JP2002360277A - New g-protein-coupled receptor protein and its dna - Google Patents

Abstract

PROBLEM TO BE SOLVED: To search for a new G protein-coupled receptor protein and to elucidate functions. SOLUTION: This new G protein-coupled receptor protein comprises an amino acid sequence which is the same or substantially the same as the amino acid sequence represented by a specific sequence or its salt. A polynucleotide encoding the same is provided. Uses thereof for medicinal uses, etc., are further provided.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a novel G protein-coupled receptor protein derived from human kidney or a salt thereof, and a DNA encoding the same.

[0002]

2. Description of the Related Art Many physiologically active substances such as hormones and neurotransmitters regulate biological functions through specific receptor proteins present in cell membranes. Many of these receptor proteins are coupled guanin
Since it transduces intracellular signals through the activation of e nucleotide-binding protein (hereinafter abbreviated as G protein) and has a common structure with seven transmembrane domains, it is a G protein-coupled receptor protein. Alternatively, a seven-transmembrane receptor protein (7
TMR). G protein-coupled receptor protein is present on the surface of each functional cell in living cells and organs, and regulates the functions of those cells and organs, for example,
It plays a physiologically important role as a target for hormones, neurotransmitters and bioactive substances. The receptor transmits a signal into a cell through binding to a physiologically active substance, and this signal causes various reactions such as activation and suppression of the cell. To clarify the relationship between substances that regulate complex functions in cells and organs of various organisms and their specific receptor proteins, especially G protein-coupled receptor proteins, it is necessary to clarify the functions of organs and cells in various organisms. It will provide a very important tool for drug development that is elucidated and closely related to their functions.

For example, in various organs of a living body, physiological functions are regulated under the regulation by many hormones, hormone-like substances, neurotransmitters or physiologically active substances. In particular, physiologically active substances are present at various sites in a living body, and regulate their physiological functions through their corresponding receptor proteins. There are many unknown hormones, neurotransmitters, and other physiologically active substances in the living body.
Many have not been reported so far. Furthermore, it is often unknown whether subtypes exist in known receptor proteins. Clarifying the relationship between a substance that regulates a complex function in a living body and its specific receptor protein is a very important means for the development of pharmaceuticals including agonists and antagonists for the receptor protein. However, conventionally, in order to efficiently screen for agonists and antagonists for receptor proteins and to develop pharmaceuticals, the functions of receptor protein genes expressed in vivo are elucidated and expressed in an appropriate expression system. It was necessary. In recent years, as a means of analyzing genes expressed in vivo, studies of randomly analyzing cDNA sequences have been actively conducted.
Registered in the database as ce Tag (EST) and published. However, many ESTs are only sequence information,
It is difficult to estimate its function.

[0004]

Heretofore, substances that inhibit the binding between a G protein-coupled receptor and its ligand, a physiologically active substance, and substances that bind to cause a signal transduction similar to that of a physiologically active substance have been disclosed. As a specific antagonist or agonist of a receptor, it has been utilized as a drug for regulating biological functions. Therefore,
As described above, finding a G protein-coupled receptor protein which is not only important in physiological expression in vivo but also a target for drug development and cloning its gene (eg, cDNA) requires a novel G protein. This is a very important means for finding specific ligands, agonists and antagonists of the coupled receptor protein. However, not all G protein-coupled receptors have been found. Even at this time, there are many unknown G protein-coupled receptors and so-called orphan receptors whose ligands have not been identified. Therefore, there is a strong need to search for new G protein-coupled receptors and elucidate their functions. G protein-coupled receptor is a new ligand (bioactive substance) whose signal transduction action is used as an index.
And the search for agonists or antagonists to the receptor. On the other hand, even if a physiological ligand is not 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 (knockout animal) of the receptor. . These ligands, agonists or antagonists to the receptor can be expected to be used as preventive / therapeutic agents or diagnostic agents for diseases related to dysfunction of G protein-coupled receptors. Furthermore, a decrease or enhancement of the function of a G protein-coupled receptor in a living body based on a gene mutation thereof often causes some disease. In this case, the present invention is applied not only to administration of an antagonist or agonist to the receptor, but also to gene therapy by introducing the receptor gene into a living body (or a specific organ) or by introducing an antisense nucleic acid to the receptor gene. You can also. In this case, the nucleotide sequence of the receptor is indispensable information for examining the presence or absence of a deletion or mutation in the gene. And diagnostics. The present invention provides a novel and useful G protein-coupled receptor protein as described above. That is, a polynucleotide containing a novel G protein-coupled receptor protein or a partial peptide thereof or a salt thereof, a polynucleotide (DNA, RNA and a derivative thereof) encoding the G protein-coupled receptor protein or a partial peptide thereof (DNA, R
NA and derivatives thereof), a recombinant vector containing the polynucleotide, a transformant carrying the recombinant vector, a method for producing the G protein-coupled receptor protein or a salt thereof, the G protein-coupled receptor protein or An antibody against the partial peptide 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, the ligand and the G
Screening method for compounds (antagonists, agonists) or salts thereof that alter the binding to protein-coupled receptor protein, the screening kit, a ligand obtainable by using the screening method or the screening kit, and the G protein-coupled receptor Compounds (antagonists, agonists) that change the binding to proteins or salts thereof, and compounds (antagonists, agonists) that change the binding between ligands and the G protein-coupled receptor proteins or compounds of the G protein-coupled receptor proteins There is provided a drug or the like containing a compound that changes the expression level or a salt thereof.

[0005]

Means for Solving the Problems As a result of intensive studies, the present inventors have isolated a cDNA encoding a novel G protein-coupled receptor protein derived from human kidney and analyzed its entire nucleotide sequence. succeeded in. Then, when this base sequence was translated into an amino acid sequence, the first to seventh transmembrane regions were confirmed on the hydrophobicity plot shown in FIG. 1, and the proteins encoded by these cDNAs were transmembrane- It was confirmed to be a G protein-coupled receptor protein. The present inventors have further studied based on these findings, and as a result, have completed the present invention.

That is, the present invention relates to (1) SEQ ID NO:
(2) 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 (1);
A G protein-coupled receptor protein or a salt thereof, comprising the amino acid sequence represented by SEQ ID NO: 1,
(3) a partial peptide of the G protein-coupled receptor protein described in (1) or a salt thereof; (4) a polynucleotide containing a polynucleotide encoding the G protein-coupled receptor protein described in (1); ) The polynucleotide according to the above (4), which is a DNA; (6) the polynucleotide according to the above (4) having the base sequence represented by SEQ ID NO: 2; and (7) the polynucleotide according to the above (4). (8) a transformant transformed with the recombinant vector according to (7), (9) a G protein-coupled receptor protein according to (1), or (3)
A pharmaceutical comprising the partial peptide of the above or a salt thereof, (10) a pharmaceutical comprising the polynucleotide of the above (4), (11) culturing the transformant of the above (8), (1) The method for producing the G protein-coupled receptor protein or the salt thereof according to (1), which comprises producing the G protein-coupled receptor protein according to (1), (12) The G protein according to (1). An antibody against the conjugated receptor protein or the partial peptide according to (3) or a salt thereof, (13) a neutralizing antibody that inactivates the signal transduction of the G protein-coupled receptor protein according to (1) above ( The antibody according to (12), (1)
4) a diagnostic agent comprising the antibody according to (12) above,
(15) a medicine comprising the antibody of (12), (16) a G protein-coupled receptor protein of (1) or a partial peptide of claim 3 or a salt thereof. A ligand for the G protein-coupled receptor protein or a salt thereof according to (1), (17)
(18) The pharmaceutical comprising the ligand according to (6), (18) the G protein-coupled receptor protein according to (1) or the partial peptide according to (3) or a salt thereof. (19) A method for determining a ligand for a G protein-coupled receptor protein or a salt thereof, (19) a ligand characterized by using the G protein-coupled receptor protein described in (1) or the partial peptide described in (3) or a salt thereof A method for screening a compound or a salt thereof that changes the binding property to the G protein-coupled receptor protein or a salt thereof according to claim 1, (20) The G protein-coupled receptor protein according to (1) or (3). Characterized by containing a partial peptide or a salt thereof A kit for screening a compound or a salt thereof that alters the binding property between a ligand that binds to the G protein-coupled receptor protein or a salt thereof according to claim 1, (21) the screening method according to (19), or the (20). A compound or a salt thereof that alters the binding property between the ligand obtainable by using the screening kit of (1) and the G protein-coupled receptor protein or salt thereof according to (1) above, (2)
2) A compound that alters the binding between the ligand obtainable by using the screening method according to (19) or the screening kit according to (20) and the G protein-coupled receptor protein or a salt thereof according to claim 1. Or a medicine containing a salt thereof, (23)
A polynucleotide that hybridizes with the polynucleotide according to the above (4) under conditions of high stringency,
(24) A polynucleotide comprising a nucleotide sequence complementary to the polynucleotide described in (4) or a part thereof, (25) A polynucleotide comprising the polynucleotide described in (4) or a part thereof. (1) the method for quantifying the mRNA of the G protein-coupled receptor protein according to (1), (26) the method for quantifying the G protein-coupled receptor protein according to (1), wherein the antibody according to (12) is used; (27) A diagnostic agent for a disease associated with the function of the G protein-coupled receptor according to (1), wherein the quantification method according to (25) or (26) is used. 25)
The method for screening a compound or a salt thereof that changes the expression level of a G protein-coupled receptor protein according to the above (1), wherein the method for quantification according to the above (26) is used. A method for screening a compound or a salt thereof that alters the amount of the G protein-coupled receptor protein in the cell membrane according to the above (1), (30) a method which can be obtained by using the screening method according to the above (28). A compound or a salt thereof that alters the expression level of the G protein-coupled receptor protein according to 1),
1) A compound or a salt thereof that alters the amount of the G protein-coupled receptor protein according to the above (1) in a cell membrane obtainable by using the screening method according to the above (29), (32) the screening method according to the above (28) And (33) a medicament containing the compound or a salt thereof that alters the expression level of the G protein-coupled receptor protein according to (1), which can be obtained by using the method described in (29). A pharmaceutical comprising a compound or a salt thereof, which alters the amount of the G protein-coupled receptor protein according to claim 1 in a cell membrane that can be obtained, (34) central disease, endocrine disease, metabolic disease, cancer, cardiovascular disease Prevention of digestive, immune, inflammatory, or infectious diseases
The medicament according to any one of the above (22), (32) or (33), which is a therapeutic agent, (35) the above (21), the above (30) or the above (3) for mammals.
A central disease, an endocrine disease, a metabolic disease, a cancer, a circulatory disease, a respiratory disease, a respiratory disease, a digestive system, wherein the compound or the salt thereof according to any one of 1) is administered in an effective amount. (36) Central disease, endocrine disease, metabolic disease, cancer cardiovascular disease, respiratory disease, digestive system disease, immune system disease, inflammatory system. (21) and (3) for producing a prophylactic / therapeutic agent for a disease or an infectious disease.
0) or the use of the compound or a salt thereof according to any of the above (31).

[0007] The present invention further relates to (37) a protein comprising the amino acid sequence represented by SEQ ID NO: 1, and one or more (preferably about 1 to 30) in the amino acid sequence represented by SEQ ID NO: 1; More preferably about 1 to 10, more preferably several (1 to 5) amino acids, the amino acid sequence represented by SEQ ID NO: 1 or 2 or more (preferably, An amino acid sequence to which about 1 to 30, more preferably about 1 to 10, and still more preferably several (1 to 5) amino acids have been added,
One or more (preferably about 1 to 30, more preferably about 1 to 10, and still more preferably several (1 to 5)) amino acids in the amino acid sequence represented by SEQ ID NO: 1 A G protein-coupled receptor protein or a salt thereof according to the above (1), which is a protein containing an amino acid sequence substituted with the amino acid or an amino acid sequence obtained by combining the amino acid sequences;
The method for determining a ligand according to the above (18), wherein the protein-coupled receptor protein or a salt thereof or the partial peptide or a salt thereof according to the above (3) is brought into contact with a test compound; For example, trace amines such as tyramine, beta-phenylethylamine, β-phenylethylamine, tryptamine and octopamine; 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, PTH, VIP (basoactive intestinal polypeptide) ), Somatostatin, dopamine, motilin, amylin, bradykinin, CGRP (calcitonin gene relayed peptide), leukotriene, pancreatastatin, prostaglandin, thromboxane, adenosine, adrenaline, chemokine superfamily (eg, IL-8, GRO
α, GROβ, GROγ, NAP-2, ENA-78, GCP-2, PF4, IP-1
CXC chemokine subfamily such as 0, Mig, PBSF / SDF-1; MCAF / MCP-1, MCP-2, MCP-3, MCP-4, eotaxin, RANT
ES, MIP-1α, MIP-1β, HCC-1, MIP-3α / LARC, MIP-3β
/ ELC, I-309, TARC, MIPF-1, MIPF-2 / eotaxin-2, MDC,
CC chemokine subfamily such as DC-CK1 / PARC, SLC;
C chemokine subfamily such as lymphotactin; fract
CX3C chemokine subfamily such as alkine), endothelin, enterogastrin, histamine, neurotensin, TRH, pancreatic polypeptide,
The method for determining a ligand according to the above (38), which is galanin, lysophosphatidic acid (LPA) or sphingosine 1-phosphate,

(40) (i) contacting a G protein-coupled receptor protein or a salt thereof described in (1) or a partial peptide or salt thereof described in (3) with a ligand; and (ii) A G protein-coupled receptor protein or a salt thereof according to the above (1) or a partial peptide or a salt thereof according to the above (3);
A screening method according to the above (19), wherein a comparison is made with a case where the ligand and a test compound are brought into contact with each other; (41) (i) a G protein-coupled receptor according to the above (1), wherein the labeled ligand is (Ii) contacting a labeled ligand and a test compound with a G protein-coupled receptor protein or a salt thereof described in (1) above; G of the labeled ligand according to the above (1) when it is brought into contact with the partial peptide or the salt thereof according to the above (3).
A ligand characterized by measuring and comparing the amount of binding to a protein-coupled receptor protein or a salt thereof or a partial peptide or a salt thereof according to the above (3), and a G protein-coupled receptor protein according to the above (1) or a salt thereof. (42) (i) a method for screening a compound or a salt thereof that changes the binding property to a salt;
When the labeled ligand is brought into contact with the cells containing the G protein-coupled receptor protein described in (1) above, (ii) the labeled ligand and the test compound are converted into the G protein-coupled receptor protein described in (1) above. Binding between the ligand and the G protein-coupled receptor protein or the salt thereof according to the above (1), wherein the amount of the labeled ligand bound to the cell when the cell is brought into contact with the cell is measured and compared. A method for screening a compound or a salt thereof that changes the sex,
(43) (i) labeling the labeled ligand with G according to (1) above;
(Ii) the labeled ligand and the test compound are contacted with the membrane fraction of the cell containing the protein-coupled receptor protein, and (ii) the membrane fraction of the cell containing the G-protein-coupled receptor protein described in (1) above. Measuring the amount of binding of the labeled ligand to the membrane fraction of the cell when contacting with the ligand, and comparing the ligand with the G protein-coupled receptor protein or the salt thereof according to (1) above. A method of screening for a compound or a salt thereof that changes the binding property,

(44) (i) when the labeled ligand is brought into contact with a G protein-coupled receptor protein expressed on the cell membrane of the transformant by culturing the transformant according to (8); ii) When the labeled ligand and the test compound are brought into contact with a G protein-coupled receptor protein expressed on the cell membrane of the transformant by culturing the transformant according to (8) above, Screening for a compound or a salt thereof that changes the binding between the ligand and the G protein-coupled receptor protein or the salt thereof according to (1) above, wherein the amount of binding to the G protein-coupled receptor protein is measured and compared. (45) (i) the G protein-coupled receptor tan according to (1) above; And if the click protein or a compound that activates a salt thereof is brought into contact with cells containing the G protein-coupled receptor protein according to (1),
(Ii) when a compound that activates the G protein-coupled receptor protein or a salt thereof according to (1) and a test compound are brought into contact with a cell containing the G protein-coupled receptor protein according to (1), A compound that changes the binding property between a ligand and a G protein-coupled receptor protein or a salt thereof according to the above (1), wherein the cell stimulating activity mediated by the G protein-coupled receptor protein is measured and compared; Salt screening method, (46) A compound that activates the G protein-coupled receptor protein or the salt thereof described in (1) above is cultured on the transformant described in (8) above on the cell membrane of the transformant. When contacted with the expressed G protein-coupled receptor protein, 1) a compound that activates a G protein-coupled receptor protein or salt thereof according and the test compound was expressed on the cell membrane of the transformant by culturing the transformant according to (8), G
A ligand characterized by measuring and comparing the cell stimulating activity mediated by the G protein-coupled receptor protein when brought into contact with the protein-coupled receptor protein, and the G protein-coupled receptor protein described in (1) or a G protein-coupled receptor protein thereof. A method for screening a compound or a salt thereof that changes the binding property to a salt,

(47) The compound that activates the G protein-coupled receptor protein according to (1) is tyramine, beta-phenylethylamine, tryptamin
e), trace amines such as octopamine; angiotensin, bombesin,
Cannabinoid, cholecystokinin, glutamine, serotonin, melatonin, neuropeptide Y, opioid,
Purine, vasopressin, oxytocin, PACAP (eg, P
ACAP27, PACAP38), 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, pancreastatin, prostaglandin, thromboxane , Adenosine, adrenaline, chemokine superfamily (eg, IL-8, GROα, GROβ, GROγ, NAP
-2, CXC chemokine subfamily such as ENA-78, GCP-2, PF4, IP-10, Mig, PBSF / SDF-1, MCAF / MCP-1, MCP-2,
MCP-3, MCP-4, eotaxin, RANTES, MIP-1α, MIP-1β, H
CC-1, MIP-3α / LARC, MIP-3β / ELC, I-309, TARC, MIPF
-1, C such as MIPF-2 / eotaxin-2, MDC, DC-CK1 / PARC, SLC
C chemokine subfamily; C chemokine subfamily such as lymphotactin; CX3C chemokine subfamily such as fractalkine), endothelin, enterogastrin, histamine, neurotensin, TRH, pancreatic polypeptide, galanin, lysophosphatidic acid (LPA) or (45) The screening method according to (45) or (46), which is sphingosine 1-phosphate; (48) a ligand obtainable by the screening method according to (40) to (47); and G according to (1).
(49) a compound or a salt thereof that changes the binding property to a protein-coupled receptor protein or a salt thereof,
A compound or a salt thereof that alters the binding between the ligand obtainable by the screening method according to any one of (40) to (47) and the G protein-coupled receptor protein or its salt according to (1). Characteristic pharmaceutical,

(50) The screening kit according to the above (20), which comprises a cell containing the G protein-coupled receptor protein according to the above (1), (51) the G according to the above (1). The screening kit according to the above (20), which comprises a membrane fraction of a cell containing a protein-coupled receptor protein, and (52) the culturing of the transformant according to the above (8) by culturing the transformant according to the above (8). G expressed on the cell membrane of the transformant
(53) The screening kit according to the above (20), which comprises a protein-coupled receptor protein; (53) the ligand obtained by using the screening kit according to the above (50) to (52); (1) a compound or a salt thereof that alters the binding property to the G protein-coupled receptor protein or a salt thereof according to (54), which can be obtained using the screening kit according to (50) to (52); A pharmaceutical comprising a compound or a salt thereof that alters the binding of the ligand to the G protein-coupled receptor protein or a salt thereof according to the above (1), (55) an antibody according to the above (12), The G protein-coupled receptor protein according to (1) or the partial peptide according to (3), or (5) a method for quantifying the G protein-coupled receptor protein or the partial peptide according to (3) or a salt thereof according to (1), wherein the antibody according to (12); The test solution and the labeled G protein-coupled receptor protein described in (1) or the partial peptide described in (3) or a salt thereof are allowed to react competitively, and the labeled The G protein-coupled receptor protein according to (1) in a test solution, wherein the ratio of the G protein-coupled receptor protein according to (1) or the partial peptide or salt thereof according to (3) is measured. Alternatively, the method for quantifying the partial peptide or a salt thereof according to the above (3), and (57) the method described in the above (12), which is insolubilized on a test solution and a carrier. Reacting the antibody of the above (12) and the labeled antibody of the above (12) simultaneously or successively, and then measuring the activity of the labeling agent on the insolubilized carrier. The present invention provides a method for quantifying the G protein-coupled receptor protein described in (3) or the partial peptide described in (3) or a salt thereof.

[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: 1. Contains receptor protein. The receptor protein of the present invention includes, for example, human and non-human mammals (eg, guinea pigs, rats, mice, rabbits, pigs, sheep, cows, monkeys, etc.), and all cells (eg, spleen cells, nerve cells, glial cells, 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 cells, hepatocytes or stromal cells, or precursors, stem cells or cancer cells of these cells ) Or cells of the blood system, or any tissue in which those cells reside, such as the brain or parts of the brain (eg,
Olfactory bulb, abdominal nucleus, basal cerebral sphere, hippocampus, thalamus, hypothalamus, hypothalamus nucleus, cerebral cortex, medulla, cerebellum, occipital lobe, frontal lobe, temporal lobe, putamen, caudate nucleus, brain stain, nigra , Spinal cord, pituitary, stomach,
Pancreas, kidney, liver, gonad, thyroid, gall bladder, bone marrow, adrenal gland, skin, muscle, lung, digestive tract (eg, large intestine, small intestine), blood vessels, heart, thymus, spleen, submandibular gland, peripheral blood, peripheral blood cells, It may be a protein derived from the prostate, testis, testis, ovary, placenta, uterus, bone, joint, skeletal muscle, or the like, or may be a synthetic protein.

The amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 1 is, for example, about 50% or more, preferably about 60% or more, and more preferably the amino acid sequence represented by SEQ ID NO: 1. Amino acid sequences having about 70% or more, more preferably about 80% or more, particularly preferably about 90% or more, and most preferably about 95% or more homology. Examples of the protein of the present invention having an amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 1 include, for example, an amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 1, A protein having substantially the same activity as the amino acid sequence represented by SEQ ID NO: 1 is preferred. Examples of substantially the same activity include a ligand binding activity and a signal transduction action. Substantially the same indicates that their activities are the same in nature. 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 2 times), but their quantitative factors such as the degree of activity and the molecular weight of the protein may be different. The activity such as the ligand binding activity and the signal information transduction can be measured according to a known method. For example, the activity can be measured according to a ligand determination method or a screening method described later.

The receptor protein of the present invention includes one of the amino acid sequences represented by SEQ ID NO: 1.
Or two or more (preferably about 1 to 30, more preferably about 1 to 10, and still more preferably several (1 to 5))
1 or 2 or more (preferably about 1 to 30, more preferably about 1 to 10, more preferably several (1) 1 to 2 or more (preferably about 1 to 30, more preferably about 1 to 10, more preferably 1 to 5) in the amino acid sequence represented by SEQ ID NO: Is several (1-5
)), An amino acid sequence in which the amino acid is replaced with another amino acid, or a protein containing an amino acid sequence obtained by combining them.

[0015] The receptor protein herein
The amino acid sequence, according to the convention of peptide labeling,
N terminal (amino terminal), right end is C terminal (carboxyl end)
End). The amino acid sequence represented by SEQ ID NO: 1
The present invention, including receptor proteins containing
Has a carboxyl group at the C-terminus
(-COOH), carboxylate (-COO^-), Amide (-
CONH_Two) Or an ester (—COOR)
Good. Here, as R in the ester, for example,
Chill, ethyl, n-propyl, isopropyl or n-
C such as butyl_1-6Alkyl groups, such as cyclopentene
C such as cyclohexyl_3-8Cycloalkyl group, for example
For example, C such as phenyl and α-naphthyl_6-12Aryl group,
For example, phenyl-C such as benzyl and phenethyl _1-2A
Α-naphthyl such as alkyl group or α-naphthylmethyl
Le-C_1-2C such as alkyl group_7-14In addition to aralkyl groups,
Pivaloyloxymethyi commonly used as an oral ester
And the like. The receptor protein of the present invention
Is a carboxyl group (or
The carboxyl group is amidated or
Is an esterified version of the receptor tan
Included in Parkin. In this case, for example,
For example, the above-mentioned C-terminal ester and the like are used. Further
In addition, the receptor protein of the present invention includes
In proteins, the amino group of the N-terminal methionine residue
Protecting group (for example, C such as formyl group and acetyl)_2-6Al
C such as a canoyl group_1-6Acyl groups)
Glutamyl group formed by cleavage of N-terminal side in vivo
Is pyroglutamine-oxidized, on the side of the amino acid in the molecule
Substituents on the chain (eg, -OH, -SH, amino group, imida
Sol, indole, guanidino, etc.)
Protecting group (for example, C such as formyl group and acetyl)_2-6Al
C such as a canoyl group_1-6Acyl groups)
Or a so-called glycoprotein with sugar chains attached
Any complex proteins are included. Receptor of the present invention
-Specific examples of the protein include, for example, SEQ ID NO: 1
A receptor protein containing an amino acid sequence represented by
Quartz is used.

The partial peptide of the receptor protein of the present invention (hereinafter sometimes abbreviated as a partial peptide) may be any peptide as long as it is a partial peptide of the 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 substantially the same activity are used. Here, “substantially the same activity” indicates, for example, ligand binding activity. The measurement of the ligand binding activity can be performed in the same manner as described above. Specifically, the partial peptide of the receptor protein having the amino acid sequence represented by SEQ ID NO: 1 was analyzed as an extracellular region (hydrophilic site) in the hydrophobicity plot analysis shown in FIG. Is a peptide comprising Hydrophobic (Hydrophobi
c) A peptide partially containing a site can also be used. A peptide containing individual domains may be used, but a peptide 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 or more, preferably 50 or more, more preferably 100 or more of the constituent amino acid sequences of the receptor protein of the present invention. A substantially identical amino acid sequence is about 50% or more, preferably about 60% or more, more preferably about 70% or more, and still more preferably about 80% or more, particularly preferably about 90% or more, of these amino acid sequences. , Most preferably about 95% or more homology.

In the partial peptide of the present invention, one or more (preferably about 1 to 10, more preferably several (1 to 5)) amino acids in the above amino acid sequence are deleted. One or more (preferably about 1 to 20, more preferably about 1 to 10, more preferably several (1 to 5)) amino acids added to the amino acid sequence, or the amino acid One or more (preferably about 1 to 10, more preferably several, and still more preferably about 1 to 5) amino acids in the sequence may be substituted with another amino acid. In the partial peptide of the present invention, the C-terminus has a carboxyl group (—COOH), a carboxylate (—COOH).
O ⁻ ), amide (—CONH ₂ ) or ester (—COOR) (R has the same meaning as described above). When the partial peptide of the present invention has a carboxyl group (or carboxylate) other than the C-terminus, those in which the carboxyl group is amidated or esterified are also included in the partial peptide of the present invention. As the ester in this case, for example, the above-mentioned C-terminal ester and the like are used. Furthermore, similarly to the above-described receptor protein of the present invention, the partial peptide of the present invention has an amino group of the N-terminal methionine residue protected by a protecting group, and is formed by cleavage of the N-terminal side in vivo. Examples include those in which Gln is pyroglutamine-oxidized, those in which the substituent on the side chain of the amino acid in the molecule is protected by an appropriate protecting group, and those in which a sugar chain is bound, such as a so-called glycopeptide. Examples of the salt of the receptor protein or its partial peptide of the present invention include a physiologically acceptable salt with an acid or a base, and a physiologically acceptable acid addition salt is particularly preferable. Examples of such salts include salts with inorganic acids (eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid),
Alternatively, organic acids (eg, acetic acid, formic acid, propionic acid,
Fumaric acid, maleic acid, succinic acid, tartaric acid, citric acid,
For example, salts with malic acid, oxalic acid, benzoic acid, methanesulfonic acid, and benzenesulfonic acid) are used.

The receptor protein of the present invention or a salt thereof can be produced from the above-mentioned method for purifying a receptor protein from human or non-human mammal cells or tissues. It can also be produced by culturing a transformant containing the encoding DNA. Also,
The protein can also be produced according to the protein synthesis method described later or according thereto. When producing from human or non-human mammal tissues or cells, after homogenizing human or non-human mammal tissues or cells, extraction is performed with an acid or the like, and the extract is subjected to reverse phase chromatography, ion exchange chromatography. Purification and isolation can be performed by combining chromatography such as described above.

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. Such resins include, for example, chloromethyl resin, hydroxymethyl resin, benzhydrylamine resin, aminomethyl resin, 4-benzyloxybenzyl alcohol resin, 4-methylbenzhydrylamine resin, PAM resin, 4-hydroxymethylmethyl Phenylacetamidomethyl resin, polyacrylamide resin, 4-
(2 ′, 4′-dimethoxyphenyl-hydroxymethyl) phenoxy resin, 4- (2 ′, 4′-dimethoxyphenyl-Fmoc
Aminoethyl) phenoxy resin and the like. 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 in accordance with the amino acid sequence of the target protein or peptide. At the end of the reaction, a protein or peptide is cleaved from the resin, and at the same time, various protecting groups are removed. Further, an intramolecular disulfide bond formation reaction is carried out in a highly diluted solution to obtain a target protein or partial peptide or an amide thereof. For the condensation of the above protected amino acids, various activating reagents that can be used for protein synthesis can be used, and carbodiimides are particularly preferable. As carbodiimides, DC
C, N, N'-diisopropylcarbodiimide, N-ethyl-
N '-(3-dimethylaminoprolyl) carbodiimide and the like are used. Activation by these involves adding a protected amino acid directly to the resin along with a racemization inhibitor additive (eg, HOBt, HOOBt), or using a symmetrical anhydride or
HOBt ester or HOOBt ester can be added to the resin after activation of the protected amino acid in advance.

The solvent used for activating the protected amino acid or condensing with the resin can be appropriately selected from solvents known to be usable for the protein condensation reaction. For example, acid amides such as N, N-dimethylformamide, N, N-dimethylacetamide, 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, or appropriate mixtures thereof Are used. The reaction temperature is appropriately selected from a range known to be usable for a protein bond formation reaction, and is usually appropriately selected from a 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, if 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 by repeating the reaction, unreacted amino acids can be acetylated using acetic anhydride or acetylimidazole.

Examples of the protecting group for the amino group of the starting material include Z, Boc, tertiary pentyloxycarbonyl,
Isobornyloxycarbonyl, 4-methoxybenzyloxycarbonyl, Cl-Z, Br-Z, adamantyloxycarbonyl, trifluoroacetyl, phthaloyl, formyl, 2-nitrophenylsulfenyl, diphenylphosphinothioyl, Fmoc and the like are used. Can be Carboxyl groups can be, for example, alkyl esterified (eg, methyl, ethyl, propyl, butyl, tertiary butyl,
Linear, branched or cyclic alkyl esterification such as cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, 2-adamantyl), aralkyl esterification (for example, benzyl ester, 4-nitrobenzyl ester, 4-methoxybenzyl ester, 4-chlorobenzyl ester, benzhydryl esterification), phenacyl esterification, benzyloxycarbonyl hydrazide, tertiary butoxycarbonyl hydrazide, trityl hydrazide, and the like. The hydroxyl group of serine can be protected, for example, by esterification or etherification. As a group suitable for the esterification, for example, a lower alkanoyl group such as an acetyl group, an aroyl group such as a benzoyl group, a group derived from carbonic acid such as a benzyloxycarbonyl group and an ethoxycarbonyl group, and the like are used. Examples of a group suitable for etherification include a benzyl group, a tetrahydropyranyl group, and a t-butyl group. Examples of the protecting group for the phenolic hydroxyl group of tyrosine include, for example, Bzl, C
l ₂ -Bzl, 2-nitrobenzyl, Br-Z, t-butyl are used. As the imidazole protecting group for histidine, for example, Tos, 4-methoxy-2,3,6-trimethylbenzenesulfonyl, DNP, benzyloxymethyl, Bum, Boc, Trt, Fmoc and the like are used.

Examples of the activated carboxyl groups of the raw materials include, for example, corresponding acid anhydrides, azides, active esters [alcohols (eg, pentachlorophenol, 2,4,5-trichlorophenol, 2,4- Dinitrophenol, cyanomethyl alcohol, paranitrophenol, HONB, N-hydroxysuccinimide, N-hydroxyphthalimide, and esters with HOBt).
As the activated amino group of the raw material, for example,
The corresponding phosphoric amide is used. As a method for removing (eliminating) the protecting group, for example, catalytic reduction in a hydrogen stream in the presence of a catalyst such as Pd-black or Pd-carbon,
Acid treatment with anhydrous hydrogen fluoride, methanesulfonic acid, trifluoromethanesulfonic acid, trifluoroacetic acid or a mixture thereof, diisopropylethylamine,
Base treatment with triethylamine, piperidine, piperazine, or the like, reduction with sodium in liquid ammonia, and the like are also used. The elimination reaction by the above-mentioned acid treatment is generally carried out at a temperature of about -20 ° C to 40 ° C. -Addition of a cation scavenger such as butanedithiol, 1,2-ethanedithiol and the like is effective. Also used as an imidazole protecting group for histidine
The 2,4-dinitrophenyl group is removed by thiophenol treatment, and the formyl group used as an indole protecting group for tryptophan is 1,2-ethanedithiol described above.
In addition to deprotection by acid treatment in the presence of 1,4-butanedithiol or the like, it is also removed by alkali treatment with dilute sodium hydroxide solution, dilute ammonia or the like.

The protection of the functional group which should not be involved in the reaction of the starting material, the protecting group, the elimination of the protective group, the 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, after protecting the α-carboxyl group of the carboxy terminal amino acid by amidation, the peptide (protein) chain is extended to a desired chain length on the amino group side, Producing a protein from which only the protecting group for the N-terminal α-amino group of the peptide chain has been removed and a protein from which only the protecting group for the C-terminal carboxyl group has been removed, and combining both proteins in a mixed solvent as described above. To condense. Details of the condensation reaction are the same as described above. After purifying the protected protein obtained by the condensation, all the protecting groups are removed by the above-mentioned method, and a desired crude protein can be obtained. The crude protein is purified using various known purification means, and the main fraction is freeze-dried to obtain an amide of the desired protein. In order to obtain an ester of a protein, for example, after condensing the α-carboxyl group of the carboxy terminal amino acid with a desired alcohol to form an amino acid ester, an ester of the desired protein is obtained in the same manner as the amide of the protein be able to.

The partial peptide of the protein of the present invention or a salt thereof can be produced according to a known peptide synthesis method, or by cleaving the protein of the present invention with an appropriate peptidase. As a method for synthesizing a peptide, for example, any of a solid phase synthesis method and a liquid phase synthesis method may be used. That is, the target peptide can be produced by condensing a partial peptide or amino acid capable of constituting the protein of the present invention with the remaining portion and, when the product has a protecting group, removing the protecting group. Known methods for condensation and elimination of protecting groups include, for example, the methods described in (1) to (4) below. M. Bodanszky and MA Ondetti, Peptide Synthesis, Interscience Publisher
s, New York (1966) Schroeder and Luebke, The Peptide,
Academic Press, NewYork (1965) Nobuo Izumiya et al., Fundamentals and experiments of peptide synthesis, Maruzen Co., Ltd.
(1975) Haruaki Yajima and Shunpei Sakakibara, Laboratory for Biochemistry 1, Protein Chemistry IV, 205, (1977) Supervised by Haruaki Yajima, Development of Continuing Drugs Volume 14 Peptide Synthesis Hirokawa Shoten The partial peptide of the present invention can be purified and isolated by a combination of purification methods such as solvent extraction, distillation, column chromatography, liquid chromatography, and recrystallization. When the partial peptide obtained by the above method is in a free form, it can be converted to an appropriate salt by a known method. Can be.

[0025] The polynucleotide encoding the receptor protein of the present invention includes a nucleotide sequence (DNA or RNA,
(Preferably DNA). Examples of the polynucleotide include DNA encoding the receptor protein of the present invention, and RNA such as mRNA.
And may be double-stranded or single-stranded. In the case of double-stranded, it may be double-stranded DNA, double-stranded RNA or DNA: RNA hybrid. In the case of a single strand, it may be a sense strand (ie, a coding strand) or an antisense strand (ie, a non-coding strand). Using a polynucleotide encoding the receptor protein of the present invention,
Known experimental medicine special edition "New PCR and its application" 15 (7), a method described in 1997 or a method analogous thereto, for example, TaqMan PCR
By the method such as, the mR of the receptor protein of the present invention
NA can be quantified. As the DNA encoding the receptor protein of the present invention, genomic DNA, genomic DNA
The library may be any of the above-mentioned cell / tissue-derived cDNA, the above-mentioned cell / tissue-derived cDNA library, 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 chain reaction was performed directly using a preparation of total RNA or mRNA fraction from the cells and tissues described above.
n (hereinafter abbreviated as RT-PCR method). Specifically, as the DNA encoding the receptor protein of the present invention, for example, a DNA containing the nucleotide sequence represented by SEQ ID NO: 2, or SEQ ID NO: 2
Having a DNA that hybridizes under high stringent conditions with a DNA having the nucleotide sequence represented by the formula (1), and having substantially the same activity as the receptor protein of the present invention (eg, ligand binding activity, signal information transduction action, etc.) Any DNA may be used as long as it encodes a receptor protein. Examples of the DNA that hybridizes with the DNA having the nucleotide sequence represented by SEQ ID NO: 2 under high stringent conditions include, for example, about 70% or more, and preferably about 80% or more with the nucleotide sequence represented by SEQ ID NO: 2. More preferably, a DNA containing a nucleotide sequence having a homology of about 90% or more, more preferably about 95% or more is used.

Hybridization can be performed 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 performed under high stringency conditions. The high stringency conditions include, for example, a sodium concentration of about 19 to 40 mM, preferably about 19 to 20 mM.
At a temperature of about 50 to 70 ° C, preferably about 60 to 65 ° C. In particular, a sodium concentration of about 19 mM and a temperature of about 65
C is most preferred. More specifically, SEQ ID NO:
As the DNA encoding the receptor protein containing the amino acid sequence represented by 1, a DNA containing the base sequence represented by SEQ ID NO: 2 or the like is used. A part of the nucleotide sequence of the DNA encoding the receptor protein of the present invention, or a polynucleotide containing a part of the nucleotide sequence complementary to the DNA, is a DNA encoding the following partial peptide of the present invention. It is used to include not only RNA but also RNA. 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 is cloned or determined and encodes a G protein-coupled receptor protein.
It can be designed and synthesized based on DNA base sequence information. Such a polynucleotide (nucleic acid) can hybridize to RNA of a G protein-coupled receptor protein gene and inhibit the synthesis or function of the RNA, or can bind to G protein-coupled receptor protein-related RNA. The expression of the G protein-coupled receptor protein gene can be regulated and controlled through the interaction. Polynucleotides that are complementary to a selected sequence of a G protein-coupled receptor protein-related RNA, and that can specifically hybridize to a G protein-coupled receptor protein-related RNA, can be used in vivo and in vitro. It is useful for regulating and controlling the expression of a protein-coupled receptor protein gene, and is also useful for treating or diagnosing a disease or the like. In addition, the 5′-end hairpin loop, 5′-end 6-base pair repeat, 5′-end untranslated region, polypeptide translation start codon, protein coding region, ORF translation stop codon of the G protein-coupled receptor protein gene,
The 3 'untranslated region, the 3' palindromic region, and the 3 'end hairpin loop may be selected as preferred target regions, but any region within the G protein-coupled receptor protein gene may be selected.

The relationship between the target nucleic acid and a polynucleotide complementary to at least a part of the target region, that is, the relationship between the target nucleic acid and a polynucleotide capable of hybridizing with the target is “antisense”. it can. Antisense polynucleotides are 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 having a non-nucleotide backbone (eg, commercially available protein nucleic acids and synthetic sequence-specific nucleic acid polymers) or other polymers containing special bonds (provided that the polymer is DNA or RNA)
(Including nucleotides having a configuration that allows base pairing and base attachment as found therein), and the like. They are double-stranded DNA, single-stranded DNA, double-stranded RN
A, which can be a single-stranded RNA, or even a DNA: RNA hybrid, further with an unmodified polynucleotide (or an unmodified oligonucleotide), or with a known modification, such as a label known in the art , Capped, methylated, one or more natural nucleotides replaced with analogs, modified intramolecularly, such as uncharged bonds (e.g., methylphosphonate, Those having a phosphotriester, phosphoramidate, carbamate, etc., those having a charged or sulfur-containing bond (eg, phosphorothioate, phosphorodithioate, etc.), such as proteins (nucleases, nuclease inhibitors, toxins, Antibody, signal peptide, poly-L-
Those having a side chain group such as lysine or sugar (for example, monosaccharide), those having an interactive compound (for example, acridine or psoralen), chelating compounds (for example, metal, having radioactivity) It may be one containing a metal, boron, an oxidizing metal, or the like, one containing an alkylating agent, or one having a modified bond (for example, α-anomeric nucleic acid). Here, "nucleoside", "nucleotide", and "nucleic acid" may include not only those containing purine and pyrimidine bases but also those having other modified heterocyclic bases. Such modifications may include methylated purines and pyrimidines, acylated purines and pyrimidines, or other heterocycles. Modified nucleotides and modified nucleotides may also be modified at the sugar moiety, for example,
One or more hydroxyl groups may be substituted with a halogen, an aliphatic group, or the like, or may be converted to a functional group such as an ether or an amine.

The antisense polynucleotide (nucleic acid) of the present invention may be RNA, DNA, or a modified nucleic acid (RN
A, DNA). Specific examples of modified nucleic acids include, but are not limited to, sulfur derivatives and thiophosphate derivatives of nucleic acids, and those that are 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, to make the antisense nucleic acid more stable in the cell, to make the antisense nucleic acid more cell permeable, to have a greater affinity for the target sense strand, and to be more toxic if it is toxic. Minimize the toxicity of sense nucleic acids.
Many such modifications are known in the art, for example, J. Kawakami et al., Pharm Tech Japan, Vol. 8, p.
p.247, 1992; Vol. 8, pp.395, 1992; ST Crooke et
al. ed., Antisense Research and Applications, CRC
Press, 1993. The antisense nucleic acid of the present invention may contain altered or modified sugars, bases or bonds, may be provided in a special form such as liposomes or microspheres, may be applied by gene therapy, It could be provided in an added form. Thus, in the form of addition, polycations such as polylysine which acts to neutralize the charge of the phosphate skeleton, lipids which enhance the interaction with the cell membrane or increase the uptake of nucleic acids ( For example, hydrophobic substances such as phospholipid and cholesterol can be mentioned. Preferred lipids for addition include cholesterol and its derivatives (eg, cholesteryl chloroformate, cholic acid, etc.). These can be attached to the 3 'or 5' end of the nucleic acid,
It can be attached via bases, sugars, intramolecular nucleoside bonds. Other groups include cap groups specifically arranged at the 3 ′ end or 5 ′ end of a nucleic acid for preventing degradation by nucleases such as exonuclease and RNase. Such capping groups include, but are not limited to, hydroxyl-protecting groups known in the art, including glycols such as polyethylene glycol and tetraethylene glycol. The antisense nucleic acid inhibitory activity can be examined 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 a G protein-coupled receptor protein. it can. The nucleic acid can be applied to cells by various known methods.

The DNA encoding the partial peptide of the present invention may be any DNA containing the above-described nucleotide sequence encoding the partial peptide of the present invention. In addition, genomic DNA, genomic DNA library, cDNA derived from the cells / tissues described above,
It may be either a cDNA library or a synthetic DNA. The vector used for the library may be any of bacteriophage, plasmid, cosmid, phagemid and the like. Alternatively, amplification can be performed directly by RT-PCR using an mRNA fraction prepared from the cells and tissues described above. Specifically, the DNA encoding the partial peptide of the present invention includes, for example, (1) DNA having a partial nucleotide sequence of DNA having the nucleotide sequence represented by SEQ ID NO: 2,
Or (2) having a DNA that hybridizes with the DNA represented by SEQ ID NO: 2 under high stringent conditions,
Activities substantially the same as those of the protein peptides of the present invention (eg, ligand binding activity, signal transduction, etc.)
For example, a DNA having a partial nucleotide sequence of a DNA encoding a protein having Examples of the DNA that hybridizes with the DNA represented by SEQ ID NO: 2 under high stringency conditions include, for example, about 70% or more, preferably about 80% or more, more preferably about 80% or more of the base sequence represented by SEQ ID NO: 2. DNA containing a nucleotide sequence having a homology of 90% or more, more preferably about 95% or more is used.

As a means for cloning a DNA completely encoding the receptor protein of the present invention or a partial peptide thereof (hereinafter, sometimes abbreviated as the receptor protein of the present invention), the peptide of the present invention is encoded.
Amplified by PCR using a synthetic DNA primer having a partial base sequence of the DNA base sequence, or a DNA fragment encoding a partial or whole region of the receptor protein of the present invention, Selection can be carried out by hybridization with a DNA-labeled DNA. Hybridization methods are described, for example, in Molecular Cloning 2nd (J. Sambrook et 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.

The DNA base sequence can be replaced by OD using PCR or a known kit such as Mutan ^™ -super Express Km (Takara Shuzo) or Mutan ^™ -K (Takara Shuzo).
A known method such as the A-LA PCR method, the gapped duplex method and the Kunkel method, or a method analogous thereto can be used. The DNA encoding the cloned receptor protein can be used as it is depending on the purpose, or can be used after digesting with a restriction enzyme or adding a linker, if desired. The DNA may have ATG as a translation initiation codon at the 5 'end and TAA, TGA or TAG as a translation stop codon at the 3' end. These translation start codons and translation stop codons should be
It can also be added using an A adapter. The expression vector of the receptor protein of the present invention includes, for example,
(A) DNA encoding the receptor protein of the present invention
For example, it can be produced by cutting out a target DNA fragment from cDNA and (ii) ligating the DNA fragment downstream of a promoter in an appropriate expression vector.

As the vector, plasmids derived from E. coli (eg, pCR4, pCR2.1, pBR322, pBR325, pUC12, pUC1
3), Bacillus subtilis-derived plasmids (eg, pUB110, pTP5, pC1
94), yeast-derived plasmids (eg, pSH19, pSH15), bacteriophages such as phage 1, animal viruses such as retrovirus, vaccinia virus, baculovirus, etc., as well as pA1-11, pXT1, pRc / CMV, pRc / RSV , PcDNAI
/ Neo is used. The promoter used in the present invention may be any promoter as long as it is appropriate for the host used for gene expression. For example, when an animal cell is used as a host, SRα promoter, SV40 promoter, LTR promoter, CMV promoter, HSV-TK promoter and the like can be mentioned. Among them, it is preferable to use the CMV promoter, SRα promoter and the like. When the host is Escherichia, trp promoter, lac promoter, recA promoter, .lambda.P _L promoter, lpp promoter, etc. When the host is Bacillus, SPO1 promoter, SP
When the host is yeast, such as O2 promoter and penP promoter, PHO5 promoter, PGK promoter, GAP
Promoters and ADH promoters are preferred. If the host is an insect cell, a polyhedrin promoter,
P10 promoter and the like are preferred.

[0033] In addition to the above, expression vectors include, if desired, an enhancer, a splicing signal, a polyA addition signal, a selection marker, and an SV40 replication origin (hereinafter referred to as SV
40 ori) may be used. As the selection marker include dihydrofolate reductase (hereinafter sometimes abbreviated as dhfr) gene [methotrexate (MTX) resistance], ampicillin resistant gene (hereinafter sometimes abbreviated as Amp ^r), neomycin resistant gene (Hereinafter, sometimes abbreviated as Neo ^r , G418 resistance) and the like. In particular, CHO (d
When the dhfr gene is used as a selection marker using hfr ⁻ ) cells, the target gene can also be selected using a thymidine-free medium. 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 a bacterium belonging to the genus Escherichia, a PhoA signal sequence, an OmpA signal sequence,
When the host is Bacillus, α-amylase signal sequence, subtilisin signal sequence, etc., when the host is yeast, MFα signal sequence, SUC2 signal sequence, etc., when the host is an animal cell For example, an insulin signal sequence, an α-interferon signal sequence, an antibody molecule / signal sequence, etc. can be used. Using the vector containing the DNA encoding the receptor protein of the present invention thus constructed, a transformant can be produced.

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 DH1 [Procedings of the National Academy
Of Sciences of the USA (Pro
Natl. Acad. Sci. USA), 60, 160 (1968)], JM1
03 [Nukuilec Acids Research, (Nucleic Ac
idsResearch, 9, 309 (1981)], JA221 [Journal of M.
olecular Biology)], 120, 517 (1978)], HB101
[Journal of Molecular Biology, 41
Vol. 459 (1969)], C600 [Genetics (Genetic
s), 39, 440 (1954)], DH5α [Inoue, H., Nojima,
H. and Okayama, H., Gene, 96, 23-28 (1990)], DH10
B [Processings of the National Academy of Sciences of the USA (Proc. Natl. Acad. Sci. USA), 87, 4645-4649]
(1990)]. Examples of Bacillus bacteria include, for example, Bacillus subtilis MI1
14 [Gene, 24, 255 (1983)], 207-21 [Journal of Biochemis
try), 95, 87 (1984)]. Examples of yeast include Saccharomyces cerevisiae (Saccha
romyces cerevisiae) AH22, AH22R, NA87-11A, DKD-5
D, 20B-12, Schizosaccharomyces pombe (Schizosac
charomyces pombe) NCYC1913, NCYC2036, Pichia pastoris and the like are used.

As insect cells, for example, the virus is Ac
In the case of NPV, a cell line derived from the larva of the armyworm, Spodopter
a frugiperda cell (Sf cell), MG1 cell derived from Trichoplusia ni midgut, High Five derived from Trichoplusia ni egg
^TM cells, cells derived from Mamestra brassicae or Estigme
Cells derived from na acrea are used. Virus is BmNP
In the case of V, a silkworm-derived cell line (Bombyx mori N; BmN
Cells) are used. As the Sf cells, for example,
Sf9 cells (ATCC CRL1711), Sf21 cells (Vaughn,
JL et al., In Vivo, 13, 213-217 (197
7)) is used. As insects, for example, silkworm larvae are used [Maeda et al., Nature (Natur
e), Volume 315, 592 (1985)]. Examples of animal cells include monkey cell COS-7, Vero, Chinese hamster cell CHO (hereinafter, CHO cells), dhfr gene deficient Chinese hamster cell CHO (hereinafter, CHO (dhfr ^-) cell), mouse L Cells, mouse AtT-20, mouse myeloma cells, rat GH3, human FL cells and the like are used.

For transformation of Escherichia, for example, Processing of the National Academy of Sciences of the USA (Proc. Natl. Acad. Sci. USA), Vol. 69 , 2110 (197
2) and Gene (17), 107 (1982). In order to transform Bacillus sp., For example, Molecular & General Genetic (Molecular & General Genetic)
s), Vol. 168, 111 (1979). To transform yeast, for example, methods in Enzymol
ogy), Volume 194, 182-187 (1991), Procsings
Proc. Natl. Acad. Sc of the National Academy of Sciences of the USA
i. USA), vol. 75, 1929 (1978). To transform insect cells or insects, for example, use Bio / Techno
logy), 6, 47-55 (1988)). To transform animal cells, for example, see Cell Engineering Separate Volume 8, New Cell Engineering Experiment Protocol. 26
3-267 (1995) (published by Shujunsha), virology (Virolo)
gy), Vol. 52, 456 (1973). Thus, a transformant transformed with the expression vector containing the DNA encoding the G protein-coupled receptor protein is obtained. When culturing a transformant whose host is a genus Escherichia or Bacillus, a liquid medium is suitable as a medium to be used for the culture. Nitrogen sources, inorganic substances, etc. are contained. As a carbon source, for example, glucose, dextrin, soluble starch, sucrose, etc.As a nitrogen source, for example, ammonium salts, nitrates, corn steep liquor, peptone, casein, meat extract, soybean meal, potato extract and the like Examples of the inorganic or organic substance and the inorganic substance include calcium chloride, sodium dihydrogen phosphate, and magnesium chloride. In addition, yeast extract, vitamins, growth promoting factors and the like may be added. The pH of the medium is about
5 to 8 is desirable.

As a medium for culturing the genus Escherichia, for example, M9 medium containing glucose and casamino acid [Miller, Journal of Experiments in Molecular Genetics (Journa)
l of Experiments in Molecular Genetics), 431-43
3, Cold Spring Harbor Laboratory, New York 1972]
Is preferred. If necessary, an agent such as 3β-indolylacrylic acid can be added in order to make the promoter work efficiently. When the host is a bacterium belonging to the genus Escherichia, the cultivation is usually carried out at about 15 to 43 ° C. for about 3 to 24 hours, and if necessary, aeration and stirring can be applied. When the host is a bacterium belonging to the genus Bacillus, the culture is usually about 30 to 40 ° C.
For about 6 to 24 hours, and if necessary, aeration and stirring can be applied. When culturing a transformant in which the host is yeast, for example, Burkholder (Burkho
lder) Minimal Medium [Bostian, KL et al., "Procedures of the National Academy of Sciences of the USA (Proc. Natl. Ac
ad. Sci. USA), Vol. 77, 4505 (1980) "and an SD medium containing 0.5% casamino acid [Bitter, GA et al. The USA (Proc.Natl. A
cad. Sci. USA), 81, 5330 (1984) ". Preferably, the pH of the medium is adjusted to about 5-8. The cultivation is usually performed at about 20 ° C. to 35 ° C. for about 24 to 72 hours, and aeration and stirring are added as necessary.

When culturing an insect cell or a transformant whose host is an insect, Grace's Insect Mediu
m (Grace, TCC, Nature, 195, 788 (1
962)) to which an additive such as 10% bovine serum immobilized as appropriate has been added. Preferably, the pH of the medium is adjusted to about 6.2-6.4. The cultivation is usually performed at about 27 ° C. for about 3 to 5 days, and aeration and stirring are added as necessary. When culturing a transformant in which the host is an animal cell, examples of the medium include a MEM medium containing about 5 to 20% fetal bovine serum [Science, Vol. 122, 501 (1952)], a DMEM medium [Virology, 8, 396 (1959)], RPMI 1640 medium [Journal of the American Medical.
Association (The Journal of the American Medi
cal Association) 199, 519 (1967)], 199 medium [Proceding of the Society for the.
Biological Medicine (Proceeding of the Soci)
ety for the Biological Medicine), 73, 1 (195
0)]. Preferably, the pH is between about 6 and 8. The cultivation is usually performed at about 30 ° C. to 40 ° C. for about 15 to 60 hours, and if necessary, aeration or stirring is added. As described above, the G protein-coupled receptor protein of the present invention can be produced in the transformant, in the cell membrane, or outside the cell.

The receptor protein of the present invention can be separated and purified from the above culture by, for example, the following method. When extracting the receptor protein of the present invention from cultured cells or cells, after culturing,
The cells or cells are collected by a known method, suspended in an appropriate buffer, and disrupted by ultrasonication, lysozyme, and / or freeze-thawing, etc., and then the crude receptor protein is centrifuged or filtered. A method of obtaining an extract is appropriately used. In a buffer, a protein denaturant such as urea or guanidine hydrochloride, or Triton X-1
00 ^TM surfactant may be included, such as. When the receptor protein is secreted into the culture solution, after the culture is completed, the supernatant is separated from the cells or cells 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 and purification methods. These known separation and purification methods include:
Methods using solubility such as salting out and solvent precipitation, dialysis, ultrafiltration, gel filtration, and methods mainly using differences in molecular weight such as SDS-polyacrylamide gel electrophoresis, ion exchange chromatography Such as methods that use differences in charge, methods that use specific affinity such as affinity chromatography, methods that use differences in hydrophobicity such as reverse-phase high-performance liquid chromatography, and methods such as isoelectric focusing. A method utilizing the difference between the electric points is used.

When the thus obtained receptor protein is obtained in a free form, it can be converted into a salt by a known method or a method analogous thereto. It can be converted to a free form or another salt by a method or a method analogous thereto. The receptor protein produced by the recombinant can be arbitrarily modified or the polypeptide can be partially removed by applying an appropriate protein modifying enzyme before or after purification. As the protein modifying enzyme, for example, trypsin, chymotrypsin, arginyl endopeptidase, protein kinase, glycosidase and the like are used. 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 or its partial peptide or its salt of the present invention may be any of polyclonal antibody and monoclonal antibody as long as it can recognize the receptor protein or its partial peptide or its salt of the present invention. Is also good. An antibody against the receptor protein of the present invention or a partial peptide thereof or a salt thereof (hereinafter, sometimes abbreviated as the receptor protein of the present invention) may be obtained by using the receptor protein of the present invention as an antigen, and using a known antibody or antiserum. It can be manufactured according to the manufacturing method.

[Preparation of Monoclonal Antibody] (a) Preparation of Monoclonal Antibody-Producing Cell The receptor protein of the present invention is administered to a mammal at a site where the antibody can be produced by administration to itself or together with a carrier and a diluent. You. Complete Freund's adjuvant or incomplete Freund's adjuvant may be administered in order to enhance the antibody-producing ability upon administration. Administration is usually
It is performed once every 2 to 6 weeks, 2 to 10 times in total. Examples of mammals used include monkeys, rabbits, dogs,
Guinea pigs, mice, rats, sheep and goats can be mentioned, and mice and rats are preferably used. When producing monoclonal antibody-producing cells, a warm-blooded animal immunized with an antigen, for example, an individual with an antibody titer was selected from a mouse and the spleen or lymph node was collected 2 to 5 days after the final immunization, and the By fusing the contained antibody-producing cells with myeloma cells, a monoclonal antibody-producing hybridoma can be prepared. The antibody titer in the antiserum can be measured, for example, by reacting a labeled receptor protein or the like described below with the antiserum, and then measuring the activity of a labeling agent bound to the antibody. The fusion operation is performed by a known method, for example, the method of Kohler and Milstein [Nature, 256, 495 (1975)].
Year)]. Examples of the fusion promoter include polyethylene glycol (PEG) and Sendai virus, but PEG is preferably used. As myeloma cells, for example, NS-1, P3U1, SP
2/0 and the like, but P3U1 is preferably used.
The preferred ratio between the number of antibody-producing cells (spleen cells) and the number of myeloma cells used is about 1: 1 to 20: 1, and PEG (preferably PEG1000 to PEG6000) is added at a concentration of about 10 to 80%. About 20 to 40 ° C, preferably about 30 to 37 ° C for about 1 to 10
The cell fusion can be performed efficiently by incubating the mixture for minutes.

Various methods can be used for screening a monoclonal antibody-producing hybridoma. For example, a hybridoma culture supernatant is added to a solid phase (eg, a microplate) on which an antigen such as a receptor protein is adsorbed directly or together with a carrier. Then, an anti-immunoglobulin antibody (anti-mouse immunoglobulin antibody is used if the cell used for cell fusion is a mouse) or protein A labeled with a radioactive substance or an enzyme is added, and the monoclonal antibody bound to the solid phase is added. Detection method: Add hybridoma culture supernatant to solid phase to which anti-immunoglobulin antibody or protein A is adsorbed, add receptor protein etc. labeled with radioactive substance or enzyme, and detect monoclonal antibody bound to solid phase And the like. The selection of the monoclonal antibody can be performed according to a known method or a method analogous thereto.
(Hypoxanthine, aminopterin, thymidine) in a medium for animal cells or the like. As a selection and breeding medium, any medium can be used as long as a hybridoma can grow. For example, 1
RPMI 164 with ~ 20%, preferably 10-20% fetal calf serum
0 medium, GIT medium containing 1-10% fetal bovine serum (Wako Pure Chemical Industries, Ltd.) or serum-free medium for hybridoma culture (SF
M-101, Nissui Pharmaceutical Co., Ltd.) can be used.
The culturing temperature is usually 20 to 40 ° C, preferably about 37 ° C.
The culturing time is usually 5 days to 3 weeks, preferably 1 week to 2 weeks. The culture can be usually performed under 5% carbon dioxide gas. The antibody titer of the hybridoma culture supernatant can be measured in the same manner as the measurement of the antibody titer in the antiserum described above.

(B) Purification of Monoclonal Antibody Separation and purification of the monoclonal antibody can be performed by the same method as in the separation and purification of normal polyclonal antibodies, for example, by separation and purification of immunoglobulin [eg, salting out method, alcohol precipitation method, isoelectric point precipitation method, Electrophoresis, adsorption / desorption with ion exchanger (eg DEAE), ultracentrifugation, gel filtration, antigen-bound solid phase or protein A
Alternatively, a specific purification method in which only the antibody is collected by an active adsorbent such as protein G and the bond is dissociated to obtain the antibody)
Can be performed according to

[Preparation of Polyclonal Antibody] The polyclonal antibody of the present invention can be produced according to a known method or a method analogous thereto. For example, a complex of an immunizing antigen (an antigen such as the protein of the present invention) and a carrier protein is formed, and a mammal is immunized in the same manner as in the above-described method for producing a monoclonal antibody. The antibody can be produced by collecting an antibody-containing substance against the antibody and separating and purifying the antibody. Regarding a complex of an immunizing antigen and a carrier protein used to immunize a mammal, the type of the carrier protein and the mixing ratio of the carrier and the hapten should be such that the antibody can be efficiently produced against the hapten immunized by crosslinking the carrier. What may be cross-linked at any ratio, for example, bovine serum albumin,
A method is used in which bovine thyroglobulin, keyhole limpet, hemocyanin and the like are coupled in a weight ratio of about 0.1 to 20, preferably about 1 to 5, with respect to 1 hapten. Various coupling agents can be used for coupling the hapten and the carrier. For example, glutaraldehyde, carbodiimide, a maleimide active ester, an active ester reagent containing a thiol group or a dithioviridyl group, or the like is used. The condensation product is administered to a warm-blooded animal itself or together with a carrier or diluent at a site where antibody production is possible. Complete Freund's adjuvant or incomplete Freund's adjuvant may be administered in order to enhance the antibody-producing ability upon administration. The administration can usually be performed once every about 2 to 6 weeks, for a total of about 3 to 10 times. The polyclonal antibody can be collected from blood, ascites, or the like, preferably from blood, of the mammal immunized by the above method. The measurement of the polyclonal antibody titer in the antiserum can be performed in the same manner as the measurement of the antibody titer in the serum described above. The 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 a salt thereof, a partial peptide thereof or a salt thereof, and a DNA encoding the receptor protein or a partial peptide thereof
(1) Determination of ligand (agonist) for G protein-coupled receptor protein of the present invention, (2)
An agent for preventing and / or treating a disease associated with dysfunction of a G protein-coupled receptor protein of the present invention,
(3) a gene diagnostic agent, (4) a method for screening a compound that changes the expression level of the receptor protein or its partial peptide of the present invention, (5) a compound that changes the expression level of the receptor protein or its partial peptide of the present invention. Preventive and / or therapeutic agent for various diseases contained therein,
(6) a method for quantifying a ligand for the G protein-coupled receptor protein of the present invention, (7) a method for screening a compound (agonist, antagonist, etc.) that changes the binding property between the G protein-coupled receptor protein and the ligand of the present invention, (8) a prophylactic and / or therapeutic agent for various diseases containing a compound (agonist, antagonist) that alters the binding property between the G protein-coupled receptor protein and the ligand of the present invention; (9) the receptor protein of the present invention or a derivative thereof. Quantification of partial peptide or salt thereof, (10) Screening method for compound that changes the amount of receptor protein of the present invention or its partial peptide in cell membrane, (11) Change of amount of receptor protein of the present invention or its partial peptide in cell membrane Compound (12) Neutralization of the receptor protein of the present invention or its partial peptide or its salt with an antibody, and (13) DNA encoding the G protein-coupled receptor protein of the present invention. Can be used for producing non-human transgenic animals having In particular, by using a receptor binding assay system using the recombinant G protein-coupled receptor protein expression system of the present invention, the binding of a ligand to a G protein-coupled receptor specific to a human or non-human mammal can be improved. Compounds to be changed (eg, agonists, antagonists, etc.) can be screened, and the agonists or antagonists can be used as agents for preventing or treating various diseases. The receptor protein or partial peptide of the present invention or a salt thereof (hereinafter sometimes abbreviated as the receptor protein of the present invention), a 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 antibodies 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 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. Examples of the test compound include known ligands (for example, trace amines such as tyramine, beta-phenylethylamine (β-phenylethylamine), tryptamine, and octopamine); angiotensin, bombesin, and cannabinoid , Cholecystokinin, glutamine, serotonin, melatonin, neuropeptide Y, opioid, purine, vasopressin, oxytocin, PACAP (eg, PACAP27, PACAP38), secretin, glucagon, calcitonin, adrenomedullin, somatostatin, GHRH, CRF, ACTH, GRP, PTH, VIP (Vasoactive Intestinal and Related Polypeptide), somatostatin, dopamine, motilin,
Amylin, bradykinin, CGRP (calcitonin gene relayed peptide), leukotriene, pancreatastatin, prostaglandin, thromboxane,
Adenosine, adrenaline, chemokine superfamily (eg, IL-8, GROα, GROβ, GROγ, NAP-2, ENA-7
8, CXC chemokine subfamily such as GCP-2, PF4, IP-10, Mig, PBSF / SDF-1; MCAF / MCP-1, MCP-2, MCP-3, MC
P-4, eotaxin, RANTES, MIP-1α, MIP-1β, HCC-1, MIP
-3α / LARC, MIP-3β / ELC, I-309, TARC, MIPF-1, MIPF-
2 / eotaxin-2, MDC, DC-CK1 / PARC, CC chemokine subfamily such as SLC; C chemokine subfamily such as lymphotactin; CX3 chemokine subfamily such as fractalkine, etc.), endothelin, enterogastrin, histamine, neurotensin, In addition to TRH, pancreatic polypeptide, galanin, lysophosphatidic acid (LPA), sphingosine 1-phosphate, etc., for example, human or non-human mammals (eg, mouse, rat, pig, cow, sheep, monkey) ), Cell culture supernatant, and the like. For example, the tissue extract,
A cell culture supernatant or the like is added to the receptor protein of the present invention, and fractionation is performed while measuring cell stimulating activity and the like, whereby a single ligand can be finally obtained.

Specifically, the ligand determination method of the present invention uses the receptor protein of the present invention or a partial peptide thereof or a salt thereof, or constructs a recombinant receptor protein expression system and uses the expression system. By using the conventional receptor binding assay system, it binds to the receptor protein of the present invention and has a cell stimulating activity (eg, arachidonic acid release, acetylcholine release, intracellular release).
Ca ²⁺ release, intracellular cAMP production, intracellular cGMP production, inositol phosphate production, cell membrane potential fluctuation, intracellular protein phosphorylation, c-fos activation, activity to promote or suppress pH reduction, etc.) (Eg, peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, etc.) or salts thereof. In the ligand determination method of the present invention, when the receptor protein of the present invention or a partial peptide thereof is brought into contact with a test compound, for example, the amount of the test compound bound to the receptor protein or the partial peptide, the cell stimulating activity, etc. Is measured.

More specifically, the present invention relates to a method for producing a labeled test compound, which comprises 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 method for determining a ligand for a receptor protein of the present invention or a salt thereof, which comprises measuring the amount of binding to a salt thereof, or the partial peptide or a salt thereof, comprising a labeled test compound containing the receptor protein of the present invention. Determining a ligand for the receptor protein of the present invention or a salt thereof, which comprises measuring the amount of a labeled test compound bound to the cell or the membrane fraction when the cell is brought into contact with the cell or the membrane fraction of the cell. Method: A labeled test compound is ligated to the receptor protein of the present invention. Culturing a transformant containing a DNA encoding the same, and contacting the receptor protein expressed on the cell membrane with a receptor protein, and measuring the amount of binding of the labeled test compound to the receptor protein or a salt thereof. A method for determining a ligand for the receptor protein of the present invention,

When a test compound is brought into contact with a cell containing the receptor protein of the present invention, cell stimulating activity via the receptor protein (eg, arachidonic acid release, acetylcholine release, intracellular Ca ²⁺ release,
Intracellular cAMP production, Intracellular cGMP production, Inositol phosphate production, Cell membrane potential fluctuation, Intracellular protein phosphorylation,
measuring the activity of promoting or suppressing c-fos, lowering pH, etc.), a method for determining a ligand for the receptor protein of the present invention or a salt thereof, and a test compound of the present invention. Cell stimulating activity through the receptor protein (eg, arachidonic acid release, acetylcholine release, cells) when the transformant containing the DNA encoding the receptor protein is cultured and then brought into contact with the receptor protein expressed on the cell membrane. Activity or suppression that promotes intracellular Ca ²⁺ release, intracellular cAMP production, intracellular cGMP production, inositol phosphate production, cell membrane potential fluctuation, intracellular protein phosphorylation, c-fos activation, pH decrease, etc. Activity of the receptor protein of the present invention or a salt thereof. Provide a method for determining the In particular, it is preferable to perform the above-mentioned tests after conducting the above-mentioned tests to confirm that the test compound binds to the receptor protein of the present invention.

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. Largely expressed receptor proteins are suitable. The above-mentioned expression method is used for producing the receptor protein of the present invention, and it is preferably carried out by expressing DNA encoding the receptor protein in mammalian cells or insect cells. Complementary DNA is usually used for the DNA fragment encoding the target protein portion, but is not necessarily limited to this. For example,
Gene fragments and synthetic DNAs may be used. In order to introduce a DNA fragment encoding the receptor protein of the present invention into host animal cells and express them efficiently, the DNA
Nuclear polyhedrosis virus (NPV) belonging to the baculovirus using the fragment as an insect host
It is preferred to incorporate the promoter into the downstream of the polyhedrin promoter, SV40-derived promoter, retrovirus promoter, metallothionein promoter, human heat shock promoter, cytomegalovirus promoter, SRα 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
Vol. 19555-19559, 1992].

Therefore, in the ligand determination method of the present invention, the receptor protein of the present invention or its partial peptide or its salt may be a receptor protein or its partial peptide or its salt purified according to a known method. Alternatively, a cell containing the receptor protein or a cell membrane fraction thereof may be used. When cells containing the receptor protein of the present invention are used in the ligand determination 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 of the present invention refers to a host cell that has expressed the receptor protein of the present invention.
Escherichia coli, Bacillus subtilis, yeast, insect cells, animal cells and the like are used. The cell membrane fraction refers to a cell membrane-rich fraction obtained by crushing cells and then obtained by a known method. As a method for crushing cells, a method of crushing cells with a Potter-Elvehjem type homogenizer, crushing with a Waring blender or a polytron (manufactured by Kinematica), crushing with ultrasonic waves, spouting cells from a thin nozzle while applying pressure with a French press, etc. Crushing and the like. For the fractionation of cell membranes, fractionation by centrifugal force such as fractionation centrifugation or density gradient centrifugation is mainly used. For example, the cell lysate is centrifuged at a low speed (500-3000 rpm) for a short time (usually about 1 minute to 10 minutes), and the supernatant is further centrifuged at a high speed (15000 to 30000 rpm), usually for 30 minutes to 2 hours. The resulting precipitate is used as the 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 cells containing the receptor protein and in the membrane fraction thereof is preferably 10 ³ to 10 ⁸ molecules per cell, more preferably 10 ⁵ to 10 ⁷ molecules per cell. is there. The higher the expression level, the higher the ligand binding activity (specific activity) per membrane fraction, which not only enables the construction of a highly sensitive screening system,
A large number of samples can be measured in the same lot. In order to carry out the above-mentioned method for determining a ligand for the receptor protein of the present invention or a salt thereof,
Appropriate receptor protein fractions and labeled test compounds are required. As the receptor protein fraction,
A natural receptor protein fraction or a recombinant receptor fraction having an activity equivalent thereto is desirable. Here, “equivalent activity” refers to equivalent ligand binding activity, signal transduction action, and the like. Examples of labeled test compounds include tyramine labeled with [ ³ H], [ ¹²⁵ I], [ ¹⁴ C], and [ ³⁵ S], β-phenylethylamine, β-phenylethylamine, and tryptamine. , Trace amines such as octopamine; angiotensin, bombesin, cannabinoid, cholecystokinin, glutamine, serotonin, melatonin, neuropeptide
Y, opioids, purines, vasopressin, oxytocin, PACAP (eg, PACAP27, PACAP38), secretin, glucagon, calcitonin, adrenomedullin, somatostatin, GHRH, CRF, ACTH, GRP, PTH, VIP (Vasoactive Intestinal and Reited) Polypeptide), somatostatin, dopamine, motilin,
Amylin, bradykinin, CGRP (calcitonin gene relayed peptide), leukotriene, pancreastatin, prostaglandin, thromboxane, adenosine, adrenaline, chemokine superfamily (eg, IL-8, GROα, GROβ, GROγ, NAP-2, ENA-78, GC
CXC chemokine subfamily such as P-2, PF4, IP-10, Mig, PBSF / SDF-1; MCAF / MCP-1, MCP-2, MCP-3, MCP-
4, eotaxin, RANTES, MIP-1α, MIP-1β, HCC-1, MIP-3
α / LARC, MIP-3β / ELC, I-309, TARC, MIPF-1, MIPF-2 /
eotaxin-2, MDC, DC-CK1 / PARC, CC chemokine subfamily such as SLC; C chemokine subfamily such as lymphotactin; CX3 chemokine subfamily such as fractalkine), endothelin, enterogastrin, histamine, neurotensin, TRH, Pancreatic polypeptide, galanin, lysophosphatidic acid (LP
A), sphingosine 1-phosphate and the like are preferred.

Specifically, in order to carry out the method for determining a ligand for the receptor protein of the present invention or a salt thereof, a cell or a membrane fraction of the cell containing the receptor protein of the present invention is first treated with a buffer suitable for the determination method. To prepare a receptor preparation. The buffer may be any buffer such as a phosphate buffer having a pH of 4 to 10 (preferably pH 6 to 8) or a buffer such as Tris-HCl buffer that does not inhibit the binding between the ligand and the receptor protein. In order to reduce non-specific binding, surfactants such as CHAPS, Tween-80 ^™ (Kao-Atlas), digitonin, deoxycholate, and various proteins such as bovine serum albumin and gelatin may be added to the buffer. it can. Furthermore, a protease inhibitor such as PMSF, leupeptin, E-64 (manufactured by Peptide Research Laboratories) and pepstatin can be added for the purpose of suppressing the degradation of the receptor or ligand by the protease. 0.
A fixed amount (5,000 to 50,000) is added to 01 to 10 ml of the receptor solution.
Test compounds labeled with [ ³ H], [ ¹²⁵ I], [ ¹⁴ C], [ ³⁵ S], etc. at 0 cpm). Non-specific binding (NS
Prepare a reaction tube containing a large excess of unlabeled test compound in order to know B). The reaction is carried out at about 0 ° C. to 50 ° C., preferably about 4 ° C. to 37 ° C., for about 20 minutes to 24 hours, preferably about 30 minutes.
Perform for minutes to 3 hours. After the reaction, the mixture is filtered with a glass fiber filter or the like, washed with an appropriate amount of the same buffer, and the radioactivity remaining on the glass fiber filter is measured with a liquid scintillation counter or a γ-counter. Count (B-NSB) obtained by subtracting the non-specific binding amount (NSB) from the total binding amount (B)
A test compound having a value of more than 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-mentioned methods (1) to (3) for determining a ligand for the receptor protein or a salt thereof of the present invention, cell stimulating activity (for example, arachidonic acid release, acetylcholine release, intracellular Ca ^{2 +} Release, intracellular cAMP production, intracellular cGMP production, inositol phosphate production, cell membrane potential fluctuation, intracellular protein phosphorylation, c-fos activation, pH reduction, etc. 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. Before performing ligand determination, replace the medium with a fresh medium or an appropriate buffer that is not toxic to cells, add test compounds, and incubate for a certain period of time.
The cells are extracted or the supernatant is collected, and the generated product is quantified according to each method. When the production of a substance (for example, arachidonic acid or the like) as an indicator of cell stimulating activity is difficult due to a degrading enzyme contained in a cell, an assay may be performed by adding an inhibitor against the degrading enzyme. In addition, activities such as cAMP production suppression can be detected as a production suppression effect on cells whose basic production amount has been increased by forskolin or the like.

The receptor protein of the present invention or its protein
The kit for determining a ligand that binds to a salt is a receptor of the present invention.
Protein or salt thereof, partial peptide of the present invention
Or a salt thereof, containing the receptor protein of the present invention.
Containing the receptor protein of the present invention.
It contains a membrane fraction of the cells to be treated. Of the present invention
Examples of ligand determination kits include:
It is. 1. Reagent for ligand determination Measurement buffer and washing buffer Hanks' Balanced Salt Solution (manufactured by Gibco) contains 0.0
5% bovine serum albumin (Sigma).
Sterilize by filtration through a 0.45 μm filter, and store at 4 ° C.
Or may be prepared at the time of use. G protein-coupled receptor protein preparation CHO cells expressing the receptor protein of the present invention
Into a 12-well plate ^FivePassaged by individual / hole, 37 ℃, 5% C
O_TwoCultured in 95% air for 2 days. Labeled test compound^ThreeH], [¹²⁵I], [¹⁴C], [³⁵S)
The compound obtained, or labeled by an appropriate method, stored in an aqueous solution at 4 ° C or -20 ° C,
Dilute to 1 μM with measurement buffer before use. Poorly soluble in water
For test compounds that show, dimethylformamide,
Dissolve in DMSO, methanol, etc. Unlabeled test compound Prepare the same as the labeled compound at a concentration 100-1000 times higher
You.

2. Measurement method CHO cells expressing the receptor protein of the present invention cultured on a 12-well tissue culture plate were washed twice with 1 ml of the measurement buffer, and 490 μl of the measurement buffer was added to each well. Add to 5 μl of the labeled test compound is added and reacted at room temperature for 1 hour. To determine the amount of non-specific binding, use an unlabeled test compound.
Add 5 μl. Remove the reaction solution and wash three times with 1 ml of wash buffer. 0.2N NaOH-1% SD labeled test compound bound to cells
Dissolve in S and add 4 ml of liquid scintillator A (Wako Pure Chemical Industries)
Mix with. Radioactivity is measured using a liquid scintillation counter (manufactured by Beckman Coulter).

The ligand capable of binding to the receptor protein of the present invention or a salt thereof includes, for example,
Examples include substances specifically present in the hypothalamus, cerebral cortex, colon cancer, lung cancer, heart, placenta, lung, and the like. Specifically, tyramine (tyramine), beta-phenylethylamine (β-phenylethylamine), Tryptam
ine), trace amines such as octopamine; angiotensin, bombesin, cannabinoid, cholecystokinin, glutamine, serotonin, melatonin, neuropeptide Y, opioid, purine, vasopressin, oxytocin, PACAP
(E.g., PACAP27, PACAP38), secretin, glucagon,
Calcitonin, adrenomedullin, somatostatin,
GHRH, CRF, ACTH, GRP, PTH, VIP (Vasoactive Intestinal and Limited polypeptide),
Somatostatin, dopamine, motilin, amylin, bradykinin, CGRP (calcitonin gene relayed peptide), leukotriene, pancreatastatin, prostaglandin, thromboxane, adenosine, adrenaline, chemokine superfamily (eg, IL-8,
GROα, GROβ, GROγ, NAP-2, ENA-78, GCP-2, PF4, IP
-10, Mig, CXC chemokine subfamily such as PBSF / SDF-1; MCAF / MCP-1, MCP-2, MCP-3, MCP-4, eotaxin, RA
NTES, MIP-1α, MIP-1β, HCC-1, MIP-3α / LARC, MIP-3
β / ELC, I-309, TARC, MIPF-1, MIPF-2 / eotaxin-2, MD
CC chemokine subfamily such as C, DC-CK1 / PARC, SLC; C chemokine subfamily such as lymphotactin; f
CX3 chemokine subfamily such as ractalkine),
Endothelin, enterogastrin, histamine, neurotensin, TRH, pancreatic polypeptide, galanin, lysophosphatidic acid (LPA), sphingosine 1-phosphate and the like are used.

(2) Preventive and / or therapeutic agent for diseases associated with dysfunction of the G protein-coupled receptor protein of the present invention In the above method (1), if the ligand for the receptor protein of the present invention becomes clear, Depending on the action of the ligand, the receptor protein of the present invention or a DNA encoding the receptor protein,
It can be used as a medicament such as an agent for preventing and / or treating a disease associated with dysfunction of the receptor protein of the present invention. For example, when there is a patient in whom the physiological activity of the ligand cannot be expected because the receptor protein of the present invention is reduced in the living body (deficiency of the receptor protein), the receptor protein of the present invention is administered to the patient and By replenishing the amount of the protein, or (a) administering and expressing DNA encoding the receptor protein of the present invention to the patient,
Alternatively, (b) increasing the amount of the receptor protein in the patient's body by inserting the DNA encoding the receptor protein of the present invention into the target cell and expressing it, and then transplanting the cell into the patient; The effect of the ligand can be sufficiently exerted. That is,
The DNA encoding the receptor protein of the present invention is useful as a safe and low-toxic agent for preventing and / or treating diseases associated with dysfunction of the receptor protein of the present invention. The receptor protein of the present invention is a novel seven-transmembrane receptor protein having about 43% homology at the amino acid sequence level to the neurotransmitter receptor which is a G protein-coupled receptor protein. The receptor protein of the present invention or a DNA encoding the receptor protein may be, for example, a central disease.
(E.g., Alzheimer's disease, dementia, eating disorders, depression, epilepsy, anxiety, etc.), endocrine diseases (e.g., Addison's disease, Cushing's syndrome, pheochromocytoma, primary aldosteronism, menopause, endometriosis, Gonadal dysfunction,
Thyroid dysfunction, pituitary dysfunction, etc.), metabolic disorders (eg, diabetes, lipid metabolism disorders, diabetic complications, obesity, gout, cataract, hyperlipidemia, etc.), cancer (eg, non-small cell lung cancer, ovarian cancer) , Prostate cancer, gastric cancer, bladder cancer, breast cancer, cervical cancer, colon cancer, rectal cancer, etc., inflammatory diseases (eg, allergy, asthma, rheumatism, arthritis, nephritis, hepatitis, retinopathy, cystitis, pneumonia, etc.) ), Cardiovascular diseases (eg, hypertension, cardiac hypertrophy, angina pectoris, myocardial infarction, arteriosclerosis, etc.), respiratory diseases (eg, cold syndrome, asthma, pneumonia, pulmonary hypertension, pulmonary thrombosis, lung cold) Plugs), digestive disorders (e.g.,
Gastric ulcer, duodenal ulcer, gastritis, reflux esophagitis, pancreatitis, etc., immune system disease (eg, autoimmune disease), infectious disease (eg, immune dysfunction, pneumonia, cytomegal virus, influenza virus, herpes virus, etc.) Virus infection, rickettsial infection, bacterial infection, etc.), migraine and the like. When the receptor protein of the present invention is used as the above-mentioned prophylactic / therapeutic agent, it can be formulated according to conventional means. On the other hand, when a DNA encoding the receptor protein of the present invention (hereinafter sometimes abbreviated as the DNA of the present invention) is used as the above-mentioned prophylactic / therapeutic agent, the DNA of the present invention may be used alone or in a retroviral vector or an adenovirus. After insertion into a suitable vector such as a vector or an adenovirus-associated virus vector, it can be carried out according to a conventional method. DNA of the present invention
Can be administered as it is or together with adjuvants for promoting uptake by a catheter such as a gene gun or a hydrogel catheter. For example, the receptor protein of the present invention or the DNA encoding the receptor protein can be used as a sugar-coated tablet, capsule, elixir, microcapsule, or the like, if necessary, or water or other pharmaceutical agent. It can be used parenterally in the form of injections, such as sterile solutions or suspensions in liquids that are acceptable. For example, the generally accepted formulation of the receptor protein of the present invention or the DNA encoding the receptor protein with known carriers, flavors, excipients, vehicles, preservatives, stabilizers, binders and the like which are physiologically recognized. By mixing in the unit dose form required. The amount of the active ingredient in these preparations is such that a proper dosage in the specified range can be obtained.

Examples of additives that can be mixed with tablets, capsules and the like include binders such as gelatin, corn starch, tragacanth and gum arabic, excipients such as crystalline cellulose, corn starch, gelatin, alginic acid. For example, a leavening agent such as sucrose, a lubricant such as magnesium stearate, a sweetening agent such as sucrose, lactose or saccharin, a flavoring agent such as peppermint, reddish oil or cherry and the like are used. When the preparation unit form is a capsule, the above type of material can further contain a liquid carrier such as oil and fat. Sterile compositions for injection can be formulated according to normal pharmaceutical practice such as dissolving or suspending the active substance in vehicles such as water for injection, and 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 auxiliary agents (eg, D-sorbitol, D-mannitol, sodium chloride, etc.) and the like are used. ,
For example, alcohol (eg, ethanol), polyalcohol (eg, propylene glycol, polyethylene glycol), nonionic surfactant (eg, polysorbate 8)
0 ^TM , HCO-50) and the like. As the oily liquid, for example, sesame oil, soybean oil and the like are used, and may be used in combination with solubilizers such as benzyl benzoate and benzyl alcohol.

The prophylactic / therapeutic agents include, for example, buffers (eg, phosphate buffer, sodium acetate buffer), soothing agents (eg, benzalkonium chloride, procaine hydrochloride, etc.), stabilizers (eg, For example, human serum albumin, polyethylene glycol, etc.), preservative (eg,
Benzyl alcohol, phenol, etc.), antioxidants and the like. The prepared injection is usually filled in a suitable ampoule. The preparations obtained in this way are safe and have low toxicity, for example, human and non-human mammals (eg, rat, mouse, rabbit, sheep, pig,
Cattle, cats, dogs, monkeys, etc.). The dose of the receptor protein of the present invention varies depending on the administration subject, target organ, condition, administration method and the like.
g) as about 0.1 to 100 mg per day, preferably about 1.0 to 50 mg, more preferably about 1.0 to 20 mg. In the case of parenteral administration, the single dose varies depending on the administration subject, target organ, symptom, administration method and the like.
g), about 0.01 to 30 mg per day,
Preferably about 0.1-20 mg, more preferably about 0.1-1
It is convenient to administer about 0 mg by intravenous injection.
In the case of other animals, the dose can be administered per 60 kg. The dose of the DNA of the present invention depends on the administration subject,
Although there are differences depending on the target organ, symptoms, administration method and the like, in the case of oral administration, for example, in a cancer patient (as 60 kg), about 0.1 to 100 mg, preferably about 1.0 to 50 mg per day is generally used. , More preferably about 1.0-20 mg.
In the case of parenteral administration, the single dose varies depending on the administration subject, target organ, symptom, administration method and the like.
g), about 0.01 to 30 mg per day,
Preferably about 0.1-20 mg, more preferably about 0.1-1
It is convenient to administer about 0 mg by intravenous injection.
In the case of other animals, the dose can be administered per 60 kg.

(3) Gene Diagnostic Agent The DNA of the present invention can be used as a probe to
A human or non-human mammal (eg, rat, mouse,
Rabbits, sheep, pigs, cows, cats, dogs, monkeys, etc.)
Can detect abnormalities (genetic abnormalities) in the DNA or mRNA encoding the receptor protein or its partial peptide of the present invention in, for example, damage, mutation, or reduced expression of the DNA or mRNA, or the DNA or mRNA. It is useful as a diagnostic agent for genes such as an increase in expression or overexpression. The above-described genetic diagnosis using the DNA of the present invention can be performed, for example, by the known Northern hybridization or PCR-SSCP method (Genomics, Vol. 5, 87).
4-879 pages (1989), The Prossings of the National Academy of Sciences of
USA (Proceedings of the National Academy)
ofSciences of the United States of America), 86th
Volume, pp. 2766-2770 (1989)).

(4) Method for Screening a Compound That Changes the Expression Level of the Receptor Protein or Partial Peptide of the Present Invention The DNA of the present invention can be used as a probe to reduce the expression level of the receptor protein or the partial peptide thereof. It can be used to screen for compounds to be altered. That is, the present invention provides, for example, the receptor protein of the present invention contained in (i) blood of a non-human mammal, a specific organ, a tissue or cell isolated from an organ, or (ii) a transformant or the like, or a part 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 measurement of the mRNA amount of the receptor protein of the present invention or its partial peptide is specifically performed as follows. (I) Normal or disease model non-human mammals (eg, mice, rats, rabbits, sheep, pigs, cows, cats, dogs, monkeys, etc., more specifically, dementia rats, obese mice, arteriosclerotic rabbits, cancer-bearing Mouse, etc.)
A drug (eg, an anti-dementia drug, a blood pressure lowering drug, an anti-cancer drug, an anti-obesity drug, etc.) or a physical stress (eg, flooding stress, electric shock, light / dark, low temperature, etc.) is given, and after a certain period of time, blood or Obtain a specific organ (eg, brain, lung, large intestine, etc.), or tissue or cells isolated from the organ. MR of the receptor protein of the present invention or a partial peptide thereof contained in the obtained cells
NA can be quantified, for example, by extracting mRNA from cells or the like by a usual method and using, for example, a technique such as TaqMan PCR, or can be analyzed by performing Northern blotting by a known means. (Ii) A transformant expressing the receptor protein of the present invention or its partial peptide is prepared according to the above method, and the mRNA of the receptor protein of the present invention or its partial peptide contained in the transformant is similarly quantified.
Can be analyzed.

The screening for a compound that alters the expression level of the receptor protein or its partial peptide of the present invention can be performed by (i) a predetermined 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 ago or after a certain time (30 minutes to 3 days later, preferably 1 hour to 2 days later,
More preferably after 1 hour to 24 hours), or a test compound is administered simultaneously with the drug or physical stress, and after a certain period of time after administration (30 minutes to 3 days, preferably 1 hour to 2 days, More preferably, after 1 hour 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, and (ii) transforming the transformant according to a conventional method. When culturing, the test compound is mixed in the medium, and after culturing for a certain period of time (1 day to 7 days, preferably 1 day to 3 days, more preferably 2 days to 3 days).
After 3 days), the amount of mRNA of the receptor protein of the present invention or its partial peptide contained in the transformant can be quantified and analyzed.

The compound obtained by using the screening method of the present invention or a salt thereof is a compound having an action of changing the expression level of the receptor protein of the present invention or a partial peptide thereof. By increasing the expression level of the receptor protein of the present invention or its partial peptide, the cell stimulating activity via G protein-coupled receptor (eg, arachidonic acid release, acetylcholine release, intracellular Ca ²⁺ release, intracellular cAMP generation,
Intracellular cGMP production, inositol phosphate production, cell membrane potential fluctuation, intracellular protein phosphorylation, c-fos activation, p
Activity that promotes or suppresses the decrease of H, etc.)
And (b) a compound that decreases the expression level of the receptor protein of the present invention or its partial peptide to thereby reduce the cell stimulating activity. Examples of the compound include a peptide, a protein, a non-peptidic compound, a synthetic compound, and a fermentation product. These compounds may be a novel compound or a known compound. The compound that enhances the cell stimulating activity is
It is useful as a safe and low toxic drug 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 drug for decreasing the physiological activity of the receptor protein of the present invention.

When a compound or a salt thereof obtained by using 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 pharmaceuticals containing the receptor protein of the present invention. Since the preparation obtained in this way is safe and low toxic, for example, human or non-human mammals (for example,
Rats, mice, rabbits, sheep, pigs, cows, cats,
Dogs, monkeys, etc.). The dose of the compound or a salt thereof varies depending on the administration subject, the target organ, the condition, the administration method, and the like. In the case of oral administration, for example, in a cancer patient (as 60 kg), one day About 0.1-100 mg, preferably about 1.0-50
mg, more preferably about 1.0-20 mg. When administered parenterally, the single dose depends on the administration target, target organ, symptoms, administration method, etc., for example,
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
It is convenient to administer about 0.1 to 20 mg, more preferably about 0.1 to 10 mg, by intravenous injection. In the case of other animals, the dose can be administered per 60 kg.

(5) Preventive and / or therapeutic agent for various diseases containing a compound that alters the expression level of the receptor protein or its partial peptide of the present invention. 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 a partial peptide thereof can be used as an agent for preventing and / or treating a disease associated with dysfunction of the receptor protein of the present invention. When the compound 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 conventional means. Diseases associated with dysfunction of the receptor protein of the present invention include, for example, central diseases (e.g., Alzheimer's disease, dementia, eating disorders, depression, epilepsy, anxiety, etc.), endocrine diseases (e.g., Addison's disease,
Cushing's syndrome, pheochromocytoma, primary aldosteronism, menopause, endometriosis, gonadal dysfunction, thyroid dysfunction, pituitary dysfunction, etc., metabolic diseases (e.g., diabetes, dyslipidemia, diabetic complications, Obesity, gout, cataract, hyperlipidemia, etc.), cancer (eg, non-small cell lung cancer, ovarian cancer, prostate cancer, gastric cancer, bladder cancer, breast cancer, cervical cancer, colon cancer, rectal cancer, etc.), inflammatory Diseases (eg, allergies,
Asthma, rheumatism, arthritis, nephritis, hepatitis, retinopathy, cystitis, pneumonia, etc., cardiovascular diseases (eg, hypertension, cardiac hypertrophy, angina, myocardial infarction, arteriosclerosis, etc.), respiratory diseases ( For example, common cold syndrome, asthma, pneumonia, pulmonary hypertension, pulmonary thrombosis, pulmonary thrombosis, etc., gastrointestinal diseases (eg, gastric ulcer,
Duodenal ulcer, gastritis, reflux esophagitis, pancreatitis, etc., immune system diseases (eg, autoimmune diseases), infectious diseases (eg, immune dysfunction, pneumonia, cytomegal virus, influenza virus, herpes virus, etc.) Disease, rickettsial infection, bacterial infection, etc.), and migraine. For example, the compound may be a sugar-coated tablet, capsule, elixir, microcapsule, or the like, as needed, orally, or aseptic solution with water or other pharmaceutically acceptable liquid, Alternatively, it can be used parenterally in the form of injections such as suspensions. For example, the compound is mixed with known physiologically acceptable carriers, flavoring agents, excipients, vehicles, preservatives, stabilizers, binders, and the like in the unit dosage form generally required for the practice of pharmaceutical preparations. Can be manufactured by The amount of the active ingredient in these preparations is such that a proper dosage in the specified range can be obtained.

Examples of additives that can be mixed with tablets, capsules and the like include binders such as gelatin, corn starch, tragacanth and gum arabic, excipients such as crystalline cellulose, corn starch, gelatin, alginic acid. Use may be made of bulking agents such as, for example, magnesium stearate, sweeteners such as sucrose, lactose or saccharin, flavoring agents such as peppermint, reddish oil or cherry. When the preparation unit form is a capsule, the above type of material can further contain a liquid carrier such as oil and fat. Sterile compositions for injection can be formulated according to normal pharmaceutical practice such as dissolving or suspending the active substance in vehicles such as water for injection, and 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 auxiliary agents (eg, D-sorbitol, D-mannitol, sodium chloride, etc.) and the like are used. ,
For example, alcohol (eg, ethanol), polyalcohol (eg, propylene glycol, polyethylene glycol), nonionic surfactant (eg, polysorbate 8)
0 ^TM , HCO-50) and the like. As the oily liquid, for example, sesame oil, soybean oil and the like are used, and may be used in combination with solubilizers such as benzyl benzoate and benzyl alcohol.

The prophylactic / therapeutic agents include, for example, buffering agents (eg, phosphate buffer, sodium acetate buffer), soothing agents (eg, benzalkonium chloride, procaine hydrochloride, etc.), stabilizers (eg, For example, human serum albumin, polyethylene glycol, etc.), preservative (eg,
Benzyl alcohol, phenol, etc.), antioxidants and the like. The prepared injection is usually filled in a suitable ampoule. The preparations obtained in this way are safe and have low toxicity, for example, human and non-human mammals (eg, rat, mouse, rabbit, sheep, pig,
Cattle, cats, dogs, monkeys, etc.). The dose of the compound or a salt thereof may be
Although there are differences depending on the target organ, symptoms, administration method and the like, in the case of oral administration, for example, in a cancer patient (as 60 kg), about 0.1 to 100 mg, preferably about 1.0 to 50 mg per day is generally used. , More preferably about 1.0-20 mg.
In the case of parenteral administration, the single dose varies depending on the administration subject, target organ, symptoms, administration method and the like.
kg)), about 0.01 to 30 mg per day, preferably about 0.1 to 20 mg, more preferably about 0.1 to 20 mg per day.
It is convenient to administer about 1 to 10 mg by intravenous injection. In the case of other animals, the dose can be administered per 60 kg.

(6) Method for Quantifying Ligands for G Protein-Coupled Receptor Proteins of the Present Invention Since the receptor proteins and the like of the present invention have binding properties to ligands, the ligand concentration in vivo can be determined with high sensitivity. can do. The quantification method of the present invention comprises:
For example, it can be used in combination with a competition method. That is, the ligand concentration in the subject can be measured by bringing the subject into contact with the receptor protein of the present invention or the like. Specifically, for example, it can be used according to the method described below or the like or a method analogous thereto. Edited by Hiro Irie "Radio Immunoassay" (Kodansha, Showa 49
Published by Hiroshi Irie, "Continuing Radio Immunoassay" (Kodansha, Showa)
(Issued in 1954)

(7) Screening method for compounds (agonists, antagonists, etc.) that alter the binding between the G protein-coupled receptor protein and the ligand of the present invention. And the like, and by using a receptor binding assay system using the expression system, a compound (e.g., peptide, protein, non-peptidic compound) that changes the binding property between the ligand and the receptor protein of the present invention. , Synthetic compounds, fermentation products, etc.) or salts thereof can be efficiently screened. Such compounds include (a) cell stimulating activity (eg, arachidonic acid release, acetylcholine release, intracellular Ca ²⁺ release,
Intracellular cAMP production, Intracellular cGMP production, Inositol phosphate production, Cell membrane potential fluctuation, Intracellular protein phosphorylation,
a compound having an activity of promoting or suppressing c-fos activation, lowering of pH, etc. (a so-called agonist for the receptor protein of the present invention); (b) a compound having no cell stimulating activity (a so-called Antagonists to the receptor protein of the present invention), (c)
Examples include compounds that enhance the binding force between the ligand and the G protein-coupled receptor protein of the present invention, or (d) compounds that decrease the binding force between the ligand and the G protein-coupled receptor protein of the present invention (in addition, It is preferable to screen the compound of (a) by the ligand determination method described above). That is, the present invention relates to (i) a case where the receptor protein of the present invention or its partial peptide or a salt thereof is brought into contact with a ligand; and (ii) a case where the receptor protein of the present invention or its partial peptide or a salt thereof, a ligand and There is provided a method for screening a compound or a salt thereof which changes the binding property between a ligand and a receptor protein or a partial peptide thereof or a salt thereof of the present invention, which is characterized by comparing with the case of contacting with a test compound. In the screening method of the present invention,
In the cases (i) and (ii), for example, the amount of binding of the ligand to the receptor protein or the like, the cell stimulating activity and the like are measured and compared.

More specifically, the present invention relates to the case where the labeled ligand is brought into contact with the receptor protein of the present invention, and the case where the labeled ligand and the test compound are brought into contact with the receptor protein of the present invention. Measuring the amount of binding of the labeled ligand to the receptor protein or the like, and comparing the measured amount, and 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. A cell containing the receptor protein or the like of the present invention or a membrane fraction of the cell, and a labeled ligand and a test compound are added to a cell or a membrane fraction of the cell containing the receptor protein or the like of the present invention. The labeled ligand, when contacted, against the cell or the membrane fraction A method for screening a compound or a salt thereof that alters the binding property between a ligand and a receptor protein or the like of the present invention, which comprises measuring and comparing the amounts of the ligands. The present invention is characterized in that, when a transformant containing the DNA of the present invention is expressed on a cell membrane by culturing a transformant containing the DNA of the present invention, the present invention is obtained by culturing a transformant containing the DNA of the present invention, when the cultured body is contacted with a receptor protein or the like expressed on the cell membrane by culturing the body. A compound that changes the binding between the ligand and the receptor protein of the present invention, which is characterized by measuring the amount of binding of the labeled ligand to the receptor protein or the like when contacted with the receptor protein or the like; Screening method for its salts,

A compound which activates the receptor protein of the present invention (eg, a ligand for the receptor protein of the present invention) is contacted with a cell containing the receptor protein of the present invention. Cell stimulating activity through the receptor (eg, arachidonic acid release, acetylcholine release, intracellular Ca ²⁺ release) when a compound that activates the like and a test compound are brought into contact with a cell containing the receptor protein or the like of the present invention. , Intracellular cAMP production, intracellular cGMP production, inositol phosphate production, cell membrane potential fluctuations, intracellular protein phosphorylation, c-fos activation, activity to promote or suppress pH reduction, etc.) Change the binding between the ligand characterized by comparison and the receptor protein of the present invention. A method for screening a compound to be caused or a salt thereof, and culturing a transformant containing the DNA of the present invention with a compound that activates the receptor protein of the present invention (eg, a ligand for the receptor protein of the present invention). When the transformant containing the DNA of the present invention is cultured with a compound that activates the receptor protein or the like of the present invention and a test compound, the cells are brought into contact with the receptor protein of the present invention expressed on the cell membrane. Receptor-mediated cell stimulating activity (eg, arachidonic acid release, acetylcholine release, intracellular Ca ²⁺ release, intracellular cAMP generation, intracellular cGMP)
Production, inositol phosphate production, cell membrane potential fluctuation, intracellular protein phosphorylation, c-fos activation, pH reduction, etc.
There is provided a method for screening a compound or a salt thereof that changes the binding property between a ligand and a receptor protein of the present invention, which is characterized by comparison.

Prior to obtaining the receptor protein or the like of the present invention, when screening for a G protein-coupled receptor agonist or antagonist, first, cells, tissues or cell membrane fractions containing the G protein-coupled receptor protein, such as rats, are used. To obtain a candidate compound (primary screening), and then a test (secondary screening) is required to confirm whether the candidate compound actually inhibits the binding of a human G protein-coupled receptor protein to a ligand. there were. If the cell, tissue, or cell membrane fraction is used as it is, other receptor proteins are also mixed, and it has been difficult to actually directly screen for an agonist or antagonist for the target receptor protein. However, for example, by using the human-derived receptor protein of the present invention, primary screening is not required, and a compound that inhibits binding between a ligand and a G protein-coupled receptor protein can be efficiently screened. Furthermore, it is possible to easily evaluate whether the screened compound is an agonist or an antagonist. The specific description of the screening method of the present invention is as follows. First, the receptor protein 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. Cell membrane fractions of mammalian organs containing are preferred. However, since it is particularly difficult to obtain human-derived organs, human-derived receptor proteins and the like, which are expressed in large amounts using recombinants, are suitable for screening.

The above-mentioned method is used for producing the receptor protein and the like of the present invention, but it is preferably carried out by expressing the DNA of the present invention in mammalian cells and insect cells. CDNA is used as the DNA fragment encoding the protein portion of interest, but is not necessarily limited thereto. For example, a gene fragment or a synthetic DNA may be used. DN encoding the receptor protein of the present invention
In order to introduce the A fragment into host animal cells and express them efficiently, the DNA fragment must be converted to a nuclear polyhedrosis virus (nuclear polyhedrosis virus belonging to baculovirus using an insect as a host).
hedrosis virus (NPV) polyhedrin promoter, S
V40-derived promoter, retrovirus promoter, metallothionein promoter, human heat shock promoter, cytomegalovirus promoter,
It is preferably incorporated downstream, such as the SRα promoter.
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. Bio
l. Chem.), 267, 19555-19559, 1992]. Therefore, in the screening method of the present invention, the protein containing the receptor protein or the like of the present invention may be a receptor protein or the like purified according to a known method,
A cell containing the receptor protein or the like may be used, or a membrane fraction of a cell containing the receptor protein or the like may be used.

When cells containing the receptor protein or the like 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 that has expressed the receptor protein or the like, and the host cell is preferably Escherichia coli, Bacillus subtilis, yeast, insect cells, animal cells, or the like. The cell membrane fraction refers to a cell membrane-rich fraction obtained by crushing cells and then obtained by a known method.
The cells can be crushed by crushing the cells with a Potter-Elvehjem homogenizer, crushing with a Waring blender or polytron (Kinematica), crushing by ultrasonic waves, or squirting the cells from a thin nozzle while applying pressure with a French press. Crushing, etc. For the fractionation of cell membranes, fractionation by centrifugal force such as fractionation centrifugation or density gradient centrifugation is mainly used. For example, the cell lysate is centrifuged at a low speed (500-3000 rpm) for a short time (usually about 1-10 minutes), and the supernatant is further centrifuged at a high speed (1
(5,000 to 30,000 rpm) for 30 minutes to 2 hours, and the resulting precipitate is used as a membrane fraction. The membrane fraction contains a large amount of expressed receptor proteins and membrane components such as cell-derived phospholipids and membrane proteins. The amount of the receptor protein in the cell or membrane fraction containing the receptor protein or the like is preferably 10 ³ to 10 ⁸ molecules per cell,
It is preferably from 0 ⁵ to 10 ⁷ molecules. The higher the expression level, the higher the ligand binding activity (specific activity) per membrane fraction, which makes 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-mentioned (1) to screen for a compound that alters the binding property between the ligand and the receptor protein of the present invention, for example, an appropriate receptor protein fraction and a labeled ligand are required. The receptor protein fraction is preferably a natural receptor protein fraction or a recombinant receptor protein fraction having an activity equivalent thereto. Here, “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],
Ligands labeled with [ ¹²⁵ I], [ ¹⁴ C], [ ³⁵ S] and the like are used. Specifically, to screen for a compound that alters the binding between a ligand and the receptor protein of the present invention, a cell or a membrane fraction of the cell containing the receptor protein of the present invention is first suitable for screening. To prepare a receptor protein standard. The buffer may be any buffer such as a phosphate buffer having a pH of 4 to 10 (preferably pH 6 to 8) or a buffer such as Tris-HCl buffer that does not inhibit the binding between the ligand and the receptor protein. For the purpose of reducing non-specific binding, a surfactant such as CHAPS, Tween-80 ^™ (Kao-Atlas), digitonin, and deoxycholate can be added to the buffer. Further, a protease inhibitor such as PMSF, leupeptin, E-64 (manufactured by Peptide Research Laboratories) and pepstatin can be added for the purpose of suppressing the degradation of the receptor or ligand by the protease. 0.01 ~
A fixed amount (5000 to 500,000 cp) is added to 10 ml of the receptor solution.
m) labeled ligand is added simultaneously with 10 ^-4 to 10 ^-10 M
Of the test compound. A reaction tube containing a large excess of unlabeled ligand is also prepared to determine the amount of non-specific binding (NSB). The reaction is carried out at about 0 ° C to 50 ° C, preferably about 4 ° C.
C. to 37.degree. C. for about 20 minutes to 24 hours, preferably about 30 minutes to 3 hours. After the reaction, the mixture is filtered with a glass fiber filter or the like, washed with an appropriate amount of the same buffer, and the radioactivity remaining on the glass fiber filter is measured with a liquid scintillation counter or a γ-counter. When the count (B ₀ -NSB) obtained by subtracting the non-specific binding amount (NSB) from the count (B ₀ ) when there is no antagonistic substance is defined as 100%, the specific binding amount (B−
For example, a test compound whose NSB) is 50% or less can be selected as a candidate substance capable of competitive inhibition.

In order to carry out the above-mentioned methods (1) and (2) for screening a compound that alters the binding property between a ligand and the receptor protein of the present invention, for example, a cell stimulating activity via the receptor protein (for example, arachidonic acid release, acetylcholine Release, intracellular Ca ²⁺ release, intracellular cAMP production, intracellular cGMP production, inositol phosphate production, cell membrane potential fluctuation, intracellular protein phosphorylation, c-
The activity of promoting or suppressing the activation of fos, lowering of pH, etc.) can be measured using a known method or a commercially available measurement kit. Specifically, cells containing the receptor protein or the like of the present invention are first cultured in a multiwell plate or the like. When performing screening, the cells were exchanged with a fresh medium or an appropriate buffer that does not show toxicity for cells in advance, and test compounds were added and incubated for a certain period of time. The product is quantified according to the respective method. When the production of a substance (for example, arachidonic acid or the like) as an indicator of cell stimulating activity is difficult due to a degrading enzyme contained in a cell, an assay may be performed by adding an inhibitor against the degrading enzyme. In addition, activities such as cAMP production suppression can be detected as a production suppression effect on cells whose basic production amount has been increased by forskolin or the like. In order to perform screening by measuring the cell stimulating activity, cells expressing an appropriate receptor protein are required.
As the cells 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 desirable. As the test compound, for example, peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, and the like are used. Or a known compound.

Ligand and receptor protein of the present invention
Of a compound or its salt that alters the binding to
Kit for the receptor protein of the present invention
Etc., a cell containing the receptor protein or the like of the present invention,
Or cells containing the receptor protein of the present invention, etc.
And the like containing a membrane fraction of The screen of the present invention
The following are examples of the kit for ning. 1. Screening reagent Measurement buffer and washing buffer Hanks' Balanced Salt Solution (manufactured by Gibco)
5% bovine serum albumin (Sigma).
Sterilize by filtration through a 0.45 μm filter, and store at 4 ° C.
Or may be prepared at the time of use. G protein-coupled receptor preparation CHO cells expressing the receptor protein of the present invention
Into a 12-well plate ^FivePassaged by individual / hole, 37 ℃, 5% C
O_TwoCultured in 95% air for 2 days. Labeled ligand^ThreeH], [¹²⁵I], [¹⁴C], [³⁵S)
Ligand stored in aqueous solution at 4 ℃ or -20 ℃
Dilute to 1 μM with measurement buffer before use. Ligand standard solution Add 0.1% bovine serum albumin (Sigma) to the ligand.
Dissolve to 1 mM in PBS and store at -20 ° C.
You.

2. Assay Method The CHO cells expressing the receptor protein of the present invention cultured on a 12-well tissue culture plate were washed twice with 1 ml of assay buffer, and 490 μl of assay buffer was added to each well. Add to After adding 5 μl of a test compound solution of 10 ⁻³ to 10 ⁻¹⁰ M,
Add 5 μl of the labeled ligand and react at room temperature for 1 hour.
To determine the amount of non-specific binding, 10
Add 5 μl of ^-3 M ligand. Remove the reaction solution and wash three times with 1 ml of wash buffer. 0.2N NaOH-1% SDS
4 ml of liquid scintillator A (Wako Pure Chemical Industries)
Mix with. The radioactivity was measured using a liquid scintillation counter (manufactured by Beckman Coulter, Inc.).
m Binding (PMB) is calculated by the following equation. PMB = [(B−NSB) / (B ₀ −NSB)] × 100 PMB: Percent Maximum Binding B: Value when a sample is added NSB: Non-specific Binding (non-specific binding amount) B ₀ : Maximum binding amount

The compound or a salt thereof obtained by using the screening method or the screening kit of the present invention is a compound having an action of changing the binding between a ligand and the receptor protein of the present invention. (A) cell stimulating activity via G protein-coupled receptor (eg, arachidonic acid release, acetylcholine release, intracellular Ca ²⁺ release, intracellular cAMP production, intracellular cGMP production, inositol phosphate production, cell membrane potential fluctuation, A compound having an activity of promoting or suppressing intracellular protein phosphorylation, activation of c-fos, lowering of pH, etc. (so-called agonist for the receptor protein of the present invention), (b) the cell stimulating activity A compound having no (a so-called antagonist to the receptor protein of the present invention), (c) Compound that potentiates the binding affinity of the G protein-coupled receptor protein of the ligand with the present invention, or (d) is a compound that reduces the binding force between the G protein-coupled receptor protein of the ligand with the present invention. Examples of the compound include a peptide, a protein, a non-peptidic compound, a synthetic compound, and a fermentation product. These compounds may be a novel compound or a known compound. Since the agonist for the receptor protein or the like of the present invention has the same activity as the physiological activity of the ligand for the receptor protein or the like of the present invention, it is useful as a safe and low-toxic drug according to the ligand activity. Since the antagonist to the receptor protein or the like of the present invention can suppress the physiological activity of the ligand to the receptor protein or the like of the present invention, it is useful as a safe and low-toxic drug for suppressing 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-toxicity drug for enhancing the physiological activity of the ligand for the receptor protein or the like of the present invention. The compound that decreases 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 drug for reducing the physiological activity of the ligand for the receptor protein of the present invention.

When a compound or a salt thereof 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 according to a conventional method. For example, tablets, capsules, elixirs, microcapsules, tablets and capsules in the same manner as the above-mentioned drug containing the receptor protein of the present invention.
It can be a sterile solution, suspension or the like. The preparations obtained in this way are safe and have low toxicity, for example, human and non-human mammals (eg, rat, rabbit,
Sheep, pigs, cows, cats, dogs, monkeys, etc.). The dose of the compound or a salt thereof varies depending on the administration subject, target organ, condition, administration method, and the like. About 0.1-100 m
g, preferably about 1.0-50 mg, more preferably about 1.0-20
mg. In the case of parenteral administration, the single dose varies depending on the administration subject, target organ, symptoms, administration method, and the like. , About 0.01-30 mg per day
Degree, preferably about 0.1-20 mg, more preferably about 0.1-20 mg.
It is convenient to administer about 0.1 to 10 mg by intravenous injection. In the case of other animals, the dose can be administered per 60 kg.

(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 and the ligand of the present invention. As described above, for example, it is considered that they play some important roles in vivo such as central function, circulatory function, digestive function, and cardiac function. Therefore, compounds (agonists, antagonists) that alter the binding between the receptor protein of the present invention and the ligand
And ligands for the receptor protein of the present invention,
It can be used as a prophylactic and / or therapeutic agent for diseases associated with dysfunction of the receptor protein of the present invention. Examples of diseases related to the dysfunction of the receptor protein of the present invention include central diseases (e.g., Alzheimer's disease, dementia, eating disorders, depression, epilepsy, anxiety, etc.), endocrine diseases (e.g., Addison's disease, Cushing's syndrome, Pheochromocytoma, primary aldosteronism, menopause, endometriosis, gonadal dysfunction, thyroid dysfunction, pituitary dysfunction, etc., metabolic disorders (e.g., diabetes, lipid metabolism, diabetes complications, obesity, gout) , Cataract, hyperlipidemia, 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., inflammatory diseases (eg, allergy, asthma, rheumatism, arthritis, nephritis, hepatitis, retinopathy, cystitis, pneumonia), cardiovascular Diseases (eg, hypertension, cardiac hypertrophy, angina, myocardial infarction, arteriosclerosis, etc.), respiratory diseases (eg, cold syndrome, asthma, pneumonia, pulmonary hypertension, pulmonary thrombus, pulmonary thrombosis, etc.), Digestive system diseases (eg, gastric ulcer, duodenal ulcer, gastritis, reflux esophagitis, pancreatitis, etc.), immune system diseases (eg, autoimmune diseases, etc.), infectious diseases (eg, immune dysfunction, pneumonia, cytomegal virus, Viral infections such as influenza virus and herpes virus, rickettsial infections, bacterial infections, etc.), and migraines. 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 conventional means. For example, the compound or ligand can be sterilized with tablets or capsules, capsules, elixirs, microcapsules, or the like, which are coated with sugar as necessary, or with water or other pharmaceutically acceptable liquids. It can be used parenterally in the form of injections, such as solutions or suspensions. For example, the compound is mixed with known physiologically acceptable carriers, flavoring agents, excipients, vehicles, preservatives, stabilizers, binders, and the like in the unit dosage form generally required for the practice of pharmaceutical preparations. Can be manufactured by The amount of the active ingredient in these preparations is such that a proper dosage in the specified range can be obtained.

Examples of additives that can be mixed with tablets, capsules and the like include binders such as gelatin, corn starch, tragacanth and gum arabic, excipients such as crystalline cellulose, corn starch, gelatin, alginic acid. For example, a leavening agent such as sucrose, a lubricant such as magnesium stearate, a sweetening agent such as sucrose, lactose or saccharin, a flavoring agent such as peppermint, reddish oil or cherry and the like are used. When the preparation unit form is a capsule, the above type of material can further contain a liquid carrier such as oil and fat. Sterile compositions for injection can be formulated according to normal pharmaceutical practice such as dissolving or suspending the active substance in vehicles such as water for injection, and 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 auxiliary agents (eg, D-sorbitol, D-mannitol, sodium chloride, etc.) and the like are used. ,
For example, alcohol (eg, ethanol), polyalcohol (eg, propylene glycol, polyethylene glycol), nonionic surfactant (eg, polysorbate 8)
0 ^TM , HCO-50) and the like. As the oily liquid, for example, sesame oil, soybean oil and the like are used, and may be used in combination with solubilizers such as benzyl benzoate and benzyl alcohol.

The prophylactic / therapeutic agents include, for example, buffers (eg, phosphate buffer, sodium acetate buffer), soothing agents (eg, benzalkonium chloride, procaine hydrochloride, etc.), stabilizers (eg, For example, human serum albumin, polyethylene glycol, etc.), preservatives (eg,
Benzyl alcohol, phenol, etc.), antioxidants and the like. The prepared injection is usually filled in a suitable ampoule. Further, the above-mentioned prophylactic / therapeutic agent can be used in combination with an appropriate drug, for example, as a DDS preparation specifically targeting an organ or tissue in which the receptor protein of the present invention is highly expressed. The preparations obtained in this way are safe and have low toxicity, for example, human and non-human mammals (eg, rat, mouse,
Rabbits, sheep, pigs, cows, cats, dogs, monkeys, etc.)
Can be administered. The dose of the compound or a salt thereof varies depending on the administration subject, the target organ, the condition, the administration method, and the like. about
0.1-100 mg, preferably about 1.0-50 mg, more preferably about 1.0-20 mg. In the case of parenteral administration, the single dose varies depending on the administration subject, target organ, symptoms, administration method, etc., for example, usually in the form of an injection, for example, in cancer patients (60 kg). It is convenient to administer about 0.01 to 30 mg, preferably about 0.1 to 20 mg, more preferably about 0.1 to 10 mg per day by intravenous injection. In the case of other animals, the dose can be administered per 60 kg.

(9) Quantification of the Receptor Protein of the Present Invention or its Partial Peptide or Its Salt Since the antibody of the present invention can specifically recognize the receptor protein or the like of the present invention, Can be used for quantification of receptor protein and the like, particularly quantification by sandwich immunoassay. That is, the present invention provides, for example, (i) reacting the antibody of the present invention with a test solution and a labeled receptor protein in a competitive manner, and measuring the ratio of the labeled receptor protein bound to the antibody. A method for quantifying the receptor protein or the like of the present invention in a test solution, and (ii) simultaneously or sequentially using the test solution and the antibody of the present invention and the labeled antibody of the present invention insolubilized on a carrier. And then measuring the activity of the labeling agent on the insolubilized carrier, thereby providing a method for quantifying the receptor protein of the present invention in a test solution. In the above (ii), it is preferable that one antibody is an antibody that recognizes the N-terminal of the receptor protein of the present invention and the other antibody is an antibody that reacts with the C-terminal of the receptor protein of the present invention. .

In addition to measuring the receptor protein and the like of the present invention using a monoclonal antibody against the receptor protein and the like of the present invention (hereinafter sometimes referred to as the monoclonal antibody of the present invention), detection by tissue staining and the like is also possible. You can also. For these purposes, the antibody molecule itself may be used, and F (ab ') ₂ , F
Ab 'or the Fab fraction may be used. The assay method using an antibody against the receptor protein or the like of the present invention is not particularly limited, and may be an antibody, an antigen, or an antibody-antigen complex corresponding to the amount of an antigen (eg, the amount of a receptor protein) in a test solution. Any measurement method may be used as long as the amount is detected by chemical or physical means and the amount is calculated from a standard curve prepared using a standard solution containing a known amount of antigen. For example, nephelometry, competition method, immunometric method and sandwich method are suitably used, but it is particularly preferable to use the sandwich method described later in terms of sensitivity and specificity. As a labeling agent used in a measurement method using a labeling substance, for example, a radioisotope, an enzyme, a fluorescent substance, a luminescent substance and the like are used. As a radioisotope, for example,
[ ¹²⁵ I], [ ¹³¹ I], [ ³ H], [ ¹⁴ C] and the like are used. As the above enzyme, a stable enzyme having a large specific activity is preferable. 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 and the like are used. As the luminescent substance, for example, luminol, a luminol derivative, luciferin, lucigenin and the like are used. Further, a biotin-avidin system can be used for binding the antibody or antigen to the labeling agent.

For the insolubilization of the antigen or antibody, physical adsorption may be used, or a method using a chemical bond usually used for insolubilizing or immobilizing proteins or enzymes may be used. As the carrier, for example, insoluble polysaccharides such as agarose, dextran, and cellulose, synthetic resins such as polystyrene, polyacrylamide, and silicon, and glass are used. In the sandwich method, the test solution is reacted with the insolubilized monoclonal antibody of the present invention (primary reaction), and further reacted with the labeled monoclonal antibody of the present invention (secondary reaction). By measuring the activity of the receptor protein, the amount of the receptor protein of the present invention in the test solution can be determined. The primary reaction and the secondary reaction may be performed in the reverse order, may be performed simultaneously, or may be performed at staggered times. The labeling agent and the method of insolubilization can be based on those described above. In the immunoassay by the sandwich method, the antibody used for the solid phase antibody or the labeling antibody is not necessarily one type, and a mixture of two or more types of antibodies is used for the purpose of improving measurement sensitivity and the like. You may. In the method for measuring a receptor protein or the like by the sandwich method of the present invention, the monoclonal antibody of the present invention used in the primary reaction and the secondary reaction is preferably an antibody 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, when the antibody used in the secondary reaction recognizes the C-terminal of the receptor protein, the antibody used in the primary reaction is preferably the C-terminal For example, an antibody that recognizes the N-terminal other than the N-terminal is used.

The monoclonal antibody of the present invention can be used in a measurement system other than the sandwich method, for example, a competition method, an immunometric method, a nephelometry and the like. In the competitive method, the antigen in the test solution and the labeled antigen are allowed to react competitively with the antibody, and then the unreacted labeled antigen is reacted with the antigen.
(F) is separated from the labeled antigen (B) bound to the antibody (B / F separation), the amount of labeling of either B or F is measured, and the amount of antigen in the test solution is quantified. In this reaction method, using a soluble antibody as an antibody, B / F separation using polyethylene glycol, a liquid phase method using a second antibody against the above antibody, or using an immobilized antibody as the first antibody, or An immobilization method using a soluble first antibody and an immobilized antibody as the second antibody is used. In the immunometric method, an antigen in a test solution and a solid-phased antigen are subjected to a competitive reaction with a fixed amount of a labeled antibody, and then the solid phase and the liquid phase are separated. Is reacted with an excess amount of the labeled antibody, and then the immobilized antigen is added to bind the unreacted labeled antibody to the solid phase, and then the solid phase and the liquid phase are separated. Next, the amount of label in any phase is measured to determine the amount of antigen in the test solution. In the nephelometry, the amount of insoluble precipitate generated as a result of the antigen-antibody reaction in a gel or in a solution is measured. Even when the amount of antigen in the test solution is small and only a small amount of sediment is obtained, laser nephelometry utilizing laser scattering is preferably used.

In applying these individual immunological measurement methods to the measurement method of the present invention, no special conditions, operations, and the like need to be set. The measuring system for the receptor protein of the present invention or a salt thereof may be constructed by adding ordinary technical considerations of those skilled in the art to ordinary conditions and operating methods in each method. For details of these general technical means, it is possible to refer to reviews, books, etc. [for example,
Edited by Hiro Irie "Radio Immunoassay" (Kodansha, Showa 49)
Ed., Edited by Hiro Irie, "Continuing Radio Immunoassay" (Kodansha, published in 1979), Eiji Ishikawa et al., "Enzyme Immunoassay" (Medical Publishing, published in 1978), Eiji Ishikawa, et al., "Enzyme Immunoassay" (2nd edition) (Medical Shoin, published in 1982),
Ed. Eiji Ishikawa et al., “Enzyme Immunoassay” (3rd edition) (Medical Publishing, published in 1987), “Methods in ENZYMOLOGY” Vol. 70 (Immunochem)
ical Techniques (Part A)), ibid.Vol. 73 (Immunoch
emicalTechniques (Part B)), ibid.Vol. 74 (Immunoc
chemical Techniques (Part C)), ibid.Vol. 84 (Immuno
chemical Techniques (Part D: Selected Immunoassay
s)), ibid.Vol. 92 (Immunochemical Techniques (Par
t E: Monoclonal Antibodies and General Immunoassay M
ethods)), ibid.Vol. 121 (Immunochemical Technique)
s (Part I: Hybridoma TechnoloGy and Monoclonal Anti
bodies)) (above, published by Academic Press). As described above, the receptor protein of the present invention or a salt thereof 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 a living body 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 a body fluid or a 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 at the time of purification, behavior of the receptor protein of the present invention in the test cells. Can be used for analysis and the like.

(10) Method for Screening Compound That Changes the Amount of the Receptor Protein of the Present Invention or Its Partial Peptide in Cell Membrane The antibody of the present invention specifically recognizes the receptor protein of the present invention or its partial peptide or a salt thereof. Therefore, it can be used for screening a compound that changes the amount of the receptor protein of the present invention or its partial peptide in the cell membrane. That is, the present invention relates to, for example, (i) the present invention, wherein the cell membrane fraction is isolated after disrupting the blood of a non-human mammal, a specific organ, tissues or cells isolated from the organ, and the like, and isolating the cell membrane fraction. 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 quantifying the receptor protein or its partial peptide, (ii) a trait that expresses the receptor protein or its partial peptide of the present invention 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) Quantifying the degree of staining of the receptor protein on the cell surface layer by using immunostaining after sectioning blood, non-human mammal blood, specific organs, tissues or cells isolated from the organs, etc. Thus, there is provided a method for screening a compound that changes the amount of the receptor protein of the present invention or a partial peptide thereof in a cell membrane by confirming the protein on the cell membrane. (Iv) Quantifying the degree of staining of the receptor protein on the cell surface by using immunostaining after transformants or the like expressing the receptor protein of the present invention or its partial peptide are sectioned, A method for screening a compound that changes the amount of the receptor protein of the present invention or a partial peptide thereof in a cell membrane by confirming the protein on the cell membrane is provided.

The quantitative determination of the receptor protein of the present invention or its partial peptide contained in the cell membrane fraction is specifically carried out as follows. (I) Normal or disease model non-human mammals (eg, mice, rats, rabbits, sheep, pigs, cows, cats, dogs, monkeys, etc., more specifically, dementia rats, obese mice, arteriosclerotic rabbits, cancer-bearing Mouse, etc.)
A drug (eg, an anti-dementia drug, a blood pressure lowering drug, an anti-cancer drug, an anti-obesity drug, etc.) or a physical stress (eg, flooding stress, electric shock, light / dark, low temperature, etc.) is given, and after a certain period of time, blood or Obtain a specific organ (eg, brain, lung, large intestine, etc.), or tissue or cells isolated from the organ. The obtained organ, tissue or cell is suspended in, for example, an appropriate buffer (eg, Tris-HCl buffer, phosphate buffer, Hepes buffer, etc.),
Destroy organs, tissues or cells and use surfactants (eg, Triton X100 ^™ , Tween-20 ^™, etc.)
Further, a cell membrane fraction is obtained by using techniques such as centrifugation, filtration, and column fractionation.

The cell membrane fraction was obtained by disrupting the cells,
It refers to a fraction containing a large amount of cell membrane obtained by a known method. The cells can be crushed by crushing the cells with a Potter-Elvehjem homogenizer, crushing with a Waring blender or polytron (Kinematica), crushing by ultrasonic waves, or squirting the cells from a thin nozzle while applying pressure with a French press. Crushing, etc. For the fractionation of cell membranes, fractionation by centrifugal force such as fractionation centrifugation or density gradient centrifugation is mainly used. For example, reduce cell lysate at low speed (500-3000 rpm)
For a short time (usually about 1 to 10 minutes), and the supernatant is further centrifuged at a high speed (15000 to 30000 rpm) for usually 30 minutes to 2 hours, and the resulting precipitate is used as a membrane fraction. The membrane fraction contains a large amount of expressed receptor proteins and 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, sandwich immunoassay using the antibody of the present invention, Western blot analysis and the like. Such a sandwich immunoassay can be performed in the same manner as described above, and the Western blot can be performed by known means.

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

Screening for a compound that alters the amount of the receptor protein of the present invention or its partial peptide in the cell membrane is carried out by (i) a predetermined 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, preferably 1 hour to 2 days, more preferably 1 hour to 24 hours), or drug or physical stress At the same time, the test compound is administered, and after a lapse of a certain time after the administration (30 minutes to 3 days, preferably 1 hour to 2 days, more preferably 1 hour to 24 hours), the receptor of the present invention in the cell membrane (Ii) When the transformant is cultured according to a conventional method, the test compound is mixed with the medium, and after culturing for a certain period of time (from 1 day to 7 days). days after,
Preferably after 1 day to 3 days, more preferably after 2 days to 3 days), it can be carried out by quantifying the amount of the receptor protein of the present invention or its partial peptide in the cell membrane.

The confirmation of the receptor protein of the present invention or its partial peptide contained in the cell membrane fraction is specifically performed as follows. (Iii) Normal or disease model non-human mammals (eg, mice, rats, rabbits, sheep, pigs, cows, cats, dogs, monkeys, etc., more specifically, dementia rats, obese mice, arteriosclerotic rabbits, cancer-bearing Mouse, etc.)
A drug (eg, an anti-dementia drug, a blood pressure lowering drug, an anti-cancer drug, an anti-obesity drug, etc.) or a physical stress (eg, flooding stress, electric shock, light / dark, low temperature, etc.) is given, and after a certain period of time, blood or Obtain a specific organ (eg, heart, placenta, lung, etc.) or tissue or cells isolated from the organ. The obtained organ, tissue or cell is cut into a tissue section according to a conventional method, and immunostaining is performed using the antibody of the present invention. The amount of the receptor protein of the present invention or its partial peptide in the cell membrane is quantitatively or qualitatively confirmed by quantifying the degree of staining of the receptor protein on the cell surface to confirm the protein on the cell membrane. can do. (Iv) It can also be confirmed by using a transformant that expresses the receptor protein of the present invention or a partial peptide thereof and the like, and performing the same procedure.

The compound or a salt thereof obtained by using the screening method of the present invention is a compound having an effect of changing the amount of the receptor protein of the present invention or a partial peptide thereof in a cell membrane.
By increasing the amount of the receptor protein of the present invention or its partial peptide in the cell membrane, the cell stimulating activity via the G protein-coupled receptor (for example,
Arachidonic acid release, acetylcholine release, intracellular Ca ²⁺ release, intracellular cAMP production, intracellular cGMP production, inositol phosphate production, cell membrane potential fluctuation, intracellular protein phosphorylation, c-fos activation, pH decrease And (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 a peptide, a protein, a non-peptidic compound, a synthetic compound, and a fermentation product. These compounds may be a novel compound or a known compound. The compound that enhances the cell stimulating activity is useful as a safe and low-toxicity drug for enhancing the physiological activity of the receptor protein or the like of the present invention. The compound that attenuates the cell stimulating activity is useful as a safe and low toxic drug for decreasing the physiological activity of the receptor protein of the present invention.

When a compound or a salt thereof obtained by using 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 pharmaceuticals containing the receptor protein of the present invention. Since the preparation obtained in this way is safe and low toxic, for example, human or non-human mammals (for example,
Rats, mice, rabbits, sheep, pigs, cows, cats,
Dogs, monkeys, etc.). The dose of the compound or a salt thereof varies depending on the administration subject, the target organ, the condition, the administration method, and the like. However, in the case of oral administration, for example, in a cancer patient (as 60 kg), for example, per day, About 0.1-100 mg, preferably about 1.0-50 m
g, more preferably about 1.0-20 mg. When administered parenterally, the single dose depends on the subject, target organ,
For example, in the form of an injection, for example, usually about 0.01 to 30 mg, preferably about 0.1 to 30 mg per day for a cancer patient (as 60 kg), depending on the condition, administration method and the like.
It is convenient to administer about 20 mg, more preferably about 0.1-10 mg, by intravenous injection. In the case of other animals, the dose can be administered per 60 kg.

(11) 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 may be, for example, a heart or a central nervous system as described above. It is thought to play some important role in vivo, such as function. Therefore, a compound that changes the amount of the receptor protein of the present invention or a partial peptide thereof in the cell membrane can be used as an agent for preventing and / or treating a disease associated with dysfunction of the receptor protein of the present invention. Examples of the disease associated with dysfunction of the receptor protein of the present invention include central diseases (e.g., Alzheimer's disease, dementia, eating disorders, depression, epilepsy, anxiety, etc.), endocrine diseases (e.g., Addison's disease, Cushing's syndrome) , Pheochromocytoma, primary aldosteronism, menopause, endometriosis, gonadal dysfunction, thyroid dysfunction, pituitary dysfunction, etc., metabolic disorders (e.g., diabetes, lipid metabolism disorders, diabetic complications, obesity, Gout, cataract, hyperlipidemia, 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., inflammatory diseases (eg, allergy, asthma, rheumatism, arthritis, nephritis, hepatitis, retinopathy, cystitis, pneumonia, etc.), cardiovascular diseases (eg, hypertension) Disease, cardiac hypertrophy, angina pectoris, myocardial infarction, arteriosclerosis, etc.), respiratory diseases (eg, cold syndrome, asthma, pneumonia, pulmonary hypertension, pulmonary thrombosis, pulmonary thrombosis, etc.), gastrointestinal diseases ( For example, gastric ulcer, duodenal ulcer, gastritis, reflux esophagitis, pancreatitis, etc., immune system diseases (eg, autoimmune diseases, etc.), infectious diseases (eg, immune dysfunction, pneumonia, cytomegal virus, influenza virus, herpes virus) Viral infections, rickettsial infections, bacterial infections, etc.), and migraines. When the compound 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 conventional means. For example, the compound may be a sugar-coated tablet, capsule,
It can be used orally as elixirs, microcapsules and the like, or parenterally in the form of injections such as sterile solutions or suspensions with water or other pharmaceutically acceptable liquids. For example, the compound is mixed with known physiologically acceptable carriers, flavoring agents, excipients, vehicles, preservatives, stabilizers, binders, and the like in the unit dosage form generally required for the practice of pharmaceutical preparations. Can be manufactured by The amount of the active ingredient in these preparations is such that a proper dosage in the specified range can be obtained.

Examples of additives which can be mixed with tablets, capsules and the like include binders such as gelatin, corn starch, tragacanth and gum arabic, excipients such as crystalline cellulose, corn starch, gelatin, alginic acid. Use may be made of bulking agents such as, for example, magnesium stearate, sweeteners such as sucrose, lactose or saccharin, flavoring agents such as peppermint, reddish oil or cherry. When the preparation unit form is a capsule, the above type of material can further contain a liquid carrier such as oil and fat. Sterile compositions for injection can be formulated according to normal pharmaceutical practice such as dissolving or suspending the active substance in vehicles such as water for injection, and 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 auxiliary agents (eg, D-sorbitol, D-mannitol, sodium chloride, etc.) and the like are used. ,
For example, alcohol (eg, ethanol), polyalcohol (eg, propylene glycol, polyethylene glycol), nonionic surfactant (eg, polysorbate 8)
0 ^TM , HCO-50) and the like. As the oily liquid, for example, sesame oil, soybean oil and the like are used, and may be used in combination with solubilizers such as benzyl benzoate and benzyl alcohol.

The prophylactic / therapeutic agents include, for example, buffers (eg, phosphate buffer, sodium acetate buffer), soothing agents (eg, benzalkonium chloride, procaine hydrochloride, etc.), stabilizers (eg, For example, human serum albumin, polyethylene glycol, etc.), preservatives (eg,
Benzyl alcohol, phenol, etc.), antioxidants and the like. The prepared injection is usually filled in a suitable ampoule. The preparations obtained in this way are safe and have low toxicity, for example, human and non-human mammals (eg, rat, mouse, rabbit, sheep, pig,
Cattle, cats, dogs, monkeys, etc.). The dose of the compound or a salt thereof may be
Although there are differences depending on the target organ, symptoms, administration method and the like, in the case of oral administration, for example, in a cancer patient (as 60 kg), about 0.1 to 100 mg, preferably about 1.0 to 50 mg per day is generally used. , More preferably about 1.0-20 mg.
In the case of parenteral administration, the single dose varies depending on the administration subject, target organ, symptom, administration method and the like.
g), about 0.01 to 30 mg per day,
Preferably about 0.1-20 mg, more preferably about 0.1-1
It is convenient to administer about 0 mg by intravenous injection.
In the case of other animals, the dose can be administered per 60 kg.

(12) Neutralization by an antibody against the receptor protein of the present invention, its partial peptide, or a salt thereof The neutralizing activity of the antibody against the receptor protein of the present invention, its partial peptide, or a salt thereof against the receptor protein and the like That is, it means an activity of inactivating a signaling function involving the receptor protein. Therefore, when the antibody has a neutralizing activity, signal transduction involving the receptor protein, for example, cell stimulating activity via the receptor protein (eg, arachidonic acid release, acetylcholine release, intracellular Ca ²⁺ release, Inactivate cAMP production, cGMP production in cells, inositol phosphate production, cell membrane potential fluctuations, phosphorylation of intracellular proteins, activation of c-fos, activity to promote or suppress pH reduction, etc.) can do. Therefore, it can be used for prevention and / or treatment of diseases caused by overexpression of the receptor protein and the like. Examples of diseases caused by overexpression of the receptor protein include, for example, central diseases
(E.g., Alzheimer's disease, dementia, eating disorders, depression, epilepsy, anxiety, etc.), endocrine diseases (e.g., Addison's disease, Cushing's syndrome, pheochromocytoma, primary aldosteronism, menopause, endometriosis, Gonadal dysfunction,
Thyroid dysfunction, pituitary dysfunction, etc.), metabolic disorders (eg, diabetes, lipid metabolism disorders, diabetic complications, obesity, gout, cataract, hyperlipidemia, etc.), cancer (eg, non-small cell lung cancer, ovarian cancer) , Prostate cancer, gastric cancer, bladder cancer, breast cancer, cervical cancer, colon cancer, rectal cancer, etc., inflammatory diseases (eg, allergy, asthma, rheumatism, arthritis, nephritis, hepatitis, retinopathy, cystitis, pneumonia, etc.) ), Cardiovascular diseases (eg, hypertension, cardiac hypertrophy, angina pectoris, myocardial infarction, arteriosclerosis, etc.), respiratory diseases (eg, cold syndrome, asthma, pneumonia, pulmonary hypertension, pulmonary thrombosis, lung cold) Plugs), digestive disorders (e.g.,
Gastric ulcer, duodenal ulcer, gastritis, reflux esophagitis, pancreatitis, etc., immune system disease (eg, autoimmune disease), infectious disease (eg, immune dysfunction, pneumonia, cytomegal virus, influenza virus, herpes virus, etc.) Viral infections, rickettsial infections, bacterial infections, etc.), and migraines.

(13) Production of Transgenic Animal Having DNA Encoding G Protein-Coupled Receptor Protein of the Present Invention Using DNA of the present invention to produce transgenic animal expressing the receptor protein of the present invention. Can be. Examples of animals include mammals (eg, rats, mice, rabbits, sheep, pigs, cows, cats, dogs, monkeys, and the like) (hereinafter sometimes abbreviated as animals), and in particular, mice and rabbits. And the like are preferred. When introducing the DNA of the present invention into a target animal, it is generally advantageous to use the DNA as a gene construct linked downstream of a promoter capable of being expressed in animal cells. For example, when a rabbit-derived DNA of the present invention is introduced, a gene construct linked to downstream of various promoters capable of expressing the DNA of the present invention derived from an animal having high homology to animal cells, for example, a rabbit fertilized egg By introducing microinjection into DNA, a DNA-transferred animal that highly produces the receptor protein of the present invention can be produced. As this promoter, for example, a ubiquitous expression promoter such as a virus-derived promoter or metallothionein can be used, but a promoter of a gene that is specifically expressed in the heart is preferably used.

The introduction of the DNA of the present invention at the fertilized egg cell stage is ensured to be present in all germ cells and somatic cells of the target animal. The presence of the receptor protein or the like of the present invention in the germinal cells of the produced animal after DNA introduction means that all the offspring of the produced animal have the receptor protein or the like of the present invention in all of its germinal and somatic cells. The progeny of this type of animal that has inherited the gene has the receptor protein of the present invention in all of its germinal and somatic cells. The DNA-introduced animal of the present invention was confirmed to stably maintain the gene by mating, and
The breeding subculture can be carried out in a normal breeding environment as a holding animal. Furthermore, by crossing male and female animals having the target DNA, a homozygous animal having the transgene on both homologous chromosomes is obtained, and by crossing the male and female animals, all progeny have the DNA. Breeding can be subcultured. Since the animal into which the DNA of the present invention has been introduced has high expression of the receptor protein of the present invention, it is useful as an animal for screening an agonist or antagonist for the receptor protein of the present invention. The DNA-introduced animal of the present invention,
It can also be used as a cell source for tissue culture.
For example, DNA in the tissue of the DNA-introduced mouse of the present invention or
The receptor protein of the present invention and the like can be analyzed by directly analyzing RNA or by analyzing a tissue in which the receptor protein of the present invention expressed by a gene is present. Culturing cells of a tissue having the receptor protein or the like of the present invention by standard tissue culture techniques, and using them to study the function of cells from a generally difficult-to-culture tissue such as, for example, from brain or peripheral tissues Can be. Also, by using the cells,
For example, it is possible to select a medicine that enhances the function of various tissues. Further, if there is a high expression cell line, the receptor protein of the present invention can be isolated and purified therefrom.

In the present specification and drawings, when bases, amino acids and the like are indicated by abbreviations, the indications are based on IUPAC-IU
This is based on the abbreviation by the B Commission on Biochemical Nomenclature or the abbreviation commonly used in this field. An example is shown below. When an amino acid may have an optical isomer, the L-form is indicated unless otherwise specified.

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: arginine His: histidine Phe yr : Tyrosine Tr: Tryptophan Pro: Proline Asn: Asparagine Gln: Glutamine pGlu: Pyroglutamic acid *: Corresponding to the stop codon Me: Methyl group Et: Ethyl group Bu: Butyl group Ph: Phenyl group TC: Thiazolidine-4 (R)- Carboxamide group

Further, substituents, protecting groups and reagents frequently 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-dicarboximide DCC: N, N'-dicyclohexylcarbodiimide

[0110] The sequence numbers in the sequence listing in the present specification indicate the following sequences. SEQ ID NO: 1 shows the amino acid sequence of the novel human-derived G protein-coupled receptor protein TGR29 of the present invention. SEQ ID NO: 2 This shows the base sequence of cDNA encoding novel human-derived G protein-coupled receptor protein TGR29 of the present invention. SEQ ID NO: 3 Primer 1 used in PCR reaction in Example 1 below
Shows the nucleotide sequence of SEQ ID NO: 4 Primer 2 used in PCR reaction in Example 1 below
Shows the nucleotide sequence of

The transformant, Escherichia coli TOP10 / pCR2.1-hTGR29, obtained in Example 1 below was
From March 5, 2001 (Heisei 13), 1-1 1-1 Higashi, Tsukuba City, Ibaraki Prefecture, Chuo No. 6 (Zip code 305-856)
6) Deposit No. FERM BP- at the National Institute of Advanced Industrial Science and Technology, National Institute of Advanced Industrial Science and Technology (NIBH) (formerly the Ministry of Economy, Trade and Industry, National Institute of Advanced Industrial Science and Technology).
7486 from February 20, 2001 (Heisei 13), 2-17-85, Jusanhoncho, Yodogawa-ku, Osaka-shi, Osaka (zip code 532-8686).
O) and deposit number IFO 16573.

[0112]

The present invention will be described in more detail with reference to the following Examples, which do not limit the scope of the present invention. In addition, the genetic manipulation method using Escherichia coli followed the method described in Molecular cloning.

Example 1 Cloning of cDNA Encoding G Protein-Coupled Receptor Protein of Human Kidney and Determination of Base Sequence Using human kidney cDNA (CLONTECH) as a template, two primers, primer 1 (SEQ ID NO: 3) And primer 2
(SEQ ID NO: 4) was used to carry out a PCR reaction. The composition of the reaction solution in the reaction was determined using the above cDNA as a 1/10 template and using Advantage-GC2 Polymerase Mix (CLONTECH) 1/5.
0 amount, primer 1 (SEQ ID NO: 3) and primer 2
(SEQ ID NO: 4) was added to each of 0.5 μM, dNTPs at 200 μM, a buffer attached to the enzyme at 1/5 volume, and GC Melt at 1/5 volume to obtain a 20 μl liquid volume. After 5 minutes at 94 ° C,
A cycle of 94 ° C for 30 seconds, 60 ° C for 30 seconds, and 68 ° C for 4 minutes was repeated 35 times, and finally an extension reaction at 68 ° C for 5 minutes was performed. The PCR
The reaction product was subcloned into a plasmid vector pCR2.1 (Invitrogen) according to the procedure of a TA cloning kit (Invitrogen). This was introduced into E. coli TOP10 and c
Clones with DNA were selected in LB agar containing ampicillin. As a result of analyzing the sequence of each clone, it encodes a novel G protein-coupled receptor protein
A cDNA sequence (SEQ ID NO: 2) was obtained. In addition, a novel G protein-coupled receptor protein containing this amino acid sequence (SEQ ID NO: 1) was named TGR29. The transformant was used as Escherichia coli TOP10 / pCR2.1-hTG
Named R29.

[0114]

The G protein-coupled receptor protein of the present invention or a partial peptide thereof or a salt thereof, and a polynucleotide (eg, DNA, RNA and derivatives thereof) encoding the receptor protein or a partial peptide thereof may be a ligand (agonist) ), Obtaining antibodies and antisera, constructing a recombinant receptor protein expression system, developing a receptor binding assay system using the expression system and screening drug candidate compounds, and examining structurally similar ligands / receptors. Implementation of drug design based on the comparison of reagents for creating probes and PCR primers in genetic diagnosis,
It can be used for the production of transgenic animals or as a drug such as a gene therapy agent.

[0115]

[Sequence List] <110> Takeda Chemical Industries, Ltd. <120> Novel G Protein-Coupled Receptor and its DNA <130> P01-0278 <150> JP 2001-6464 <151> 2001-01-15 <150> JP 2001-102483 <151> 2001-03-30 <160> 4 <210> 1 <211> 342 <212> PRT <213> Human <400> 1 Met Thr Ser Asn Phe Ser Gln Pro Val Val Gln Leu Cys Tyr Glu Asp 5 10 15 Val Asn Gly Ser Cys Ile Glu Thr Pro Tyr Ser Pro Gly Ser Arg Val 20 25 30 Ile Leu Tyr Thr Ala Phe Ser Phe Gly Ser Leu Leu Ala Val Phe Gly 35 40 45 Asn Leu Leu Val Met Thr Ser Val Leu His Phe Lys Gln Leu His Ser 50 55 60 Pro Thr Asn Phe Leu Ile Ala Ser Leu Ala Cys Ala Asp Phe Leu Val 65 70 75 80 Gly Val Thr Val Met Leu Phe Ser Met Val Arg Thr Val Glu Ser Cys 85 90 95 Trp Tyr Phe Gly Ala Lys Phe Cys Thr Leu His Ser Cys Cys Asp Val 100 105 110 Ala Phe Cys Tyr Ser Ser Val Leu His Leu Cys Phe Ile Cys Ile Asp 115 120 125 Arg Tyr Ile Val Val Thr Asp Pro Leu Val Tyr Ala Thr Lys Phe Thr 130 135 140 Val Ser Val Ser Gly Ile Cys Ile Ser Val Ser Trp Ile Leu Pro Leu 145 150 155 160 T hr Tyr Ser Gly Ala Val Phe Tyr Thr Gly Val Asn Asp Asp Gly Leu 165 170 175 Glu Glu Leu Val Ser Ala Leu Asn Cys Val Gly Gly Cys Gln Ile Ile 180 185 190 Val Ser Gln Gly Trp Val Leu Ile Asp Phe Leu Leu Phe Phe Ile Pro 195 200 205 Thr Leu Val Met Ile Ile Leu Tyr Ser Lys Ile Phe Leu Ile Ala Lys 210 215 220 Gln Gln Ala Ile Lys Ile Glu Thr Thr Ser Ser Lys Val Glu Ser Ser 225 230 235 240 Ser Glu Ser Tyr Lys Ile Arg Val Ala Lys Arg Glu Arg Lys Ala Ala 245 250 255 Lys Thr Leu Gly Val Thr Val Leu Ala Phe Val Ile Ser Trp Leu Pro 260 265 270 Tyr Thr Val Val Asp Ile Leu Ile Asp Ala Phe Met Gly Phe Leu Thr Pro 275 280 285 Ala Tyr Ile Tyr Glu Ile Cys Cys Trp Ser Ala Tyr Tyr Asn Ser Ala 290 295 300 Met Asn Pro Leu Ile Tyr Ala Leu Phe Tyr Pro Trp Phe Arg Lys Ala 305 310 310 315 320 Ile Lys Leu Ile Leu Ser Gly Asp Val Leu Lys Ala Ser Ser Ser Thr 325 330 335 Ile Ser Leu Phe Leu Glu 340 <210> 2 <211> 1026 <212> DNA <213> Human <400> 2 atgaccagca atttttccca acctgttgtg cagctttgct atgaggatgt gaatggatct 60 tgtattgaaa ctccctattc tcctgggtcc cgggtaattc tgtacacggc gtttagcttt 120 gggtctttgc tggctgtatt tggaaatctc ttagtaatga cttctgttct tcattttaag 180 cagctgcact ctccaaccaa ttttctcatt gcctctctgg cctgtgctga cttcttggta 240 ggtgtgactg tgatgctttt cagcatggtc aggacggtgg agagctgctg gtattttgga 300 gccaaatttt gtactcttca cagttgctgt gatgtggcat tttgttactc ttctgtcctc 360 cacttgtgct tcatctgcat cgacaggtac attgtggtta ctgatcccct ggtctatgct 420 accaagttca ccgtgtctgt gtcgggaatt tgcatcagcg tgtcctggat tctgcctctc 480 acgtacagcg gtgctgtgtt ctacacaggt gtcaatgatg atgggctgga ggaattagta 540 agtgctctca actgcgtagg tggctgtcaa attattgtaa gtcaaggctg ggtgttgata 600 gattttctgt tattcttcat acctaccctt gttatgataa ttctttacag taagattttt 660 cttatagcta aacaacaagc tataaaaatt gaaactacta gtagcaaagt agaatcatcc 720 tcagagagtt ataaaatcag agtggccaag agagagagga aagcagctaa aaccctgggg 780 gtcacggtac tagcatttgt tatttcatgg ttaccgtata cagttgatat attaattgat 840 gcctttatgg gcttcctgac ccctgcctat atctatgaaa tttgctgttg gagtgcttat 900 tataactcag ccatgaatcc tttgattta t gctctatttt atccttggtt taggaaagcc 960 ataaaactta ttttaagtgg agatgtttta aaggctagtt catcaaccat tagtttattt 1020 ttagaa 1026 <210> 3 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 3 gtcgacatt cccagcacagca 4 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 4 gctagcttat tctaaaaata aactaatggt tg 32

[Brief description of the drawings]

FIG. 1 is a hydrophobicity plot of TGR29.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61K 48/00 A61P 3/00 A61P 1/00 5/00 3/00 9/00 5/00 11/00 9/00 25/00 11/00 29/00 25/00 31/00 29/00 35/00 31/00 37/00 35/00 C07K 14/705 37/00 16/28 C07K 14/705 C12N 1 / 15 16/28 1/19 C12N 1/15 1/21 1/19 C12P 21/02 C 1/21 C12Q 1/02 5/10 1/68 A C12P 21/02 G01N 33/15 Z C12Q 1/02 33 / 50 Z 1/68 33/53 D G01N 33/15 M 33/50 33/566 33/53 C12N 15/00 ZNAA 5/00 A 33/566 A61K 37/02 (72) Inventor Yasushi Shintani Osaka, Osaka, Japan No. 6-14-8-606, Shintaka 6-chome, Yodogawa-ku, Ichikawa 72) Inventor Nobuyuki Miyajima 4-16-4, Azuma, Tsukuba-shi, Ibaraki Preview Azuma 403

Claims (36)

[Claims] 1. A G protein-coupled receptor protein or a salt thereof, comprising an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1. 2. A G protein-coupled receptor protein comprising the amino acid sequence represented by SEQ ID NO: 1, or a salt thereof. 3. A partial peptide of the G protein-coupled receptor protein according to claim 1, or a salt thereof. 4. A polynucleotide comprising a polynucleotide encoding the G protein-coupled receptor protein according to claim 1. 5. The polynucleotide according to claim 4, which is a DNA. 6. The polynucleotide according to claim 4, which has the nucleotide sequence represented by SEQ ID NO: 2. 7. A recombinant vector containing the polynucleotide according to claim 4. A transformant transformed with the recombinant vector according to claim 7. 9. A medicament comprising the G protein-coupled receptor protein according to claim 1, or the partial peptide according to claim 3, or a salt thereof. A pharmaceutical comprising the polynucleotide according to claim 4. 11. culturing the transformant according to claim 8,
A method for producing the G protein-coupled receptor protein or a salt thereof according to claim 1, wherein the G protein-coupled receptor protein according to claim 1 is produced. 12. An antibody against the G protein-coupled receptor protein according to claim 1, the partial peptide according to claim 3, or a salt thereof. 13. The antibody according to claim 12, which is a neutralizing antibody that inactivates signal transduction of the G protein-coupled receptor protein according to claim 1. (14) A diagnostic agent comprising the antibody according to (12). A pharmaceutical comprising the antibody according to claim 12. [16] The present invention can be obtained by using the G protein-coupled receptor protein according to [1], the partial peptide according to [3], or a salt thereof.
A ligand for the described G protein-coupled receptor protein or a salt thereof. A pharmaceutical comprising the ligand according to claim 16. 18. A ligand for the G protein-coupled receptor protein or a salt thereof according to claim 1, wherein the G protein-coupled receptor protein according to claim 1 or the partial peptide according to claim 3 or a salt thereof is used. How to determine. 19. A ligand comprising the G protein-coupled receptor protein according to claim 1 or the partial peptide according to claim 3 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 a salt thereof that changes the binding property to a salt. 20. A ligand comprising the G protein-coupled receptor protein according to claim 1 or the partial peptide according to claim 3 or a salt thereof, and the G protein-coupled receptor protein according to claim 1 or A kit for screening a compound or a salt thereof that changes the binding property to the salt. 21. Alter the binding between the ligand obtainable by the screening method according to claim 19 or the screening kit according to claim 20 and the G protein-coupled receptor protein or a salt thereof according to claim 1. Compound or salt thereof. 22. Alter the binding between the ligand obtainable by using the screening method according to claim 19 or the screening kit according to claim 20 and the G protein-coupled receptor protein or a salt thereof according to claim 1. A medicament comprising a compound or a salt thereof. 23. A polynucleotide that hybridizes with the polynucleotide of claim 4 under high stringency conditions. 24. A polynucleotide comprising a nucleotide sequence complementary to the polynucleotide according to claim 4 or a part thereof. 25. The G according to claim 1, wherein the polynucleotide according to claim 4 or a part thereof is used.
Method for quantifying mRNA of protein-coupled receptor protein. 26. The method for quantifying a G protein-coupled receptor protein according to claim 1, wherein the antibody according to claim 12 is used. 27. The method for diagnosing a disease associated with the function of a G protein-coupled receptor according to claim 1, wherein the quantification method according to claim 25 or 26 is used. 28. A method for screening a compound or a salt thereof that alters the expression level of a G protein-coupled receptor protein according to claim 1, wherein the method according to claim 25 is used. 29. A method for screening a compound or a salt thereof that alters the amount of the G protein-coupled receptor protein according to claim 1 in a cell membrane, wherein the method uses the quantification method according to claim 26. 30. 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 28. 31. A compound or a salt thereof, which alters the amount of the G protein-coupled receptor protein of claim 1 in a cell membrane obtainable by using the screening method of claim 29. 32. A medicament comprising a compound capable of changing the expression level of the G protein-coupled receptor protein according to claim 1 or a salt thereof, which can be obtained by using the screening method according to claim 28. 33. A pharmaceutical comprising a compound or a salt thereof, which alters the amount of the G protein-coupled receptor protein according to claim 1 in a cell membrane obtainable by using the screening method according to claim 29. 34. Central diseases, endocrine diseases, metabolic diseases,
34. The method according to claim 22, which is an agent for preventing or treating cancer, a circulatory disease, a respiratory disease, a digestive disease, an immune disease, an inflammatory disease, or an infectious disease. Medicine. 35. A central disease in a mammal, which comprises administering to the mammal an effective amount of the compound according to any one of claims 21, 30 or 31 or a salt thereof. Endocrine disease, metabolic disease, cancer, cardiovascular disease Respiratory disease Digestive disease, immune system disease,
A method for preventing or treating inflammatory diseases or infectious diseases. 36. A method for producing a preventive / therapeutic agent for a central disease, an endocrine disease, a metabolic disease, a cancer cardiovascular disease, a respiratory disease, a gastrointestinal disease, an immune system disease, an inflammatory disease, or an infectious disease. Claim 21, Claim 30, or Claim 31
Or a salt thereof.