JP2003180376A - New g protein-coupled receptor protein and its dna - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new protein useful for screening agonists/antagonists, and the like. <P>SOLUTION: This human being-originated protein or its salt. The DNA encoding the protein. A method for determining a ligand for the protein. A method for screening a compound for screening the bondability of the ligand to the protein. A kit for screening the compound for screening the bondability of the ligand to the protein. The compound obtained by the screening or its salt, and the like. The human being-originated protein or the DNA encoding the protein can be used for (1) the determination of the ligand for the protein, (2) a medicine for preventing and/or treating a disease related to the function failure of the protein, (3) the screening of a compound for changing the bondability of the ligand to the protein, and the like. <P>COPYRIGHT: (C)2003,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel G protein-coupled receptor protein derived from human heart or a salt thereof and a polynucleotide encoding the same. Further, the present invention relates to a screening method using the novel G protein-coupled receptor protein of the present invention or a salt thereof, a screening kit, a compound obtained by using the screening method or the screening kit, a salt thereof, and the like.

[0002]

BACKGROUND OF THE INVENTION Many physiologically active substances such as hormones and neurotransmitters regulate the function of the living body through specific receptor proteins present in the cell membrane. Many of these receptor proteins are guanin coupled
Intracellular signal transduction is carried out through activation of e-nucleotide-binding protein (hereinafter sometimes abbreviated as G protein), and since it has a common structure with 7 transmembrane regions, it is G protein-coupled. Type receptor protein or 7-transmembrane receptor protein (7TMR). G protein-coupled receptor proteins are present on the surface of functional cells of living cells and organs, and are physiologically used as targets for molecules that regulate the functions of those cells and organs, such as hormones, neurotransmitters, and physiologically active substances. It plays an important role. The receptor transmits a signal into the cell through binding with a physiologically active substance, and this signal causes various reactions such as activation and inhibition of the cell. To elucidate the relationship between substances that regulate complex functions in cells and organs of various living organisms and their specific receptor proteins, particularly G protein-coupled receptor proteins, is the function of cells and organs of various living organisms. And provide a very important means for drug development closely related to these functions.

For example, in various organs of the living body, physiological functions are regulated under the control of many hormones, hormone-like substances, neurotransmitters or physiologically active substances. In particular, physiologically active substances are present in various parts of the body and regulate their physiological functions through corresponding receptor proteins. There are many unknown hormones, neurotransmitters and other physiologically active substances in the body, and the structures of their receptor proteins
Many have not been reported so far. Furthermore, even in known receptor proteins, many do not know whether subtypes exist. Clarifying the relationship between substances that regulate complex functions in the living body and their specific receptor proteins is a very important means for drug development. Further, in order to efficiently screen for agonists and antagonists to receptor proteins and to develop pharmaceuticals, it is necessary to elucidate the functions of genes of receptor proteins expressed in vivo and express them in an appropriate expression system. Was needed. In recent years, as a means for analyzing a gene expressed in a living body, research on randomly analyzing the sequence of cDNA has been actively carried out, and thus obtained c
DNA fragment sequence is Expressed Sequence Tag (EST)
Has been registered and published in the database as. However, many ESTs have only sequence information, and it is difficult to estimate their function.

[0004]

[Problems to be Solved by the Invention] Conventionally, G protein-coupled receptors and physiologically active substances (ie, ligands)
Substances that inhibit the binding with and those that cause signal transduction similar to physiologically active substances (that is, ligands) are used as specific antagonists or agonists of these receptors and as drugs that regulate biological functions. Came. Therefore, it is new to find a G protein-coupled receptor protein that is not only important for physiological expression in vivo, but can also be a target for drug development, and to clone the gene (for example, cDNA). It becomes a very important means for finding a specific ligand, agonist or antagonist of the G protein-coupled receptor protein. However, not all G protein-coupled receptors have been found, and at present, there are many unknown G protein-coupled receptors and so-called orphan receptors whose corresponding ligands have not been identified. Therefore, search for new G protein-coupled receptors and elucidation of their functions have been earnestly desired. The G protein-coupled receptor is useful for searching for a new physiologically active substance (that is, ligand) using its signal transduction action as an index, and for searching for an agonist or antagonist for the receptor. On the other hand, even if no physiological ligand is found, it is also possible to prepare an agonist or antagonist for the receptor by analyzing the physiological action of the receptor from an inactivation experiment of the receptor (knockout animal). . Ligands, agonists or antagonists for these receptors can be expected to be utilized as preventive / therapeutic agents and diagnostic agents for diseases associated with G protein-coupled receptor dysfunction. Furthermore, in many cases, a decrease or enhancement of the function of the G protein-coupled receptor gene in the living body, which is caused by a gene mutation, causes some disease. In this case, not only administration of an antagonist or agonist for the receptor,
In vivo (or a specific organ) of the receptor gene
It can also be applied to gene therapy by introducing into the gene or an antisense nucleic acid to the receptor gene. In this case, the nucleotide sequence of the receptor is indispensable information for investigating the presence or absence of a deletion or mutation on the gene, and the gene of the receptor is a prophylactic / therapeutic drug for diseases associated with dysfunction of the receptor. It can also be applied to diagnostic agents. The present invention provides a novel G protein-coupled receptor protein useful as described above. That is, a novel G protein-coupled receptor protein or a partial peptide thereof or a salt thereof, a polynucleotide (DNA, RNA) encoding the G protein-coupled receptor protein or a partial peptide thereof
And a derivative thereof) (DNA, RNA and derivatives thereof), a recombinant vector containing the polynucleotide, a transformant carrying the recombinant vector, the G protein-coupled receptor protein or the same. A method for producing a salt, an antibody against the G protein-coupled receptor protein or a partial peptide thereof or a salt thereof, a compound that changes the expression level of the G protein-coupled receptor protein, a method for determining a ligand for the G protein-coupled receptor,
A screening method for a compound (antagonist, agonist) or a salt thereof that changes the binding property between a ligand and the G protein-coupled receptor protein, the screening kit, the ligand obtainable by using the screening method or the screening kit, and the G Compounds (antagonists, agonists) or salts thereof that change the binding properties to protein-coupled receptor proteins, and compounds (antagonists, agonists) or the G protein couplings that change the binding properties of ligands to the G protein-coupled receptor proteins The present invention provides a drug or the like comprising a compound or a salt thereof that changes the expression level of the type I receptor protein.

[0005]

Means for Solving the Problems As a result of intensive studies, the present inventors isolated a cDNA encoding a novel G protein-coupled receptor protein derived from human adipocytes and analyzed the entire base sequence thereof. Was successful. When this base sequence was translated into an amino acid sequence, the first to seventh transmembrane regions were confirmed on the hydrophobicity plot, and the proteins encoded by these cDNAs were 7-transmembrane G protein-coupled receptors. It was confirmed to be a protein. The present inventors have completed the present invention as a result of further research based on these findings.

That is, the present invention provides (1) SEQ ID NO:
A G protein-coupled receptor protein or a salt thereof, which contains the same or substantially the same amino acid sequence as the amino acid sequence represented by 5 (2)
The G protein-coupled receptor protein or salt thereof according to (1) above, which contains the amino acid sequence represented by SEQ ID NO: 5, (3) above (1) containing the amino acid sequence represented by SEQ ID NO: 1. G protein-coupled receptor protein or salt thereof, (4) Partial peptide of G protein-coupled receptor protein or salt thereof according to (1) above, (5) G protein-coupled receptor protein according to (1) above, A polynucleotide containing a polynucleotide to
(6) The polynucleotide according to (5) above, which is DNA, (7) the polynucleotide according to (6) above, which comprises the nucleotide sequence represented by SEQ ID NO: 2 or SEQ ID NO: 6, (8) above ( 5) A recombinant vector containing the polynucleotide described in (9), a transformant transformed with the recombinant vector described in (8) above, (10) a transformant described in (9) above is cultured, G described in (1) above
(11) A method for producing a G protein-coupled receptor protein or a salt thereof according to (1) above, which comprises producing a protein-coupled receptor protein.
An antibody against the G protein-coupled receptor protein according to the above (1) or the partial peptide or a salt thereof according to the above (4), (12) inactivating signal transduction of the G protein coupled receptor protein according to the above (1). The antibody according to (11) above, which is a neutralizing antibody, (1
3) A diagnostic agent comprising the antibody according to (11) above,
(14) A medicine comprising the antibody according to (11) above,
(15) The above (1) which can be obtained by using the G protein-coupled receptor protein according to the above (11) or the partial peptide according to the above (4) or a salt thereof.
(16) A ligand for the G protein-coupled receptor protein or a salt thereof, (16) a medicament comprising the ligand for the G protein-coupled receptor protein according to (15) above, (17) the G protein according to (1) above A method for determining a ligand for a G protein-coupled receptor protein salt thereof according to (1) above, which comprises using a coupled receptor protein or the partial peptide according to (4) above or a salt thereof, (18) above (1) ) The G protein-coupled receptor protein described above or the partial peptide described in (4) above or a salt thereof is used to bind to the G protein-coupled receptor protein described above (1) or salt thereof. How to screen for changing compounds or their salts (19) A ligand characterized by containing the G protein-coupled receptor protein according to (1) above, or the partial peptide according to (4) above or a salt thereof, and the G protein-coupled receptor according to (1) above. A screening kit for a compound or a salt thereof that changes the binding property to a protein or a salt thereof, (20) a ligand obtainable using the screening method according to (18) above or the screening kit according to (19) above, and (1) A compound or a salt thereof that changes the binding property to the G protein-coupled receptor protein or a salt thereof according to (1), (2) above
0) A pharmaceutical comprising the compound or salt thereof according to item 0,
(22) A polynucleotide which hybridizes with the polynucleotide according to (5) under high stringent conditions, (23) A polynucleotide containing a base sequence complementary to the polynucleotide according to (5) or a part thereof. Nucleotide, (24) The polynucleotide described in (5) above or a part thereof is used, (1) above
MR of the described G protein-coupled receptor protein
Method for quantifying NA, (25) Method for quantifying G protein-coupled receptor protein according to (1) above, which uses the antibody according to (11) above, (26) above (24) or above (25) (27) A method for diagnosing a disease associated with the function of a G protein-coupled receptor according to (1) above, which comprises using the quantification method described above.
The method for screening a compound or a salt thereof which changes the expression level of the G protein-coupled receptor protein according to the above (1), characterized by using the quantification method according to the above (24), (28) the above (25) A method for screening a compound or a salt thereof that changes the amount of G protein-coupled receptor protein in the cell membrane according to the above (1), characterized by using a quantification method;
9) A compound or a salt thereof that changes the expression level of the G protein-coupled receptor protein according to the above (1), which can be obtained using the screening method according to the above (27), (30) the screening method according to the above (28) G in the cell membrane which can be obtained by using
A compound or a salt thereof that changes the amount of protein-coupled receptor protein, (31) a medicament comprising the compound or a salt thereof according to (29) above, (32) a compound or a salt thereof according to (30) above (33) Central diseases, inflammatory diseases, cardiovascular diseases,
The drug according to (21) 21, 31 or 32 above, which is a preventive / therapeutic agent for cancer, metabolic disease, immune system disease or digestive system disease, (34) Screening against mammals according to (18) above Administering an effective amount of a compound or a salt thereof that alters the binding property between the ligand obtainable by the method or the screening kit according to (19) above and the G protein-coupled receptor protein according to (1) above or a salt thereof. A method for preventing / treating a central disease, inflammatory disease, cardiovascular disease, cancer, metabolic disease, immune system disease or digestive system disease, characterized in that (35) An effective amount of a compound or a salt thereof that changes the expression level of the G protein-coupled receptor protein according to (1) above, which can be obtained using the screening method according to the above, is administered. Central diseases characterized by,
Prophylactic / therapeutic method for inflammatory disease, cardiovascular disease, cancer, metabolic disease, immune system disease or digestive system disease, (36) Obtained using the screening method described in (28) above for mammals G in the cell membrane of erythrocyte
Of central diseases, inflammatory diseases, cardiovascular diseases, cancer, metabolic diseases, immune system diseases or digestive system diseases characterized by administering an effective amount of a compound or a salt thereof that changes the amount of protein-coupled receptor protein Prevention / treatment methods,
(37) The screening method according to the above (18) or the above (19) for producing a prophylactic / therapeutic agent for a central disease, an inflammatory disease, a cardiovascular disease, a cancer, a metabolic disease, an immune system disease or a digestive system disease. Use of a compound or a salt thereof that alters the binding property between the ligand obtainable by using the screening kit according to (1) and the G protein-coupled receptor protein according to (1) above or a salt thereof, (38)
Central diseases, inflammatory diseases, cardiovascular diseases, cancer, metabolic diseases,
Changing the expression level of the G protein-coupled receptor protein according to (1) above, which can be obtained by using the screening method according to (27) above for producing a prophylactic / therapeutic agent for immune system diseases or digestive system diseases. (39) Use of a compound or a salt thereof, (39) The above-mentioned (28) for producing a prophylactic / therapeutic agent for central diseases, inflammatory diseases, cardiovascular diseases, cancer, metabolic diseases, immune system diseases or digestive system diseases Use of a compound or a salt thereof that alters the amount of G protein-coupled receptor protein described in (1) above in a cell membrane obtainable by using the screening method of

Further, the (40) protein has 1 or 2 or more amino acids in the amino acid sequence represented by SEQ ID NO: 5 or the amino acid sequence represented by SEQ ID NO: 1 (preferably,
1 to 30 amino acids, more preferably 1 to 9 amino acids, and even more preferably several (1 to 5) amino acids deleted, 1 or 2 in the amino acid sequence represented by SEQ ID NO: 5. 1 or more (preferably about 1 to 30, more preferably about 1 to 10 and even more preferably several (1
~ 5)) amino acid sequence, 1 or 2 or more (preferably 1 to 30 or so, more preferably 1 to 1) in the amino acid sequence represented by SEQ ID NO: 5.
The G protein according to the above (1), which is a protein containing an amino acid sequence in which about 0, more preferably several (1 to 5) amino acids are substituted with other amino acids, or an amino acid sequence in which they are combined. (41) The test compound is contacted with a coupled receptor protein or a salt thereof, (41) the G protein-coupled receptor protein according to (1) or a salt thereof, or the partial peptide according to (4) or a salt thereof. The method for determining a ligand according to (18) above, (42) wherein the ligand is, for example, angiotensin, bombesin, cannabinoid, cholecystokinin, glutamine, serotonin, melatonin, neuropeptide Y, opioid, purine,
Vasopressin, oxytocin, PACAP (eg PA
CAP27, PACAP38), secretin, glucagon, calcitonin, adrenomedullin, somatostatin, GHRH, CRF, ACTH, GRP, PTH, V
IP (vasoactive intestinal polypeptide), somatostatin, dopamine, motilin, amylin, bradykinin, CGRP (calcitonin gene relayed peptide), leukotriene, pancreatatin, prostaglandin, thromboxane, adenosine, adrenaline, chemokine superfamily (Eg, IL-8, GROα, GROβ, GROγ, NA
P-2, ENA-78, GCP-2, PF4, IP-1
0, Mig, PBSF / SDF-1 and other CXC chemokine subfamilies; MCAF / MCP-1, MCP-
2, MCP-3, MCP-4, eotaxin, RAN
TES, MIP-1α, MIP-1β, HCC-1, M
IP-3α / LARC, MIP-3β / ELC, I-3
09, TARC, MIPF-1, MIPF-2 / eot
axin-2, MDC, DC-CK1 / PARC, SL
CC chemokine subfamily such as C; lympho
C chemokine subfamily such as tactin; fr
(CX3C chemokine subfamily such as actalkine), endothelin, enterogastrin, histamine, neurotensin, TRH, pancreatic polypeptide, galanin, lysophosphatidic acid (LPA) or sphingosine 1-phosphate. Ligand determination method,

(43) (i) When the ligand is contacted with the G protein-coupled receptor protein or salt thereof described in (1) above or the partial peptide or salt thereof described in (4) above, (ii) G described in (1) above
The screening method according to (18) above, wherein the protein-coupled receptor protein or salt thereof or the partial peptide or salt thereof described in (4) above is contacted with a ligand and a test compound. (44) (i) when the labeled ligand is contacted with the G protein-coupled receptor protein or salt thereof described in (1) above, or the partial peptide or salt thereof described in (4) above, and (ii) labeled The labeled ligand G as described in (1) above, which is obtained by contacting the ligand and the test compound with the G protein-coupled receptor protein or salt thereof described in (1) above or the partial peptide or salt thereof described in (4) above.
The amount of binding to the protein-coupled receptor protein or salt thereof or the partial peptide or salt thereof described in (4) above is measured and compared with the ligand and the G protein-coupled receptor protein described in (1) above or to the same. A method for screening a compound or salt thereof that changes the binding property to a salt, (45) (i) contacting the labeled ligand with a cell containing the G protein-coupled receptor protein according to (1) above, ii) When the labeled ligand and the test compound are contacted with the cells containing the G protein-coupled receptor protein according to (1) above, the binding amount of the labeled ligand to the cells is measured and compared. And a G protein-coupled receptor tan described in (1) above A method of screening a compound or its salt that alters the binding property between click protein or a salt thereof,
(46) (i) The labeled ligand is the G described in (1) above.
When the membrane fraction of a cell containing the protein-coupled receptor protein is contacted, (ii) the labeled ligand and the test compound are added to the membrane fraction of the cell containing the G protein-coupled receptor protein described in (1) above. The amount of the labeled ligand bound to the membrane fraction of the cell is measured and compared with the ligand and the G protein-coupled receptor protein or salt thereof according to (1) above. A method for screening a compound or a salt thereof that changes the binding property,

(47) (i) When the labeled ligand is brought into contact with the G protein-coupled receptor protein expressed on the cell membrane of the transformant by culturing the transformant according to (9) above, ii) When the labeled ligand and the test compound are brought into contact with the G protein-coupled receptor protein expressed on the cell membrane of the transformant by culturing the transformant described in (9) above, Screening for a compound or a salt thereof that changes the binding property between the ligand and the G protein-coupled receptor protein or the salt thereof according to (1), characterized in that the amount of binding to the G protein-coupled receptor protein is measured and compared. Method, (48) (i) G protein-coupled receptor tamper according to (1) above When a compound that activates a protein or a salt thereof is contacted with a cell containing the G protein-coupled receptor protein according to (1) above,
i) A compound that activates the G protein-coupled receptor protein or the salt thereof according to (1) above and a test compound are brought into contact with cells containing the G protein-coupled receptor protein according to (1) above,
A compound which changes the binding property between the ligand and the G protein-coupled receptor protein or the salt thereof according to (1) above, which comprises measuring and comparing cell stimulating activities mediated by the G protein-coupled receptor protein. (49) Screening method for salt, (49) A compound that activates the G protein-coupled receptor protein or salt thereof according to (1) above is added to the cell membrane of the transformant by culturing the transformant according to (9) above. When the expressed G protein-coupled receptor protein is brought into contact, the compound activating the G protein-coupled receptor protein or salt thereof described in (1) above and a test compound are cultured in the transformant described in (9) above. G protein-coupled receptor expressed on the cell membrane of the transformant by Binding of a ligand to the G protein-coupled receptor protein or a salt thereof according to (1) above, which comprises measuring and comparing cell stimulating activities mediated by the G protein-coupled receptor protein when brought into contact with a protein. A method for screening a compound or its salt that alters sex,

(50) The compound that activates the G protein-coupled receptor protein according to (1) above is angiotensin, bombesin, cannabinoid, cholecystokinin, glutamine, serotonin, melatonin, neuropeptide Y, opioid, purine, vasopressin. ,
Oxytocin, PACAP (eg, PACAP27, PA
CAP38), secretin, glucagon, calcitonin, adrenomedullin, somatostatin, GHRH,
CRF, ACTH, GRP, PTH, VIP (vasoactive intestinal polypeptide), somatostatin, dopamine, motilin, amylin, bradykinin, CGRP (calcitonin gene relayed peptide), leukotriene, pancreatatin, prostaglandin, thromboxane. , Adenosine, adrenaline, chemokine superfamily (eg, IL-8,
GROα, GROβ, GROγ, NAP-2, ENA-
78, GCP-2, PF4, IP-10, Mig, PB
CXC chemokine subfamily such as SF / SDF-1; MCAF / MCP-1, MCP-2, MCP-3,
MCP-4, eotaxin, RANTES, MIP-
1α, MIP-1β, HCC-1, MIP-3α / LA
RC, MIP-3β / ELC, I-309, TARC,
MIPF-1, MIPF-2 / eotaxin-2, M
CC chemokine subfamily such as DC, DC-CK1 / PARC, SLC; C chemokine subfamily such as lymphoactin; fractalkin
(CX3C chemokine subfamily such as e), endothelin, enterogastrin, histamine, neurotensin, TRH, pancreatic polypeptide, galanin, lysophosphatidic acid (LPA) or sphingosine 1-phosphate (47) or ( 48) the screening method described above, (51) the above (4)
3) to a compound capable of changing the binding property between the ligand obtainable by the screening method described in (50) and the G protein-coupled receptor protein described in (1) above or a salt thereof, (52) above (43) ~ Above (5
0) A pharmaceutical comprising a compound or a salt thereof that changes the binding property between the ligand obtainable by the screening method according to 0) and the G protein-coupled receptor protein according to (1) or a salt thereof,

(53) The screening kit according to the above (19), which comprises cells containing the G protein-coupled receptor protein according to the above (1), (54) the G according to the above (1) The screening kit according to the above (19), which comprises a membrane fraction of cells containing a protein-coupled receptor protein, (55) the trait obtained by culturing the transformant according to the above (9) A screening kit according to the above (19), which contains a G protein-coupled receptor protein expressed on the cell membrane of the transformant,
(56) A compound which can be obtained by using the screening kit described in (53) to (55) and which changes the binding property between the ligand and the G protein-coupled receptor protein described in (1) or a salt thereof, or a compound thereof Salt, (5
7) A compound or a salt thereof which can be obtained by using the screening kit according to any one of (53) to (55) and which changes the binding property between the ligand and the G protein-coupled receptor protein according to (1) or a salt thereof. (58) The antibody according to (11) above is contacted with the G protein-coupled receptor protein according to (1) above or the partial peptide or salt thereof according to (4) above. A method for quantifying the G protein-coupled receptor protein according to (1) above, or the partial peptide according to (4) above or a salt thereof, (5)
9) Competitively reacting the antibody according to (11) with the test solution and the labeled G protein-coupled receptor protein according to (1) or the partial peptide according to (4) or a salt thereof. And a labeled G protein-coupled receptor protein according to the above (1) or a partial peptide according to the above (4) or a salt thereof, which is labeled with the antibody, is measured. (1) The method for quantifying the G protein-coupled receptor protein or the partial peptide or salt thereof according to (4) above, and (60) The antibody and labeling according to (11) above, which are insolubilized on a test solution and a carrier. The above (1
The G protein-coupled receptor protein according to (1) above in a test solution, which comprises reacting with the antibody according to 1) simultaneously or continuously and then measuring the activity of the labeling agent on the insolubilized carrier. Alternatively, a method for quantifying the partial peptide described in (4) above or a salt thereof is provided.

[0012]

BEST MODE FOR CARRYING OUT THE INVENTION The G protein-coupled receptor protein of the present invention (hereinafter sometimes abbreviated as receptor protein) has the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 5. Is a receptor protein containing The receptor protein of the present invention is, for example, a mammal (eg, human, guinea pig, rat, mouse, rabbit, pig, sheep, cow, monkey, etc.) (eg, splenocyte, nerve cell, glial cell, pancreatic β). Cells, bone marrow cells, mesangial cells, Langerhans cells, epidermal cells, epithelial cells, endothelial cells, fibroblasts, fiber cells, muscle cells, adipocytes, immune cells (eg, macrophages, T cells, B cells, natural killer cells, Mast cells, neutrophils, basophils, eosinophils, monocytes), megakaryocytes, synovial cells, chondrocytes, osteocytes, osteoblasts, osteoclasts, mammary gland cells, hepatocytes or stromal cells, Alternatively, progenitor cells of these cells, stem cells or cancer cells, cells of hematopoietic lineage, or any tissue in which these cells exist, for example, the brain, various parts of the brain (eg, olfactory bulb, flat cells). Nucleus, basal sphere, hippocampus, thalamus, hypothalamus, subthalamic nucleus, cerebral cortex, medulla oblongata, cerebellum, occipital lobe, frontal lobe, temporal lobe, putamen, caudate nucleus, brain stain, substantia nigra, spinal cord, inferior Pituitary, stomach,
Pancreas, kidney, liver, gonad, thyroid, gallbladder, bone marrow, adrenal gland, skin, muscle, lung, digestive tract (eg, large intestine, small intestine), blood vessel, heart, thymus, spleen, submandibular gland, peripheral blood, peripheral blood cell, It may be a protein derived from prostate, testis, testis, ovary, placenta, uterus, bone, joint, skeletal muscle or the like, or may be a synthetic protein.

The amino acid sequence which is substantially the same as the amino acid sequence represented by SEQ ID NO: 5 is, for example, about 50% or more, preferably about 60% or more, more preferably the amino acid sequence represented by SEQ ID NO: 5. It is preferably about 70% or more, particularly preferably about 80% or more, and particularly preferably about 90%.
% And most preferably about 95% or more homologous amino acid sequences and the like. SEQ ID NO: 5 of the present invention
As a protein containing an amino acid sequence substantially identical to the amino acid sequence represented by, for example, SEQ ID NO: 5
A protein containing an amino acid sequence substantially the same as the amino acid sequence represented by and having substantially the same activity as the amino acid sequence represented by SEQ ID NO: 5 is preferable. Examples of substantially the same activity include ligand binding activity and signal transduction activity. "Substantially the same quality" means that their activities are qualitatively the same. Therefore, the activities such as ligand binding activity and signal transduction activity are equivalent (eg, about 0.01 to 100 times, preferably about 0.5 to 20 times, more preferably about 0.5 to 10 times).
2 times), but quantitative factors such as the degree of these activities and the molecular weight of the protein may be different. The activity such as the ligand binding activity and the signal transduction activity can be measured according to a known method,
For example, it can be measured according to the ligand determination method and screening method described later. SEQ ID NO: 5
As a protein containing an amino acid sequence substantially the same as the amino acid sequence represented by, specifically, SEQ ID NO:
Examples thereof include a protein containing the amino acid sequence represented by 1.

As the receptor protein of the present invention, 1 or 2 or more (preferably about 1 to 30, more preferably about 1 to 10) in the amino acid sequence represented by SEQ ID NO: 5 is further used. Preferably several (1-5
1) or 2 or more (preferably 1 to 30 or so, more preferably 1 to 10 or so, still more preferably in the amino acid sequence represented by SEQ ID NO: 5). 1 or 2 or more (preferably 1 to 3) in the amino acid sequence represented by SEQ ID NO: 5 by adding several (1 to 5) amino acids
A protein containing an amino acid sequence in which about 0, more preferably about 1 to 10, more preferably several (1 to 5) amino acids are substituted with other amino acids, or an amino acid sequence combining them, etc. Is also used.

In the receptor protein in the present specification, the left end is the N-terminal (amino terminal) and the right end is the C-terminal (carboxyl terminal) according to the convention of peptide notation.
The receptor protein of the present invention, including the receptor protein containing the amino acid sequence represented by SEQ ID NO: 5, has a C-terminal carboxyl group (-COOH),
Carboxylate (-COO ^-), amide (-CON
H ₂ ) or ester (-COOR). Here, R in the ester is, for example, methyl, ethyl, n-propyl, isopropyl or n.
A C _1-6 alkyl group such as butyl, for example, a C _3-8 cycloalkyl group such as cyclopentyl and cyclohexyl,
For example, C _6-12 aryl groups such as phenyl and α-naphthyl, for example, phenyl-C such as benzyl and phenethyl.
_1-2 alkyl group or α-, such as α-naphthylmethyl
In addition to a C _7-14 aralkyl group such as naphthyl-C _1-2 alkyl group, a pivaloyloxymethyl group commonly used as an oral ester is used. When the receptor protein of the present invention has a carboxyl group (or carboxylate) in addition to the C-terminal, those in which the carboxyl group is amidated or esterified are also included in the receptor protein of the present invention. As the ester in this case, for example, the above-mentioned C-terminal ester or the like is used. Furthermore, in the receptor protein of the present invention, in the above-mentioned protein, the amino group of the N-terminal methionine residue is a protecting group (eg, formyl group, C _1-6 acyl group such as C _2-6 alkanoyl group such as acetyl, etc.). Etc.), a glutamyl group produced by cleavage at the N-terminal side in vivo is pyroglutamine-oxidized, a substituent on the side chain of an amino acid in the molecule (for example, —OH, —SH, amino group). , Imidazole group, indole group, guanidino group, etc.) are suitable protecting groups (for example, formyl group, C _1-6 acyl group such as C _2-6 alkanoyl group such as acetyl, etc.)
Those which are protected by, or complex proteins such as so-called glycoproteins to which sugar chains are bound.
Specific examples of the receptor protein of the present invention include a receptor protein containing the amino acid sequence represented by SEQ ID NO: 5 and a receptor protein containing the amino acid sequence represented by SEQ ID NO: 1.

The partial peptide of the receptor protein of the present invention (hereinafter sometimes abbreviated as a partial peptide) may be any partial peptide of the above-mentioned receptor protein of the present invention. ,
Among the receptor protein molecules of the present invention, those which are exposed outside the cell membrane and have receptor binding activity are used. Specifically, SEQ ID NO: 5
The partial peptide of the receptor protein containing the amino acid sequence represented by is a peptide containing the portion analyzed to be the extracellular region (hydrophilic region) in the hydrophobicity plot analysis. Further, a peptide partially containing a hydrophobic site can also be used. A peptide containing each domain individually may be used, but a peptide of a part containing a plurality of domains at the same time may be used. The number of amino acids of the partial peptide of the present invention is preferably a peptide having an amino acid sequence of at least 20 amino acids, preferably 50 amino acids or more, more preferably 100 amino acids or more among the constituent amino acid sequences of the receptor protein of the present invention described above. . A substantially identical amino acid sequence means about 50% or more, preferably about 60% or more, more preferably about 70% or more, further preferably about 80% or more, and particularly preferably about 90% or more with these amino acid sequences. , Most preferably about 95%
The amino acid sequences having the above homology are shown. here,
The "substantially the same quality of activity" has the same meaning as described above.
The "substantially the same quality of activity" can be measured in the same manner as above.

The partial peptide of the present invention has one or more (preferably 1 to 10) amino acids in the above amino acid sequence.
About one, more preferably several (1 to 5) amino acids are deleted, or 1 or 2 or more (preferably about 1 to 20, more preferably 1) in the amino acid sequence.
-10, more preferably several (1-5) amino acids are added, or 1 or 2 or more (preferably 1-10, more preferably several) in the amino acid sequence. , And more preferably about 1 to 5 amino acids) may be substituted with other amino acids. Further, the partial peptide of the present invention has a carboxyl group (-COO at the C-terminal).
H), carboxylate (-COO ^-), amide (-C
ONH ₂₎ or may be any of the ester (-COOR). Further, in the partial peptide of the present invention, as in the above-described receptor protein of the present invention, the amino group of the N-terminal methionine residue is protected by a protective group, and the N-terminal side is cleaved in vivo to generate the partial peptide. Also included are those in which Gln is pyroglutamine-oxidized, those in which the substituents on the side chains of amino acids in the molecule are protected by appropriate protecting groups, or complex peptides such as so-called glycopeptides to which sugar chains are bound. Examples of the salt of the receptor protein of the present invention or its partial peptide include physiologically acceptable salts with acids or bases, and physiologically acceptable acid addition salts are particularly preferable. Examples of such salts include salts with inorganic acids (eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid), or organic acids (eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, succinic acid). Acid, tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid) and salts thereof are used.

The receptor protein of the present invention or a salt thereof can be produced from the above-mentioned mammalian cells or tissues by a known method for purifying the receptor protein, or a DNA encoding the receptor protein of the present invention described later can be prepared. It can also be produced by culturing the transformant containing it. In addition, it can also be produced by a protein synthesis method described later or a method similar thereto. When produced from mammalian tissue or cells,
After homogenizing mammalian tissues or cells, extraction with acid or the like is carried out, and the extract can be purified and isolated by combining chromatography such as reverse phase chromatography and ion exchange chromatography.

For the synthesis of the receptor protein of the present invention, its partial peptide, its salt or its amide, a commercially available resin for protein synthesis can be used. Examples of such a resin include chloromethyl resin, hydroxymethyl resin, benzhydrylamine resin, aminomethyl resin, 4-benzyloxybenzyl alcohol resin, 4-methylbenzhydrylamine resin, PAM resin, 4-hydroxymethylmethyl resin. Phenylacetamide methyl resin, polyacrylamide resin, 4
Examples thereof include-(2 ', 4'-dimethoxyphenyl-hydroxymethyl) phenoxy resin and 4- (2', 4'-dimethoxyphenyl-Fmocaminoethyl) phenoxy resin. Using such a resin, an amino acid having an α-amino group and a side chain functional group appropriately protected is condensed on the resin according to various known condensation methods according to the sequence of the target protein. At the end of the reaction, the protein is cleaved from the resin and at the same time various protective groups are removed, and then an intramolecular disulfide bond forming reaction is carried out in a highly diluted solution to obtain the target protein or its amide.
Regarding the condensation of the above-mentioned protected amino acids, various activation reagents that can be used for protein synthesis can be used, but carbodiimides are particularly preferable. As the carbodiimides, DCC, N, N′-diisopropylcarbodiimide, N-ethyl-N ′-(3-dimethylaminoprolyl) carbodiimide and the like are used. For activation by these, additives for suppressing racemization (eg, HOBt, H
The protected amino acid can be directly added to the resin together with (OOBt), or can be added to the resin after previously performing activation of the protected amino acid as a symmetrical acid anhydride or HOBt ester or HOOBt ester.

The solvent used for activation of the protected amino acid or condensation with the resin can be appropriately selected from solvents known to be usable for protein condensation reaction. For example, acid amides such as N, N-dimethylformamide, N, N-dimethylacetamide and N-methylpyrrolidone, halogenated hydrocarbons such as methylene chloride and chloroform, alcohols such as trifluoroethanol,
Sulfoxides such as dimethyl sulfoxide, ethers such as pyridine, dioxane, and tetrahydrofuran, nitriles such as acetonitrile and propionitrile, esters such as methyl acetate and ethyl acetate, and appropriate mixtures thereof are used. The reaction temperature is appropriately selected from the range known to be applicable to protein bond-forming reactions, and is usually appropriately selected from the range of about -20 ° C to 50 ° C. The activated amino acid derivative is usually used in a 1.5 to 4-fold excess. As a result of the test using the ninhydrin reaction, when the condensation is insufficient, sufficient condensation can be performed by repeating the condensation reaction without removing the protecting group. When sufficient condensation cannot be obtained even after repeating the reaction, unreacted amino acid can be acetylated using acetic anhydride or acetylimidazole.

Examples of the protective group for the amino group of the raw material include Z, Boc, tertiary pentyloxycarbonyl, isobornyloxycarbonyl, 4-methoxybenzyloxycarbonyl, Cl-Z, Br-Z, adamantyloxycarbonyl, Trifluoroacetyl, phthaloyl, formyl, 2-nitrophenylsulfenyl, diphenylphosphinothioyl oil, Fmoc and the like are used. The carboxyl group is, for example, an alkyl esterified (for example, linear, branched or cyclic alkyl ester such as methyl, ethyl, propyl, butyl, tert-butyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, 2-adamantyl and the like. ), Aralkyl esterification (eg benzyl ester, 4-
Nitrobenzyl ester, 4-methoxybenzyl ester, 4-chlorobenzyl ester, benzhydryl esterification), phenacyl esterification, benzyloxycarbonyl hydrazide formation, tertiary butoxycarbonyl hydrazide formation, trityl hydrazide formation, etc. . The hydroxyl group of serine can be protected by, for example, esterification or etherification.
Examples of groups suitable for this esterification include lower alkanoyl groups such as acetyl group, aroyl groups such as benzoyl group, groups derived from carbonic acid such as benzyloxycarbonyl group and ethoxycarbonyl group.
Moreover, examples of a group suitable for etherification include a benzyl group, a tetrahydropyranyl group, and a t-butyl group. As a protective group for the phenolic hydroxyl group of tyrosine,
For example, Bzl, Cl ₂ -Bzl, 2-nitrobenzyl, Br-Z, tertiary butyl and the like are used.
Examples of the protecting group for imidazole of histidine include:
Tos, 4-methoxy-2,3,6-trimethylbenzenesulfonyl, DNP, benzyloxymethyl, Bum,
Boc, Trt, Fmoc, etc. are used.

The activated carboxyl group of the raw material includes, for example, corresponding acid anhydrides, azides, active esters [alcohols (for example, pentachlorophenol, 2,4,5-trichlorophenol, 2,4- Dinitrophenol, cyanomethyl alcohol, paranitrophenol, HONB, N-hydroxysuccinide, N
-Hydroxyphthalimide, ester with HOBt)]
Are used. As the activated amino group of the raw material, for example, the corresponding phosphoric acid amide is used. Examples of the method for removing (eliminating) the protecting group include Pd
-Catalytic reduction in a hydrogen stream in the presence of a catalyst such as black or Pd-carbon, or acid with anhydrous hydrogen fluoride, methanesulfonic acid, trifluoromethanesulfonic acid, trifluoroacetic acid, or a mixture thereof. Treatment, base treatment with diisopropylethylamine, triethylamine, piperidine, piperazine, etc., and reduction with sodium in liquid ammonia are also used. The elimination reaction by the acid treatment is generally carried out at a temperature of about -20 ° C to 40 ° C. In the acid treatment, for example, anisole, phenol, thioanisole, metacresol,
The addition of cation trapping agents such as para-cresol, dimethyl sulfide, 1,4-butanedithiol, 1,2-ethanedithiol is effective. Further, the 2,4-dinitrophenyl group used as the imidazole protecting group of histidine is removed by thiophenol treatment, and the formyl group used as the indole protecting group of tryptophan is the above 1,2-ethanedithiol, 1,4-butane group. In addition to deprotection by acid treatment in the presence of dithiol or the like, it is also removed by alkali treatment with dilute sodium hydroxide solution, dilute ammonia or the like.

Protection of a functional group which should not be involved in the reaction of the raw material, removal of the protective group, removal of the protective group, activation of the functional group involved in the reaction and the like can be appropriately selected from known groups or known means. . As another method for obtaining an amide form of a protein, for example, first, the α-carboxyl group of a carboxy-terminal amino acid is amidated and protected, and then a peptide (protein) chain is extended to the amino group side to a desired chain length. , A protein from which only the protecting group of the α-amino group at the N-terminal of the peptide chain has been removed and a protein from which only the protecting group of the carboxyl group at the C-terminal has been removed, and both proteins are prepared in a mixed solvent as described above. To condense. The details of the condensation reaction are the same as above. After purifying the protected protein obtained by condensation, all the protecting groups can be removed by the above method to obtain the desired crude protein. This crude protein can be purified by making full use of various known purification means, and the main fraction can be lyophilized to obtain the amide form of the desired protein. To obtain an ester form of a protein, for example, the α-carboxyl group of a carboxy-terminal amino acid is condensed with a desired alcohol to form an amino acid ester, and then an ester form of the desired protein is obtained in the same manner as the amide form of the protein. be able to.

The partial peptide of the protein of the present invention or a salt thereof can be produced by a known peptide synthesis method or by cleaving the protein of the present invention with an appropriate peptidase. The peptide synthesis method may be, for example, either a solid phase synthesis method or a liquid phase synthesis method. That is, the target peptide can be produced by condensing a partial peptide or amino acid that can form the protein of the present invention with the remaining portion and removing the protecting group when the product has a protecting group. Examples of known condensation methods and removal of protective groups include the methods described in the following (1) to (3). M. Bodanszky and MA Ondetti, Peptide Synthesis, Interscience Publisher
s, New York (1966) Schroeder and Luebke, The Peptide,
Academic Press, New York (1965) Nobuo Izumiya et al., Fundamentals and experiments of peptide synthesis, Maruzen Co., Ltd.
(1975) Haruaki Yajima and Shunpei Sakakibara, Laboratory of Biochemistry 1, Chemistry of Proteins IV, 205, (1977) Supervised by Haruaki Yajima, Development of Pharmaceuticals Volume 14 Peptide Synthesis Hirokawa Shoten The partial peptide of the present invention can be purified and isolated by a combination of the above-mentioned purification methods such as solvent extraction, distillation, column chromatography, liquid chromatography, and recrystallization. When the partial peptide obtained by the above method is a free form, it can be converted into an appropriate salt by a known method, and conversely, when it is obtained as a salt, it can be converted into a free form by a known method. You can

The polynucleotide encoding the receptor protein of the present invention includes a nucleotide sequence (DNA or R) encoding the above-mentioned receptor protein of the present invention.
It may be any as long as it contains NA, preferably DNA). The polynucleotide may be DN that encodes the receptor protein of the present invention.
A, RNA such as mRNA, which may be double-stranded or single-stranded. In the case of double-stranded, double-stranded DNA,
It may be double-stranded RNA or a DNA: RNA hybrid. In the case of a single strand, it may be a sense strand (that is, a coding strand) or an antisense strand (that is, a non-coding strand). By using the polynucleotide encoding the receptor protein of the present invention, for example, the publicly known experimental medicine special issue “New PCR and its application” 15 (7), 19
By the method described in 97 or a method analogous thereto, mRNA of the receptor protein of the present invention can be quantified. DN encoding the receptor protein of the present invention
A may be any of genomic DNA, genomic DNA library, cDNA derived from the cells and tissues described above, cDNA library derived from the cells and tissues described above, and synthetic DNA. The vector used for the library may be any of bacteriophage, plasmid, cosmid, phagemid and the like. In addition, reverse Transcriptase Polymerase Chai can be directly used by preparing total RNA or mRNA fraction from the above cells / tissues.
It can also be amplified by n Reaction (hereinafter abbreviated as RT-PCR method). Specifically, examples of the DNA encoding the receptor protein of the present invention include:
DNA containing the nucleotide sequence represented by SEQ ID NO: 6, or DNA containing the nucleotide sequence represented by SEQ ID NO: 6
And a DNA that hybridizes under high stringency conditions with a receptor protein containing the amino acid sequence represented by SEQ ID NO: 5 and having substantially the same activity (eg, ligand binding activity, signal transduction activity, etc.) )
Any DNA may be used as long as it encodes a receptor protein having The DNA that hybridizes with the DNA containing the base sequence represented by SEQ ID NO: 6 under high stringent conditions is, for example, about 70% or more, preferably about 70% or more, of the base sequence represented by SEQ ID NO: 6. 80% or more, more preferably about 90% or more, and most preferably about 95% or more homologous DNA containing a base sequence is used.
Examples thereof include DNA containing the base sequence represented by 2.

Hybridization can be carried out by a known method or a method analogous thereto, for example, Molecular Cloning 2nd (J. Sambrook et al.
al., Cold Spring Harbor Lab. Press, 1989). When a commercially available library is used, it can be performed according to the method described in the attached instruction manual. More preferably, it can be carried out under high stringent conditions. The high stringent condition is, for example, a sodium concentration of about 19 to 40 mM, preferably about 19 to 20 mM.
And the temperature is about 50-70 ° C, preferably about 60-65 ° C.
The conditions of are shown. Particularly, the case where the sodium concentration is about 19 mM and the temperature is about 65 ° C. is most preferable. More specifically,
As the DNA encoding the receptor protein containing the amino acid sequence represented by SEQ ID NO: 5, the DNA containing the nucleotide sequence represented by SEQ ID NO: 6 contains the amino acid sequence represented by SEQ ID NO: 1. No. 2 as a DNA encoding a receptor protein
A DNA containing a base sequence represented by is used. DNA encoding the receptor protein of the present invention
The polynucleotide containing a part of the nucleotide sequence of, or a part of the nucleotide sequence complementary to the DNA includes not only DNA encoding the following partial peptide of the present invention but also RNA. Used in the inclusive sense. According to the present invention, an antisense polynucleotide (nucleic acid) capable of inhibiting the replication or expression of a G protein-coupled receptor protein gene encodes a cloned or determined G protein-coupled receptor protein. It can be designed and synthesized based on the nucleotide sequence information of DNA. Such a polynucleotide (nucleic acid) can hybridize with RNA of the G protein-coupled receptor protein gene,
Or the expression of the G protein-coupled receptor protein gene can be regulated / controlled via the interaction with the G protein-coupled receptor protein-related RNA. G
A polynucleotide complementary to a selected sequence of a protein-coupled receptor protein-related RNA and a polynucleotide capable of specifically hybridizing with a G protein-coupled receptor protein-related RNA are G proteins in vivo and in vitro. It is useful for regulating and controlling the expression of the coupled receptor protein gene, and also useful for treating or diagnosing diseases and the like. The term "corresponding" means having homology or being complementary to a specific sequence of nucleotides, base sequences or nucleic acids including genes. “Corresponding” between a nucleotide (base sequence or nucleic acid) and a peptide (protein) means a peptide (protein) in a command derived from the nucleotide (nucleic acid) sequence or its complement.
Usually refers to amino acids. 5'end hairpin loop of G protein-coupled receptor protein gene, 5'end 6-base pair repeat, 5'end untranslated region, polypeptide translation initiation codon, protein coding region, ORF
The translation stop codon, the 3'-end untranslated region, the 3'-end palindromic region, and the 3'-end hairpin loop can be selected as preferred regions of interest, but any region within the G protein-coupled receptor protein gene can be selected as a region of interest. sell.

It can be said that the relationship between the target nucleic acid and the polynucleotide complementary to at least a part of the target region, and the relationship between the polynucleotide capable of hybridizing with the target region are "antisense". . Antisense polynucleotides include polynucleotides containing 2-deoxy-D-ribose, polynucleotides containing D-ribose, other types of polynucleotides that are N-glycosides of purine or pyrimidine bases, Alternatively, other polymers with non-nucleotide backbones (eg, commercially available protein nucleic acids and synthetic sequence-specific nucleic acid polymers) or other polymers containing special linkages, provided that such polymers are found in DNA or RNA. Containing a nucleotide having a configuration that allows pairing of bases and attachment of bases) and the like. They are double-stranded DNA, single-stranded DN
A, double-stranded RNA, single-stranded RNA, and further DNA: RN
A hybrid, which can be an unmodified polynucleotide (or an unmodified oligonucleotide), and also an addition of a known modification, for example, a label known in the art, a capped one, Methylated, substituted with one or more natural nucleotides by analogs, modified with an intramolecular nucleotide, such as an uncharged bond (eg, methylphosphonate,
With phosphotriesters, phosphoramidates, carbamates, etc., with charged bonds or with sulfur-containing bonds (eg phosphorothioates, phosphorodithioates, etc.), eg proteins (nucleases, nuclease inhibitors, toxins, Antibodies, signal peptides, poly-L-lysine, etc.), sugars (eg, monosaccharides, etc.) having side chain groups, intercurrent compounds (eg, acridine, etc.)
Those having psoralen, etc., chelate compounds (eg,
Metal, radioactive metal, boron, oxidizing metal, etc.), alkylating agent, modified bond (eg, α-anomer type nucleic acid, etc.) Good. As used herein, the term "nucleoside", "nucleotide" and "nucleic acid" may include those having not only purine and pyrimidine bases but also other modified heterocyclic bases. Such modifications include methylated purines and pyrimidines,
It may include acylated purines and pyrimidines, or other heterocycles. The modified nucleotide and modified nucleotide may also have a modified sugar moiety, for example, one or more hydroxyl groups are substituted with halogen, an aliphatic group, or a functional group such as ether or amine. It may have been converted.

The antisense polynucleotide (nucleic acid) of the present invention is RNA, DNA or a modified nucleic acid (RNA, DNA). Specific examples of the modified nucleic acid include, but are not limited to, sulfur derivatives and thiophosphate derivatives of nucleic acids, and those resistant to degradation of polynucleoside amides and oligonucleoside amides. The antisense nucleic acid of the present invention can be preferably designed according to the following policy. That is, it makes the antisense nucleic acid more stable in the cell, increases the cell permeability of the antisense nucleic acid, has a greater affinity for the target sense strand, and if it is toxic, Make sense nucleic acid less toxic. Thus many modifications are known in the art, for example J. Kawakami et al., Pharm Tech Japan, Vo.
l. 8, pp. 247, 1992; Vol. 8, pp. 395, 1992; ST Cr
ooke et al. ed., Antisense Research and Applicatio
ns, CRC Press, 1993 etc. The antisense nucleic acid of the present invention has an altered or modified sugar,
It may contain a base or a bond, and may be provided in a special form such as liposome, microsphere, applied by gene therapy, or provided in an added form. Thus, as an additive form, polycationic substances such as polylysine that act to neutralize the charge of the phosphate group, lipids that enhance the interaction with cell membranes and increase the uptake of nucleic acids ( Examples thereof include hydrophobic ones such as phospholipids and cholesterol). Preferred lipids to add include cholesterol and its derivatives (eg cholesteryl chloroformate, cholic acid, etc.). These can be attached to the 3'-end or 5'-end of the nucleic acid, and can be attached via a base, a sugar, or an intramolecular nucleoside bond. Examples of the other group include a capping group specifically arranged at the 3'-end or the 5'-end of a nucleic acid, which is for preventing decomposition by nucleases such as exonuclease and RNase. Examples of such a capping group include, but are not limited to, hydroxyl group-protecting groups known in the art including glycols such as polyethylene glycol and tetraethylene glycol. The inhibitory activity of the antisense nucleic acid can be examined by using the transformant of the present invention, the in vivo or in vitro gene expression system of the present invention, or the in vivo or in vitro translation system of G protein-coupled receptor protein. it can. The nucleic acid can be applied to cells by various known methods.

DNA encoding the partial peptide of the present invention
Any may be used as long as it contains the nucleotide sequence encoding the partial peptide of the present invention. Further, any of genomic DNA, genomic DNA library, cDNA derived from the cells / tissues described above, cDNA library derived from the cells / tissues described above, and synthetic DNA may be used. The vector used for the library may be any of bacteriophage, plasmid, cosmid, phagemid and the like. In addition, using the mRNA fraction prepared from the cells / tissues described above, the reverse
Transcriptase Polymerase Chain Reaction (hereinafter RT
-Abbreviated as PCR method). Specifically, D encoding the partial peptide of the present invention
Examples of NA include (1) DN having a partial base sequence of DNA containing the base sequence represented by SEQ ID NO: 6.
A, or (2) a receptor having a DNA that hybridizes with a DNA containing the base sequence represented by SEQ ID NO: 6 under high stringent conditions and containing the amino acid sequence represented by SEQ ID NO: 5 A DNA having a partial base sequence of a DNA encoding a receptor protein having substantially the same activity as the protein (eg, ligand binding activity, signal transduction activity, etc.) is used. DNA containing the nucleotide sequence represented by SEQ ID NO: 6
As the DNA that hybridizes under high stringency conditions with, for example, about 70% or more, preferably about 80% or more, more preferably about 90% or more, and most preferably the nucleotide sequence represented by SEQ ID NO: 6. Is a DNA containing a base sequence having a homology of about 95% or more, and a specific example thereof is a DNA containing the base sequence represented by SEQ ID NO: 2.

As a means for cloning the DNA which completely encodes the receptor protein of the present invention or a partial peptide thereof (hereinafter sometimes abbreviated as the receptor protein of the present invention), the means for cloning the DNA encoding the receptor protein of the present invention is A DNA fragment or synthetic DNA that encodes a part or the whole region of the receptor protein of the present invention is obtained by amplifying DNA by a PCR method using a synthetic DNA primer having a partial base sequence or incorporating it into an appropriate vector. It can be selected by hybridization with the one used and labeled. Hybridization methods include, for example, molecular cloning (Molecular Clonin
g) 2nd (J. Sambrook et al., Cold Spring Harbor La
b. Press, 1989). When a commercially available library is used, it can be performed according to the method described in the attached instruction manual.

The conversion of the nucleotide sequence of DNA is carried out by PCR or a known kit such as Mutan ^™ -superExpress Km (Takara Shuzo), Mutan ^™ -K (Takara Shuzo) or the like, using the ODA-LA PCR method or Gapped duplex method. It can be performed according to a known method such as the Kunkel method or a method similar thereto. The cloned DNA encoding the receptor protein can be used as it is, or if desired after digestion with a restriction enzyme or addition of a linker, depending on the purpose. The DNA may have ATG as a translation initiation codon at the 5′-terminal side and TAA, TGA or TAG as a translation termination codon at the 3′-terminal side. These translation initiation codon and translation termination codon can be added using an appropriate synthetic DNA adaptor. The expression vector of the receptor protein of the present invention is
For example, (a) cutting out a target DNA fragment from the DNA encoding the receptor protein of the present invention,
(B) It can be produced by ligating the DNA fragment downstream of the promoter in an appropriate expression vector.

As a vector, a plasmid derived from E. coli (eg, pCR4, pCR2.1, pBR322, pB
R325, pUC12, pUC13), plasmids derived from Bacillus subtilis (eg, pUB110, pTP5, pC19)
4), yeast-derived plasmid (eg, pSH19, pSH1
5), bacteriophages such as λ phage, animal viruses such as retrovirus, vaccinia virus, baculovirus, etc., as well as pA1-11, pXT1, pR
c / CMV, pRc / RSV, pcDNAI / Neo and the like are used. The promoter used in the present invention may be any promoter as long as it is suitable for the host used for gene expression. For example, when an animal cell is used as a host, SRα promoter,
Examples include SV40 promoter, LTR promoter, CMV promoter, HSV-TK promoter and the like. Among these, it is preferable to use the CMV promoter, the SRα promoter and the like. When the host is a bacterium belonging to the genus Escherichia, the trp promoter, the lac promoter, the recA promoter, the λP _L promoter,
The lpp promoter and the like are SPO1 promoter, SPO2 promoter, penP promoter and the like when the host is Bacillus, and the PHO5 promoter, PGK promoter and GA when the host is yeast.
P promoter, ADH promoter and the like are preferable.
When the host is an insect cell, polyhedrin promoter, P10 promoter and the like are preferable.

In addition to the above, the expression vector may optionally contain an enhancer, a splicing signal, a poly A addition signal, a selection marker, an SV40 replication origin (hereinafter sometimes abbreviated as SV40 ori) and the like. Can be used. Examples of selection markers include dihydrofolate reductase (hereinafter, dhfr).
The gene [methotrexate (M
TX) resistance], ampicillin resistance gene (hereinafter referred to as Amp
^r ), neomycin resistance gene (hereinafter sometimes abbreviated as Neo ^r , G418 resistance) and the like. In particular, when the dhfr gene is used as a selection marker using CHO (dhfr ⁻ ) cells, the target gene can also be selected with a thymidine-free medium. In addition, if necessary, a signal sequence suitable for the host is added to the N-terminal side of the receptor protein of the present invention. When the host is Escherichia, Ph
oA / signal sequence, OmpA / signal sequence, etc.
When the host is a bacterium of the genus Bacillus, α-amylase signal sequence, subtilisin signal sequence, and the like, when the host is yeast, MFα signal sequence, SUC2
-When the host is an animal cell such as a signal sequence,
Insulin signal sequence, α-interferon
A signal sequence, an antibody molecule, a signal sequence, etc. can be used respectively. A transformant can be produced using the vector containing the DNA encoding the receptor protein of the present invention thus constructed.

As the host, for example, Escherichia, Bacillus, yeast, insect cells, insects, animal cells and the like are used. Specific examples of Escherichia bacteria include:
Escherichia coli K12 ・ DH
1 [Proc. Natl. Acad. Sci. USA], Volume 60, 160 (1) [Procedures of the National Academy of Sciences of the USA]
968)], JM103 [Nucleic Acids Research], Volume 9, 309.
(1981)], JA221 [Journal of Molecular Biolog (Journal of Molecular Biolog
y), 120, 517 (1978)], HB101 [Journal of Molecular Biology, 41.
Vol., 459 (1969)], C600 [Genetics, 39, 440 (1954)], DH5α
(Inoue, H., Nojima, H. and Okayama, H., Gene, 96, 23-28 (1
990)], DH10B [Procedures of the National Academy of Sciences of
The USA (Proc. Natl. Acad. Sci. USA), 8
7 vol., 4645-4649 (1990)] and the like. Examples of the bacterium of the genus Bacillus include Bacillus subtilis MI114 [Gene, 24
Vol. 255 (1983)], 207-21 [Journal
Journal of Biochemistr
y), Vol. 95, 87 (1984)]. Examples of the yeast include Saccharomyces cerevisiae AH22 and AH22.
^{R -, NA87-11A, DKD-5D} , 20B-1
2. Schizosaccharomy
ces pombe) NCYC1913, NCYC2036, Pichia pastoris and the like are used.

Examples of insect cells include virus A
In the case of cNPV, the cell line derived from the larva of Spodoptera frugiperda (Spod
optera frugiperda cell; Sf cell), Trichoplusia n
MG1 cells from the midgut of i, from the eggs of Trichoplusia ni
High Five ^™ cells, Mamestra brassicae-derived cells, Estigmena acrea-derived cells and the like are used. When the virus is BmNPV, the silkworm-derived cell line (Bombyx m
ori N; BmN cells) and the like are used. Examples of the Sf cells include Sf9 cells (ATCC CRL1711), Sf2
1 cell (above, Vaughn, JL et al., In Viv
o), 13, 213-217, (1977)) are used. Examples of insects include silkworm larvae [Maeda et al., Nature, 315, 592 (198).
5)]. Animal cells include, for example, monkey cells COS-
7, Vero, Chinese hamster cell CHO (hereinafter,
CHO cells), dhfr gene-deficient Chinese hamster cells CHO (hereinafter abbreviated as CHO (dhfr ⁻ ) cells), mouse L cells, mouse AtT-20, mouse myeloma cells, rat GH3, human FL cells and the like are used.

To transform Escherichia, for example, Proc. Natl. Acad. Sci. USA, Volume 69, Procedures of the National Academy of Sciences of the USA. , 2110
(1972) and Gene, Volume 17, 107 (198)
It can be performed according to the method described in 2) or the like. To transform Bacillus, for example, molecular
And General Genetics (Molecular &
General Genetics), 168, 111 (1979) and the like. For transforming yeast, for example, Methods in Enzymology, 194, 182-
187 (1991), Procedures of the National Academy of Sciences of.
The USA (Proc. Natl. Acad. Sci. USA), 7
5 volume, 1929 (1978)) and the like. For transforming insect cells or insects, for example, Bio / Technolog
y), 6, 47-55 (1988) and the like. To transform animal cells, see, eg, Cell Engineering Supplement 8, New Cell Engineering Experimental Protocol. 263-26
7 (1995) (published by Shujunsha), Virology
gy), 52, 456 (1973). Thus, a transformant transformed with the expression vector containing the DNA encoding the G protein-coupled receptor protein can be obtained.
When the host is culturing a transformant that is a bacterium of the genus Escherichia, a bacterium of the genus Bacillus, a liquid medium is suitable as a medium used for the culture, and among them, a carbon source necessary for the growth of the transformant, A nitrogen source, an inorganic substance, etc. are contained.
Examples of carbon sources include glucose, dextrin,
Examples of nitrogen sources such as soluble starch and sucrose include ammonium salts, nitrates, corn steep liquor,
Examples of inorganic or organic substances such as peptone, casein, meat extract, soybean meal, and potato extract, and inorganic substances include calcium chloride, sodium dihydrogen phosphate, magnesium chloride and the like. In addition, yeast extract, vitamins, growth promoting factor and the like may be added. P of medium
H is preferably about 5-8.

As a medium for culturing Escherichia, for example, M9 medium containing glucose and casamino acid [Miller, Journal of Experiments in Molecular Genetics (Journa
l of Experiments in Molecular Genetics), 431-
433, Cold Spring Harbor Laboratory, New York1
972] is preferable. If necessary, a drug such as 3β-indolylacrylic acid can be added to the promoter so as to work efficiently. When the host is a bacterium belonging to the genus Escherichia, the culture is usually carried out at about 15 to 43 ° C. for about 3 to 24 hours, and if necessary, aeration and stirring can be added. When the host is a bacterium of the genus Bacillus, the culture is usually carried out at about 30 to 40 ° C. for about 6 to 24 hours, and aeration and stirring can be added if necessary. When culturing a transformant whose host is yeast, examples of the medium include Burkholder's minimum medium [Bostian, KL
Et al., "Proc. Natl. Acad. Sci. USA", Vol. 77, 4505. "Procedures of the National Academy of Sciences of the USA".
(1980) "or SD medium containing 0.5% casamino acid [Bitter, GA et al.," Procedures of the.
National Academy of Sciences of the USA (Proc. Natl. Acad. Sci. USA),
81, 5330 (1984). P of medium
H is preferably adjusted to about 5-8. Culture is usually about 2
It is carried out at 0 ° C to 35 ° C for about 24 to 72 hours, and aeration and stirring are added if necessary.

When culturing a transformant whose host is an insect cell or an insect, the medium is Grace's Insect Mediu
m (Grace, TCC, Nature, 19
5, 788 (1962)) to which an additive such as immobilized 10% bovine serum is appropriately added. The pH of the medium is preferably adjusted to about 6.2 to 6.4. Culturing is usually performed at about 27 ° C. for about 3 to 5 days, and aeration and agitation are added if necessary. When culturing a transformant whose host is an animal cell, the medium is, for example, MEM medium containing about 5 to 20% fetal bovine serum [Science,
122, 501 (1952)], DMEM medium [Virology, 8th, 396 (1959)], RP
MI 1640 medium [The Journal of the American Medical Association (The Journal of t
he American Medical Association) Volume 199, 519
(1967)], 199 medium [Proceeding of the Society for the Biological Medicine (Proceeding of the Society for the Biological Medicine
Medicine), 73, 1 (1950)]. The pH is preferably about 6-8. Culturing is usually performed at about 30 ° C to 40 ° C for about 15 to 60 hours, and aeration and agitation are added if necessary. As described above, the G protein-coupled receptor protein of the present invention can be produced inside the transformant cells, on the cell membrane or outside the cells.

The receptor protein of the present invention can be separated and purified from the above-mentioned culture, for example, by the following method. When the receptor protein of the present invention is extracted from cultured bacterial cells or cells, after the culture, the bacterial cells or cells are collected by a known method, suspended in a suitable buffer solution, and then subjected to ultrasonic wave, lysozyme and / or freeze-thawing. A method of obtaining a crude extract of the receptor protein by centrifuging or filtering after appropriately destroying the bacterial cells or cells by, for example, is appropriately used. In the buffer solution, protein denaturants such as urea and guanidine hydrochloride, and Triton X-1
A surfactant such as 00 ^™ may be included. When the receptor protein is secreted into the culture medium, after the culture is completed, the cells or cells are separated from the supernatant by a known method, and the supernatant is collected. The culture supernatant thus obtained,
Alternatively, the receptor protein contained in the extract can be purified by appropriately combining known separation / purification methods. Known separation and purification methods for these are methods utilizing solubility such as salting out and solvent precipitation, dialysis,
Ultrafiltration, gel filtration, SDS-polyacrylamide gel electrophoresis and other methods that mainly utilize the difference in molecular weight, ion exchange chromatography and other methods that utilize the difference in charge, affinity chromatography and other specific methods A method of using affinity, a method of utilizing difference in hydrophobicity such as reversed-phase high performance liquid chromatography, a method of utilizing difference in isoelectric point such as isoelectric focusing are used.

When the receptor protein thus obtained is obtained as a free form, it can be converted into a salt by a known method or a method analogous thereto. Conversely, when it is obtained as a salt, a known method or It can be converted into a free form or other salt by a similar method.
The receptor protein produced by the recombinant can be optionally modified or partially removed by reacting it with an appropriate protein-modifying enzyme before or after purification. Examples of the protein-modifying enzyme include trypsin, chymotrypsin, arginyl endopeptidase, protein kinase, glycosidase and the like. The activity of the receptor protein of the present invention or a salt thereof thus produced can be measured by a binding experiment with a labeled ligand, an enzyme immunoassay using a specific antibody, or the like.

The antibody against the receptor protein of the present invention or its partial peptide or its salt may be either a polyclonal antibody or a monoclonal antibody as long as it is an antibody capable of recognizing the receptor protein of the present invention or its partial peptide or its salt. Good. An antibody against the receptor protein of the present invention or a partial peptide thereof or a salt thereof (hereinafter sometimes abbreviated as “receptor protein of the present invention”) is a known antibody or antiserum prepared by using the receptor protein of the present invention as an antigen. It can be manufactured according to the manufacturing method.

[Preparation of Monoclonal Antibody] (a) Preparation of Monoclonal Antibody-Producing Cell The receptor protein or the like of the present invention is administered to a mammal at a site where antibody production can be carried out by itself or together with a carrier or diluent. It Complete Freund's adjuvant or incomplete Freund's adjuvant may be administered to enhance the antibody-producing ability upon administration. The administration is usually performed once every 2 to 6 weeks, about 2 to 10 times in total.
Examples of mammals used include monkeys, rabbits,
Dogs, guinea pigs, mice, rats, sheep and goats are mentioned, but mice and rats are preferably used. For preparation of monoclonal antibody-producing cells, warm-blooded animals immunized with an antigen, for example, individuals with an antibody titer were selected from mice, and 2 to 5 days after the final immunization, spleens or lymph nodes were collected and By fusing the antibody-producing cells contained therein with myeloma cells, a monoclonal antibody-producing hybridoma can be prepared. The antibody titer in the antiserum can be measured, for example, by reacting the labeled receptor protein described below with the antiserum and then measuring the activity of the labeling agent bound to the antibody. The fusion operation is a known method, for example, the method of Koehler and Milstein [Nature]]],
256, 495 (1975) "". Examples of the fusion accelerator include polyethylene glycol (PEG) and Sendai virus, and PEG is preferably used. Examples of myeloma cells include NS-1, P3U1, SP2 / 0.
Etc., but P3U1 is preferably used.
The preferred ratio of the number of antibody-producing cells (spleen cells) to the number of myeloma cells used is about 1: 1 to 20: 1.
G (preferably PEG1000 to PEG6000) is added at a concentration of about 10 to 80%, and the temperature is about 20 to 40 ° C.
Cell fusion can be efficiently carried out by preferably incubating at about 30 to 37 ° C. for about 1 to 10 minutes.

Although various methods can be used for screening monoclonal antibody-producing hybridomas, for example, the hybridoma culture supernatant is added to a solid phase (eg, a microplate) on which an antigen such as a receptor protein is directly or adsorbed with a carrier. Then, add an anti-immunoglobulin antibody labeled with a radioactive substance or an enzyme (if the cell used for cell fusion is a mouse, an anti-mouse immunoglobulin antibody is used) or protein A, and add the monoclonal antibody bound to the solid phase. Method for detection, hybridoma culture supernatant is added to a solid phase on which anti-immunoglobulin antibody or protein A is adsorbed, and receptor protein labeled with a radioactive substance or enzyme is added to detect the monoclonal antibody bound to the solid phase Method etc. are mentioned. The selection of the monoclonal antibody can be performed according to a known method or a method similar thereto, but usually HAT
(Hypoxanthine, aminopterin, thymidine) can be added to the medium for animal cells. As the medium for selection and breeding, any medium may be used as long as the hybridoma can grow. For example, 1
R containing 20%, preferably 10-20% fetal calf serum
A PMI 1640 medium, a GIT medium containing 1 to 10% fetal bovine serum (Wako Pure Chemical Industries, Ltd.), a serum-free medium for hybridoma culture (SFM-101, Nissui Pharmaceutical Co., Ltd.) or the like can be used. . The culture temperature is usually 20 to 40 ° C, preferably about 37 ° C. Cultivation time is usually 5 days to 3 weeks, preferably 1 week to 2 weeks. Culturing can usually be performed under 5% carbon dioxide gas.
The antibody titer of the hybridoma culture supernatant can be measured in the same manner as the above antibody titer measurement in antiserum.

(B) Purification of Monoclonal Antibody The isolation and purification of the monoclonal antibody can be carried out in the same manner as the usual isolation and purification of a polyclonal antibody [eg, salting out method, alcohol precipitation method, isoelectric focusing method, Electrophoresis method, adsorption / desorption method with ion exchanger (eg, DEAE), ultracentrifugation method, gel filtration method, antigen-binding solid phase or active antibody such as protein A or protein G is used to dissociate the antibody. Specific Purification Method of Obtaining Antibody to Obtain Antibody]

[Preparation of Polyclonal Antibody] The polyclonal antibody of the present invention can be manufactured by publicly known methods or modifications thereof. For example, a complex of an immune antigen (an antigen such as the receptor protein of the present invention) and a carrier protein is formed, and a mammal is immunized in the same manner as in the above-mentioned method for producing a monoclonal antibody. Etc. can be produced by collecting the antibody-containing substance against the above and separating and purifying the antibody. Regarding a complex of an immunizing antigen and a carrier protein used for immunizing a mammal, the kind of the carrier protein and the mixing ratio of the carrier and the hapten are such that the antibody can efficiently be against the hapten immunized by crosslinking with the carrier. Any kind may be cross-linked at any ratio, but, for example, bovine serum albumin, bovine thyroglobulin, keyhole limpet hemocyanin, etc. in a weight ratio of about 0.1 to hapten 1.
A method of coupling at a ratio of 1 to 20, preferably about 1 to 5 is used. Although various condensing agents can be used for coupling the hapten and carrier, glutaraldehyde, carbodiimide, maleimide active ester, active ester reagent containing thiol group, dithiopyridyl group, etc. are used. The condensation product is administered to a warm-blooded animal at a site where antibodies can be produced, by itself or together with a carrier and a diluent. Complete Freund's adjuvant or incomplete Freund's adjuvant may be administered to enhance the antibody-producing ability upon administration. The administration can be usually performed once every about 2 to 6 weeks, about 3 to 10 times in total. The polyclonal antibody can be collected from blood, ascites, etc., preferably blood of a mammal immunized by the above method. The polyclonal antibody titer in the antiserum can be measured in the same manner as the above-mentioned antibody titer in the serum. Separation and purification of the polyclonal antibody can be performed according to the same immunoglobulin separation and purification method as the above-described separation and purification of the monoclonal antibody.

The receptor protein of the present invention or its salt, its partial peptide or its salt, and DN encoding the receptor protein or its partial peptide.
A is (1) determination of a ligand (agonist) for the G protein-coupled receptor protein of the present invention,
(2) a preventive and / or therapeutic agent for a disease associated with dysfunction of the G protein-coupled receptor protein of the present invention, (3) a gene diagnostic agent, (4) an expression level of the receptor protein of the present invention or a partial peptide thereof. (5) Preventive and / or therapeutic agent for various diseases containing a compound that changes the expression level of the receptor protein of the present invention or its partial peptide, (6) G protein-coupled receptor of the present invention Method for quantifying ligand for protein, (7) G of the present invention
A method for screening a compound (agonist, antagonist, etc.) that changes the binding property between a protein-coupled receptor protein and a ligand, (8) a compound that changes the binding property between a G protein-coupled receptor protein of the present invention and a ligand (an agonist, Antagonist)
A preventive and / or therapeutic agent for various diseases containing
(9) Quantification of the receptor protein of the present invention or a partial peptide thereof or a salt thereof, (10) a method for screening a compound that changes the amount of the receptor protein of the present invention or a partial peptide thereof in the cell membrane, (1
1) A preventive and / or therapeutic agent for various diseases containing a compound that alters the amount of the receptor protein of the present invention or its partial peptide in the cell membrane, (12) an antibody against the receptor protein of the present invention or its partial peptide or its salt Neutralization, (13) G of the present invention
D encoding a protein-coupled receptor protein
It can be used for production of non-human animals having NA. In particular, a compound that changes the binding property of a ligand to a mammalian-specific G protein-coupled receptor by using a receptor binding assay system using the expression system of the recombinant G protein-coupled receptor protein of the present invention ( (Eg, agonist, antagonist, etc.), and the agonist or antagonist can be used as a preventive / therapeutic agent for various diseases. The receptor protein or partial peptide of the present invention or a salt thereof (hereinafter sometimes abbreviated as the receptor protein or the like of the present invention), DNA encoding the receptor protein of the present invention or a partial peptide thereof (hereinafter abbreviated as the DNA of the present invention) The use of an antibody against the receptor protein or the like of the present invention (hereinafter sometimes abbreviated as the antibody of the present invention) will be specifically described below.

(1) Determination of ligand (agonist) for the G protein-coupled receptor protein of the present invention The receptor protein of the present invention or a salt thereof or the partial peptide of the present invention or a salt thereof is used for the receptor protein of the present invention or a salt thereof. It is useful as a reagent for searching or determining a ligand (agonist). That is, the present invention provides a method for determining a ligand for the receptor protein of the present invention, which comprises contacting the receptor protein of the present invention or a salt thereof or the partial peptide of the present invention or a salt thereof with a test compound. As the test compound, a known ligand (for example, angiotensin, bombesin, cannabinoid, cholecystokinin, glutamine, serotonin,
Melatonin, neuropeptide Y, opioid, purine, vasopressin, oxytocin, PACAP (eg,
PACAP27, PACAP38), secretin, glucagon, calcitonin, adrenomedullin, somatostatin, GHRH, CRF, ACTH, GRP, PT
H, VIP (vasoactive intestinal and related polypeptide), somatostatin, dopamine, motilin, amylin, bradykinin, CGR
P (calcitonin gene relayed peptide), leukotriene, pancreatatin, prostaglandin, thromboxane, adenosine, adrenaline, chemokine superfamily (eg, IL-8, GROα,
GROβ, GROγ, NAP-2, ENA-78, GC
P-2, PF4, IP-10, Mig, PBSF / SD
CXC chemokine subfamily such as F-1; MCA
F / MCP-1, MCP-2, MCP-3, MCP-
4, eotaxin, RANTES, MIP-1α, M
IP-1β, HCC-1, MIP-3α / LARC, M
IP-3β / ELC, I-309, TARC, MIPF
-1, MIPF-2 / eotaxin-2, MDC, D
CC chemokine subfamily such as C-CK1 / PARC, SLC; C chemokine subfamily such as lymphoactin; CX3C chemokine subfamily such as fractalkine), endothelin, enterogastrin, histamine, neurotensin, TRH, pancreatic polypeptide, In addition to galanin, lysophosphatidic acid (LPA), sphingosine 1-phosphate, etc.), for example, tissue extracts of mammals (eg, human, mouse, rat, pig, cow, sheep, monkey, etc.), on cell culture Kiyoshi etc. are used.
For example, the tissue extract, cell culture supernatant and the like can be added to the receptor protein of the present invention and fractionated while measuring the cell stimulating activity and the like to finally obtain a single ligand.

Specifically, the method for determining the ligand of the present invention uses the receptor protein of the present invention or its partial peptide or its salt, or constructs an expression system for a recombinant receptor protein and uses the expression system. Cell stimulating activity by binding to the receptor protein of the present invention (for example, arachidonic acid release, acetylcholine release, intracellular Ca ²⁺ release, intracellular cAMP production, intracellular cAMP production inhibition, Intracellular cGMP production, inositol phosphate production,
Cell membrane potential fluctuation, intracellular protein phosphorylation, cf
The method is for determining a compound (for example, a peptide, a protein, a non-peptidic compound, a synthetic compound, a fermentation product, etc.) or a salt thereof having os activation, an activity of promoting or suppressing a decrease in pH, etc.). In the method for determining a ligand of the present invention, when the test compound is brought into contact with the receptor protein of the present invention or its partial peptide, for example, the binding amount of the test compound to the receptor protein or the partial peptide, cell stimulating activity, etc. It is characterized by measuring.

More specifically, the present invention provides the labeled test compound, which is obtained by contacting the labeled test compound with the receptor protein of the present invention or a salt thereof or the partial peptide of the present invention or a salt thereof. Or a salt thereof, or a method for determining a ligand for the receptor protein of the present invention or a salt thereof, which comprises measuring the amount of binding to the partial peptide or a salt thereof, and a labeled test compound containing the receptor protein of the present invention Determination of a ligand for the receptor protein of the present invention or a salt thereof, which comprises measuring the binding amount of a labeled test compound to the cell or the membrane fraction when contacted with the cell or the membrane fraction of the cell Method, labeled test compound is a receptor protein of the invention Characterized in that the amount of labeled test compound bound to the receptor protein or a salt thereof is measured when the transformant containing the DNA encoding the is contacted with the receptor protein expressed on the cell membrane. A method for determining a ligand for the receptor protein of the present invention,

When the test compound is contacted with cells containing the receptor protein of the present invention, the cell-stimulating activity mediated by the receptor protein (eg, arachidonic acid release, acetylcholine release, intracellular Ca ²⁺ release, intracellular cAMP production, intracellular cAMP production inhibition, intracellular cGMP production, inositol phosphate production, cell membrane potential fluctuation, intracellular protein phosphorylation, c-fos activation, activity promoting or suppressing pH decrease, etc. On the cell membrane by culturing a transformant containing the DNA encoding the receptor protein of the present invention, and a method for determining a ligand for the receptor protein of the present invention or a salt thereof, and a test compound. When contacted with the receptor protein expressed in Cell stimulating activity mediated by the receptor protein (e.g., arachidonic acid release, acetylcholine release, intracellular Ca ²⁺ release, intracellular cAMP production, intracellular cAMP production suppression, intracellular cGMP production, inositol phosphate production, change in cell membrane potential, A method for determining a ligand for the receptor protein or a salt thereof according to the present invention, which comprises measuring the phosphorylation of intracellular proteins, the activation of c-fos, the activity of promoting or suppressing the decrease of pH, and the like. provide. In particular, it is preferable to perform the above-mentioned tests to confirm that the test compound binds to the receptor protein of the present invention, and then perform the above-mentioned tests.

First, the receptor protein used in the ligand determination method may be any as long as it contains the above-described receptor protein of the present invention or the partial peptide of the present invention. Receptor proteins expressed in large amounts are suitable. The above-mentioned expression method is used to produce the receptor protein of the present invention, but it is preferable to express the DNA encoding the receptor protein in mammalian cells or insect cells. Although complementary DNA is usually used for the DNA fragment encoding the target protein portion, it is not necessarily limited thereto. For example, a gene fragment or synthetic DNA may be used. To introduce the DNA fragment encoding the receptor protein of the present invention into host animal cells and express them efficiently, in order to efficiently express them, a nuclear polyhedrosis virus (nuclear polyhedrosis v
irus; NPV) polyhedrin promoter, SV40
Origin promoter, retrovirus promoter,
Metallothionein promoter, human heat shock promoter, cytomegalovirus promoter, SR
It is preferably incorporated downstream of the α promoter and the like. The amount and quality of the expressed receptor can be examined by a known method. For example, the literature [Nambi, P. Et al., The Journal of Biological Chemistry (J. Biol.
Chem.), 267, 19555-19559, 1992].

Therefore, in the method for determining a ligand of the present invention, the receptor protein of the present invention or its partial peptide or its salt is a receptor protein or its partial peptide or its salt purified by a known method. Alternatively, cells containing the receptor protein or cell membrane fractions thereof may be used. When a cell containing the receptor protein of the present invention is used in the method for determining a ligand of the present invention, the cell may be immobilized with glutaraldehyde, formalin or the like. The immobilization method can be performed according to a known method. The cells containing the receptor protein of the present invention refer to host cells expressing the receptor protein of the present invention, and examples of the host cells include Escherichia coli, Bacillus subtilis, yeast, insect cells and animal cells. The cell membrane fraction refers to a fraction containing a large amount of cell membrane obtained by a known method after crushing cells. As a method of crushing cells, a method of crushing cells with a Potter-Elvehjem type homogenizer, crushing with a Waring blender or polytron (Kinematica), crushing with ultrasonic waves, and blasting cells from a thin nozzle while applying pressure with a French press etc. Examples include crushing by things. For the fractionation of the cell membrane, a fractionation method by centrifugal force such as a fractionation centrifugation method or a density gradient centrifugation method is mainly used. For example, the cell lysate may be mixed at a low speed (500 rpm to 300 rpm).
Centrifuge at 0 rpm for a short time (usually about 1 to 10 minutes),
Supernatant at even higher speed (15,000 rpm to 30,000 rp)
Centrifugation in m) is usually performed for 30 minutes to 2 hours, and the resulting precipitate is used as a membrane fraction. The membrane fraction is rich in expressed receptor proteins and membrane components such as cell-derived phospholipids and membrane proteins.

The amount of the receptor protein in the cell containing the receptor protein or its membrane fraction is preferably 10 ³ to 10 ⁸ molecules per cell, and 10 ⁵ to 1
It is preferably 0 ⁷ molecules. It should be noted that the higher the expression level, the higher the ligand binding activity (specific activity) per membrane fraction, making it possible not only to construct a highly sensitive screening system, but also to measure a large number of samples in the same lot. . In order to carry out the above methods of determining the ligand for the receptor protein or salt thereof of the present invention, a suitable receptor protein fraction and a labeled test compound are required. As the receptor protein fraction, a natural receptor protein fraction or a recombinant receptor fraction having an activity equivalent to that is desirable. Here, the equivalent activity refers to equivalent ligand binding activity, signal transduction action, and the like. The labeled test compounds include [ ³ H], [ ¹²⁵ I],
Angiotensin labeled with [ ¹⁴ C], [ ³⁵ S], etc.,
Bombesin, cannabinoid, cholecystokinin, glutamine, serotonin, melatonin, neuropeptide Y,
Opioid, purine, vasopressin, oxytocin,
PACAP (eg, PACAP27, PACAP38),
Secretin, glucagon, calcitonin, adrenomedullin, somatostatin, GHRH, CRF, ACT
H, GRP, PTH, VIP (vasoactive intestinal and limited polypeptide), somatostatin, dopamine, motilin, amylin, bradykinin, CGRP (calcitonin gene relayed peptide), leukotriene, pancreatatin,
Prostaglandins, thromboxane, adenosine, adrenaline, chemokine superfamily (eg IL
-8, GROα, GROβ, GROγ, NAP-2, E
NA-78, GCP-2, PF4, IP-10, Mi
g, CXC chemokine subfamily such as PBSF / SDF-1; MCAF / MCP-1, MCP-2, MC
P-3, MCP-4, eotaxin, RANTES,
MIP-1α, MIP-1β, HCC-1, MIP-3
α / LARC, MIP-3β / ELC, I-309, T
ARC, MIPF-1, MIPF-2 / eotaxin
CC chemokine subfamily such as -2, MDC, DC-CK1 / PARC, SLC; lymphoact
C chemokine subfamily such as in; fracta
CX3C chemokine subfamily such as lkine), endothelin, enterogastrin, histamine, neurotensin, TRH, pancreatic polypeptide, galanin, lysophosphatidic acid (LP
A), sphingosine 1-phosphate and the like are preferable.

Specifically, in order to carry out the method for determining a ligand for the receptor protein of the present invention or a salt thereof, first, a cell or a membrane fraction of cells containing the receptor protein of the present invention is buffered with a buffer suitable for the determination method. A receptor preparation is prepared by suspending in. Any buffer may be used as long as it does not inhibit the binding between the ligand and the receptor protein, such as a phosphate buffer having a pH of 4 to 10 (preferably a pH of 6 to 8) and a Tris-hydrochloric acid buffer. Further, in order to reduce non-specific binding, surfactants such as CHAPS, Tween-80 ^™ (Kao-Atlas), digitonin and deoxycholate, and various proteins such as bovine serum albumin and gelatin may be added to the buffer. it can. Further, a protease inhibitor such as PMSF, leupeptin, E-64 (manufactured by Peptide Laboratories), pepstatin or the like can be added for the purpose of suppressing the decomposition of the receptor and the ligand by the protease. A fixed amount (5000 cpm-5000) was added to 0.01 ml-10 ml of the receptor solution.
00 cpm) [ ³ H], [ ¹²⁵ I], [ ¹⁴ C],
A test compound labeled with [ ³⁵ S] or the like is allowed to coexist. A reaction tube containing a large excess of unlabeled test compound is also prepared in order to know the non-specific binding amount (NSB). Reaction is about 0
℃ ~ 50 ℃, preferably about 4 ℃ ~ 37 ℃, about 20 minutes ~
It is carried out for 24 hours, preferably for about 30 minutes to 3 hours. After the reaction
After filtering with glass fiber filter paper and the like and washing with an appropriate amount of the same buffer, the radioactivity remaining on the glass fiber filter paper is measured with a liquid scintillation counter or a γ-counter. A test compound having a count (B-NSB) obtained by subtracting the non-specific binding amount (NSB) from the total binding amount (B) exceeding 0 cpm can be selected as a ligand (agonist) for the receptor protein of the present invention or a salt thereof. .

In order to carry out the above methods (1) to (3) for determining a ligand for the receptor protein of the present invention or a salt thereof, cell-stimulating activity mediated by the receptor protein (eg, arachidonic acid release, acetylcholine release, intracellular Ca ^{2 +} Activity to promote release, intracellular cAMP production, intracellular cAMP production suppression, intracellular cGMP production, inositol phosphate production, cell membrane potential fluctuation, intracellular protein phosphorylation, c-fos activation, pH decrease, etc. Or suppressive activity) can be measured using a known method or a commercially available measurement kit. Specifically, first, cells containing the receptor protein are cultured in a multiwell plate or the like. To determine the ligand, exchange with fresh medium or an appropriate buffer that does not show toxicity to cells in advance, add a test compound etc. and incubate for a certain period of time, then extract the cells or collect the supernatant and generate The amount of the obtained product is quantified according to each method. When the production of a substance that serves as an index of cell stimulating activity (for example, arachidonic acid) is difficult to assay with a degrading enzyme contained in cells, an inhibitor for the degrading enzyme may be added to the assay. Further, the activity of suppressing cAMP production and the like can be detected as a production suppressing action on cells in which the basic production amount of cells has been increased by forskolin or the like.

The kit for determining a ligand that binds to the receptor protein of the present invention or a salt thereof comprises a receptor protein of the present invention or a salt thereof, a partial peptide of the present invention or a salt thereof, a cell containing the receptor protein of the present invention, or It contains a membrane fraction of cells containing the receptor protein of the invention. Examples of the ligand determination kit of the present invention include the following. 1. Ligand determination reagent In measuring buffer and washing buffer Hanks' Balanced Salt Solution (manufactured by Gibco),
To which 05% bovine serum albumin (manufactured by Sigma) was added. Sterilize by filtration with a 0.45μm filter at 4 ℃
It may be stored at or prepared at the time of use. G protein-coupled receptor protein preparation CHO cells expressing the receptor protein of the present invention were subcultured on a 12-well plate at 5 × 10 ⁵ cells / well, and 37
Cultivated at 5 ° C., 5% CO ₂ , 95% air for 2 days. Labeled test compound Commercially available compound labeled with [ ³ H], [ ¹²⁵ I], [ ¹⁴ C], [ ³⁵ S] or the like, labeled with an appropriate method in the form of an aqueous solution at 4 ° C or -20 Store at 0 ° C and dilute to 1 μM with measurement buffer before use. Test compounds that are poorly soluble in water are dissolved in dimethylformamide, DMSO, methanol and the like. Unlabeled test compound The same as the labeled compound is prepared at a concentration 100 to 1000 times higher.

2. Assay method Receptor protein-expressing CHO cells of the present invention cultivated in a 12-well tissue culture plate were assayed with a measurement buffer of 1 m.
After washing twice with 1, 490 μl of measuring buffer is added to each well. 5 μl of the labeled test compound is added, and the mixture is reacted at room temperature for 1 hour. To know the amount of non-specific binding, 5 μl of unlabeled test compound is added. Remove the reaction solution and wash 3 times with 1 ml of wash buffer. The labeled test compound bound to the cells was treated with 0.2 N NaO.
It is dissolved in H-1% SDS and mixed with 4 ml of liquid scintillator A (manufactured by Wako Pure Chemical Industries). Liquid scintillation counter (Beckman)
Is used to measure the radioactivity.

The ligand capable of binding to the receptor protein of the present invention or a salt thereof is, for example,
Examples include substances specifically present in the brain, pituitary gland, heart, pancreas, adipose tissue, mammary gland, testis, etc., and specifically, angiotensin, bombesin, cannabinoids, cholecystokinin, glutamine, serotonin, melatonin, and neuro. Peptide Y, opioid, purine, vasopressin, oxytocin, PACAP (eg, PACAP27,
PACAP38), secretin, glucagon, calcitonin, adrenomedullin, somatostatin, GHR
H, CRF, ACTH, GRP, PTH, VIP (vasoactive intestinal and related polypeptide), somatostatin, dopamine, motilin, amylin, bradykinin, CGRP (calcitonin gene related peptide), leukotriene,
Pancreatatin, prostaglandins, thromboxane, adenosine, adrenaline, chemokine superfamily (eg, IL-8, GROα, GROβ, GR
Oγ, NAP-2, ENA-78, GCP-2, PF
4, CXC chemokine subfamily such as IP-10, Mig, PBSF / SDF-1; MCAF / MCP-
1, MCP-2, MCP-3, MCP-4, eotax
in, RANTES, MIP-1α, MIP-1β, H
CC-1, MIP-3α / LARC, MIP-3β / E
LC, I-309, TARC, MIPF-1, MIPF
-2 / eotaxin-2, MDC, DC-CK1 / P
CC chemokine subfamily such as ARC and SLC;
C chemokine subfamily such as lymphoactin; CX3C chemokine subfamily such as fractalkine), endothelin, enterogastrin, histamine, neurotensin, TRH, pancreatic polypeptide, galanin, lysophosphatidic acid (LPA), sphingosine 1-phosphate. Are used.

(2) Preventive and / or therapeutic agent for diseases associated with dysfunction of G protein-coupled receptor protein of the present invention If the ligand for the receptor protein of the present invention is clarified in the method of (1) above, The receptor protein of the present invention or a DNA encoding the receptor protein according to the action of the ligand
Can be used as a medicament such as a prophylactic and / or therapeutic agent for diseases associated with dysfunction of the receptor protein of the present invention. For example, in the case where there is a patient in whom the physiological action of the ligand cannot be expected due to a decrease in the receptor protein of the present invention in vivo (deficiency of the receptor protein), the receptor protein of the present invention is administered to the patient and the receptor protein By supplementing the amount of the protein, (a) by administering the DNA encoding the receptor protein of the present invention to the patient and expressing it, or (b) adding the DNA encoding the receptor protein of the present invention to target cells. Increasing the amount of receptor protein in the patient's body, such as by transplanting the cells into the patient after insertion and expression,
The action of the ligand can be fully exerted. That is, DN encoding the receptor protein of the present invention
A is useful as a prophylactic and / or therapeutic agent for diseases associated with dysfunction of the receptor protein of the present invention that is safe and has low toxicity. The receptor protein of the present invention is G
HOT7T00, a protein-coupled receptor protein
9 (JP-A-2000-279183), a novel 7-transmembrane receptor protein having a homology of about 35.7% at the amino acid sequence level. The receptor protein of the present invention or the DNA encoding the receptor protein is a central disease (for example, Alzheimer's disease, dementia, eating disorder, etc.), an inflammatory disease (for example, allergy,
Asthma, rheumatism, etc., cardiovascular disease (for example, hypertension, cardiac hypertrophy, angina, arteriosclerosis, etc.), cancer (for example, non-small cell lung cancer, ovarian cancer, prostate cancer, gastric cancer, bladder cancer, breast cancer,
Cervical cancer, colon cancer, rectal cancer etc.), metabolic diseases (eg diabetes, diabetic complications, obesity, arteriosclerosis, gout, cataracts etc.), immune system diseases (eg autoimmune diseases etc.),
Gastrointestinal disorders (eg gastric ulcer, duodenal ulcer, gastritis,
It is useful for prevention and / or treatment of reflux esophagitis and the like. When the receptor protein of the present invention is used as the above-mentioned preventive / therapeutic agent, it can be prepared into a pharmaceutical preparation according to a conventional method. On the other hand, when the DNA encoding the receptor protein of the present invention (hereinafter sometimes abbreviated as the DNA of the present invention) is used as the prophylactic / therapeutic agent, the DNA of the present invention is used alone or in a retrovirus vector or adenovirus. After insertion into a suitable vector such as a vector or an adenovirus associated virus vector, it can be carried out by a conventional method. The DNA of the present invention can be administered as it is or together with an auxiliary agent for promoting uptake by a gene gun or a catheter such as a hydrogel catheter. For example, the receptor protein of the present invention or the DNA encoding the receptor protein is orally administered as tablets, capsules, elixirs, microcapsules, etc., which are optionally coated with sugar, or water or other pharmaceutical agents. It can be used parenterally in the form of an injectable solution such as a sterile solution with an acceptable liquid or a suspension. For example,
The receptor protein of the present invention or the DNA encoding the receptor protein is required for the generally accepted formulation practice together with known physiologically acceptable carriers, flavoring agents, excipients, vehicles, preservatives, stabilizers, binders and the like. It can be manufactured by mixing in the unit dosage form. The amount of active ingredient in these preparations is such that a suitable dose within the indicated range can be obtained.

Additives that can be mixed with tablets, capsules and the like include, for example, binders such as gelatin, corn starch, tragacanth and acacia, excipients such as crystalline cellulose, corn starch, gelatin, alginic acid. Puffing agents such as and the like, lubricants such as magnesium stearate, sweeteners such as sucrose, lactose or saccharin, flavoring agents such as peppermint, red oil or cherry, and the like are used. When the unit dosage form is a capsule, a liquid carrier such as fat may be included in addition to materials of the above type. Sterile compositions for injection can be formulated according to conventional pharmaceutical practices such as dissolving or suspending the active substance in a vehicle such as water for injection, naturally occurring vegetable oils such as sesame oil, coconut oil and the like. As the aqueous solution for injection, for example, physiological saline, isotonic solution containing glucose and other adjuvants (for example, D-sorbitol, D-mannitol, sodium chloride, etc.) and the like are used. For example, it may be used in combination with alcohol (eg, ethanol), polyalcohol (eg, propylene glycol, polyethylene glycol), nonionic surfactant (eg, Polysorbate 80 ^™ , HCO-50). As the oily liquid, for example, sesame oil, soybean oil and the like are used, and may be used in combination with solubilizing agents such as benzyl benzoate and benzyl alcohol.

The above-mentioned prophylactic / therapeutic agents include, for example, buffers (eg, phosphate buffer, sodium acetate buffer), soothing agents (eg, benzalkonium chloride, procaine hydrochloride, etc.), stabilizers ( For example, human serum albumin, polyethylene glycol, etc.), preservatives (eg,
(Benzyl alcohol, phenol, etc.), antioxidant, etc. The prepared injection solution is usually filled in a suitable ampoule. Since the preparation thus obtained is safe and has low toxicity, for example, a mammal (for example,
Human, rat, mouse, rabbit, sheep, pig, cow,
(Cats, dogs, monkeys, etc.). The dose of the receptor protein of the present invention varies depending on the administration subject, target organ, symptom, administration method and the like, but in the case of oral administration, generally, for example, cancer patients (60 k
(as g) about 0.1 mg to 10 per day
0 mg, preferably about 1.0 to 50 mg, more preferably about 1.0 to 20 mg. When administered parenterally, the single dose varies depending on the administration subject, target organ, symptom, administration method, etc .; About 0.01 to 30 mg per day, preferably about 0.
1 to 20 mg, more preferably about 0.1 to 10 mg
It is convenient to administer the degree intravenously. In the case of other animals, the dose converted per 60 kg can be administered. The dose of the DNA of the present invention is
Although there are differences depending on the target organs, symptoms, administration method, etc., in the case of oral administration, generally, for example, in a cancer patient (as 60 kg), about 0.1 mg to 100 m per day
g, preferably about 1.0 to 50 mg, more preferably about 1.0 to 20 mg. When administered parenterally,
Although the single dose varies depending on the administration subject, target organ, symptom, administration method, etc., it is usually in the form of an injection, for example, about 0.01 to 30 mg per day in a cancer patient (as 60 kg). Degree, preferably about 0.1
It is convenient to administer about 20 mg, more preferably about 0.1 to 10 mg by intravenous injection. In the case of other animals, the dose converted per 60 kg can be administered.

(3) Genetic Diagnostic Agent The DNA of the present invention can be used in a mammal (eg, human, rat, mouse, rabbit, sheep, pig, cow, cat, dog, monkey, etc.) by using it as a probe. Since it is possible to detect an abnormality (genetic abnormality) of DNA or mRNA encoding the receptor protein of the invention or its partial peptide, for example, the DNA
Or mRNA damage, mutation or decreased expression,
It is useful as a gene diagnostic agent for increasing or overexpressing the DNA or mRNA. The above-mentioned gene diagnosis using the DNA of the present invention is carried out, for example, by known Northern hybridization or PCR-SSCP method (Genomics, Vol. 5, pp. 874-879 (1989).
,), Proceedings of the National Academy of Sciences o
f the United States of America), Vol. 86, 276
6 to 2770 (1989)) and the like.

(4) Screening method for compounds that change the expression level of the receptor protein of the present invention or its partial peptide: By using the DNA of the present invention as a probe,
It can be used for screening a compound that changes the expression level of the receptor protein of the present invention or its partial peptide. That is, the present invention includes, for example, (i) blood of a non-human mammal, a specific organ, a tissue or cell isolated from an organ, or (ii) a receptor protein of the present invention contained in a transformant or a portion thereof. A method for screening a compound that changes the expression level of the receptor protein of the present invention or a partial peptide thereof by measuring the mRNA level of the peptide is provided.

The mRNA amount of the receptor protein of the present invention or its partial peptide is specifically measured as follows. (I) Normal or disease model Non-human mammal (eg, mouse, rat, rabbit, sheep, pig, cow, cat, dog, monkey, and more specifically, demented rat, obese mouse, arteriosclerotic rabbit, cancer bearing) Mouse)
Blood ( Obtaining a specific organ (eg, adipose tissue, mammary gland, testis, etc.), or tissue or cells isolated from the organ. The mRNA of the receptor protein or its partial peptide of the present invention contained in the obtained cells can be quantified, for example, by extracting mRNA from cells or the like by an ordinary method, and using a technique such as TaqMan PCR, It can also be analyzed by performing Northern blotting by a known means. (Ii) A transformant expressing the receptor protein of the present invention or a partial peptide thereof is produced according to the above method, and the mRNA of the receptor protein of the present invention or a partial peptide thereof contained in the transformant is similarly quantified, Can be analyzed.

Screening for a compound that changes the expression level of the receptor protein of the present invention or its partial peptide is carried out by (i) a certain period of time before a drug or physical stress is applied to a normal or disease model non-human mammal ( 30 minutes to 24 hours ago, preferably 30 minutes to 12 hours ago, more preferably 1 hour to 6 hours before) or after a certain time (30 minutes to 3 days later, preferably 1 hour to 2 days later), More preferably 1 hour to 24 hours later), or the test compound is administered at the same time as the drug or physical stress, and after a lapse of a certain time after the administration (30 minutes to 3 hours).
Days later, preferably 1 hour to 2 days later, more preferably 1 hour
After 24 hours to 24 hours), it can be carried out by quantifying and analyzing the mRNA amount of the receptor protein of the present invention or its partial peptide contained in the cells. After the test compound is mixed in the medium and cultured for a certain period of time (1 day to 7 days later, preferably 1 day to 3 days later, more preferably 2 days to 3 days later),
This can be performed by quantifying and analyzing the amount of mRNA of the receptor protein of the present invention or its partial peptide contained in the transformant.

The compound or its salt obtained by using the screening method of the present invention is a compound having an action of changing the expression level of the receptor protein of the present invention or its partial peptide, and specifically, (a) By increasing the expression level of the receptor protein of the invention or a partial peptide thereof, cell stimulating activity mediated by G protein-coupled receptors (eg, arachidonic acid release, acetylcholine release, intracellular Ca ²⁺ release, intracellular cAMP)
Production, intracellular cAMP production suppression, intracellular cGMP production,
(B) a compound that enhances inositol phosphate production, cell membrane potential fluctuation, phosphorylation of intracellular protein, activation of c-fos, activity of suppressing or lowering pH, etc.), (b) receptor protein of the present invention Alternatively, it is a compound that reduces the cell stimulating activity by reducing the expression level of the partial peptide. Examples of the compound include peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, etc. These compounds may be novel compounds or known compounds. The compound that enhances the cell stimulating activity is useful as a safe and low toxic pharmaceutical for enhancing the physiological activity of the receptor protein of the present invention. The compound that attenuates the cell stimulating activity is useful as a safe and low toxic pharmaceutical for reducing the physiological activity of the receptor protein of the present invention.

When the compound or its salt obtained by the screening method of the present invention is used as a pharmaceutical composition, it can be carried out according to a conventional method. For example,
Tablets, capsules, elixirs, microcapsules, sterile solutions, suspensions and the like can be prepared in the same manner as the above-mentioned pharmaceutical containing the receptor protein of the present invention. Since the preparation thus obtained is safe and has low toxicity, it should be administered to, for example, mammals (eg, humans, rats, mice, rabbits, sheep, pigs, cows, cats, dogs, monkeys). You can The dose of the compound or its salt varies depending on the administration subject, target organ, symptom, administration method, etc., but in the case of oral administration, generally, for example, in a cancer patient (as 60 kg), About 0.1 to 100 mg, preferably about 1.0 to 50 mg, more preferably about 1.0 to 20 m
It is g. When administered parenterally, the single dose varies depending on the administration subject, target organ, symptom, administration method, etc .; About 0.01 per day
~ 30 mg, preferably about 0.1-20 mg,
More preferably, about 0.1 to 10 mg is conveniently administered by intravenous injection. 60 for other animals
An amount converted per kg can be administered.

(5) Preventive and / or therapeutic agent for various diseases containing a compound that changes the expression level of the receptor protein of the present invention or a partial peptide thereof. It is thought to play some important role in the body. Therefore, the compound that changes the expression level of the receptor protein of the present invention or its partial peptide can be used as a prophylactic and / or therapeutic agent for diseases associated with dysfunction of the receptor protein of the present invention. When the compound is used as a prophylactic and / or therapeutic agent for diseases associated with dysfunction of the receptor protein of the present invention, it can be formulated according to a conventional method. For example, the compound is orally as a sugar-coated tablet, capsule, elixir, microcapsule, or the like, or a sterile solution with water or another pharmaceutically acceptable liquid, Alternatively, it can be used parenterally in the form of injection such as suspension. For example, admixing the compound with a known physiologically acceptable carrier, flavoring agent, excipient, vehicle, preservative, stabilizer, binder and the like in a unit dose form generally required for the implementation of the formulation. Can be manufactured by.
The amount of active ingredient in these preparations is such that a suitable dose within the indicated range can be obtained.

Additives that can be mixed with tablets, capsules and the like include, for example, binders such as gelatin, corn starch, tragacanth and gum arabic, excipients such as crystalline cellulose, corn starch, gelatin, alginic acid. Puffing agents such as and the like, lubricants such as magnesium stearate, sweeteners such as sucrose, lactose or saccharin, flavoring agents such as peppermint, red oil or cherry, and the like are used. When the unit dosage form is a capsule, a liquid carrier such as fat may be included in addition to materials of the above type. Sterile compositions for injection can be formulated according to conventional pharmaceutical practices such as dissolving or suspending the active substance in a vehicle such as water for injection, naturally occurring vegetable oils such as sesame oil, coconut oil and the like. As the aqueous solution for injection, for example, physiological saline, isotonic solution containing glucose and other adjuvants (for example, D-sorbitol, D-mannitol, sodium chloride, etc.) and the like are used. For example, it may be used in combination with alcohol (eg, ethanol), polyalcohol (eg, propylene glycol, polyethylene glycol), nonionic surfactant (eg, Polysorbate 80 ^™ , HCO-50). As the oily liquid, for example, sesame oil, soybean oil and the like are used, and may be used in combination with solubilizing agents such as benzyl benzoate and benzyl alcohol.

The above-mentioned prophylactic / therapeutic agents include, for example, buffers (eg, phosphate buffer, sodium acetate buffer), soothing agents (eg, benzalkonium chloride, procaine hydrochloride, etc.), stabilizers ( For example, human serum albumin, polyethylene glycol, etc.), preservatives (eg,
(Benzyl alcohol, phenol, etc.), antioxidant, etc. The prepared injection solution is usually filled in a suitable ampoule. Since the preparation thus obtained is safe and has low toxicity, for example, a mammal (for example,
Human, rat, mouse, rabbit, sheep, pig, cow,
(Cats, dogs, monkeys, etc.). The dose of the compound or its salt varies depending on the administration subject, target organ, condition, administration method, etc., but in the case of oral administration, it is generally, for example, a cancer patient (60 kg).
In the above, about 0.1 to 100 mg, preferably about 1.0 to 50 mg, more preferably about 1.0 to 2 per day.
It is 0 mg. When administered parenterally, the single dose varies depending on the administration subject, target organ, symptom, administration method, etc., but is usually in the form of an injection, for example, in a cancer patient (60 kg), About 0 per day.
It is convenient to administer about 01 to 30 mg, preferably about 0.1 to 20 mg, more preferably about 0.1 to 10 mg by intravenous injection. For other animals,
The amount converted per 60 kg can be administered.

(6) Method for Quantifying Ligand for G Protein-Coupled Receptor Protein of the Present Invention Since the receptor protein and the like of the present invention have binding ability to a ligand, the ligand concentration in vivo can be quantified with high sensitivity. can do. The quantitative method of the present invention is
For example, it can be used in combination with a competitive method. That is, the concentration of the ligand in the sample can be measured by bringing the sample into contact with the receptor protein of the present invention. Specifically, for example, it can be used according to the method described below or the like or a method similar thereto. Hiroshi Irie "Radioimmunoassay" (Kodansha, Showa 4
Published in 9 years) Hiroshi Irie “Radioimmunoassay” (Kodansha, 1979)

(7) Screening Method for Compounds (Agonist, Antagonist, etc.) that Change the Binding between the G Protein-Coupled Receptor Protein of the Present Invention and a Ligand The receptor protein of the present invention is used, or a recombinant receptor protein A compound that changes the binding property between the ligand and the receptor protein or the like of the present invention by constructing an expression system such as, and using a receptor binding assay system using the expression system (for example, peptide, protein, non-peptide compound , Synthetic compounds, fermentation products, etc.) or salts thereof can be efficiently screened. Such compounds include (a) cell stimulating activity via G protein-coupled receptors (eg, arachidonic acid release, acetylcholine release, intracellular Ca ²⁺ release, intracellular cAMP production, intracellular cAMP production inhibition, cell Internal cGMP production, inositol phosphate production, cell membrane potential fluctuation, intracellular protein phosphorylation, c-fos activation, activity to promote or suppress pH decrease, etc. (so-called, of the present invention (A) an agonist for the receptor protein, (b) a compound having no cell stimulating activity (a so-called antagonist for the receptor protein of the present invention), (c) enhancing the binding power between the ligand and the G protein-coupled receptor protein of the present invention Compound, or (d) ligand and G protein-coupled ligand of the present invention Reducing the bonding force between Puta proteins and the like compounds (the compound of the above (b) is preferably screened by the ligand determination methods described above). That is, the present invention provides (i) a case where the receptor protein of the present invention or a partial peptide thereof or a salt thereof is contacted with a ligand, and (ii) a receptor protein of the present invention or a partial peptide thereof or a salt thereof, a ligand and Provided is a method for screening a compound or a salt thereof, which changes the binding property between a ligand and the receptor protein of the present invention or a partial peptide thereof or a salt thereof, which is characterized by performing a comparison with the case of contacting with a test compound.
In the screening method of the present invention, (i) and (i
In the case of i), for example, the amount of ligand binding to the receptor protein or the like, the cell stimulating activity, etc. are measured and compared.

More specifically, the present invention relates to a case where the labeled ligand is brought into contact with the receptor protein of the present invention and a case where the labeled ligand and the test compound are brought into contact with the receptor protein of the present invention. A method for screening a compound or a salt thereof which changes the binding property between the ligand and the receptor protein or the like of the present invention, characterized in that the amount of the labeled ligand bound to the receptor protein or the like is measured and compared. When a cell containing the receptor protein of the present invention or a membrane fraction of the cell is contacted, the labeled ligand and the test compound are added to the cell containing the receptor protein of the present invention or the membrane fraction of the cell. Labeled ligand to the cell or membrane fraction when contacted A method for screening a compound or a salt thereof that changes the binding property between the ligand and the receptor protein or the like of the present invention, characterized by measuring and comparing the total amount, and the labeled ligand is transformed with the DNA of the present invention. The present invention expressed on the cell membrane by contacting the receptor protein expressed on the cell membrane by culturing the body and by culturing the transformant containing the labeled ligand and the test compound containing the DNA of the present invention Or a compound which changes the binding property between the ligand and the receptor protein or the like of the present invention, which is characterized by measuring and comparing the amount of binding of the labeled ligand to the receptor protein or the like when brought into contact with the receptor protein or the like. The salt screening method,

A compound that activates the receptor protein of the present invention (for example, a ligand for the receptor protein of the present invention) is brought into contact with cells containing the receptor protein of the present invention, and the receptor protein of the present invention. When a compound containing a receptor protein or the like of the present invention is brought into contact with a compound that activates etc. and a test compound, a cell-stimulating activity mediated by the receptor (eg, arachidonic acid release, acetylcholine release, intracellular Ca ²⁺ release) , Intracellular cAMP production, intracellular c
AMP production inhibition, intracellular cGMP production, inositol phosphate production, cell membrane potential fluctuation, intracellular protein phosphorylation, c-fos activation, activity promoting or suppressing pH decrease, etc.), A method for screening a compound or a salt thereof that changes the binding property between the ligand and the receptor protein or the like of the present invention, and a compound that activates the receptor protein or the like of the present invention (for example, the receptor protein of the present invention Etc.) and a compound that activates the receptor protein etc. of the present invention, by contacting the receptor protein etc. of the present invention expressed on the cell membrane by culturing the transformant containing the DNA of the present invention And a test compound for culturing a transformant containing the DNA of the present invention. Cell stimulating activities (e.g., via in when contacted with the receptor protein or the like of the present invention expressed on the cell membrane, the receptor by, arachidonic acid release, acetylcholine release, intracellular Ca ²⁺ release, intracellular cAMP production,
Inhibition of intracellular cAMP production, intracellular cGMP production, inositol phosphate production, cell membrane potential fluctuation, intracellular protein phosphorylation, c-fos activation, activity promoting or suppressing pH decrease, etc.) The present invention also provides a method for screening a compound or a salt thereof that changes the binding property between the ligand and the receptor protein or the like of the present invention, which is characterized by comparison.

Before the receptor protein etc. of the present invention was obtained, when screening a G protein-coupled receptor agonist or antagonist, first, cells, tissues or cell membrane fractions thereof containing G protein-coupled receptor protein such as rat were used. Therefore, a test (secondary screening) is required to obtain candidate compounds (primary screening) and then confirm whether the candidate compounds actually inhibit the binding of human G protein-coupled receptor protein to the ligand. there were. If the cell, tissue or cell membrane fraction is used as it is, other receptor proteins also coexist, so it was difficult to actually screen an agonist or antagonist for the target receptor protein. However, for example, by using the human-derived receptor protein of the present invention, the need for primary screening is eliminated, and compounds that inhibit the binding between the ligand and the G protein-coupled receptor protein can be efficiently screened. Furthermore, whether the screened compound is an agonist or an antagonist can be easily evaluated. A detailed description of the screening method of the present invention is given below. First, the receptor protein or the like of the present invention used in the screening method of the present invention may be any as long as it contains the above-described receptor protein of the present invention. The cell membrane fraction of the mammalian organ containing is preferred. However, since human-derived organs are extremely difficult to obtain, human-derived receptor proteins and the like that are expressed in large amounts using recombinants are suitable for use in screening.

The above-mentioned method is used to produce the receptor protein and the like of the present invention, but it is preferable to express the DNA of the present invention in mammalian cells or insect cells. DNA encoding the desired protein portion
Although complementary DNA is used for the fragment, it is not necessarily limited thereto. For example, gene fragments and synthetic DN
A may be used. To introduce the DNA fragment encoding the receptor protein of the present invention into host animal cells and express them efficiently, in order to efficiently express them, a nuclear polyhedrosis virus belonging to a baculovirus whose insect is a host is used. (NPV) polyhedrin promoter, SV40-derived promoter, retrovirus promoter, metallothionein promoter, human heat shock promoter, cytomegalovirus promoter, SRα promoter and the like are preferably incorporated downstream. The amount and quality of the expressed receptor can be examined by a known method. For example, the literature [Nambi, P. Et al., The Journal of Biological Chemistry (J. Biol. Chem.), Vol. 267, 19555-19.
559, 1992]. Therefore, in the screening method of the present invention, the receptor protein or the like of the present invention may be a receptor protein or the like purified according to a known method, or a cell containing the receptor protein or the like may be used. Alternatively, a membrane fraction of cells containing the receptor protein or the like may be used.

When cells containing the receptor protein of the present invention are used in the screening method of the present invention, the cells may be immobilized with glutaraldehyde, formalin or the like. The immobilization method can be performed according to a known method. The cell containing the receptor protein or the like of the present invention refers to a host cell expressing the receptor protein or the like. Examples of the host cell include Escherichia coli,
Bacillus subtilis, yeast, insect cells, animal cells and the like are preferable. The cell membrane fraction refers to a fraction containing a large amount of cell membrane obtained by a known method after crushing cells. As a method of crushing cells, a method of crushing cells with a Potter-Elvehjem type homogenizer, crushing with a Waring blender or polytron (Kinematica), crushing with ultrasonic waves, ejection of cells from a thin nozzle while applying pressure with a French press, etc. For example, crushing by allowing it to occur. For the fractionation of the cell membrane, a fractionation method by centrifugal force such as a fractionation centrifugation method or a density gradient centrifugation method is mainly used. For example, the cell lysate may be fed at a low speed (500 rpm to 3000 rp).
m) for a short time (usually about 1 minute to 10 minutes), and the supernatant is further centrifuged at a higher speed (15000 rpm to 30000 rpm) for usually 30 minutes to 2 hours, and the resulting precipitate is used as a membrane fraction. The membrane fraction is rich in expressed receptor proteins and other membrane components such as cell-derived phospholipids and membrane proteins. The amount of the receptor protein in the cell or the membrane fraction containing the receptor protein or the like is preferably 10 ³ to 10 ⁸ molecules per cell, and 10 ⁵ to 10 ⁷
It is preferably a molecule. The higher the expression level, the higher the ligand binding activity (specific activity) per membrane fraction,
Not only will it be possible to construct a highly sensitive screening system, but it will also be possible to measure a large number of samples in the same lot.

In order to carry out the above steps (1) to (3) for screening a compound that alters the binding property between the ligand and the receptor protein or the like of the present invention, for example, an appropriate receptor protein fraction and a labeled ligand are required. As the receptor protein fraction, a natural receptor protein fraction or a recombinant receptor protein fraction having an activity equivalent to that is desirable. Here, the equivalent activity refers to equivalent ligand binding activity, signal transduction action, and the like. As the labeled ligand, a labeled ligand, a labeled ligand analog compound or the like is used. For example, [ ³ H],
A ligand labeled with [ ¹²⁵ I], [ ¹⁴ C], [ ³⁵ S] or the like is used. Specifically, in order to screen a compound that changes the binding property between the ligand and the receptor protein or the like of the present invention, first, cells or cell membrane fractions containing the receptor protein or the like of the present invention are
A receptor protein preparation is prepared by suspending in a buffer suitable for screening. Any buffer may be used as long as it does not inhibit the binding between the ligand and the receptor protein, such as a phosphate buffer having a pH of 4 to 10 (preferably a pH of 6 to 8) and a Tris-hydrochloric acid buffer. Further, for the purpose of reducing non-specific binding, a surfactant such as CHAPS, Tween-80 ^™ (Kao-Atlas), digitonin and deoxycholate may be added to the buffer. Further, a protease inhibitor such as PMSF, leupeptin, E-64 (manufactured by Peptide Laboratories), pepstatin or the like can be added for the purpose of suppressing the decomposition of the receptor or the ligand by the protease. A fixed amount (5000 cpm to 500,000 cp) was added to 0.01 ml to 10 ml of the receptor solution.
m) labeled ligand was added, and at the same time 10 ⁻⁴ M to 1 was added.
Coexist with 0 ^-10 M of test compound. A reaction tube containing a large excess of unlabeled ligand is also prepared in order to know the amount of non-specific binding (NSB). The reaction is about 0 to 50
C., preferably about 4 to 37.degree. C. for about 20 minutes to 24.
The time is preferably about 30 minutes to 3 hours. After the reaction, the mixture is filtered through a glass fiber filter paper or the like, washed with an appropriate amount of the same buffer, and the radioactivity remaining on the glass fiber filter paper is measured with a liquid scintillation counter or a γ-counter. Count (B ₀ -NS) obtained by subtracting the amount of non-specific binding (NSB) from the count (B ₀ ) in the absence of an antagonist.
Specific binding amount (B-NSB) when B) is 100%
However, for example, a test compound having 50% or less can be selected as a candidate substance capable of competitive inhibition.

In order to carry out the above methods (1) to (3) for screening a compound that changes the binding property between a ligand and the receptor protein or the like of the present invention, for example, cell stimulating activity mediated by the receptor protein (eg, arachidonic acid release, acetylcholine). Release, intracellular Ca ²⁺ release,
Intracellular cAMP production, intracellular cAMP production inhibition, intracellular cGMP production, inositol phosphate production, cell membrane potential fluctuation, intracellular protein phosphorylation, c-fos activation, pH promoting activity or inhibition, etc. Activity etc.) can be measured using a known method or a commercially available measurement kit. Specifically, first, cells containing the receptor protein or the like of the present invention are cultured in a multiwell plate or the like. For screening, the medium was exchanged with a fresh medium or an appropriate buffer that did not show toxicity to cells in advance, and after adding a test compound or the like and incubating for a certain period of time, cells were extracted or supernatant was collected to generate The product is quantified according to the respective method. When the production of a substance that serves as an index of cell stimulating activity (for example, arachidonic acid) is difficult to assay with a degrading enzyme contained in cells, an inhibitor for the degrading enzyme may be added to the assay. Further, the activity of suppressing cAMP production and the like can be detected as a production suppressing action on cells in which the basic production amount of cells has been increased by forskolin or the like. In order to measure the cell stimulating activity and perform screening, cells expressing an appropriate receptor protein are required. As a cell expressing the receptor protein of the present invention, a cell line having the natural receptor protein of the present invention, a cell line expressing the above-mentioned recombinant receptor protein or the like is preferable. As the test compound, for example, peptides, proteins, non-peptide compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, etc. are used, and these compounds may be novel compounds. It may be a known compound.

A kit for screening a compound or a salt thereof that alters the binding property between a ligand and the receptor protein or the like of the present invention comprises a cell containing the receptor protein or the like of the present invention, a cell containing the receptor protein or the like of the present invention,
Alternatively, it contains a membrane fraction of cells containing the receptor protein or the like of the present invention. Examples of the screening kit of the present invention include the following. 1. To the screening reagent measurement buffer and washing buffer Hanks' Balanced Salt Solution (manufactured by Gibco), 0.
To which 05% bovine serum albumin (manufactured by Sigma) was added. Sterilize by filtration with a 0.45μm filter at 4 ℃
It may be stored at or prepared at the time of use. G protein-coupled receptor preparation CHO cells expressing the receptor protein of the present invention were subcultured on a 12-well plate at 5 × 10 ⁵ cells / well, and 37
Cultivated at 5 ° C., 5% CO ₂ , 95% air for 2 days. Labeled ligand Commercially available ligands labeled with [ ³ H], [ ¹²⁵ I], [ ¹⁴ C], [ ³⁵ S], etc. are stored at 4 ° C or -20 ° C and used as a measurement buffer at the time of use. Dilute to 1 μM with solution. Ligand standard solution 0.1% bovine serum albumin (manufactured by Sigma)
Dissolve to 1 mM in PBS containing and store at -20 ° C.

2. Assay method Receptor protein-expressing CHO cells of the present invention cultivated in a 12-well tissue culture plate were assayed with a measurement buffer of 1 m.
After washing twice with 1, 490 μl of measuring buffer is added to each well. After adding 5 μl of a test compound solution of 10 ⁻³ to 10 ⁻¹⁰ M, 5 μl of a labeled ligand is added, and the mixture is reacted at room temperature for 1 hour. To determine the amount of non-specific binding, 5 μl of 10 ⁻³ M ligand was added in place of the test compound. Remove the reaction solution and wash 3 times with 1 ml of wash buffer. Labeled ligand bound to cells is 0.2N NaOH
Dissolve in -1% SDS, 4 ml of liquid scintillator A
Mix with (Wako Pure Chemical Industries). Liquid scintillation counter (Beckman)
Radioactivity was measured using a Percent Maximum Binding
(PMB) is calculated by the following formula. PMB = [(B-NSB) / (B 0 -NSB)] × 10
0 PMB: Percent Maximum Binding B: Value when a sample is added NSB: Non-specific Binding B ₀ : Maximum binding amount

The compound or its salt obtained by using the screening method or the screening kit of the present invention is a compound having an action of changing the binding property between the ligand and the receptor protein or the like of the present invention. (A) Cell stimulating activity via G protein-coupled receptors (eg, arachidonic acid release, acetylcholine release, intracellular Ca ²⁺ release, intracellular cAMP production, intracellular cAMP production inhibition, intracellular cGMP production, inositol phosphate A compound (so-called agonist for the receptor protein of the present invention) having an activity of promoting or suppressing production, cell membrane potential fluctuation, phosphorylation of intracellular protein, activation of c-fos, lowering of pH, etc., ( B) A compound that does not have the cell stimulating activity (so-called the receptor of the present invention). -An antagonist to a protein), (c) a compound that enhances the binding force between the ligand and the G protein-coupled receptor protein of the present invention, or (d) a reduction in the binding force between the ligand and the G protein-coupled receptor protein of the present invention It is a compound that causes. Examples of the compound include peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, etc. These compounds may be novel compounds or known compounds. The agonist for the receptor protein or the like of the present invention has the same physiological activity as that of the ligand for the receptor protein or the like of the present invention, and is therefore useful as a safe and low-toxic pharmaceutical according to the ligand activity. Since the antagonist for the receptor protein etc. of the present invention can suppress the physiological activity possessed by the ligand for the receptor protein etc. of the present invention, it is useful as a safe and low toxic pharmaceutical that suppresses the ligand activity. The compound that enhances the binding force between the ligand and the G protein-coupled receptor protein of the present invention is useful as a safe and low toxic pharmaceutical for enhancing the physiological activity of the ligand for the receptor protein of the present invention. The compound that reduces the binding force between the ligand and the G protein-coupled receptor protein of the present invention is useful as a safe and low-toxic pharmaceutical agent for reducing the physiological activity of the ligand for the receptor protein of the present invention.

When the compound or its salt obtained by using the screening method or the screening kit of the present invention is used as the above-mentioned pharmaceutical composition, it can be carried out by a conventional means. For example, tablets, capsules, elixirs, microcapsules, similar to the drug containing the receptor protein of the present invention described above,
It can be a sterile solution, suspension or the like. Since the preparation thus obtained is safe and has low toxicity, it should be administered to, for example, mammals (eg, humans, rats, mice, rabbits, sheep, pigs, cows, cats, dogs, monkeys). You can The dose of the compound or its salt varies depending on the administration subject, target organ, condition, administration method, etc., but in the case of oral administration, generally, for example, in a cancer patient (as 60 kg), the daily dose is about 0.
The amount is 1 to 100 mg, preferably about 1.0 to 50 mg, more preferably about 1.0 to 20 mg. When administered parenterally, the single dose varies depending on the administration subject, target organ, symptom, administration method, etc., but is usually in the form of an injection, for example, in a cancer patient (as 60 kg), About 0.01 to 30 mg per day, preferably about 0.1 to 20 mg, more preferably about 0.1 to
It is convenient to administer about 10 mg by intravenous injection. In the case of other animals, the dose converted per 60 kg can be administered.

(8) Preventive and / or therapeutic agent for various diseases containing a compound (agonist, antagonist) that changes the binding property between the G protein-coupled receptor protein of the present invention and a ligand. The receptor protein of the present invention is as described above. As described above, it is considered that they play some important roles in the body such as central function, circulatory function and digestive function. Therefore, the compound (agonist, antagonist) that changes the binding property between the receptor protein of the present invention and the ligand and the ligand for the receptor protein of the present invention are used for the prevention and / or treatment of diseases associated with the dysfunction of the receptor protein of the present invention. It can be used as an agent. When the compound or ligand is used as a prophylactic and / or therapeutic agent for a disease associated with dysfunction of the receptor protein of the present invention, it can be formulated according to a conventional method. For example, the compound or ligand may be orally administered as a sugar-coated tablet, capsule, elixir, microcapsule, etc., or aseptic with water or other pharmaceutically acceptable liquid. It can be used parenterally in the form of injection such as solution or suspension. For example, admixing the compound with a known physiologically acceptable carrier, flavoring agent, excipient, vehicle, preservative, stabilizer, binder and the like in a unit dose form generally required for the implementation of the formulation. Can be manufactured by. The amount of active ingredient in these preparations is such that a suitable dose within the indicated range can be obtained.

Additives that can be mixed with tablets, capsules and the like include, for example, binders such as gelatin, corn starch, tragacanth and acacia, excipients such as crystalline cellulose, corn starch, gelatin, alginic acid. Puffing agents such as and the like, lubricants such as magnesium stearate, sweetening agents such as sucrose, lactose or saccharin, flavoring agents such as peppermint, red oil or cherry and the like are used. When the unit dosage form is a capsule, a liquid carrier such as fat may be included in addition to materials of the above type. Sterile compositions for injection can be formulated according to conventional pharmaceutical practices such as dissolving or suspending the active substance in a vehicle such as water for injection, naturally occurring vegetable oils such as sesame oil, coconut oil and the like. As the aqueous solution for injection, for example, physiological saline, isotonic solution containing glucose and other adjuvants (for example, D-sorbitol, D-mannitol, sodium chloride, etc.) and the like are used. For example, it may be used in combination with alcohol (eg, ethanol), polyalcohol (eg, propylene glycol, polyethylene glycol), nonionic surfactant (eg, Polysorbate 80 ^™ , HCO-50). As the oily liquid, for example, sesame oil, soybean oil and the like are used, and may be used in combination with solubilizing agents such as benzyl benzoate and benzyl alcohol.

The above-mentioned prophylactic / therapeutic agents include, for example, buffers (eg, phosphate buffer, sodium acetate buffer), soothing agents (eg, benzalkonium chloride, procaine hydrochloride), stabilizers ( For example, human serum albumin, polyethylene glycol, etc.), preservatives (eg,
(Benzyl alcohol, phenol, etc.), antioxidant, etc. The prepared injection solution is usually filled in a suitable ampoule. Furthermore, the above-mentioned prophylactic / therapeutic agents can be used in combination with an appropriate agent as a DDS preparation specifically targeting an organ or tissue in which the receptor protein of the present invention is highly expressed. Since the preparation thus obtained is safe and has low toxicity, it should be administered to, for example, mammals (eg, humans, rats, mice, rabbits, sheep, pigs, cows, cats, dogs, monkeys). You can The dose of the compound or its salt varies depending on the administration subject, target organ, condition, administration method, etc., but in the case of oral administration, generally, for example, in a cancer patient (as 60 kg), the daily dose is about 0.
The amount is 1 to 100 mg, preferably about 1.0 to 50 mg, more preferably about 1.0 to 20 mg. When administered parenterally, the single dose varies depending on the administration subject, target organ, symptom, administration method, etc., but is usually in the form of an injection, for example, in a cancer patient (as 60 kg), About 0.01 to 30 mg per day, preferably about 0.1 to 20 mg, more preferably about 0.1 to
It is convenient to administer about 10 mg by intravenous injection. In the case of other animals, the dose converted per 60 kg can be administered.

(9) Quantification of Receptor Protein of the Present Invention or Partial Peptide or Salt thereof Since the antibody of the present invention can specifically recognize the receptor protein of the present invention, the present invention in a test solution Can be used for the quantification of the receptor protein, etc., especially by the sandwich immunoassay. That is, the present invention comprises, for example, (i) measuring the ratio of the labeled receptor protein or the like bound to the antibody by competitively reacting the antibody of the present invention with a test solution, a labeled receptor protein or the like. A method for quantifying the receptor protein or the like of the present invention in a test liquid, which comprises (ii) the test liquid and the antibody of the present invention insolubilized on a carrier and the labeled antibody of the present invention simultaneously or successively The method for quantifying the receptor protein or the like of the present invention in a test liquid is characterized by measuring the activity of the labeling agent on the insolubilized carrier after the reaction with the above. In the above (ii), it is preferable that one antibody recognizes the N-terminal portion of the receptor protein of the present invention and the other antibody reacts with the C-terminal portion of the receptor protein of the present invention. .

In addition to the measurement of the receptor protein of the present invention using a monoclonal antibody against the receptor protein of the present invention (hereinafter sometimes referred to as the monoclonal antibody of the present invention), detection by tissue staining or the like is also performed. You can also For these purposes, the antibody molecule itself may be used, or F (ab ′) _{2 of} the antibody molecule,
Fab 'or Fab fraction may be used. The measuring method using an antibody against the receptor protein or the like of the present invention is not particularly limited, and the amount of the antibody, the antigen or the antibody-antigen complex corresponding to the amount of the antigen in the liquid to be measured (for example, the amount of the receptor protein) is not limited. Any measuring method may be used as long as it is a method of detecting the amount by chemical or physical means and calculating it from a standard curve prepared using a standard solution containing a known amount of antigen. For example, nephrometry, competitive method, immunometric method and sandwich method are preferably used, but from the viewpoint of sensitivity and specificity, the sandwich method described later is particularly preferable.
As the labeling agent used in the assay method using a labeling substance,
For example, radioisotopes, enzymes, fluorescent substances, luminescent substances, etc. are used. Examples of radioisotopes include, for example, [
¹²⁵ I], [ ¹³¹ I], [ ³ H], [ ¹⁴ C] and the like are used. As the above-mentioned enzyme, those having a stable and large specific activity are preferable, and for example, β-galactosidase, β-glucosidase, alkaline phosphatase, peroxidase, malate dehydrogenase and the like are used. As the fluorescent substance, for example, fluorescamine, fluorescein isothiocyanate, etc. are used. Luminol, a luminol derivative, luciferin, lucigenin, etc. are used as a luminescent substance, for example. Furthermore, the biotin-avidin system can be used for binding the antibody or the antigen to the labeling agent.

For the insolubilization of an antigen or an antibody, physical adsorption may be used, or a method using a chemical bond which is usually used for insolubilizing or immobilizing a protein, an enzyme or the like may be used. As the carrier, for example, insoluble polysaccharides such as agarose, dextran and cellulose, synthetic resins such as polystyrene, polyacrylamide and silicon, or glass is used. In the sandwich method, the insolubilized monoclonal antibody of the present invention is reacted with a test solution (first reaction), and further the labeled monoclonal antibody of the present invention is reacted (secondary reaction), and then the labeling agent on the insolubilized carrier is used. The amount of the receptor protein of the present invention in the test liquid can be quantified by measuring the activity of the. The primary reaction and the secondary reaction may be performed in reverse order, may be performed simultaneously, or may be performed at different times. The labeling agent and the method of insolubilization can be the same as those described above. In the immunoassay by the sandwich method, the antibody used as the solid phase antibody or the labeling antibody does not necessarily have to be one type, and a mixture of two or more types of antibodies may be used for the purpose of improving the measurement sensitivity. May be. In the method for measuring a receptor protein and the like by the sandwich method of the present invention, the monoclonal antibodies of the present invention used in the primary reaction and the secondary reaction are preferably antibodies having different binding sites for the receptor protein and the like. That is, the antibody used in the primary reaction and the secondary reaction is, for example, the antibody used in the secondary reaction,
When recognizing the C-terminal portion of the receptor protein, the antibody used in the primary reaction is preferably an antibody that recognizes other than the C-terminal portion, for example, the N-terminal portion.

The monoclonal antibody of the present invention can be used in a measuring system other than the sandwich method, for example, a competitive method, an immunometric method or nephrometry. In the competitive method, the antigen in the test liquid and the labeled antigen are reacted competitively with the antibody, and then the unreacted labeled antigen is used.
(F) and the labeled antigen (B) bound to the antibody are separated (B
/ F separation), the labeled amount of either B or F is measured to quantify the amount of antigen in the test liquid. In this reaction method, a soluble antibody is used as an antibody, and B / F separation is performed using polyethylene glycol,
A liquid phase method using a second antibody or the like against the above antibody, and using a solid phase antibody as the first antibody, or
The first antibody is soluble and the second antibody is a solid-phased antibody. In the immunometric method, the antigen in the test liquid and the solid-phased antigen are competitively reacted with a fixed amount of the labeled antibody and then the solid phase and the liquid phase are separated, or the antigen in the test liquid is separated. After reacting with an excess amount of the labeled antibody and then adding the immobilized antigen to bind the unreacted labeled antibody to the solid phase, the solid phase and the liquid phase are separated. Next, the labeled amount of either phase is measured to quantify the amount of antigen in the test liquid. In nephrometry, the amount of insoluble precipitate generated as a result of the antigen-antibody reaction in the gel or in the solution is measured. Even when the amount of antigen in the test liquid is small and only a small amount of precipitate can be obtained, laser nephrometry utilizing laser scattering is preferably used.

In applying these individual immunological assay methods to the assay method of the present invention, it is not necessary to set special conditions, operations and the like. The assay system for the receptor protein of the present invention or a salt thereof may be constructed by adding ordinary technical consideration to those skilled in the art to the ordinary conditions and operating methods in each method. For details of these general technical means, reference can be made to reviews, textbooks, etc. [eg,
Hiroshi Irie "Radioimmunoassay" (Kodansha, Showa 4
(Published in 1997), edited by Hiroshi Irie, "Continued Radioimmunoassay"
(Kodansha, published in 1979), Eiji Ishikawa et al., “Enzyme Immunoassay” (medical bookstore, published in 1978), Eiji Ishikawa, et al., “Enzyme Immunoassay” (second edition) (medical study, 1982) Issue), Eiji Ishikawa et al. "Enzyme Immunoassay" (3rd edition)
(Medical Shoin, published in 1987), "Methods in ENZYMOLOGY" Vol. 70 (Im
munochemical Techniques (Part A)), ibid Vol. 73 (Im
munochemical Techniques (Part B)), ibid Vol. 74 (Im
munochemical Techniques (Part C)), ibid Vol. 84 (Im
munochemical Techniques (Part D: Selected Immunoass
ays)), ibid.Vol. 92 (Immunochemical Techniques (Par
t E: Monoclonal Antibodies and General Immunoassay
Methods)), ibid.Vol. 121 (Immunochemical Techniques)
(Part I: Hybridoma Technology and Monoclonal Antib
odies)) (issued by Academic Press, etc.). As described above, the receptor protein of the present invention or its salt can be quantified with high sensitivity by using the antibody of the present invention. Furthermore, by quantifying the receptor protein of the present invention or a salt thereof in vivo using the antibody of the present invention, various diseases associated with dysfunction of the receptor protein of the present invention can be diagnosed. Further, the antibody of the present invention can be used for specifically detecting the receptor protein or the like of the present invention present in a subject such as body fluid or tissue. In addition, preparation of an antibody column used for purifying the receptor protein of the present invention, detection of the receptor protein of the present invention in each fraction during purification, of the behavior of the receptor protein of the present invention in a test cell It can be used for analysis etc.

(10) Method for screening compound that changes amount of receptor protein of the present invention or partial peptide thereof in cell membrane The antibody of the present invention specifically recognizes receptor protein of the present invention or partial peptide thereof or a salt thereof. Therefore, it can be used for screening a compound that alters the amount of the receptor protein of the present invention or its partial peptide in the cell membrane. That is, the present invention provides, for example, (i) the present invention, which comprises the cell membrane fraction after destroying blood of a non-human mammal, a specific organ, a tissue or cell isolated from the organ, and the like, and isolating the cell membrane fraction. Method for screening a compound that changes the amount of the receptor protein of the present invention or its partial peptide in the cell membrane by quantifying the receptor protein of the present invention or its partial peptide, (ii) a trait expressing the receptor protein of the present invention or its partial peptide After disrupting the transformant etc., the cell membrane fraction is isolated, and the amount of the receptor protein of the present invention or its partial peptide in the cell membrane is determined by quantifying the receptor protein of the present invention or its partial peptide contained in the cell membrane fraction. A method for screening a compound to be changed, (ii
i) Quantify the degree of staining of the receptor protein on the cell surface by using immunostaining after sectioning blood, specific organs, tissues or cells isolated from organs of non-human mammals Thus, a method for screening a compound that changes the amount of the receptor protein of the present invention or its partial peptide in the cell membrane by confirming the protein on the cell membrane is provided. (Iv) After sectioning a transformant or the like expressing the receptor protein of the present invention or a partial peptide thereof,
The amount of the receptor protein of the present invention or its partial peptide in the cell membrane by confirming the protein on the cell membrane by quantifying the degree of staining of the receptor protein on the cell surface by using an immunostaining method. A method for screening a compound that changes

The receptor protein of the present invention or its partial peptide contained in the cell membrane fraction is quantified specifically as follows. (I) Normal or disease model Non-human mammal (eg, mouse, rat, rabbit, sheep, pig, cow, cat, dog, monkey, and more specifically, demented rat, obese mouse, arteriosclerotic rabbit, cancer bearing) Mouse)
Blood ( Obtaining a specific organ (eg, adipose tissue, mammary gland, testis, etc.), or tissue or cells isolated from the organ. The obtained organ, tissue or cell is suspended in, for example, an appropriate buffer solution (eg, Tris-HCl buffer solution, phosphate buffer solution, Hepes buffer solution, etc.) to destroy the organ, tissue or cell, A cell membrane fraction is obtained by using an activator (for example, Triton X-100 ^™ , Tween 20 ^™, etc.) and the like, and further by a method such as centrifugation, filtration, or column fractionation.

As the cell membrane fraction, after crushing the cells,
It means a fraction containing a large amount of cell membranes obtained by a known method. As a method of crushing cells, a method of crushing cells with a Potter-Elvehjem type homogenizer, crushing with a Waring blender or polytron (Kinematica), crushing with ultrasonic waves, ejection of cells from a thin nozzle while applying pressure with a French press, etc. For example, crushing by allowing it to occur. For the fractionation of the cell membrane, a fractionation method by centrifugal force such as a fractionation centrifugation method or a density gradient centrifugation method is mainly used. For example, the cell lysate may be mixed at low speed (500 rpm to 3
Centrifuge at 000 rpm for a short time (usually about 1 to 10 minutes), and then the supernatant is spun at a higher speed (15,000 to 30,000 rpm).
It is usually centrifuged for 30 minutes to 2 hours at (rpm) and the resulting precipitate is used as a membrane fraction. The membrane fraction is rich in expressed receptor proteins and other membrane components such as cell-derived phospholipids and membrane proteins.

The receptor protein of the present invention or its partial peptide contained in the cell membrane fraction can be quantified by, for example, a sandwich immunoassay using the antibody of the present invention, Western blot analysis and the like. Such sandwich immunoassay can be performed in the same manner as the above method, and Western blotting can be performed by a known means.

(Ii) A transformant expressing the receptor protein of the present invention or its partial peptide can be prepared according to the above method, and the receptor protein of the present invention contained in the cell membrane fraction or its partial peptide can be quantified. .

Screening for a compound that changes the amount of the receptor protein of the present invention or its partial peptide in the cell membrane is carried out by (i) a certain period of time before a drug or physical stress is applied to a normal or disease model non-human mammal. (30 minutes to 24 hours ago, preferably 30 minutes to 12 hours ago, more preferably 1 hour ago to 6 hours
Before time) or after a certain period of time (after 30 minutes to 3 days, preferably after 1 hour to 2 days, more preferably after 1 hour to 2)
4 hours later), or the test compound is administered at the same time as the drug or physical stress, and after a lapse of a certain period of time (30 minutes to 3 days later, preferably 1 hour to 2 days later, more preferably 1 hour later). 24 hours later), the amount of the receptor protein of the present invention or its partial peptide in the cell membrane can be quantified, and (ii) the test compound is mixed with the medium when the transformant is cultured according to a conventional method. After culturing for a certain period of time (after 1 to 7 days, preferably after 1 to 3 days, more preferably after 2 to 3 days), the amount of the receptor protein of the present invention or its partial peptide in the cell membrane is quantified. be able to.

The receptor protein of the present invention or its partial peptide contained in the cell membrane fraction is specifically confirmed as follows. (Iii) normal or disease model non-human mammal (eg, mouse, rat, rabbit,
For sheep, pigs, cows, cats, dogs, monkeys, and the like, more specifically, for demented rats, obese mice, arteriosclerotic rabbits, cancer-bearing mice, etc., drugs (eg, anti-dementia drugs, blood pressure lowering drugs, anti-cancer drugs) , Anti-obesity drugs) or physical stress (eg flooding stress, electric shock, light and dark, low temperature, etc.) and after a certain period of time, blood or specific organs (eg adipose tissue, mammary gland, testis etc.) ), Or tissue or cells isolated from an organ. The obtained organs, tissues, cells or the like are cut into tissue sections according to a conventional method and immunostained with the antibody of the present invention. By quantifying the degree of staining of the receptor protein on the cell surface, by confirming the protein on the cell membrane, the amount of the receptor protein of the present invention or its partial peptide on the cell membrane can be quantitatively or qualitatively determined. You can check. (Iv) It can also be confirmed by the same procedure using a transformant expressing the receptor protein of the present invention or its partial peptide.

The compound or its salt obtained by using the screening method of the present invention is a compound having an action of changing the amount of the receptor protein of the present invention or its partial peptide in the cell membrane, and specifically, (a)
By increasing the amount of the receptor protein of the present invention or a partial peptide thereof in the cell membrane, cell stimulating activity mediated by G protein-coupled receptors (eg,
Arachidonic acid release, acetylcholine release, intracellular Ca ²⁺
Release, intracellular cAMP production, intracellular cAMP production suppression,
(B) A compound that enhances intracellular cGMP production, inositol phosphate production, cell membrane potential fluctuation, intracellular protein phosphorylation, c-fos activation, activity promoting or suppressing pH decrease, etc. (b) A compound that reduces the cell stimulating activity by reducing the amount of the receptor protein of the present invention or its partial peptide in the cell membrane. Examples of the compound include peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, etc. These compounds may be novel compounds or known compounds. The compound that enhances the cell stimulating activity is useful as a safe and low toxic pharmaceutical for enhancing the physiological activity of the receptor protein of the present invention. The compound that attenuates the cell stimulating activity is useful as a safe and low toxic pharmaceutical for reducing the physiological activity of the receptor protein of the present invention.

When the compound or its salt obtained by the screening method of the present invention is used as a pharmaceutical composition, it can be carried out according to a conventional method. For example,
Tablets, capsules, elixirs, microcapsules, sterile solutions, suspensions and the like can be prepared in the same manner as the above-mentioned pharmaceutical containing the receptor protein of the present invention. Since the preparation thus obtained is safe and has low toxicity, it should be administered to, for example, mammals (eg, humans, rats, mice, rabbits, sheep, pigs, cows, cats, dogs, monkeys). You can The dose of the compound or its salt varies depending on the administration subject, target organs, symptoms, administration method, etc., but in the case of oral administration, generally, for example, in a cancer patient (as 60 kg), the daily dose is about 0.1-100 mg, preferably about 1.0-50 mg, more preferably about 1.0-20 mg
Is. When administered parenterally, the single dose varies depending on the administration subject, target organ, symptom, administration method, etc .; About 0.01 per day
~ 30 mg, preferably about 0.1-20 mg,
More preferably, about 0.1 to 10 mg is conveniently administered by intravenous injection. 60 for other animals
An amount converted per kg can be administered.

(11) A preventive and / or therapeutic agent for various diseases containing a compound that alters the amount of the receptor protein of the present invention or its partial peptide in the cell membrane. The receptor protein of the present invention is as described above, for example, the central function and the like. It is considered to play some important role in the living body. Therefore, the compound that changes the amount of the receptor protein of the present invention or its partial peptide in the cell membrane can be used as a prophylactic and / or therapeutic agent for diseases associated with dysfunction of the receptor protein of the present invention. When the compound is used as a prophylactic and / or therapeutic agent for diseases associated with dysfunction of the receptor protein of the present invention, it can be formulated according to a conventional method. For example, the compound is orally as a sugar-coated tablet, capsule, elixir, microcapsule, or the like, or a sterile solution with water or another pharmaceutically acceptable liquid, Alternatively, it can be used parenterally in the form of injection such as suspension. For example, admixing the compound with a known physiologically acceptable carrier, flavoring agent, excipient, vehicle, preservative, stabilizer, binder and the like in a unit dose form generally required for the implementation of the formulation. Can be manufactured by. The amount of active ingredient in these preparations is such that a suitable dose within the indicated range can be obtained.

Additives that can be mixed with tablets, capsules and the like include, for example, binders such as gelatin, corn starch, tragacanth and acacia, excipients such as crystalline cellulose, corn starch, gelatin, alginic acid. Puffing agents such as and the like, lubricants such as magnesium stearate, sweetening agents such as sucrose, lactose or saccharin, flavoring agents such as peppermint, red oil or cherry and the like are used. When the unit dosage form is a capsule, a liquid carrier such as fat may be included in addition to materials of the above type. Sterile compositions for injection can be formulated according to conventional pharmaceutical practices such as dissolving or suspending the active substance in a vehicle such as water for injection, naturally occurring vegetable oils such as sesame oil, coconut oil and the like. As the aqueous solution for injection, for example, physiological saline, isotonic solution containing glucose and other adjuvants (for example, D-sorbitol, D-mannitol, sodium chloride, etc.) and the like are used. For example, it may be used in combination with alcohol (eg, ethanol), polyalcohol (eg, propylene glycol, polyethylene glycol), nonionic surfactant (eg, Polysorbate 80 ^™ , HCO-50). As the oily liquid, for example, sesame oil, soybean oil and the like are used, and may be used in combination with solubilizing agents such as benzyl benzoate and benzyl alcohol.

The above-mentioned prophylactic / therapeutic agents include, for example, buffers (eg, phosphate buffer, sodium acetate buffer), soothing agents (eg, benzalkonium chloride, procaine hydrochloride), stabilizers ( For example, human serum albumin, polyethylene glycol, etc.), preservatives (eg,
(Benzyl alcohol, phenol, etc.), antioxidant, etc. The prepared injection solution is usually filled in a suitable ampoule. Since the preparation thus obtained is safe and has low toxicity, for example, a mammal (for example,
Human, rat, mouse, rabbit, sheep, pig, cow,
(Cats, dogs, monkeys, etc.). The dose of the compound or its salt varies depending on the administration subject, target organ, condition, administration method, etc., but in the case of oral administration, it is generally, for example, a cancer patient (60 kg).
In the above, about 0.1 to 100 mg, preferably about 1.0 to 50 mg, more preferably about 1.0 to 2 per day.
It is 0 mg. When administered parenterally, the single dose varies depending on the administration subject, target organ, symptom, administration method, etc., but is usually in the form of an injection, for example, in a cancer patient (60 kg), About 0 per day.
It is convenient to administer about 01 to 30 mg, preferably about 0.1 to 20 mg, more preferably about 0.1 to 10 mg by intravenous injection. For other animals,
The amount converted per 60 kg can be administered.

(12) The receptor protein of the present invention,
Neutralization by Antibodies to Partial Peptides or Salts Thereof The neutralizing activity of the antibodies to the receptor protein of the present invention or partial peptides or salts thereof to those receptor proteins means the signal transduction function involving the receptor proteins. Means the activity of inactivating. Therefore, when the antibody has a neutralizing activity, signal transduction involving the receptor protein, for example, cell stimulating activity mediated by the receptor protein (eg, arachidonic acid release, acetylcholine release, intracellular Ca ²⁺ release, cell Intracellular cAMP production, intracellular c
AMP production inhibition, intracellular cGMP production, inositol phosphate production, cell membrane potential fluctuation, intracellular protein phosphorylation, activation of c-fos, activity promoting or suppressing pH decrease, etc.) can do. Therefore, it can be used for prevention and / or treatment of diseases caused by overexpression of the receptor protein.

(13) Preparation of Animal Having DNA Encoding G Protein-Coupled Receptor Protein of the Present Invention Using the DNA of the present invention, a transgenic animal expressing the receptor protein of the present invention can be prepared. . Animals include mammals (eg, rat,
Mouse, rabbit, sheep, pig, cow, cat, dog, monkey, etc.) (hereinafter sometimes abbreviated as animal) and the like, and particularly mouse, rabbit and the like are preferable. When the DNA of the present invention is introduced into a target animal, it is generally advantageous to use the DNA as a gene construct linked downstream of a promoter that can be expressed in animal cells. For example, when a rabbit-derived DNA of the present invention is introduced, a gene construct in which the DNA of the present invention having a high homology with this is linked to the downstream of various promoters capable of expressing in animal cells, for example, a rabbit fertilized egg. By microinjection into DNA, a DNA-introduced animal that highly produces the receptor protein of the present invention can be produced. As this promoter, for example, a virus-derived promoter and a ubiquitous expression promoter such as metallothionein can be used, but preferably an NGF gene promoter or an enolase gene promoter which is specifically expressed in the brain is used.

The introduction of the DNA of the present invention at the fertilized egg cell stage is ensured to be present in all germinal cells and somatic cells of the target animal. The presence of the receptor protein or the like of the present invention in the embryonic cell of the produced animal after the introduction of DNA means that all the progeny of the produced animal have the receptor protein of the present invention in all of the embryonic cells and somatic cells. The offspring of this type of animal that has inherited the gene has the receptor protein of the present invention in all of its germ cells and somatic cells. By confirming that the DNA-introduced animal of the present invention stably retains the gene by mating, the DNA-introduced animal can be passaged in the usual breeding environment as the DNA-bearing animal. Furthermore, a male and female animal carrying the target DNA is mated to obtain a homozygous animal having the transgene on both homologous chromosomes. By mating the male and female animals, all offspring are made to have the DNA. Can be bred and passaged. DN of the present invention
The animal into which A has been introduced has high expression of the receptor protein of the present invention, and is therefore useful as an animal for screening agonists or antagonists of the receptor protein of the present invention. The DNA-introduced animal of the present invention can also be used as a cell source for tissue culture. For example, by directly analyzing DNA or RNA in the tissue of the DNA-introduced mouse of the present invention,
Alternatively, the receptor protein of the present invention and the like can be analyzed by analyzing a tissue in which the receptor protein of the present invention expressed by a gene is present. To cultivate cells of a tissue having the receptor protein or the like of the present invention by a standard tissue culture technique, and use these to study the function of cells from generally difficult-to-cultivate tissues such as those derived from the brain or peripheral tissues. You can Further, by using the cells, for example, it is possible to select a drug that enhances the functions of various tissues. Further, if there is a highly expressing cell line, the receptor protein of the present invention can be isolated and purified from the cell line.

In the present specification and drawings, when an abbreviation is used for a base or amino acid, IUPAC-IUB is used.
It is based on the abbreviations by the Commission on Biochemical Nomenclature or the abbreviations commonly used in this field, and examples are given below. When amino acids may have optical isomers, the L form is shown unless otherwise specified. DNA: deoxyribonucleic acid cDNA: complementary deoxyribonucleic acid A: adenine T: thymine G: guanine C: cytosine RNA: ribonucleic acid mRNA: messenger ribonucleic acid dATP: deoxyadenosine triphosphate dTTP: deoxythymidine triphosphate dGTP: deoxyguanosine tri Phosphate dCTP: Deoxycytidine triphosphate ATP: Adenosine triphosphate EDTA: Ethylenediaminetetraacetic acid SDS: Sodium dodecyl sulfate

Gly: glycine Ala: alanine Val: valine Leu: leucine Ile: isoleucine Ser: serine Thr: threonine Cys: cysteine Met: methionine Glu: glutamic acid Asp: aspartic acid Lys: lysine Arg: argininePhis: arginine HisP: arginine Hisin. : Tyrosine Trp: Tryptophan Pro: Proline Asn: Asparagine Gln: Glutamine pGlu: Pyroglutamic acid Me: Methyl group Et: Ethyl group Bu: Butyl group Ph: Phenyl group TC: Thiazolidine-4 (R) -carboxamide group

Further, substituents, protecting groups and reagents commonly used in the present specification are represented by the following symbols. Tos: p-toluenesulfonyl CHO: formyl Bzl: benzyl Cl ₂ Bzl: 2,6-dichlorobenzyl Bom: benzyloxymethyl Z: benzyloxycarbonyl Cl-Z: 2-chlorobenzyloxycarbonyl Br-Z: 2-bromobenzyl Oxycarbonyl Boc: t-butoxycarbonyl DNP: dinitrophenol Trt: trityl Bum: t-butoxymethyl Fmoc: N-9-fluorenylmethoxycarbonyl HOBt: 1-hydroxybenztriazole HOOBt: 3,4-dihydro-3-hydroxy -4-Oxo-1,2,3-benzotriazine HONB: 1-Hydroxy-5-norbornene-2,3-dicarboxyimide DCC: N, N'-dicyclohexylcarbodiimide

The sequence numbers in the sequence listing of the present specification show the following sequences. SEQ ID NO: 1 shows the amino acid sequence of human-derived novel G protein-coupled receptor protein TGR39 of the present invention. SEQ ID NO: 2 This shows the base sequence of cDNA encoding the human-derived novel G protein-coupled receptor protein TGR39 of the present invention.

SEQ ID NO: 3 This shows the base sequence of primer 1 used in the PCR reaction of Example 1 below. SEQ ID NO: 4 Shows the base sequence of primer 2 used in the PCR reaction in the following Example 1 and Example 2. SEQ ID NO: 5 This shows the amino acid sequence of human-derived novel G protein-coupled receptor protein hTGR39A of the present invention. SEQ ID NO: 6 This shows the base sequence of cDNA encoding the human-derived novel G protein-coupled receptor protein hTGR39A of the present invention. SEQ ID NO: 7 This shows the base sequence of primer 3 used in the PCR reaction of Example 2 below. SEQ ID NO: 8 This shows the base sequence of primer 1 used in the PCR reaction of Example 3 below. SEQ ID NO: 9 This shows the base sequence of primer 2 used in the PCR reaction of Example 3 below. SEQ ID NO: 10 Shows the base sequence of the probe used in the PCR reaction in Example 3 below.

Transformant E obtained in Example 1 described below
scherichia coli TOP10 / pCR
2.1-hTGR39 is a foundation of the Fermentation Research Institute (IF) of 2-1785, 13-Honchocho, Yodogawa-ku, Osaka-shi, Osaka Prefecture, Japan (postal code 532-8686) from September 13, 2001.
O) as the consignment number IFO 16702 from September 25, 2001, 1-1, Higashi 1-1-chome, Tsukuba, Ibaraki, Japan
They have been deposited under the deposit number FERM BP-7753 at the Patent Organism Depositary Center, National Institute of Advanced Industrial Science and Technology, Central No. 6 (Postal Code 305-8566). Transformant Esch obtained in Example 2 described below
erichia coli TOP10 / pCR2.1-
hTGR39A has been available from September 18th, 2001, 2-17-5, Jusohonmachi, Yodogawa-ku, Osaka-shi, Osaka (postal code 53
2-8686), the Institute for Fermentation (IFO), with the accession number IFO 16706, October 1, 2001
From Japan to 1-1-1, Higashi 1-1-chome, Tsukuba, Ibaraki, Japan Depositary number FE to the Patent Biological Depositary Center, National Institute of Advanced Industrial Science and Technology, Central No. 6 (zip code 305-8566)
Deposited as RM BP-7760, respectively.

[0113]

The present invention will be described in more detail with reference to the following examples, which are not intended to limit the scope of the present invention. In addition, the gene using Escherichia coli followed the method described in Molecular cloning. Example 1 Cloning of cDNA encoding human protein G-coupled receptor protein and determination of nucleotide sequence Using human heart cDNA (CLONTECH) as a template,
A PCR reaction was performed using two primers, primer 1 (SEQ ID NO: 3) and primer 2 (SEQ ID NO: 4). The composition of the reaction solution in the reaction was such that 1/10 amount of the above cDNA was used as a template and Advantage-G was used.
C2 Polymerase Mix (CLONTEC
H company) 1/50 amount, 0.5 μM each of primer 1 (SEQ ID NO: 3) and primer 2 (SEQ ID NO: 4), dN
200 μM of TPs and 1 buffer of enzyme
/ 5 volume and 1/5 volume of GC Melt were added to make a volume of 20 μl. PCR reaction at 94 ℃ ・ 5 minutes, then 94 ℃ ・
3 cycles of 30 seconds, 60 ° C, 30 seconds, 68 ° C, 2 minutes
This was repeated 5 times, and finally the extension reaction was carried out at 68 ° C. for 5 minutes. The PCR reaction product was cloned into a TA cloning kit (Invi
plasmid vector pCR2.
1 (Invitrogen) was subcloned. This was introduced into E. coli TOP10, and a clone having a cDNA was selected in LB agar medium containing ampicillin. As a result of analyzing the sequence of each clone, a nucleotide sequence (SEQ ID NO: 2) of a cDNA encoding a novel G protein-coupled receptor protein was obtained. The novel G protein-coupled receptor protein having this amino acid sequence (SEQ ID NO: 1) was named TGR39. In addition, the transformant was transformed into Escherichia coli TOP10 / pCR.
It was named 2.1-hTGR39. A hydrophobicity plot of TGR39 is shown in FIG.

Example 2 Cloning of cDNA encoding human heart G protein-coupled receptor protein and determination of nucleotide sequence Using human heart cDNA (CLONTECH) as a template,
A PCR reaction was performed using two primers, primer 1 (SEQ ID NO: 7) and primer 2 (SEQ ID NO: 4). The composition of the reaction solution in the reaction was such that 1/10 amount of the above cDNA was used as a template and Advantage-G was used.
C2 Polymerase Mix (CLONTEC
H company) 1/50 amount, 0.5 μM each of primer 1 (SEQ ID NO: 7) and primer 2 (SEQ ID NO: 4), dN
200 μM of TPs and 1 buffer of enzyme
/ 5 volume and 1/5 volume of GC Melt were added to make a volume of 20 μl. PCR reaction at 94 ℃ ・ 5 minutes, then 94 ℃ ・
3 cycles of 30 seconds, 60 ° C, 30 seconds, 68 ° C, 2 minutes
This was repeated 5 times, and finally the extension reaction was carried out at 68 ° C. for 5 minutes. The PCR reaction product was labeled with the TA cloning kit (In
Subcloning into the plasmid vector pCR2.1 (Invitrogen) according to the prescription of Vitrogen). This was introduced into Escherichia coli TOP10, and cDNA was added.
Clones with A were selected in LB agar containing ampicillin. As a result of analyzing the sequences of the individual clones, the nucleotide sequence of cDNA encoding the novel G protein-coupled receptor protein (SEQ ID NO: 6) was obtained. Also,
The novel G protein-coupled receptor protein having this amino acid sequence (SEQ ID NO: 5) was named hTGR39A. In addition, transformants were transformed into Escherichia coli (Escheric).
hia coli) TOP10 / pCR2.1-hTG
It was named R39A. In the amino acid sequence represented by SEQ ID NO: 5, the 9th Asp in the amino acid sequence represented by SEQ ID NO: 1 is replaced with Glu. In the base sequence represented by SEQ ID NO: 6, the 27th C in the base sequence represented by SEQ ID NO: 2 is replaced with A. A hydrophobicity plot of hTGR39A is shown in FIG.

Example 3 Expression distribution analysis of TGR39 in human tissue Expression analysis of TGR39 in human tissue was performed using TaqMan P
It was investigated by using the CR method. As a template, Human
Using a Multiple Tissue cDNA Panel (Clontech), primer 1 (SEQ ID NO:
TaqMan PCR was carried out using 8) and primer 2 (SEQ ID NO: 9), and a probe having the nucleotide sequence represented by SEQ ID NO: 10. The composition of the reaction solution in the reaction is
12.5 μl of TaqMan Universal PCR Master Mix (Applied Biosystems Japan), 0.5 μl each of 10 μM primer 1 and primer 2 and 1 μl of 5 μM probe,
2 μl of template and 8.5 μl of distilled water for a total of 25 μl
For the R reaction, hold at 50 ° C for 2 minutes and 95 ° C for 10 minutes, then
A cycle of 15 seconds at 60 ° C for 1 minute was repeated 40 times. The results calculated as the copy number per 1 μl of cDNA based on the obtained results are shown in FIG. From this, the expression level of TGR39 is
It was found to be highly expressed in the heart.

[0116]

INDUSTRIAL APPLICABILITY The G protein-coupled receptor protein of the present invention or a partial peptide thereof or a salt thereof, a polynucleotide (for example, DNA, RNA and derivatives thereof) encoding the receptor protein or a partial peptide thereof is used to determine the ligand. , Obtaining antibodies and antisera, constructing a recombinant receptor protein expression system, developing a receptor binding assay system using the expression system, screening drug candidate compounds (agonists, antagonists, etc.), structurally similar ligands It can be used as a drug for carrying out drug design based on comparison with a receptor, a reagent for preparing a probe or a PCR primer in gene diagnosis, a transgenic animal, or a drug such as a gene prophylactic / therapeutic agent.

[0117]

[Sequence list] SEQUENCE LISTING <110> Takeda Chemical Industries, Ltd. <120> Novel G Protein-Coupled Receptor Protein and its DNA <130> P02-0070 <150> JP2001-193399 <151> 2001-06-26 <150> JP2001-311880 <151> 2001-10-09 <160> 10 <210> 1 <211> 300 <212> PRT <213> Human <400> 1 Met Ala Ser Ala Arg Gly Arg Val Asp Ser Pro Glu Leu Ser Gly Pro                  5 10 15 Cys Phe Pro Ser Ala Gly Arg Lys Arg Arg Arg Gly Leu Leu Ala Asn              20 25 30 Ser Ser Leu Gly Asn Gly Ser Glu Asn Thr Ser Pro Ala Arg Ser Pro          35 40 45 Arg Ser Leu His Gly Leu Asp Gly Val Ala Gly Ala Val Arg Ala Pro      50 55 60 Ala Leu Gln Ala Leu Ile Ala Gly Ala Tyr Trp Ala Leu Cys Ala Val  65 70 75 80 Gly Leu Val Gly Asn Val Leu Val Leu Val Arg Val Arg Ser Gln Gln                  85 90 95 Trp Arg Arg His Trp Leu Leu Asn Cys Phe Leu Leu Asn Leu Ala Ala             100 105 110 Thr Asp Leu Gln Phe Val Leu Thr Leu Pro Phe Trp Ala Val Asp Thr         115 120 125 Val Arg Asp Phe Ser Trp Pro Phe Gly Gly Ala Ile Cys Lys Val Met     130 135 140 Leu Thr Leu Thr Val Pro Asn Met Tyr Ala Ser Ile Phe Leu Phe Ser 145 150 155 160 Ala Met Ser Val Ala Arg Tyr Cys Ile Val Thr Gly Ala Leu Pro Pro                 165 170 175 Ser His Arg Gly Ala Ser Arg Ala Ser Cys Val Cys Cys Leu Leu Trp             180 185 190 Ala Met Ala Val Leu Ala Thr Ala Pro Thr Ala Leu Phe Ala Thr Ala         195 200 205 Ala Arg Val Gly Gly Asn His Ser Cys Leu Leu Arg Phe Pro Ala Gly     210 215 220 Gly Pro Lys Trp Gln Val Leu Tyr His Leu Gln Lys Ile Ala Val Ala 225 230 235 240 Phe Val Leu Pro Leu Ala Thr Leu Gly Thr Cys Ser Leu Leu Leu Arg                 245 250 255 Phe Leu Arg Leu Trp Cys Ala Arg Trp Pro Ser Arg Val Arg Arg Arg             260 265 270 Leu Arg Ser Arg Val Thr Cys Ala Leu Ala Cys Val Leu Leu Ala Phe         275 280 285 Val Leu Cys Trp Leu Pro Ser Gln Ala Phe Thr Leu     290 295 300 <210> 2 <211> 900 <212> DNA <213> Human <400> 2 atggcatccg ccagaggcag ggtcgactcg ccggagctgt ctgggccgtg cttcccttca 60 gctggccgga agaggcgtcg ggggctgctc gctaacagct ccctgggcaa cggatcagag 120 aacactagcc cggcccggag tccccgcagc ctgcatggcc tggacggggt ggcgggggcc 180 gtccgggcgc cggcgctgca ggcgctgatt gcgggcgcct actgggccct gtgcgctgta 240 ggcctggtgg gcaacgtgct ggtgctagtc cgggtgaggt cccagcagtg gcgccgccac 300 tggctgctca attgcttcct cctcaacctg gcagccactg acctgcagtt tgtgctaacg 360 ctgccctttt gggccgtgga cacggtgcgc gactttagct ggcccttcgg gggtgccatc 420 tgcaaggtga tgctgacgct caccgtgccc aacatgtatg ccagcatctt cctcttcagt 480 gccatgagcg tggcacgcta ttgcattgtg actggcgcgc tgcctccgag ccatcggggc 540 gcatcacggg ccagctgtgt gtgctgcctg ctctgggcta tggccgtcct ggctacggcg 600 cccaccgccc tgttcgccac ggcagctagg gtggggggaa atcactcgtg cctgctgcgc 660 ttccccgccg gcggccccaa atggcaggtg ctctaccacc tgcagaagat cgcagtagcc 720 ttcgtgctgc cgctggccac gctgggcacc tgttcgctgc tgctgcgctt cctgcgactg 780 tggtgcgcac gctggccgtc gagggtcagg cgccgactgc gttcccgagt cacctgtgcg 840 ctggcctgcg tgctactggc cttcgtgctc tgctggctgc ccagccaagc gttcacactc 900 <210> 3 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> Designed oligonucleotide primer to amplify DNA encoding TGR39 <400> 3 gtcgacatgg catccgccag aggcagggtc g 31 <210> 4 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Designed oligonucleotide primer to amplify DNA encoding TGR39 and hTGR39A <400> 4 tactagttca gagtgtgaac gcttggctgg gc 32 <210> 5 <211> 300 <212> PRT <213> Human <400> 5 Met Ala Ser Ala Arg Gly Arg Val Glu Ser Pro Glu Leu Ser Gly Pro                  5 10 15 Cys Phe Pro Ser Ala Gly Arg Lys Arg Arg Arg Gly Leu Leu Ala Asn              20 25 30 Ser Ser Leu Gly Asn Gly Ser Glu Asn Thr Ser Pro Ala Arg Ser Pro          35 40 45 Arg Ser Leu His Gly Leu Asp Gly Val Ala Gly Ala Val Arg Ala Pro      50 55 60 Ala Leu Gln Ala Leu Ile Ala Gly Ala Tyr Trp Ala Leu Cys Ala Val  65 70 75 80 Gly Leu Val Gly Asn Val Leu Val Leu Val Arg Val Arg Ser Gln Gln                  85 90 95 Trp Arg Arg His Trp Leu Leu Asn Cys Phe Leu Leu Asn Leu Ala Ala             100 105 110 Thr Asp Leu Gln Phe Val Leu Thr Leu Pro Phe Trp Ala Val Asp Thr         115 120 125 Val Arg Asp Phe Ser Trp Pro Phe Gly Gly Ala Ile Cys Lys Val Met     130 135 140 Leu Thr Leu Thr Val Pro Asn Met Tyr Ala Ser Ile Phe Leu Phe Ser 145 150 155 160 Ala Met Ser Val Ala Arg Tyr Cys Ile Val Thr Gly Ala Leu Pro Pro                 165 170 175 Ser His Arg Gly Ala Ser Arg Ala Ser Cys Val Cys Cys Leu Leu Trp             180 185 190 Ala Met Ala Val Leu Ala Thr Ala Pro Thr Ala Leu Phe Ala Thr Ala         195 200 205 Ala Arg Val Gly Gly Asn His Ser Cys Leu Leu Arg Phe Pro Ala Gly     210 215 220 Gly Pro Lys Trp Gln Val Leu Tyr His Leu Gln Lys Ile Ala Val Ala 225 230 235 240 Phe Val Leu Pro Leu Ala Thr Leu Gly Thr Cys Ser Leu Leu Leu Arg                 245 250 255 Phe Leu Arg Leu Trp Cys Ala Arg Trp Pro Ser Arg Val Arg Arg Arg             260 265 270 Leu Arg Ser Arg Val Thr Cys Ala Leu Ala Cys Val Leu Leu Ala Phe         275 280 285 Val Leu Cys Trp Leu Pro Ser Gln Ala Phe Thr Leu     290 295 300 <210> 6 <211> 900 <212> DNA <213> Human <400> 6 atggcatccg ccagaggcag ggtcgaatcg ccggagctgt ctgggccgtg cttcccttca 60 gctggccgga agaggcgtcg ggggctgctc gctaacagct ccctgggcaa cggatcagag 120 aacactagcc cggcccggag tccccgcagc ctgcatggcc tggacggggt ggcgggggcc 180 gtccgggcgc cggcgctgca ggcgctgatt gcgggcgcct actgggccct gtgcgctgta 240 ggcctggtgg gcaacgtgct ggtgctagtc cgggtgaggt cccagcagtg gcgccgccac 300 tggctgctca attgcttcct cctcaacctg gcagccactg acctgcagtt tgtgctaacg 360 ctgccctttt gggccgtgga cacggtgcgc gactttagct ggcccttcgg gggtgccatc 420 tgcaaggtga tgctgacgct caccgtgccc aacatgtatg ccagcatctt cctcttcagt 480 gccatgagcg tggcacgcta ttgcattgtg actggcgcgc tgcctccgag ccatcggggc 540 gcatcacggg ccagctgtgt gtgctgcctg ctctgggcta tggccgtcct ggctacggcg 600 cccaccgccc tgttcgccac ggcagctagg gtggggggaa atcactcgtg cctgctgcgc 660 ttccccgccg gcggccccaa atggcaggtg ctctaccacc tgcagaagat cgcagtagcc 720 ttcgtgctgc cgctggccac gctgggcacc tgttcgctgc tgctgcgctt cctgcgactg 780 tggtgcgcac gctggccgtc gagggtcagg cgccgactgc gttcccgagt cacctgtgcg 840 ctggcctgcg tgctactggc cttcgtgctc tgctggctgc ccagccaagc gttcacactc 900 <210> 7 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Designed oligonucleotide primer to amplify DNA encoding hTGR39A <400> 7 gtcgacatgg catccgccag aggcagggt 29 <210> 8 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 8 tcttcctctt cagtgccatg a 21 <210> 9 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 9 ctcggaggca gcgcg 15 <210> 10 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Probe <400> 10 cgtggcacgc tattgcattg tgactg 26

[Brief description of drawings]

FIG. 1 is a hydrophobicity plot of TGR39.

FIG. 2 is a hydrophobicity plot of hTGR39A.

FIG. 3 is a distribution map of TGR39 expression.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) A61P 3/00 A61P 9/00 4C084 9/00 25/00 4C085 25/00 25/28 4H045 25/28 29 / 00 29/00 35/00 35/00 37/00 37/00 43/00 111 43/00 111 C07K 14/705 C07K 14/705 16/28 16/28 C12N 1/15 C12N 1/15 1/19 1/19 1/21 1/21 C12P 21/02 C 5/10 C12Q 1/02 C12P 21/02 1/68 A C12Q 1/02 G01N 33/15 Z 1/68 33/50 Z G01N 33/15 33 / 53 D 33/50 M 33/53 33/566 C12N 15/00 ZNAA 33/566 5/00 A (72) Inventor Yasushi Shintani 2-9-1 501 Nishinidoka, Toyonaka City, Osaka Prefecture (72) Invention Nobuyuki Miyajima 4-16-4 Azuma Tsukuba, Ibaraki Pref. Azuma 403 F Term (Preview) 2G045 AA40 DA12 DA13 DA1 4 DA36 FB02 FB03 4B024 AA01 AA11 BA63 CA04 CA09 CA12 DA02 DA06 EA04 GA11 GA18 GA19 HA03 HA14 4B063 QA01 QA18 QQ20 QQ53 QR32 QR38 QR55 QR82 QS25 QS34 QS38 Q44 01 MA17 MA23 MA35 MA37 MA38 MA52 MA55 NA14 ZA022 ZA362 ZA662 ZB072 ZB112 ZB352 ZC412 ZC422 4C085 AA14 AA15 BB11 CC22 CC23 EE01 GG01 GG08 4H045 AA10 AA11 AA20 AA30 BA10 CA40 DA50 DA75 DA86 EA21 EA22 EA23 EA25 EA27 EA28 EA50 FA71 FA74

Claims (39)

[Claims] 1. A G protein-coupled receptor protein or a salt thereof, which contains the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 5. 2. The G protein-coupled receptor protein or the salt thereof according to claim 1, which contains the amino acid sequence represented by SEQ ID NO: 5. 3. The G protein-coupled receptor protein or the salt thereof according to claim 1, which contains the amino acid sequence represented by SEQ ID NO: 1. 4. A partial peptide of the G protein-coupled receptor protein according to claim 1, or a salt thereof. 5. A polynucleotide containing a polynucleotide encoding the G protein-coupled receptor protein according to claim 1. 6. The polynucleotide according to claim 5, which is DNA. 7. The polynucleotide according to claim 6, which comprises the base sequence represented by SEQ ID NO: 2 or SEQ ID NO: 6. 8. A recombinant vector containing the polynucleotide according to claim 5. 9. A transformant transformed with the recombinant vector according to claim 8. 10. The G protein-coupled receptor protein or salt thereof according to claim 1, which is characterized in that the transformant according to claim 9 is cultured to produce the G protein-coupled receptor protein according to claim 1. Manufacturing method. 11. An antibody against the G protein-coupled receptor protein according to claim 1, the partial peptide according to claim 4, or a salt thereof. 12. The antibody according to claim 11, which is a neutralizing antibody that inactivates signal transduction of the G protein-coupled receptor protein according to claim 1. 13. A diagnostic agent comprising the antibody according to claim 11. 14. A medicine comprising the antibody according to claim 11. 15. A G protein-coupled receptor protein or a salt thereof according to claim 1, which can be obtained by using the G protein-coupled receptor protein according to claim 1, the partial peptide according to claim 4, or a salt thereof. Ligand. 16. A medicine comprising a ligand for the G protein-coupled receptor protein according to claim 15. 17. The G protein-coupled receptor protein according to claim 1, the partial peptide according to claim 4, or a salt thereof is used.
A method for determining a ligand for the G protein-coupled receptor protein or a salt thereof described. 18. A ligand comprising the G protein-coupled receptor protein according to claim 1, the partial peptide according to claim 4, or a salt thereof, and a G protein-coupled receptor protein according to claim 1 or a salt thereof. A method for screening a compound or its salt, which changes the binding property to a salt. 19. A ligand comprising the G protein-coupled receptor protein according to claim 1, the partial peptide according to claim 4, or a salt thereof, and the G protein-coupled receptor protein according to claim 1. A kit for screening a compound or a salt thereof that changes the binding property to the salt. 20. The binding property between a ligand obtainable by using the screening method according to claim 18 or the screening kit according to claim 19 and the G protein-coupled receptor protein according to claim 1 or a salt thereof is changed. A compound or salt thereof. 21. A medicine comprising the compound according to claim 20 or a salt thereof. 22. A polynucleotide which hybridizes with the polynucleotide according to claim 5 under high stringent conditions. 23. A polynucleotide containing a base sequence complementary to the polynucleotide of claim 5 or a part thereof. 24. The polynucleotide according to claim 1, wherein the polynucleotide according to claim 5 or a part thereof is used.
Method for quantifying mRNA of protein-coupled receptor protein. 25. The method for quantifying a G protein-coupled receptor protein according to claim 1, wherein the antibody according to claim 11 is used. 26. The method for diagnosing a disease associated with the function of a G protein-coupled receptor according to claim 1, which uses the quantification method according to claim 24 or 25. 27. A method for screening a compound or a salt thereof which changes the expression level of a G protein-coupled receptor protein according to claim 1, characterized in that the quantification method according to claim 24 is used. 28. A method for screening a compound or a salt thereof that alters the amount of G protein-coupled receptor protein according to claim 1 in a cell membrane, characterized by using the quantification method according to claim 25. 29. A compound or a salt thereof which can be obtained by using the screening method according to claim 27 and which changes the expression level of the G protein-coupled receptor protein according to claim 1. 30. A compound or salt thereof, which alters the amount of G protein-coupled receptor protein according to claim 1 in a cell membrane obtainable by using the screening method according to claim 28. 31. A medicine comprising the compound according to claim 29 or a salt thereof. 32. A medicine comprising the compound according to claim 30 or a salt thereof. 33. Central diseases, inflammatory diseases, cardiovascular diseases,
33. The medicine according to claim 21, 31 or 32, which is a prophylactic / therapeutic agent for cancer, metabolic disease, immune system disease or digestive system disease. 34. A ligand obtainable by using the screening method according to claim 18 or the screening kit according to claim 19 for a mammal, and the G protein-coupled receptor protein or salt thereof according to claim 1. A central disease characterized by administering an effective amount of a compound or a salt thereof that changes the binding property of
A method for preventing or treating cardiovascular disease, cancer, metabolic disease, immune system disease or digestive system disease. 35. An effective amount of a compound or a salt thereof which alters the expression level of the G protein-coupled receptor protein according to claim 1, which can be obtained by using the screening method according to claim 27, is administered to a mammal. A method for preventing / treating a central disease, an inflammatory disease, a cardiovascular disease, a cancer, a metabolic disease, an immune system disease or a digestive system disease, which comprises: 36. An effective amount of the compound or its salt that alters the amount of G protein-coupled receptor protein according to claim 1 in the cell membrane obtainable by using the screening method according to claim 28 is administered to a mammal. A method for preventing / treating a central disease, an inflammatory disease, a cardiovascular disease, a cancer, a metabolic disease, an immune system disease or a digestive system disease, which comprises: 37. Central diseases, inflammatory diseases, cardiovascular diseases,
A ligand obtainable using the screening method according to claim 18 or the screening kit according to claim 19 for producing a prophylactic / therapeutic agent for cancer, metabolic disease, immune system disease or digestive system disease. Use of the compound or a salt thereof that changes the binding property to the G protein-coupled receptor protein or the salt thereof according to 1. 38. Central diseases, inflammatory diseases, cardiovascular diseases,
Expression of the G protein-coupled receptor protein according to claim 1, which can be obtained by using the screening method according to claim 27 for producing a prophylactic / therapeutic agent for cancer, metabolic disease, immune system disease or digestive system disease. Use of a compound or a salt thereof which changes the amount. 39. Central diseases, inflammatory diseases, cardiovascular diseases,
The G protein-coupled receptor protein according to claim 1 in a cell membrane obtainable by using the screening method according to claim 28 for producing a prophylactic / therapeutic agent for cancer, metabolic disease, immune system disease or digestive system disease. Use of a compound or a salt thereof which changes the amount.