therapeutic agents for diseases, preventing, ameliorating, and preventing various diseases.
There is a need to identify new receptors that may play important roles in treatment and to elucidate the link to the disease.

[0005] Among them, the central nervous system transmits and controls various kinds of information using physiologically active substances represented by neurotransmitters, and G protein-coupled receptors are used for transmitting and controlling the information. Plays an important role. The presence of many types of G protein-coupled receptors in the central nervous system makes them important therapeutic targets for diseases of the central nervous system. For example, the G protein-coupled receptor for the neurotransmitter dopamine is schizophrenia (Seeman, P. e.
t al. (1997) Neuropsychopharmacology, 16,93-11
0), serotonin G protein-coupled receptor is depressed (C
owen, PJ (1991) Br. J. Psychiatry, 159 (Suppl.
12), 7-14), G protein-coupled receptor for neuropeptide Y is an eating disorder (Blomqvist, AG and Herzog, H. et al.
(1997) Trends Neurosci., 20, 294-298). G protein-coupled receptors also play an important role in the inflammatory immune system. Inflammation and immune response play a beneficial role in protecting yourself from foreign body invasion and tissue damage, while excessive and abnormal reactions can cause inflammatory and autoimmune diseases. (Gallin, JI et al. (19
92) Inflammation Basic Principles and Clinical Cor
relates Raven Press (New York), Roitt, IM et al.
(1993) IMMUNOLOGY Mosby-Year Book Europe Limited.
(London)). Many of these substances that trigger an inflammatory immune response are called mediators. Among the mediators, many of the mediators that work via G protein-coupled receptors such as prostaglandins, leukotrienes, platelet activators, chemokines, bradykinins, and anaphylatoxins are diseases caused by abnormal inflammation and immunity (eg, asthma, allergy) , Is considered as a therapeutic target for rheumatoid arthritis, inflammatory bowel disease, infectious diseases, etc. (Halushka, PV et al. (1989) Annu. Rev. Pharm
acol. Toxicol. 29, 213-239, Hosford, D. et al. (19
90) Prog. Med. Chem. 27, 325-380, Oppenheim, JJ
et al. (1991) Annu. Rev. Immunol. 9, 617-648). The functions of leukocytes that control immune and inflammatory reactions are controlled by G protein-coupled receptors, which are receptors for chemokines, anaphylatoxins, lipid metabolites such as PAF and eicosanoids. In addition, lipid metabolites such as PAF and eicosanoids produced by leukocytes are modified via receptors in tissues surrounding leukocytes (translated by Yoshio Aso, Immunology at a glance (1993), published by Medical Science International, 62 -73, Proost, P. et al. (1996) Int.
J. Clin. Lab. Res. 26, 211-23, Scott, DT et a
l. (1994) Gen. Pharmacol. 25, 1285-96). For these reasons, G protein-coupled receptors of chemokines, anaphylatoxins, PAF and eicosanoids are considered to be therapeutic targets for diseases caused by abnormal immunity and inflammation. However, not all G protein-coupled receptors involved in diseases of the central nervous system and / or diseases caused by abnormal immune inflammation (but not limited thereto) and molecules acting on them are understood.

[0006]

[Problems to be Solved by the Invention] A novel drug required for the development of a preventive / therapeutic agent for a disease targeted by a G protein-coupled receptor, particularly for diseases of the central nervous system (schizophrenia, Parkinson's disease, pain). It is an object of the present invention to provide a recombinant protein for isolating and identifying a gene encoding a novel G protein-coupled receptor family, constructing an expression production system for the receptor, and searching for a substance that modifies the receptor activity.

[0007]

Under these circumstances, the present inventors have conducted intensive studies and as a result, have found that a gene encoding a novel G protein-coupled receptor expressed at a site involved in the central nervous system is obtained. Successfully isolated and its full-length ORF
(open reading frame) was determined. Furthermore, a novel G protein-coupled receptor is expressed to enable production of a recombinant protein, a vector containing the gene, a host cell containing the vector, the G protein-coupled receptor using the host cell, the G protein-coupled receptor. A method for producing antibodies against the type receptor was established. This enabled screening of the G protein-coupled receptor and a substance that modulates the activity of the G protein-coupled receptor, thereby completing the present invention.

That is, the present invention relates to (1) a G protein-coupled receptor having the amino acid sequence of SEQ ID NO: 2, or a G protein-coupled receptor which is an equivalent of the receptor; A gene encoding the amino acid sequence of the G protein-coupled receptor, (3) a vector containing the gene described in (2), (4) a host cell containing the vector described in (3), (5) a host cell described in (4) The method for producing a G protein-coupled receptor according to (1), wherein
(6) an antibody against the G protein-coupled receptor according to (1); (7) a substance that contacts the G protein-coupled receptor according to (1) with a test compound to modify the activity of the G protein-coupled receptor How to screen for
About.

[0009]

DESCRIPTION OF THE PREFERRED EMBODIMENTS Terms used in the present invention will be described below. As used herein, “G protein-coupled receptor” refers to “G protein-coupled receptor protein”. "Equivalent substance" of the G protein-coupled receptor of the present invention
Is any one of the amino acid sequences described in SEQ ID NO: 2.
Or a G protein-coupled receptor having the same activity as the G protein-coupled receptor, wherein one or several amino acid residues may be substituted, deleted, and / or inserted at a plurality of sites. Say. It preferably has an amino acid sequence having 1 to 10, more preferably 1 to 7, particularly preferably 1 to 5 amino acids in the amino acid sequence represented by SEQ ID NO: 2, with amino acid substitution, deletion or insertion, and And a G protein-coupled receptor protein having the same activity as the protein represented by the amino acid sequence of SEQ ID NO: 2, and a polypeptide having the amino acid sequence of SEQ ID NO: 2 is most suitable.

[0010] The gene having a nucleotide sequence encoding the novel G protein-coupled receptor protein of the present invention may be a G protein-coupled receptor protein represented by the amino acid sequence of SEQ ID NO: 2 or an equivalent thereof. Any gene having a coding base sequence may be used. Preferably, it is a gene having a base sequence encoding the amino acid sequence of SEQ ID NO: 2. More preferably, it is a gene having the first to 1002th nucleotides of the nucleotide sequence described in SEQ ID NO: 1.

(Production method) The gene encoding the G protein-coupled receptor of the present invention, the vector of the present invention, the host cell of the present invention, the G protein-coupled receptor of the present invention, and the activity of the G protein-coupled receptor of the present invention A method for screening for a substance that modifies G, and a method for producing an antibody that reacts with a G protein-coupled receptor are described in 1) to 4) below.

1) Method for Producing New G Protein-Coupled Receptor Gene a) First Production Method mRNA is extracted from a human cell or tissue having the ability to produce the G protein-coupled receptor of the present invention. Next, using this mRNA as a template, the G protein-coupled receptor mRNA
Alternatively, two types of primers sandwiching a part of the mRNA region are prepared. Performing reverse transcriptase-polymerase chain reaction (hereinafter referred to as RT-PCR) suitable for a protein containing a part of the amino acid sequence represented by SEQ ID NO: 2 of the present invention by improving denature temperature, conditions for adding a denaturant, and the like. Thus, the G protein-coupled receptor cDNA or a part thereof can be obtained. Furthermore, by incorporating the obtained G protein-coupled receptor cDNA or a part thereof into an appropriate expression vector, it can be expressed in host cells to produce the receptor. First, mRNAs including those encoding the receptor are extracted from cells or tissues capable of producing the G protein-coupled receptor of the present invention, for example, human spleen by a known method. As the extraction method, guanidine
Thiocyanate hot phenol method, guanidine
A thiocyanate-guanidine / hydrochloric acid method may be mentioned, and a guanidine / thiocyanate cesium chloride method is preferred. Cells or tissues capable of producing the receptor are identified by Northern blotting using a gene having a nucleotide sequence encoding the receptor or a part thereof, Western blotting using an antibody specific to the receptor, or the like. be able to.

[0013] mRNA may be purified according to a conventional method.
NA can be adsorbed and eluted on an oligo (dT) cellulose column and purified. Furthermore, mRNA can be further fractionated by sucrose density gradient centrifugation or the like. Alternatively, a commercially available extracted mRNA may be used without extracting the mRNA. Next, the purified mRNA is subjected to a reverse transcriptase reaction in the presence of a random primer or an oligo dT primer to synthesize a first-strand cDNA. This synthesis can be performed by a conventional method. The obtained first-strand cDNA is subjected to PCR using two types of primers sandwiching a partial region of the target gene to amplify the target novel G protein-coupled receptor DNA. The obtained DNA is fractionated by agarose gel electrophoresis or the like. If desired, cut the DNA with restriction oxygen or the like and connect
Fragments can also be obtained.

B) Second Production Method The gene of the present invention can be produced by a conventional genetic engineering technique in addition to the above-mentioned production method. First, using the mRNA obtained by the above method as a template and a single-stranded cD
After synthesizing the NA, a double-stranded cDNA is synthesized from the single-stranded cDNA. The method includes the S1 nuclease method (Efstrati
adis, A. et al. (1976) Cell, 7, 279-288), Land method (L
and, H. et al. (1981) Nucleic Acids Res., 9, 2251-
2266), O. Joon Yoo method (Yoo, OJ et al. (1983) Pro
c. Natl. Acad. Sci. USA, 79, 1049-1053), Okayama-B
erg method (Okayama, H. and Berg, P. (1982) Mol. Cell. B
iol., 2, 161-170). Next, the recombinant plasmid obtained by the above-mentioned method is
The strain can be introduced into a strain and transformed, and a recombinant can be selected using tetracycline resistance or ampicillin resistance as an index. For example, when the host cell is E. coli, the method of Hanahan (Hanahan, D. (198
3) J. Mol. Biol., 166, 557-580), ie, CaCl ₂ or MgC
competent cells prepared in the presence of a l ₂ or RbCl can be carried out by a method of adding the recombinant DNA material. In addition, a phage vector such as a lambda system can be used in addition to the plasmid.

As a method for selecting a strain having the DNA of the desired novel G protein-coupled receptor from the transformants obtained as described above, for example, the following various methods can be adopted. Screening Method Using Synthetic Oligonucleotide Probes Oligonucleotides corresponding to all or a part of the G protein-coupled receptor of the present invention were synthesized (in this case, nucleotide sequences derived using codon usage or possible nucleotide sequences were combined. Any of a plurality of nucleotide sequences may be used, and in the latter case, inosine may be included to reduce the number of nucleotides.) Using this as a probe (labeled with ³² P or ³³ P), the DNA of the transformed strain is used.
Is hybridized with a denatured and fixed nitrocellulose filter, and the obtained positive strain is searched for and selected. Screening Method Using Probe Prepared by Polymerase Chain Reaction Sense and antisense primer oligonucleotides corresponding to a part of the G protein-coupled receptor protein of the present invention are synthesized, and these are combined to perform polymerase chain reaction (Saiki, RK et al. (1988) Science 239,
487-491) to amplify a DNA fragment encoding all or part of the desired G protein-coupled receptor protein.
As the template DNA used here, cDNA synthesized by reverse transcription reaction from mRNA of a cell producing the G protein-coupled receptor protein or genomic DNA can be used. The DNA thus prepared is labeled with a fragment of ³² P or ³³ P, and colony hybridization or plaque hybridization is performed using the fragment as a probe to select a target clone.

Method for Screening by Producing a Novel G Protein-Coupled Receptor Protein in Other Animal Cells A transformant is cultured, the gene is amplified, and the gene is transfected into an animal cell (in this case, self-replicating Or a plasmid that can be integrated into the chromosome of an animal cell), and the protein encoded by the gene is produced on the cell surface. By detecting the protein using the antibody against the G protein-coupled receptor protein of the present invention, a strain having cDNA encoding the desired G protein-coupled receptor protein is selected from the original transformant. Method for Selection Using Antibody Against G Protein-Coupled Receptor Protein of the Present Invention An antibody against the G protein-coupled receptor protein of the present invention and a cDNA produced in advance in an expression vector to produce a protein on the surface of a transformant, and the antibody A desired G protein-coupled receptor protein-producing strain is detected using a secondary antibody against, and a target strain is selected. Method using selective hybridization translation system cDNA obtained from the transformant, blotted on a nitrocellulose filter or the like, and hybridized with mRNA from the G protein-coupled receptor protein-producing cell of the present invention. The mRNA bound to is dissociated and recovered. The recovered mRNA is translated into protein by a protein translation system, for example, injection into Xenopus oocytes or a cell-free system such as rabbit reticulocyte lysate or wheat germ. The target strain is selected by detection using an antibody against the G protein-coupled receptor protein of the present invention. DNA encoding the G protein-coupled receptor of the present invention from the obtained target transformant
Can be collected by a known method (Maniatis, T. et al. (1)
982): “MolecularCloning-A Laboratory Manual” Cold
Spring Harbor Laboratory, NY). For example, a fraction corresponding to plasmid DNA is separated from cells,
It can be carried out by cutting out a cDNA region from the plasmid DNA.

C) Third Production Method The G protein-coupled receptor gene of the present invention can also be produced by binding a DNA fragment produced by a chemical synthesis method. Each DNA is prepared using a DNA synthesizer (for example, Oligo 1000).
M DNA Synthesizer (Beckman) or 394 DNA /
RNA Synthesizer (Applied Biosystems) or the like).

D) Fourth Production Method The G protein-coupled receptor gene of the present invention can be prepared, for example, by the phosphite triester method (Hunkapiller, M. et al. (1984)
Nature, 10, 105-111) and the like, and can also be produced by chemical synthesis of nucleic acids. The codon for the desired amino acid is known per se, and the selection may be arbitrarily selected.
ic Acids Res., 9 r43-r74). Furthermore, partial modification of the codons of these nucleotide sequences can be performed by a conventional method using a site-specific mutagenesis (site) using a primer consisting of a synthetic oligonucleotide encoding the desired modification.
specific mutagenesis) (Mark, DF et al. (1984) Pro
c. Natl. Acad. Sci. USA, 81, 5662-5666). As described above, the sequencing of the DNA obtained by a) to d) is performed by, for example, the Maxam-Gilbert chemical modification method.
(Maxam, AM and Gilbert, W. (1980): “Methods in
Enzymology "65, 499-559) and dideoxynucleotide chain termination using M13 (Messing, J. and Vieira, J
(1982) Gene, 19, 269-276).

2) Method for Producing Recombinant Protein of G Protein-Coupled Receptor of the Present Invention The isolated fragment containing the gene encoding the G protein-coupled receptor of the present invention is re-incorporated into an appropriate vector DNA, Eukaryotic and prokaryotic host cells can be transformed. Furthermore, by introducing an appropriate promoter and a sequence related to expression into these vectors, the gene can be expressed in each host cell. For example, eukaryotic host cells include cells such as vertebrates, insects, and yeast, and vertebrate cells include, for example, monkey cells such as CO.
Dihydrofolate reductase-deficient strains of S cells (Gluzman, Y. (1981) Cell, 23, 175-182) and Chinese hamster ovary cells (CHO) (Urlaub, G. and Chasin, LA (198
0) Proc. Natl. Acad. Sci. USA, 77, 4216-4220), HEK293 cells derived from human fetal kidney and EpsteinBarr
293-EBNA cells transfected with Virus EBNA-1 gene (Invitro
gen) is often used, but is not limited thereto. As a vertebrate cell expression vector, those having a promoter, an RNA splice site, a polyadenylation site, a transcription termination sequence, and the like, which are usually located upstream of the gene to be expressed, can be used. May have. As an example of the expression vector, pSV2dhfr having the early promoter of SV40 (Subramani, S. et al. (1981) Mol. Cel)
l. Biol., 1, 854-864), pEF-BOS having a human elongation factor promoter (Mizushima, S. and Nagata,
S. (1990) Nucleic Acids Res., 18, 5322), cytomega
Examples include, but are not limited to, pCEP4 (Invitrogen) having a lovirus promoter.

As an example, when a COS cell is used as a host cell, the expression vector has an SV40 origin of replication, is capable of autonomous growth in COS cells, and has a transcription promoter, a transcription termination signal and an RNA splice. One with a site can be used, for example, p
ME18S, (Maruyama, K. and Takebe, Y. (1990) Med.Imm
unol., 20, 27-32), pEF-BOS (Mizushima, S. and Naga
ta, S. (1990) Nucleic Acids Res., 18, 5322), pCDM
8 (Seed, B. (1987) Nature, 329, 840-842). The expression vector is a DEAE-dextran method (Luthma
n, H. and Magnusson, G. (1983) Nucleic Acids Res.,
11, 1295-1308), calcium phosphate-DNA coprecipitation method (Gra
ham, FL and van der Ed, AJ (1973) Virology,
52, 456-457), a method using FuGENE6 (Boeringer Mannheim), and an electric pulse perforation method (Neumann, E. et a
l. (1982) EMBO J., 1, 841-845) and the like, and can be used to obtain desired transformed cells. When a CHO cell is used as a host cell, a vector capable of expressing a neo gene functioning as a G418 resistance marker together with an expression vector, such as p
RSVneo (Sambrook, J. et al. (1989): “Molecular Cl
oning-A Laboratory Manual "Cold Spring Harbor Labo
ratory, NY) and pSV2-neo (Southern, PJ and Berg, P.
(1982) J. Mol. Appl. Genet., 1, 327-341), etc., and transformants that stably produce novel G protein-coupled receptors are selected by selecting G418-resistant colonies. Obtainable. When 293-EBNA cells are used as host cells, Epstein Barr Virus
PC, which has an origin of replication and is capable of self-replication in 293-EBNA cells
Desired transformed cells can be obtained using an expression vector such as EP4 (Invitrogen).

The desired transformant obtained above can be cultured according to a conventional method, and the G protein-coupled receptor of the present invention is produced intracellularly or on the cell surface by the culture. The medium used for the culture can be appropriately selected from various ones commonly used depending on the host cell used.For example, the above-mentioned COS cells include RPMI-1640 medium and Dulbecco's modified Eagle minimum essential medium (DMEM). A medium to which serum components such as fetal bovine serum (FBS) are added as necessary can be used. Also, if the above 293-EBNA cells, fetal bovine serum (FBS)
A medium obtained by adding G418 to a medium such as Dulbecco's modified Eagle's minimum essential medium (DMEM) to which a serum component such as the above has been added can be used. As described above, the G protein-coupled receptor of the present invention produced in the cells of the transformant or on the cell surface can be prepared by various known separation procedures utilizing the physical properties and chemical properties of the receptor. It can be further separated and purified. As the method, specifically, for example, after solubilizing a membrane fraction containing the receptor, treatment with a usual protein precipitant, ultrafiltration, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange Examples include various types of liquid chromatography such as body chromatography, affinity chromatography, high performance liquid chromatography (HPLC), dialysis, and combinations thereof. The membrane fraction can be obtained according to a conventional method.
For example, it can be obtained by culturing cells expressing the G protein-coupled receptor of the present invention on the surface thereof, suspending them in a buffer, homogenizing, and centrifuging. In addition, by solubilizing the G protein-coupled receptor protein with a solubilizing agent as mild as possible (CHAPS, Triton X-100, dichitonin, etc.), the properties of the receptor can be maintained after solubilization.

The G protein-coupled receptor of the present invention is expressed by fusing it in-frame with a marker sequence.
Confirmation of expression of the G protein-coupled receptor protein, confirmation of intracellular localization, purification, and the like can be performed. Marker sequences include, for example, FLAG epitope, Hexa-Histidine tag, He
There are magglutinin tag and myc epitope. Further, by inserting a specific sequence recognized by a protease such as enterokinase, factor Xa, or thrombin between the marker sequence and the G protein-coupled receptor protein, the marker sequence can be cleaved and removed by these proteases. Is possible. For example, there is a report that a muscarinic acetylcholine receptor and a Hexa-Histidine tag are linked by a thrombin recognition sequence (Hayashi, MK and Hag
a, T. (1996) J. Biochem., 120, 1232-1238)

3) Screening method for substances (compounds, peptides and antibodies) that modify the activity of the G protein-coupled receptor of the present invention The screening method of the present invention
Using a protein-coupled receptor, a test substance is added to a system for measuring an index of modification of the G protein-coupled receptor according to the physiological characteristics of the G protein-coupled receptor,
Means for measuring the index. Specific examples of the measurement system include the following screening methods a) to d). The test substance is known to have a G protein-coupled receptor ligand activity, but it is not known whether the test substance selectively modifies the activity of the novel G protein-coupled receptor or a chemical file. Various registered G protein-coupled receptor ligand activities are unknown and known compounds and peptides, and combinatorial chemistry techniques (Terrett, NK, et al.
(1995) Tetrahedron, 51, 8135-8137) and the phage display method (Felici, F.,
et al. (1991) J. Mol. Biol., 222, 301-310) can be used. In addition, culture supernatants of microorganisms, natural components derived from plants and marine organisms, animal tissue extracts, and the like are also targets for screening. Alternatively, a compound or peptide obtained by chemically or biologically modifying the compound or peptide selected by the screening method of the present invention can be used.

A) Screening method using ligand binding assay A compound that binds to the G protein-coupled receptor of the present invention,
Peptides and antibodies (collectively, ligands) can be screened by ligand binding assays. A cell membrane expressing the receptor or a purified preparation of the receptor is prepared, and the purified ligand is radiolabeled (50-2000 Ci / mmol) for a ligand binding assay. The assay conditions such as buffer, ion and pH are optimized, and the cell membrane expressing the receptor or the purified preparation of the receptor is incubated with the radiolabeled ligand for a certain period of time in the optimized buffer. After the reaction, the mixture is filtered through a glass filter or the like and washed with an appropriate amount of buffer, and the radioactivity remaining on the filter is measured with a liquid scintillation counter or the like (total binding amount). The amount of non-specific binding is measured by adding a large excess of a non-radiolabeled ligand to the reaction solution, and the amount of specific binding is obtained by subtracting the amount of non-specific binding from the total amount of binding. A ligand that specifically binds to a cell membrane expressing the receptor protein or a purified preparation of the receptor protein can be selected as a ligand for the G protein-coupled receptor of the present invention. Further, compounds, peptides and antibodies having agonist activity of the G protein-coupled receptor, compounds, peptides and antibodies having antagonist activity can be screened using the obtained inhibition of the binding of the radioactive ligand as an index.

B) Screening Method Using GTPγS Binding Method Compounds, peptides and antibodies that modify the activity of the G protein-coupled receptor of the present invention can be screened by the GTPγS binding method (Lazareno, S. and Birds).
all, NJM (1993) Br. J. Pharmacol. 109, 1120-112
7). Cell membrane expressing the receptor was washed with 20 mM HEPES.
(pH 7.4), 100 mM NaCl, 10 mM MgCl ₂ , mixed with 400 pM of GTPγS labeled with ³⁵ S in a 50 mM GDP solution. After incubation in the presence and absence of the test drug, the mixture is filtered with a glass filter or the like, and the radioactivity of the bound GTPγS is measured with a liquid scintillation counter or the like. A compound having an agonist activity of the G protein-coupled receptor, based on an increase in specific GTPγS binding in the presence of the test drug,
Peptides and antibodies can be screened.
In addition, compounds, peptides and antibodies having the antagonistic activity of the G protein-coupled receptor can be screened using the obtained compounds having agonist activity, peptides and antibodies to suppress the increase in GTPγS binding as an index.

C) Screening Method Utilizing Changes in Intracellular Ca ⁺⁺ and cAMP Concentrations Many G protein-coupled receptors cause an increase in intracellular Ca ⁺⁺ and / or an increase or decrease in cAMP concentration upon agonist stimulation. . Therefore, compounds, peptides and antibodies that modify the activity of the G protein-coupled receptor of the present invention can be screened by utilizing the fluctuation of intracellular Ca ⁺⁺ or cAMP concentration. Ca ⁺⁺ concentration was measured using fura2, etc., and cAMP concentration was measured using a commercially available cAMP measurement kit (Amersha
m). Further, it is possible to measure indirectly Ca ⁺⁺ and cAMP concentrations by detecting the transcription activity of a gene whose transcription amount depending on the Ca ⁺⁺ and cAMP concentrations are adjusted. A compound, peptide, tissue extract or the like is allowed to act on cells expressing the receptor and host cells not expressing the receptor (control cells) for a certain period of time, and the Ca ⁺⁺ and cAMP concentrations are measured directly or indirectly. Compared with control cells, compounds, peptides and antibodies having an agonist activity can be screened based on an increase in cell-specific Ca ⁺⁺ expressing the receptor and / or an increase or decrease in cAMP concentration. Further, a compound having the obtained agonist activity,
Elevation of Ca ⁺⁺ and / or cAMP by peptides and antibodies
Compounds, peptides and antibodies having an antagonist activity of the G protein-coupled receptor can be screened using the increase or decrease in the concentration as an index.

D) Screening method using microphysiometer When cells perform various signal responses, a small outflow of hydrogen ions outside the cells is detected. Most of this hydrogen ion efflux occurs when there is an increase in metabolites resulting from fuel consumption to gain energy for the cells to respond, or when cell signals are transmitted directly to the hydrogen ion pump. Since the G protein-coupled receptor of the present invention requires energy for signal transmission, hydrogen ion efflux occurs when the receptor is activated. CYTOSE
NSOR microphysiometer (Molecular Devices)
Can detect such a pH change due to the slight outflow of hydrogen ions in the culture medium near the cells.
It can be used to detect the activation of such energy-consuming receptors. Cells expressing the receptor and host cells not expressing the receptor (control cells)
, A compound, peptide, tissue extract or the like is allowed to act for a certain period of time, and the change in pH due to the outflow of hydrogen ions is measured. Compared with control cells, compounds, peptides and antibodies having agonist activity can be screened using pH change caused by cell-specific hydrogen ion efflux expressing the receptor as an indicator. In addition, compounds, peptides and antibodies having the antagonistic activity of the G protein-coupled receptor can be screened using the pH change caused by the efflux of hydrogen ions by the resulting compounds, peptides and antibodies having the agonist activity as an index.

4) Method for Preparing Antibody Reacting with G Protein-Coupled Receptor of the Present Invention Antibodies that react with the G protein-coupled receptor of the present invention, such as polyclonal antibodies and monoclonal antibodies, can be used in various animals to produce the novel G protein-coupled receptor. It can be obtained by directly administering a receptor or a fragment of the G protein-coupled receptor. Further, a DNA vaccine method (Raz, E. et al. (1994) Proc. Natl. Acad. Sci. U.) was carried out using a plasmid into which a gene encoding the G protein-coupled receptor of the present invention was introduced.
SA, 91, 9519-9523; Donnelly, JJ et al. (1996)
J. Infect. Dis., 173, 314-320). A polyclonal antibody is prepared by emulsifying the G protein-coupled receptor or a fragment thereof in a suitable adjuvant such as Freund's complete adjuvant and immunizing the peritoneal cavity, subcutaneously or intravenously, and sensitizing animals such as rabbits, rats, goats, chickens, etc. Manufactured from serum or eggs.
Polyclonal antibodies can be separated and purified from the serum or eggs thus produced by conventional protein isolation and purification methods. Examples of conventional protein isolation and purification methods include centrifugation, dialysis, and salting out with ammonium sulfate. , DEAE-cellulose, hydroxyapatite, protein A agarose and the like.

The monoclonal antibody was obtained by the cell fusion method of Kohler and Milstein (Kohler, G. and Milstein, C. (1)
975) Nature, 256, 495-497), which can be easily produced by those skilled in the art. That is, the G protein-coupled receptor of the present invention or a fragment thereof is immunized by repeatedly inoculating a mouse intraperitoneally, subcutaneously, or vein several times with an emulsion prepared by emulsifying it in an appropriate adjuvant such as Freund's complete adjuvant every few weeks. . After the final immunization, spleen cells are removed and fused with myeloma cells to produce hybridomas. As a myeloma cell for obtaining a hybridoma, a myeloma cell having a marker such as hypoxanthine-guanine-phosphoribosyltransferase deficiency or thymidine kinase deficiency, for example, a mouse myeloma cell line P3X63Ag8.U1, is used. In addition, polyethylene glycol is used as a fusion agent. Furthermore, Eagle's minimum essential medium, Dulbecco's modified minimum essential medium, RP
10 to 3 as appropriate for commonly used ones such as MI-1640
Use with 0% fetal calf serum. The fusion strain is selected by the HAT selection method. Hybridoma screening is performed by using well-known methods such as ELISA method and immunohistochemical staining method or the above-mentioned screening method using the culture supernatant, and a clone of the hybridoma secreting the desired antibody is selected. In addition, the monoclonality of the hybridoma is guaranteed by repeating subcloning by the limiting dilution method. The hybridoma obtained in this way is BALB / c pretreated with the medium for 2 to 4 days or with pristane.
By culturing the strain mouse intraperitoneally for 10 to 20 days, a purifiable amount of antibody is produced. The monoclonal antibody thus produced can be separated and purified from the culture supernatant or ascites by a conventional protein isolation and purification method. Alternatively, an antibody fragment containing a monoclonal antibody or a part thereof can be produced by incorporating all or a part of the gene encoding the antibody into an expression vector and introducing the gene into Escherichia coli, yeast, or animal cells.

With respect to the antibody separated and purified as described above,
Proteolytic enzymes such as pepsin, papain, etc.
Therefore, digestion is performed, and then the protein isolation and purification method is continued.
By separating and purifying more,
Antibody fragments, e.g., F (ab ') _Two, Fab, Fab ', Fv
Can be. Further, the G protein-coupled receptor of the present invention
Antibodies that react with
(Clackson, T. et al. (1991) Nature, 352, 624-628;
 Zebedee, S. et al. (1992) Proc. Natl. Acad. Sci.
USA, 89, 3175-3179) as single chain Fv or Fab.
It is also possible to obtain. In addition, mouse antibody genes
Transgenic mouse replaced with human antibody gene
(Lonberg, N. et al. (1994) Nature, 368, 856-859)
It is also possible to obtain a human antibody by immunizing the subject.

The present invention encompasses a drug containing as an active ingredient a substance that significantly modifies the activity of a G protein-coupled receptor protein or a G protein-coupled receptor protein selected by the above screening method. The preparation containing the G protein-coupled receptor activity-modifying compound, peptide, antibody or antibody fragment of the present invention as an active ingredient may be, depending on the type of the active ingredient, carriers or excipients usually used for the preparation of the active ingredient. Can be prepared using the following additives. For administration, tablets, pills, capsules, granules, fine granules, powders, oral liquids, etc., or injections such as intravenous and intramuscular injections, suppositories, transdermal preparations, transmucosal preparations And parenteral administration. In particular, parenteral administration such as intravenous injection is desired for peptides digested in the stomach.

The solid composition for oral administration according to the present invention comprises one or more active substances which contain at least one inert diluent, such as lactose, mannitol, glucose,
It is mixed with microcrystalline cellulose, hydroxypropylcellulose, starch, polyvinylpyrrolidone, magnesium aluminate metasilicate and the like. The composition may contain additives other than the inert diluent, such as a lubricant, a disintegrant, a stabilizer, and a solubilizing or solubilizing agent, according to a conventional method. Tablets and pills may be coated with sugar coating or a film of a gastric or enteric substance, if necessary. Liquid compositions for oral use include emulsions, solutions, suspensions, syrups, and elixirs, and include commonly used inert diluents such as purified water and ethanol. The composition may contain additives other than inert diluents, such as wetting agents, suspending agents, sweetening agents, flavoring agents, preservatives.
Parenteral injections include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. The water-soluble solutions and suspensions include, for example, distilled water for injection, physiological saline, and the like as diluents. Examples of diluents for non-aqueous solutions and suspensions include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, alcohols such as ethanol, and polysorbate 80. The composition may further contain a wetting agent, an emulsifier, a dispersant, a stabilizer, a solubilizing or solubilizing agent, a preservative and the like. The composition is sterilized by, for example, filtration through a bacteria-retaining filter, blending of a bactericide, or irradiation. In addition, a sterile solid composition can be produced and dissolved in sterile water or another sterile injectable medium before use. The dose is appropriately determined in consideration of the activity intensity, symptoms, age, sex, and the like of the active ingredient selected by the screening method.

[0033]

EXAMPLES Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited to the examples. Unless otherwise specified, a known method (Maniatis, T. at al. (1982): "Molecular Clonin
g-A Laboratory Manual "Cold Spring Harbor Labora
tory, NY). Example 1 Isolation of a Gene Encoding a Novel G Protein-Coupled Receptor The full-length cDNA encoding the G protein-coupled receptor protein HORK1 of the present invention is a human spleen-derived poly A + RNA (Clont
ech) as a template by RT-PCR. The oligonucleotide represented by SEQ ID NO: 3 was
As er, an oligonucleotide represented by SEQ ID NO: 4
Used as reverse primer (Xb at each 5 'end
aI site is added). RT-PCR is Pyrobest DNA poly
Merase (Takara Shuzo) in the presence of 5% DMS0 at 98 ° C (10 ° C)
Cycle) / 58 ° C (30 seconds) / 72 ° C (2 minutes) was repeated 34 times. As a result, a DNA fragment of about 1.0 kbp was amplified. After digesting this fragment with XbaI, pEF-BOS plasmid (M
izushima, S. and Nagata, S. (1990) Nucleic Acids Re
s., 18, 5322). The nucleotide sequence of the obtained clone was determined by the dideoxy terminator method.
Analysis was performed using ABI377 DNA Sequencer (manufactured by Applied Biosystems). The elucidated sequence is shown in SEQ ID NO: 1. The nucleotide sequence represented by SEQ ID NO: 1 has an ORF of 1002 bases. The amino acid sequence (333 amino acids) predicted from the ORF is shown in SEQ ID NO: 2. Since the predicted amino acid sequence has a hydrophobic region which seems to be seven transmembrane domains characteristic of a G protein-coupled receptor, it was found that this gene encodes a G protein-coupled receptor. The G protein-coupled receptor encoded by this gene was named HORK1. Novel G protein-coupled receptor
HORK1 has an amino acid sequence of 30% or less in homology with a known G protein-coupled receptor family corresponding to a ligand.

(Example 2) Expression distribution of a novel human G protein-coupled receptor family gene in tissues The expression distribution of the G protein-coupled receptor gene of the present invention was analyzed by RT-PCR. Human organs (brain (amygdala,
Caudate nucleus, hippocampus, corpus callosum, substantia nigra, cerebellum), spinal cord, pituitary, heart, placenta, lung, trachea, liver, kidney, pancreas, small intestine, stomach, spleen, bone marrow, thymus, thyroid, salivary gland, adrenal gland, 5 mg of poly A + RNA from mammary gland, prostate, testis and ovary (Clontec
h) using DNase (Nippon Gene) at 37 ° C for 15 minutes.
Minutes. MMLV Reverse Transcriptase (Clontech) from 4 mg of DNase-treated poly A + RNA
The reaction was performed sequentially at 60 ° C. for 60 minutes and at 94 ° C. for 5 minutes to synthesize cDNA.
The synthesized cDNA was dissolved in 800 ml of sterilized water. The expression distribution of HORK1 was determined using the cDNA of each of the above human organs as a template.
For the imer set, an oligonucleotide represented by SEQ ID NO: 5 and an oligonucleotide represented by SEQ ID NO: 6 were used.
5% PCR using Pyrobest DNA polymerase (Takara Shuzo)
98 ° C (10 seconds) / 56 ° C (30 seconds) / 72 ° C in the presence of DMS0
(1 minute) cycle was repeated 30 times. In addition, as an internal standard, Huma
Glyceraldehyde-3-Phosphate Dehy by PCR under the same conditions using nG3PDH Control Amplimer Set (manufactured by Clontech)
The gene of drogenase (G3PDH) was amplified. 1% reaction product
Analysis was performed by electrophoresis on an agarose gel (FIG. 1). HO
The amplified product of about 400 bp of RK1 is expressed in the brain (amygdala, caudate nucleus, hippocampus,
Corpus callosum, substantia nigra), spinal cord, placenta, spleen, and bone marrow.
Among them, the substantia nigra, spinal cord and spleen showed relatively strong signals. From the above results, it was found that HORK1 was expressed at a site involved in the central nervous system. In other words, the present G protein-coupled receptor is expressed in the substantia nigra-striatal system, and its site contains dopaminergic neurons. This indicates that degeneration of dopaminergic neurons is useful for Parkinson's disease. In addition, the spinal cord has a site related to transmission of pain sensation, which indicates that it is useful for pain-related diseases.

Example 3 Confirmation of Expression of Novel G Protein-Coupled Receptor Family Proteins pEF-B was used as an expression vector for expressing human HORK1.
OS was used. At that time, in order to fuse a FLAG epitope as a marker sequence at the N-terminus of human HORK1, an oligonucleotide represented by SEQ ID NO: 7 was inserted at the 5 ′ end of the HORK1 protein coding sequence. Each of the plasmids thus constructed was designated as pEFBOS-FL-HORK1. By using this plasmid, it is expressed as a polypeptide in which the polypeptide of SEQ ID NO: 8 is fused to the N-terminal of the human HORK1 polypeptide. 293-EBNA (In the 10cm Petri dish)
After inoculation with 1x10 ⁶ cells and culturing for 1 day, 8 mg
Add pEF-BOS-FL-HORK1 and pEF-BOS-FL (empty vector) to Fu
The gene was transfected using GENE6 (Boeringer Mannheim). After gene transfer, collect and wash cells cultured for one day,
Suspended in 1% BSA / PBS. Place this under ice temperature shading, 5x10
^A mouse anti-FLAG monoclonal antibody (M2; Sigma) or normal mouse IgG (Zymed) was added as a primary antibody to a final concentration of 2 mg / ml for every ⁵ cells, and incubated for 1 hour. After washing with PBS, FITC-labeled goat anti-mouse Ig (Biosource) was further added as a secondary antibody so as to be diluted 200 times,
Incubated for 1 hour in the dark protected from ice. Measurements of fluorescence intensity were carried out on an EPICS ^¬ XL-MCL (COULTER Co.) (Fig. 2). FIG. 2 shows the results of measuring 10,000 cells, in which the vertical axis represents the number of cells and the horizontal axis represents the fluorescence intensity. Only when M2 is used as the primary antibody for HORK1 transfected cells, the FLTC epitope is included because it is shifted in the direction of increasing FITC fluorescence intensity
It was confirmed that HORK1 was expressed on the cell membrane surface.

[0036]

Industrial Applicability The G protein-coupled receptor of the present invention is useful as a screening tool for a preventive / therapeutic agent for central nervous system diseases (particularly schizophrenia, Parkinson's disease, pain). Specifically, by contacting a test substance with the G protein-coupled receptor of the present invention, a substance (compound, peptide and antibody) that modulates the activity of the G protein-coupled receptor is screened, and a new drug, particularly a new drug, This means screening for a prophylactic / therapeutic agent for a central nervous system disease that cannot be completely controlled yet. Further, the gene encoding the G protein-coupled receptor of the present invention or an antibody against the receptor is useful as a tool for diagnosing the susceptibility of a disease that can be caused by mutation of the gene or abnormal expression.

[0037]

[Brief description of the drawings]

FIG. 1 shows the results of analysis of the distribution of expression of HORK1 in human organs.

FIG. 2 shows the results of confirming the expression of HORK1.

[Sequence List] SEQUENCE LISTING <110> Yamanouchi Pharmaceutical Co., Ltd. <120> Novel G protein-coupled receptor <130> 0000002898 <160> 8 <170> PatentIn Ver. 2.0 <210> 1 <211> 1002 < 212> DNA <213> Homo sapiens <400> 1 atgaacacca cagtgatgca aggcttcaac agatctgagc ggtgccccag agacactcgg 60 atagtacagc tggtattccc agccctctac acagtggttt tcttgaccgg catcctgctg 120 aatactttgg ctctgtgggt gtttgttcac atccccagct cctccacctt catcatctac 180 ctcaaaaaca ctttggtggc cgacttgata atgacactca tgcttccttt caaaatcctc 240 tctgactcac acctggcacc ctggcagctc agagcttttg tgtgtcgttt ttcttcggtg 300 atattttatg agaccatgta tgtgggcatc gtgctgttag ggctcatagc ctttgacaga 360 ttcctcaaga tcatcagacc tttgagaaat atttttctaa aaaaacctgt ttttgcaaaa 420 acggtctcaa tcttcatctg gttctttttg ttcttcatct ccctgccaaa tatgatcttg 480 agcaacaagg aagcaacacc atcgtctgtg aaaaagtgtg cttccttaaa ggggcctctg 540 gggctgaaat ggcatcaaat ggtaaataac atatgccagt ttattttctg gactgttttt 600 atcctaatgc ttgtgtttta tgtggttatt gcaaaaaaag tatatgattc ttatagaaag 660 tccaaaagta aggacagaaa aaacaacaaa aagctggaag gcaaagtatt tgttgtcgtg 720 gctgtcttct ttgtgtgttt tgctccattt cattttgcca gagttccata tactcacagt 780 caaaccaaca ataagactga ctgtagactg caaaatcaac tgtttattgc taaagaaaca 840 actctctttt tggcagcaac taacatttgt atggatccct taatatacat attcttatgt 900 aaaaaattca cagaaaagct accatgtatg caagggagaa agaccacagc atcaagccaa 960 gaaaatcata gcagtcagac agacaacata accttaggct ga 1002 <210> 2 <211> 333 < 212> PRT <213> Homo sapiens <400> 2 Met Asn Thr Thr Val Val Met Gln Gly Phe Asn Arg Ser Glu Arg Cys Pro 1 5 10 15 Arg Asp Thr Arg Ile Val Gln Leu Val Phe Pro Ala Leu Tyr Thr Val 20 25 30 Val Phe Leu Thr Gly Ile Leu Leu Asn Thr Leu Ala Leu Trp Val Phe 35 40 45 Val His Ile Pro Ser Ser Ser Thr Phe Ile Ile Tyr Leu Lys Asn Thr 50 55 60 Leu Val Ala Asp Leu Ile Met Thr Leu Met Leu Pro Phe Lys Ile Leu 65 70 75 80 Ser Asp Ser His Leu Ala Pro Trp Gln Leu Arg Ala Phe Val Cys Arg 85 90 95 Phe Ser Ser Val Ile Phe Tyr Glu Thr Met Tyr Val Gly Ile Val Leu 100 105 110 Leu Gly Leu Ile Ala Phe Asp Arg Phe Leu Lys Ile Ile Arg Pro Leu 115 120 125 Arg Asn Ile Phe Leu Lys Lys Pro Val Phe Ala Lys Thr Val Ser Ile 130 135 140 Phe Ile Trp Phe Phe Leu Phe Phe Ile Ser Leu Pro Asn Met Ile Leu 145 150 155 160 Ser Asn Lys Glu Ala Thr Pro Ser Ser Val Lys Lys Cys Ala Ser Leu 165 170 175 Lys Gly Pro Leu Gly Leu Lys Trp His Gln Met Val Asn Asn Ile Cys 180 185 190 Gln Phe Ile Phe Trp Thr Val Phe Ile Leu Met Leu Val Phe Tyr Val 195 200 205 Val Ile Ala Lys Lys Val Tyr Asp Ser Tyr Arg Lys Ser Lys Ser Lys 210 215 220 Asp Arg Lys Asn Asn Lys Lys Leu Glu Gly Lys Val Phe Val Val Val 225 230 235 240 Ala Val Phe Phe Val Cys Phe Ala Pro Phe His Phe Ala Arg Val Pro 245 250 255 Tyr Thr His Ser Gln Thr Asn Asn Lys Thr Asp Cys Arg Leu Gln Asn 260 265 270 Gln Leu Phe Ile Ala Lys Glu Thr Thr Leu Phe Leu Ala Ala Thr Asn 275 280 285 Ile Cys Met Asp Pro Leu Ile Tyr Ile Phe Leu Cys Lys Lys Phe Thr 290 295 300 Glu Lys Leu Pro Cys Met Gln Gly Arg Lys Thr Thr Ala Ser Ser Gln 305 310 315 320 Glu Asn His Ser Ser Gln Thr Asp Asn Ile Thr Leu Gly 325 330 <210> 3 <211> 39 <212> DNA <213> Homo sapiens <400> 3 gggtctagaa tgaacaccac agtgatgcaa ggcttcaac 39 <210> 4 <211> 37 <212> DNA <213> Homo sapiens <400> 4 ccctctagat cagcctaagg ttatgttgtc tgtctga 37 <210> 5 <211> 40 <212> DNA <213> Homo sapiens <400> 5 ctgtccttac ttttggactt tctataagaa tcatatactt 40 <210> 6 <211> 40 <212> DNA < 213> Homo sapiens <400> 6 ctggcaccct ggcagctcag agcttttgcg tgtcgttttt 40 <210> 7 <211> 36 <212> DNA <213> Homo sapiens <400> 7 atggactaca aggacgacga tgacaagggg atcctg 36 <210> 8 <211> PRT <213> Homo sapiens <400> 8 Met Asp Tyr Lys Asp Asp Asp Asp Lys Gly Ile Leu 1 5 10

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61P 25/04 A61P 25/16 4C084 25/16 25/18 4C085 25/18 C07K 14/705 4H045 C07K 14 / 705 C12P 21/02 C C12N 5/10 21/08 C12P 21/02 C12Q 1/68 A 21/08 G01N 33/15 Z C12Q 1/68 33/50 Z G01N 33/15 33/53 D 33/50 33 / 566 33/53 A61K 37/02 33/566 C12N 5/00 B (72) Inventor: Sugimoto Kan, 21 Miyukigaoka, Tsukuba, Ibaraki Pref. Yamanouchi Pharmaceutical Co., Ltd. (72) Inventor: Masazumi Kambara, Miyuki, Tsukuba, Ibaraki Gaoka 21 Yamanouchi Pharmaceutical Company Limited (72) Inventor Tetsu Saito 21 Miyukigaoka Tsukuba City, Ibaraki Prefecture Yamanouchi Pharmaceutical Company Limited F-term (reference) 2G045 AA34 BB20 CB01 CB21 DA36 FB03 4B024 AA01 AA11 BA 43 BA63 CA04 DA02 EA04 GA05 GA11 HA14 HA15 4B063 QA01 QA08 QQ02 QQ43 QR33 QR56 QS25 QS34 QX07 4B064 AG01 AG27 CA10 CA19 CA20 CC24 DA01 DA13 4B065 AA90X AA92X AA933 AB01 AC10 BA02 A23 CA18 A26 CA46 A18 CA18 A14 MA31 MA35 MA37 MA41 MA43 MA52 MA56 MA63 MA66 NA14 ZA022 ZA052 ZA082 ZA892 4C085 AA13 AA14 BB11 CC04 CC13 DD22 DD32 DD38 DD41 EE01 FF20 GG02 GG08 4H045 AA10 AA11 AA20 AA30 BA10 CA40 DA50 DA76 EA

Claims (7)

[Claims] 1. G having the amino acid sequence of SEQ ID NO: 2.
A protein-coupled receptor, or a G protein-coupled receptor which is the same as the receptor. 2. A gene encoding the amino acid sequence of the G protein-coupled receptor according to claim 1. [3] a vector comprising the gene according to [2]; 4. A host cell comprising the vector according to claim 3. 5. The method for producing a G protein-coupled receptor according to claim 1, wherein the host cell according to claim 4 is used. 6. An antibody against the G protein-coupled receptor according to claim 1. 7. A method for contacting the G protein-coupled receptor according to claim 1 with a test compound and screening for a substance that modifies the activity of the G protein-coupled receptor.