JP2000175691A - New g protein coupled receptor protein and its dna - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new G protein coupled receptor which consists of a G protein coupled receptor protein containing a specific amino acid sequence and is useful in the screening for agonist or antagonist and the prevention and treatment or the like of diseases relevant to the dysfunction of the above protein. SOLUTION: This G protein is a new G protein coupled receptor protein (salt) containing an amino acid sequence homologous or substantially homologous with the amino acid sequence represented by the formula and is useful in screening out agonist or antagonist for this protein and as a preventive and/or therapeutic agent for diseases relevant to the dysfunction of this protein or as an agent for gene diagnosis or the like by using its gene. The protein is obtained by using a cDNA arising from rat cerebellum as a template, carrying out PCR reaction by the use of two primers comprising a partial sequence of the gene coding for G protein coupled receptor protein, integrating the gene coding for the resultant G protein coupled receptor protein arising from rat cerebellum into a vector and cultivating a transformant obtained by transducing it into a hose cell.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a novel G protein-coupled receptor protein derived from rat cerebellum, 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 the functions of living organisms through specific receptor proteins present on cell membranes. Many of these receptor proteins are conjugated guanine nucleo
It performs intracellular signal transduction through activation of tide-binding protein (hereinafter sometimes abbreviated as G protein), and has a common structure having seven transmembrane domains. It is collectively called a protein or a seven-transmembrane receptor protein (7TMR). G protein-coupled receptor proteins are present on the surface of various functional cells of living cells and organs, and are physiologically important as targets for molecules that regulate the functions of those cells and organs, such as hormones, neurotransmitters and bioactive substances. Role. 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 cells and organs in various organisms. And provide a very important means for drug development 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 exist at various sites in a living body, and regulate their physiological functions through their corresponding receptor proteins. There are still many unknown hormones, neurotransmitters and other physiologically active substances in living organisms, and many of the structures of their receptor proteins have not yet been reported. In addition, many of the known receptor proteins do not know whether subtypes are present. It is a very important tool for drug development to clarify the relationship between substances that regulate complex functions in living organisms and their specific receptor proteins. Also, efficiently screen for agonists and antagonists for the receptor protein,
In order to develop pharmaceuticals, it was necessary to elucidate the functions of the receptor protein genes expressed in vivo and to express them in an appropriate expression system. In recent years, as a means for analyzing genes expressed in vivo, cDNA
Research on the random analysis of the sequence of cDNA has been actively conducted.
It is registered in the database as an essed Sequence Tag (EST) and is open to the public. However, many ESTs have only sequence information, and it is difficult to estimate their functions.

[0004]

Heretofore, substances that inhibit the binding between a G protein-coupled receptor and a physiologically active substance (that is, a ligand), and signal transmission that binds to the same as a physiologically active substance (that is, a ligand) Have been utilized as specific antagonists or agonists of these receptors as pharmaceuticals that regulate biological functions. Therefore, it is important to newly discover a G protein-coupled receptor protein which is not only important in physiological expression in vivo but also a target of drug development and to clone its gene (eg, cDNA). This is a very important means for finding specific ligands, agonists and antagonists of G protein-coupled receptor proteins. However, not all G protein-coupled receptors have been found. At present, there are many unknown G protein-coupled receptors and so-called orphan receptors for which no corresponding ligand has been identified. Therefore, there is a long-awaited search for new G protein-coupled receptors and elucidation of their functions. G
The protein-coupled receptor is useful for searching for a new physiologically active substance (that is, a ligand) and for searching for an agonist or antagonist for the receptor using the signal transduction action as an index. 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 or diagnostic agents for diseases associated with dysfunction of G protein-coupled receptors. Furthermore, a decrease or increase in 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 useful G protein-coupled receptor protein as described above. That is, a novel G protein-coupled receptor protein or a partial peptide thereof or a salt thereof, a polynucleotide encoding the G protein-coupled receptor protein or a partial peptide thereof (DN
A, RNA and their derivatives) -containing polynucleotides (DNA, RNA and their derivatives), recombinant vectors containing the polynucleotides, transformants carrying the recombinant vectors, G-protein coupled receptors Method for producing protein or salt thereof, antibody against G protein-coupled receptor protein or partial peptide thereof or salt thereof, compound capable of changing expression level of G protein-coupled receptor protein, determination of ligand for G protein-coupled receptor Methods, screening methods for compounds (antagonists, agonists) or salts thereof that alter the binding between ligands and the G protein-coupled receptor protein, kits for screening, ligands obtained using the screening methods or screening kits, Compounds (antagonists, agonists) or salts thereof that change the binding to protein-coupled receptor proteins, and compounds (antagonists, agonists) or compounds that change the binding of ligands to the G protein-coupled receptor proteins And a pharmaceutical comprising a compound that changes the expression level of a type receptor protein or a salt thereof.

[0005]

Means for Solving the Problems The inventors of the present invention have conducted intensive studies and as a result, have obtained E. coli prepared by the degenerated PCR method.
Based on the ST information, a cDNA encoding a novel G protein-coupled receptor protein derived from rat cerebellum was isolated, and the entire base sequence was successfully analyzed. When this base sequence was translated into an amino acid sequence, the first to seventh transmembrane regions were confirmed on a hydrophobicity plot, and the protein encoded by these cDNAs was a seven-transmembrane G protein-coupled receptor. It was confirmed to be a protein. The present inventors have further studied based on these findings, and as a result, completed the present invention.

That is, the present invention relates to (1) SEQ ID NO: 1.
A G protein-coupled receptor protein or a salt thereof, which comprises the same or substantially the same amino acid sequence as the amino acid sequence represented by (2).
(3) a polynucleotide comprising a polynucleotide having a nucleotide sequence encoding the G protein-coupled receptor protein according to (1), (4) DNA;
(5) a polynucleotide comprising the nucleotide sequence represented by SEQ ID NO: 2; (6) a polynucleotide containing the polynucleotide according to (3); A recombinant vector, (7) a transformant transformed with the recombinant vector according to the above (6),
(8) The G protein-coupled receptor protein of (1) or a G protein-coupled receptor protein of (1), wherein the transformant of (7) is cultured to produce the G protein-coupled receptor protein of (1). A method for producing a salt, (9) an antibody against the G protein-coupled receptor protein according to (1) or the partial peptide according to (2) or a salt thereof, (10) a G protein-coupled receptor protein according to (1). The antibody according to the above (9), which is a neutralizing antibody that inactivates the signal transduction of (9), (11) the diagnostic agent comprising the antibody according to the above (9), (12) the G protein according to the above (1) The G protein-coupled receptor protein or the salt thereof according to the above (1), which can be obtained by using the coupled receptor protein or the partial peptide or the salt thereof as described in the above (2). (13) a medicine comprising the ligand of the G protein-coupled receptor protein of (12), (14) the G protein-coupled receptor protein of (1) or the partial peptide of (2) Or (1) characterized in that a salt thereof is used.
(15) A method for determining a ligand for the G protein-coupled receptor protein or a salt thereof according to the above (15), wherein the G protein-coupled receptor protein according to the above (1), the partial peptide according to the above (2), or a salt thereof is used. (1) a method for screening a compound or a salt thereof that alters the binding property between a ligand to be bound and the G protein-coupled receptor protein or a salt thereof according to the above (1);
The binding between the G protein-coupled receptor protein described above or the ligand comprising the partial peptide described in (2) or a salt thereof and the G protein-coupled receptor protein described in (1) or a salt thereof is A kit for screening a compound to be changed or a salt thereof, (1
7) Changes in binding between the ligand and the G protein-coupled receptor protein or the salt thereof according to (1), which can be obtained using the screening method according to (15) or the screening kit according to (16). Or a salt thereof, (18) a ligand and a G protein-coupled receptor protein according to (1) or a G protein-coupled receptor protein according to (1), which can be obtained using the screening method according to (15) or the screening kit according to (16). (19) a medicament comprising a compound that changes the binding property to a salt or a salt thereof, (19) a polynucleotide that hybridizes under high stringent conditions to the polynucleotide described in (3), (20) a A) a polynucleotide comprising a complementary nucleotide sequence to the polynucleotide and a portion thereof; 21) (3), wherein the polynucleotide or mRN of G protein-coupled receptor protein of the above (1), wherein the use of a portion thereof
A method for quantifying A, (22) the method for quantifying a G protein-coupled receptor protein according to (1), wherein the antibody according to (9) is used, (23) the method (21) or (22). The method for diagnosing a disease associated with the function of the G protein-coupled receptor protein according to the above (1), wherein the method for quantification according to the above (21) is used, wherein the method for quantification according to the above (21) is used. A method for screening a compound or a salt thereof that alters the expression level of a G protein-coupled receptor protein according to (1),
5) a method for screening a compound or a salt thereof that alters the amount of the G protein-coupled receptor protein described in (1) above in a cell membrane, which comprises using the quantification method described in (22); A) a 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 screening method according to (1); The present invention also relates to a compound that changes the amount of the G protein-coupled receptor protein described in (1) above in a cell membrane, or a salt thereof.

[0007] Furthermore, (28) the amino acid sequence represented by SEQ ID NO: 1 or 1 or 2 or more (preferably about 1 to 30, more preferably 1) in the amino acid sequence represented by SEQ ID NO: 1; Is an amino acid sequence in which about 1 to 9, more preferably several (1 to 5) amino acids have been deleted, and one or more (preferably 1 to 30) in the amino acid sequence represented by SEQ ID NO: 1. Amino acid sequence to which about 1, more preferably about 1 to 10, and even more preferably several (1 to 5) amino acids have been added, and one or two or more (preferably Is about 1 to 30, more preferably about 1 to 10, and still more preferably several (1 to 5
G) protein-coupled receptor protein or a salt thereof according to the above (1), which is a protein containing an amino acid sequence in which the amino acid of (1) is substituted with another amino acid, or an amino acid sequence obtained by combining them. (14) The method according to (14), wherein the test compound is brought into contact with the G protein-coupled receptor protein or the salt thereof according to 1) or the partial peptide or the salt thereof according to (2).
The method for determining a ligand as described in (30), wherein the ligand is, for example, angiotensin, bombesin, cannabinoid, cholecystokinin, glutamine, serotonin, melatonin, neuropeptide Y, opioid, purine, vasopressin, oxytocin, PACAP, secretin, glucagon, calcitonin, address Nomedulin, somatostatin, GHRH, CRF, ACTH, GRP, PTH, V
IP (basoactive intestinal polypeptide), somatostatin, dopamine, motilin, amylin, bradykinin, CGRP (calcitonin gene-related peptide), leukotriene, pancreastatin, prostaglandin, thromboxane, adenosine, adrenaline, α and β -Chemokines (chemok
ine) (e.g., IL-8, GROα, GROβ, GR
Oγ, NAP-2, ENA-78, PF4, IP10,
GCP-2, MCP-1, HC14, MCP-3, I-
309, MIP1α, MIP-1β, RANTES, etc.), endothelin, enterogastrin, histamine, neurotensin, TRH, pancreatic polypeptide or galanin, the method for determining a ligand according to the above (29),

(31) (i) contacting a G protein-coupled receptor protein or a salt thereof described in the above (1) or a partial peptide or a salt thereof described in the above (2) with a ligand; and (ii) The G protein-coupled receptor protein described in (1) or a salt thereof or the partial peptide described in (2) or a salt thereof is compared with a ligand and a test compound. (15) The screening method described in (15), (32) (i) when the labeled ligand is brought into contact with the G protein-coupled receptor protein described in (1) or a salt thereof or the partial peptide described in (2) or a salt thereof. And (ii) labeling the labeled ligand and test compound with the G protein-coupled receptor protein described in (1) above or a salt thereof, or (2) Binding of the labeled ligand to the G protein-coupled receptor protein or the salt thereof described in (1) above or the partial peptide or the salt thereof described in (2) above when brought into contact with the partial peptide or the salt thereof described in (2). (33) a method for screening a compound or a salt thereof that changes the binding property between the ligand and the G protein-coupled receptor protein or the salt thereof according to (1) above, wherein the amount is measured and compared;
(I) the case where the labeled ligand is brought into contact with the cells containing the G protein-coupled receptor protein described in the above (1); A ligand characterized by measuring and comparing the amount of a labeled ligand bound to a cell containing the receptor protein when the cell is brought into contact with the cell, and comparing the ligand with the G protein-coupled receptor protein or a salt thereof according to the above (1). (34) (i) when a labeled ligand is brought into contact with the membrane fraction of a cell containing the G protein-coupled receptor protein described in (1) above, And (ii) contacting the labeled ligand and the test compound with the membrane fraction of a cell containing the G protein-coupled receptor protein described in (1) above. In which the amount of the labeled ligand bound to the membrane fraction of the cell is measured and compared, and the binding between the ligand and the G protein-coupled receptor protein or a salt thereof according to (1) above. A method for screening a compound or a salt thereof that changes

(35) (i) When the labeled ligand is brought into contact with the G protein-coupled receptor protein expressed on the cell membrane of the transformant by culturing the transformant according to (7), 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 (7) 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 the above (1), wherein the amount of binding to the G protein-coupled receptor protein is measured and compared. Method (36) (i) The above (1)
Contacting a compound that activates the G protein-coupled receptor protein or a salt thereof according to the above (1) with a cell containing the G protein-coupled receptor protein according to (1) above;
(Ii) when a compound that activates the G protein-coupled receptor protein described in (1) or a salt thereof and a test compound are brought into contact with cells containing the G protein-coupled receptor protein described in (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 via the G protein-coupled receptor protein is measured and compared. Salt screening method,
(37) A G protein-coupled receptor protein described in (1) or a compound that activates a salt thereof is cultured on the transformant according to (7), and the G protein-coupled receptor is expressed on the cell membrane of the transformant. And a compound that activates the G protein-coupled receptor protein or a salt thereof described in (1) above and a test compound, by culturing the transformant described in (7) above. 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 G protein-coupled receptor protein expressed on the cell membrane of the transformant; A method for screening a compound or a salt thereof that changes the binding property to a G protein-coupled receptor protein or a salt thereof,

(38) The compound that activates the G protein-coupled receptor protein according to (1) is an angiotensin, a bombesin, a cannabinoid, a cholecystokinin,
Glutamine, serotonin, melatonin, neuropeptide Y, opioid, purine, vasopressin, oxytocin, PACAP, secretin, glucagon, calcitonin, adrenomedullin, somatostatin, GHR
H, CRF, ACTH, GRP, PTH, VIP (vasoactive intestinal polypeptide), somatostatin, dopamine, motilin, amylin, bradykinin, CGRP (calcitonin gene-related peptide), leukotriene, pancreastatin, prostaglandin, Thromboxane, adenosine, adrenaline, α and β-chemokines (eg, IL-8, GROα, GROβ, GROγ, NA
P-2, ENA-78, PF4, IP10, GCP-
2, MCP-1, HC14, MCP-3, I-309,
MIP1α, MIP-1β, RANTES, etc.), endothelin, enterogastrin, histamine, neurotensin, TRH, pancreatic polypeptide or galanin, the screening method according to (36) or (37), (39) ) To (3)
8) A compound or a salt thereof that changes the binding property between the ligand and the G protein-coupled receptor protein or the salt thereof according to (1), which can be obtained by the screening method described in (8), (40) the above (31) to (38) A) a ligand obtainable by the screening method described in (1) and G
A medicament comprising a compound or a salt thereof that changes the binding property to a protein-coupled receptor protein or a salt thereof,

(41) The screening kit according to (16), which comprises a cell containing the G protein-coupled receptor protein according to (1).
(42) the screening kit according to (16), which comprises a membrane fraction of a cell containing the G protein-coupled receptor protein according to (1);
3) The above-mentioned (16), which comprises a G protein-coupled receptor protein expressed on the cell membrane of the transformant by culturing the transformant of the above (7).
(44) The kit for screening described above.
To (43) a compound or a salt thereof that alters the binding property between the ligand and the G protein-coupled receptor protein or a salt thereof according to (1) above, which can be obtained using the screening kit according to (43); 41) A compound or a salt thereof, which can be obtained by using the screening kit according to (43), which changes the binding property between the ligand and the G protein-coupled receptor protein or the salt thereof according to (1) above. (46) A medicament characterized by contacting the antibody according to (9) with the G protein-coupled receptor protein according to (1) or the partial peptide according to (2) or a salt thereof. (1) the method for quantifying the G protein-coupled receptor protein or the partial peptide or the salt thereof according to (2);
7) The antibody of the above (9) is allowed to competitively react with the test solution and the labeled G protein-coupled receptor protein of the above (1) or the partial peptide of the above (2) or a salt thereof. Measuring the ratio of the labeled G protein-coupled receptor protein described in (1) above or the partial peptide described in (2) or a salt thereof bound to the antibody. G described in (1)
(48) a method for quantifying a protein-coupled receptor protein or a partial peptide according to the above (2) or a salt thereof;
After reacting the test solution with the antibody of (9) insolubilized on the carrier and the labeled antibody of (9) simultaneously or continuously, the activity of the labeling agent on the insolubilized carrier is measured. The G protein-coupled receptor protein according to the above (1) or the above (2) in a test solution.
And a method for quantifying the partial peptide 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 an amino acid sequence represented by SEQ ID NO: 1 (FIG. 1).
And the same or substantially the same amino acid sequence as the amino acid sequence in FIG. 2). The receptor protein of the present invention includes, for example, humans and mammals (eg, guinea pigs, rats, mice, rabbits,
Pigs, sheep, cows, monkeys, etc.) Fiber cells, muscle cells, fat cells, immune cells (eg, macrophages, T cells, B cells, natural killer cells, mast cells, neutrophils, basophils, eosinophils, monocytes), megakaryocytes, synovium Cells, chondrocytes, osteocytes, osteoblasts, osteoclasts, mammary cells, hepatocytes or stromal cells, or precursors of these cells, stem cells or cancer cells), blood cells, or those cells Any tissue present, for example, the brain, parts of the brain (eg, olfactory bulb, nucleus pulposus, basal sphere, hippocampus, thalamus,
Hypothalamus, hypothalamus nucleus, cerebral cortex, medulla oblongata, cerebellum, occipital lobe,
Frontal lobe, temporal lobe, putamen, caudate nucleus, brain stain, substantia nigra), spinal cord,
Pituitary, stomach, pancreas, kidney, liver, gonad, thyroid, gall bladder, bone marrow, adrenal gland, skin, muscle, lung, gastrointestinal tract (eg, colon,
Small intestine), blood vessels, heart, thymus, spleen, submandibular gland, peripheral blood, peripheral blood cells, prostate, testicle, testis, ovary, placenta, uterus, bone,
It may be a protein derived from a joint, skeletal muscle or the like (particularly, brain or various parts of the brain), 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 70% or more, more preferably the amino acid sequence represented by SEQ ID NO: 1. About 80% or more, more preferably about 90% or more, most preferably about 95%
Amino acid sequences having the above homology are exemplified.
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, activities such as ligand binding activity and signal transduction activity are equivalent (eg, about 0.01 to 100 times, preferably about 0.5 to 2 times).
(0 times, more preferably about 0.5 to 2 times), but the quantitative factors such as the degree of activity and the molecular weight of the protein may be different. The measurement of the activity such as the ligand binding activity and the signal information transduction can be carried out according to a method known per se. For example, the activity can be measured according to a ligand determination method and a screening method described later.

[0014] The receptor protein of the present invention may be one or more (preferably about 1 to 30, more preferably about 1 to 10, more preferably about 1 or more) in the amino acid sequence represented by SEQ ID NO: 1. Are several (1-5
Amino acid sequence), the amino acid sequence represented by SEQ ID NO: 1 or 2 or more (preferably about 1 to 30, more preferably 1 to 1)
About 0, more preferably several (1 to 5) amino acids added, 1 or 2 or more (preferably, in the amino acid sequence represented by SEQ ID NO: 1)
It contains an amino acid sequence in which about 1 to 30, more preferably about 1 to 10, and still more preferably several (1 to 5) amino acids have been substituted with other amino acids, or an amino acid sequence obtained by combining them. Proteins and the like are also used.

[0015] The receptor protein in the present specification has the N-terminus (amino terminus) at the left end and the C-terminus (carboxyl terminus) at the right end in accordance with the convention of peptide labeling. The receptor protein of the present invention, including the receptor protein containing the amino acid sequence represented by SEQ ID NO: 1, usually has a carboxyl group (—COOH) or a carboxylate (—COO ⁻ ) at the C-terminus. May be an amide (—CONH ₂ ) or an ester (—COOR). Here, as R in the ester, for example, a C _1-6 alkyl group such as methyl, ethyl, n-propyl, isopropyl or n-butyl, for example, a C _3-8 cycloalkyl group such as cyclopentyl and cyclohexyl, for example, phenyl And C _6-12 such as α-naphthyl
Aryl groups, for example, phenyl-C _1-2 alkyl groups such as benzyl and phenethyl or C _7-14 aralkyl groups such as α-naphthyl-C _1-2 alkyl groups such as α-naphthylmethyl, and as oral esters A commonly used pivaloyloxymethyl group or the like is used. When the receptor protein 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 receptor protein of the present invention. As the ester in this case, for example, the above-mentioned C-terminal ester and the like are used. Furthermore, in the receptor protein of the present invention, the amino group of the methionine residue at the N-terminus may be a protective group (for example, a C _1-6 acyl group such as a C _2-6 alkanoyl group such as formyl group or acetyl). ), A glutamyl group formed by cleavage of the N-terminal side in vivo and pyroglutamine oxidation, a substituent on the side chain of an amino acid in the molecule (eg, -OH, -SH, amino group) , Imidazole group, indole group, guanidino group, etc.) are suitable protecting groups (eg, C _1-6 acyl groups such as C _2-6 alkanoyl groups such as formyl group and acetyl)
Or a complex protein such as a so-called glycoprotein to which a sugar chain is bound.
Specific examples of the receptor protein of the present invention include, for example,
A rat-derived (more preferably, rat cerebellum-derived) receptor protein containing the amino acid sequence represented by SEQ ID NO: 1 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 receptor binding activity are used. Specifically, the partial peptide of the receptor protein having the amino acid sequence represented by SEQ ID NO: 1 was analyzed to be an extracellular region (hydrophilic site) in the hydrophobicity plot analysis shown in FIG. Is a peptide comprising Further, a peptide partially containing a hydrophobic (Hydrophobic) 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 amino acid sequences constituting the receptor protein of the present invention. . A substantially identical amino acid sequence is defined as about 50% or more, preferably about 70% or more, more preferably about 80% or more, more preferably about 90% or more, and most preferably about 95% or more, of these amino acid sequences. 1 shows an amino acid sequence having homology. Here, “substantially the same activity” has the same meaning as described above. "Substantially the same activity" can be measured in the same manner as described above.

Further, the partial peptide of the present invention may comprise one or more (preferably 1 to 10)
About, more preferably several (1 to 5) amino acids are deleted, or one or more (preferably about 1 to 20, more preferably 1 to 2)
About 10 to 10, more preferably several (1 to 5) amino acids are added, or 1 or 2 or more (preferably about 1 to 10 and more preferably several) in the amino acid sequence. , More preferably about 1 to 5 amino acids) may be substituted with another amino acid. In the partial peptide of the present invention, the C-terminus usually has a carboxyl group (-C
OOH) or carboxylate (—COO ⁻ ), but the C-terminal may be amide (—CONH ₂ ) or ester (—COOR) as in the protein of the present invention described above. 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. Also included are those obtained by pyroglutamine oxidation of Gln, those in which the substituent on the side chain of the amino acid in the molecule is protected with an appropriate protecting group, and those so-called glycopeptides having a sugar chain bonded thereto. The partial peptide of the present invention usually has a carboxyl group (—COOH) or a carboxylate (—COO ⁻ ) at the C-terminus, but has an amide (—CONH ₂ ) or ester at the C-terminus as in the protein of the present invention described above. (-COOR). Examples of the salt of the receptor protein or its partial peptide of the present invention include physiologically acceptable salts with acids or bases, and particularly preferred are physiologically acceptable acid addition salts. Examples of such salts include salts with inorganic acids (eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid) and organic acids (eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, succinic acid) , Tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid,
For example, salts with methanesulfonic acid and benzenesulfonic acid) are used.

The receptor protein of the present invention or a salt thereof can be produced from the aforementioned human or mammalian cells or tissues by a known method for purifying a receptor protein, or encodes the receptor protein of the present invention described later. It can also be produced by culturing a transformant containing DNA. Further, it can also be produced by the protein synthesis method described below or according to it. When producing from human or mammalian tissues or cells, the homogenized human or mammalian tissues or cells are extracted with an acid or the like, and the extract is subjected to chromatography such as reverse phase chromatography or ion exchange chromatography. Can be purified and isolated.

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-hydroxymethylmethylphenylacetamidomethyl resin, polyacrylamide resin, 4-
(2 ′, 4′-dimethoxyphenyl-hydroxymethyl) phenoxy resin, 4- (2 ′, 4′-dimethoxyphenyl-Fmocaminoethyl) phenoxy resin and the like can be mentioned. 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 in accordance with the sequence of the target protein according to various known condensation methods. At the end of the reaction, the protein 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 an amide thereof. For the condensation of the protected amino acids described above, various activating reagents that can be used for protein synthesis can be used, and carbodiimides are particularly preferable. As carbodiimides, DCC,
N, N'-diisopropylcarbodiimide, N-ethyl-N '-(3-
(Dimethylaminoprolyl) carbodiimide and the like are used. For these activations, the protected amino acid was directly added to the resin together with a racemization inhibitor additive (eg, HOBt, HOOBt), or the protected amino acid was previously activated as a symmetric acid anhydride or HOBt ester or HOOBt ester. It can be added later to the resin.

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; and an appropriate mixture thereof are used. The reaction temperature is appropriately selected from a range known to be usable for a protein bond forming 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. 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, etc., aralkyl esterification (for example, benzyl ester, 4-nitrobenzyl ester, 4-methoxybenzyl ester) 4-chlorobenzyl ester, benzhydryl esterification), phenacyl esterification, benzyloxycarbonyl hydrazide, tertiary butoxycarbonyl hydrazide,
It can be protected by trityl hydrazide or 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 the 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, Cl ₂
-Bzl, 2-nitrobenzyl, Br-Z, tert-butyl and the like are used. Examples of the protecting group for imidazole of histidine include Tos, 4-methoxy-2,3,6-trimethylbenzenesulfonyl, DNP, benzyloxymethyl, B
um, 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, para-nitrophenol, HONB, N-hydroxysuccinimide, N-hydroxyphthalimide, and esters with HOBt). As the activated amino group of the raw material, for example, a 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, or hydrogen fluoride anhydride, methanesulfonic acid, trifluoromethane or the like can be used. Acid treatment with dichloromethane, trifluoroacetic acid or a mixture thereof, base treatment with diisopropylethylamine, triethylamine, piperidine, piperazine, etc., reduction with sodium in liquid ammonia, and the like are also used. The elimination reaction by the acid treatment is as follows:
Generally, the reaction is carried out at a temperature of about -20 ° C to 40 ° C. In the acid treatment, for example, anisole, phenol, thioanisole, metacresol, paracresol, dimethylsulfide, 1,4-butanedithiol, 1,2-ethane Addition of a cation scavenger such as dithiol is effective. In addition, the 2,4-dinitrophenyl group used as the imidazole protecting group of histidine is removed by thiophenol treatment, and the formyl group used as the indole protecting group of tryptophan is 1,2-ethanedithiol, 1,4-butane described above. In addition to deprotection by acid treatment in the presence of dithiol or the like, it is also removed by alkali treatment with dilute sodium hydroxide solution, dilute ammonia or the like.

The protection of the functional group which should not be involved in the reaction of the raw materials, 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, after amidating and protecting the α-carboxyl group of the carboxy terminal amino acid, a peptide (protein) chain is extended to a desired chain length on the amino group side, and 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 carboxyl group at the C-terminal has been removed, and these proteins are mixed 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 peptide synthesis method known per se 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
Peptide), Academic Press, New York (1965) Nobuo Izumiya et al., Fundamentals and experiments of peptide synthesis, Maruzen Co., Ltd.
(1975) Haruaki Yajima and Shunpei Sakakibara, Laboratory of Biochemistry 1, 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 a free form, it can be converted to an appropriate salt by a known method, and conversely, when it is obtained by a salt, it can be converted to a free form by a known method. Can be.

The polynucleotide encoding the receptor protein of the present invention may be any polynucleotide as long as it contains the above-described nucleotide sequence (DNA or RNA, preferably DNA) encoding the receptor protein of the present invention. Good. The polynucleotide is RNA such as DNA or mRNA encoding the receptor protein of the present invention, 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 the polynucleotide encoding the receptor protein of the present invention, for example, by the method described in the known experimental medicine special edition "New PCR and its application" 15 (7), 1997, or a method analogous thereto, the receptor protein of the present invention mRNA can be quantified. The DNA encoding the receptor protein of the present invention may be any of a genomic DNA, a genomic DNA library, the above-described cell / tissue-derived cDNA, the above-described cell / tissue-derived cDNA library, and a 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 was directly used by using a total RNA or mRNA fraction prepared from the cells and tissues described above.
It can also be amplified by Reaction (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 a DNA having the nucleotide sequence represented by SEQ ID NO: 2 under high stringency conditions Any DNA can be used as long as it has a nucleotide sequence that hybridizes with the above, and encodes a receptor protein having substantially the same activity (eg, ligand binding activity, signal transduction action, etc.) as the receptor protein of the present invention. Good. Examples of the DNA capable of hybridizing with the nucleotide sequence represented by SEQ ID NO: 2 include, for example, SEQ ID NO:
2 and about 70 or more, preferably about 8
D containing a nucleotide sequence having 0% or more, more preferably about 90% or more, and most preferably about 95% or more homology.
NA or the like is used.

Hybridization is carried out by a method known per se or a method analogous thereto, for example, molecular
Cloning (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. More preferably, it can be performed under high stringent conditions. The high stringent conditions include, for example,
The sodium concentration is about 19-40 mM, preferably about 19
２０20 mM at a temperature of about 50-70 ° C., preferably about 6
The condition of 0 to 65 ° C is shown. In particular, when the sodium concentration is about 1
Most preferred is 9 mM at a temperature of about 65 ° C. More specifically, as the DNA encoding the receptor protein having the amino acid sequence represented by SEQ ID NO: 1, a DNA having the base sequence represented by SEQ ID NO: 2 or the like is used. D encoding the receptor protein of the present invention
The polynucleotide comprising a part of the base sequence of NA or a part of the base sequence complementary to the DNA includes not only DNA encoding the following partial peptide of the present invention, but also RNA. Is also used in a sense that also encompasses
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 a DNA encoding the G protein-coupled receptor protein is determined. Can be designed and synthesized based on the nucleotide sequence information of Such a polynucleotide (nucleic acid) can hybridize with RNA of a G protein-coupled receptor protein gene and can inhibit the synthesis or function of the RNA, or can bind to a 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 complementary to a selected sequence of G protein-coupled receptor protein-related RNA, and polynucleotides capable of specifically hybridizing to G protein-coupled receptor protein-related RNA, are available 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. The term "corresponding" means having homology or being complementary to a specific sequence of nucleotides, base sequences or nucleic acids including genes. "Corresponding" between a nucleotide, a base sequence or a nucleic acid and a peptide (protein) means a nucleotide (nucleic acid)
Or the amino acid of the peptide (protein) in the instructions derived from its sequence or its complement. A 5′-end hairpin loop of a G protein-coupled receptor protein gene,
5 'end 6-base pair repeat, 5' end untranslated region,
Polypeptide translation initiation codon, protein coding region, OR
The F translation initiation codon, the 3′-end untranslated region, the 3′-end palindrome region, and the 3′-end hairpin loop can be selected as preferred regions of interest, but any region within the G protein-coupled receptor protein gene can be selected as the region of interest. Can.

The relationship between the target nucleic acid and the polynucleotide complementary to at least a part of the target region is that the relationship between the target nucleic acid and the polynucleotide capable of hybridizing with the target is "antisense". . Antisense polynucleotides are polydeoxynucleotides containing 2-deoxy-D-ribose, D-
Ribose-containing polydeoxynucleotides, other types of polynucleotides that are N-glycosides of purine or pyrimidine bases, or other polymers having a non-nucleotide backbone (eg, commercially available protein nucleic acids and synthetic sequence-specific nucleic acids) Polymer) or other polymer containing a special bond, provided that the polymer is D
NA or a pair of nucleotides found in RNA or containing a nucleotide having a configuration permitting base attachment). They include double-stranded DNA, single-stranded DNA, double-stranded RNA, single-stranded RNA, and DN.
A: It can be an RNA hybrid, further unmodified polynucleotides (or unmodified oligonucleotides), or those with known modifications, such as those with labels known in the art, capped , Methylated, one or more natural nucleotides replaced by analogs, modified intramolecular nucleotides, such as uncharged bonds (eg, methylphosphonates, phosphotriesters, phosphoramidates) , Carbamates, etc.), those having a charged bond or a sulfur-containing bond (eg, phosphorothioate, phosphorodithioate, etc.), such as proteins (nucleases, nuclease inhibitors, toxins, antibodies,
Those having a side chain group such as a signal peptide, poly-L-lysine or the like, or a sugar (eg, a monosaccharide), an interactive compound (eg, acridine,
Psoralen), those containing chelating compounds (eg, metals, radioactive metals, boron, oxidizable metals, etc.), those containing alkylating agents,
Those having a modified bond (for example, α-anomeric nucleic acid) may be used. Where "nucleoside",
The terms “nucleotide” and “nucleic acid” may include not only those containing the purine and pyrimidine bases described above, but also those having other modified heterocyclic bases.
Such modifications may include methylated purines and pyrimidines, acylated purines and pyrimidines, or other heterocycles. The modified nucleotides and modified nucleotides may also be modified at the sugar moiety, for example, one or more hydroxyl groups are replaced by halogens, aliphatic groups, etc., or converted to functional groups such as ethers, amines, etc. May have been.

The antisense polynucleotide (nucleic acid) of the present invention is RNA, DNA, or a modified nucleic acid (RNA, 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. Thus, many modifications are known in the art. For example, J. Kawakami et al., Pharm Tech Japan, Vo
l. 8, pp. 247, 1992; Vol. 8, pp. 395, 1992; ST Cr
ooke et al. ed., Antisense Research and Applicatio
ns, CRC Press, 1993. The antisense nucleic acid of the present invention has an altered or modified sugar,
It may contain a base or a bond, and may be provided in a special form such as liposome or microsphere, applied by gene therapy, or 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, crude water-based substances such as phospholipids and cholesterol are exemplified. Preferred lipids for addition include cholesterol and its derivatives (eg, cholesteryl chloroformate, cholic acid, etc.). These can be attached to the 3 'end or 5' end of the nucleic acid, and can be attached via a base, sugar, or intramolecular nucleoside bond. Other groups include:
A cap group specifically arranged at the 3 ′ end or 5 ′ end of a nucleic acid, for preventing degradation by a nuclease 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 itself can be applied to cells by various methods known per se.

DNA encoding the partial peptide of the present invention
May be any as long as it contains the nucleotide sequence encoding the partial peptide of the present invention described above. In addition, any of genomic DNA, genomic DNA library, cDNA derived from the above-mentioned cells / tissues, cDNA library derived from the aforementioned cells / tissues, and synthetic DNA may be used. The vector used for the library may be any of bacteriophage, plasmid, cosmid, phagemid and the like. In addition, using the mRNA fraction prepared from the cells and tissues described above,
Transcriptase Polymerase Chain Reaction (hereinafter, RT
-PCR method). Specifically, D encoding the partial peptide of the present invention
Examples of NA include, for example, (1) DN having a partial base sequence of DNA having a base sequence represented by SEQ ID NO: 2.
A or (2) has a nucleotide sequence that hybridizes under high stringent conditions to the nucleotide sequence represented by SEQ ID NO: 2, and has substantially the same activity as the receptor protein peptide of the present invention (eg, ligand binding) DNA having a partial nucleotide sequence of a DNA encoding a receptor protein having activity, signal transduction action, etc.). Examples of the DNA capable of hybridizing with the nucleotide sequence represented by SEQ ID NO: 2 include, for example, about 70% or more, preferably about 80% or more, more preferably about 90% or more, most preferably the nucleotide sequence represented by SEQ ID NO: 2. Preferably about 9
DNA containing a base sequence having a homology of 5% or more is used.

As a means for cloning a DNA completely encoding the receptor protein of the present invention or its partial peptide (hereinafter sometimes abbreviated as the receptor protein of the present invention), the partial nucleotide sequence of the receptor protein of the present invention is used. Amplification by the PCR method using the synthetic DNA primers, or the DNA incorporated into an appropriate vector and labeled with a DNA fragment encoding a partial or entire region of the receptor protein of the present invention or a synthetic DNA. Selection can be performed by hybridization. The hybridization method is
For example, molecular cloning (Molecular Clon
ing) 2nd (J. Sambrook et al., ColdSpring Harbor L
ab. Press, 1989). When a commercially available library is used, it can be performed according to the method described in the attached instruction manual.

The conversion of the nucleotide sequence of DNA can be performed by a known kit, for example, Mutan ^™ -G (Takara Shuzo Co., Ltd.), Mutan ^™ -K
(Takara Shuzo Co., Ltd.) using the Gupped duplex method and Kun
It can be carried out according to a method known per se such as the kel method or a method analogous thereto. 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 has an ATG as a translation initiation codon at its 5 'end,
TAA and T as translation termination codons are located at the 3 'end.
It may have GA or TAG. These translation initiation codon and translation termination codon can also be added using a suitable synthetic DNA adapter. The expression vector for the receptor protein of the present invention can be obtained, for example, by (A) converting the DNA encoding the receptor protein of the present invention into a desired DN.
A fragment can be prepared by cutting out the A fragment and (ii) ligating the DNA fragment downstream of a promoter in an appropriate expression vector.

As a vector, a plasmid derived from Escherichia coli (eg, pBR322, pBR325, pUC12,
UC13), a plasmid derived from Bacillus subtilis (eg, pUB11)
0, pTP5, pC194), yeast-derived plasmids (eg, pSH19, pSH15), bacteriophages such as λ phage, animal viruses such as retrovirus, vaccinia virus, baculovirus, etc.
A1-11, pXT1, pRc / CMV, pRc / RS
V, pcDNAI / Neo, etc. are 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 animal cells are used as hosts, the SRα promoter, SV40 promoter, L
TR promoter, CMV promoter, HSV-TK
Promoters and the like. Of these, CMV
It is preferable to use a promoter, SRα promoter or the like. When the host is Escherichia, trp
Promoter, lac promoter, recA promoter, λP _L promoter, lpp promoter, etc., when the host is Bacillus sp., SPO1 promoter, SPO2 promoter, penP promoter, etc .; , GAP promoter, ADH
Promoters and the like are preferred. When the host is an insect cell, a polyhedrin promoter, a P10 promoter and the like are preferable.

[0033] In addition to the above, the expression vector may optionally contain an enhancer, a splicing signal, a poly-A addition signal, a selection marker, an SV40 replication origin (hereinafter sometimes abbreviated as SV40 ori) and the like. Can be used. As a selection marker, for example, dihydrofolate reductase (hereinafter, dhfr)
Gene (sometimes abbreviated as “methotrexate (M
TX) resistance], an ampicillin resistance gene (hereinafter referred to as Amp
^r ), a neomycin resistance gene (hereinafter sometimes abbreviated as Neo ^r , G418 resistance), and the like. In particular, when the dhfr gene is used as a selection marker using CHO (dhfr ⁻ ) cells, the target gene can be selected using a thymidine-free medium. Also, if necessary, a signal sequence suitable for the host is added to the N-terminal side of the receptor protein of the present invention. When the host is Escherichia, PhoA.
When the host is Bacillus, an α-amylase signal sequence, a subtilisin signal sequence, or the like is used. When the host is yeast, an MFα signal sequence, an SUC2 signal, or the like is used. When the host is an animal cell, such as a sequence, an insulin signal sequence, an α-interferon signal sequence, an antibody molecule / signal sequence, etc. can be used, respectively. A transformant can be produced using the vector containing the DNA encoding the receptor protein of the present invention thus constructed.

As the host, for example, Escherichia, Bacillus, yeast, insect cells, insects, animal cells and the like are used. Specific examples of Escherichia bacteria include
Escherichia coli K12 DH
1 [Processings of the National Academy of Sciences of the USA (Proc. Natl. Acad. Sci. USA), 60, 160]
(1968)], JM103 [Nukuilec Acids
Research, (Nucleic Acids Research), 9, 309
(1981)], JA221 [Journal of Molecular Biolog
y)], 120, 517 (1978)], HB101
[Journal of Molecular Biology, 4
1, 459 (1969)], C600 [Genetics, 39, 440 (1954)] and the like. Examples of Bacillus bacteria include, for example, Bacillus
Bacillus subtilis MI114 [Gene, 24, 255 (1983)], 207-21 [Journal of Biochemistry
emistry), Vol. 95, 87 (1984)]. Examples of yeast include, for example, Saccharomyces cerevisiae AH22, AH22
^{R -, NA87-11A, DKD-5D} , 20B-1
2. Schizosaccharomy
ces pombe) NCYC1913, NCYC2036, Pichia pastoris and the like are used.

As insect cells, for example, virus is A
In the case of cNPV, a cell line derived from night larvae of moth (Spodop
tera frugiperda cell (Sf cell), Trichoplusia ni
MG1 cells from the midgut, H from the eggs of Trichoplusia ni
igh Five ^TM cells, cells derived from Mamestrabrassicae or
Estigmena acrea-derived cells and the like are used. When the virus is BmNPV, a silkworm-derived cell line (Bombyx mor
iN; BmN cells). Examples of the Sf cells include Sf9 cells (ATCC CRL1711), Sf21
Cells (Vaughn, JL et al., In Viv
o), 13, 213-217, (1977)). As insects, for example, silkworm larvae and the like are used [Maeda et al., Nature, 315, 592 (198).
5)]. As animal cells, for example, monkey cells COS-
7, Vero, Chinese hamster cell CHO (hereinafter abbreviated as CHO cell), dhfr gene-deficient Chinese hamster cell CHO (hereinafter, CHO (dhf)
r ⁻ ) cells), mouse L cells, mouse AtT-2
0, mouse myeloma cells, rat GH3, human FL cells, and the like.

For transforming Escherichia sp., For example, Procagings of the National Academy of Sciences of the USA (Proc. Natl. Acad. Sci. USA), Vol. 69 , 21
10 (1972) and Gene, 17, 107 (1)
982). For transformation of Bacillus sp., For example, Molecular and General Genetics (Mo
lecular & General Genetics), 168, 111 (1
979) and the like. To transform yeast, see, for example, Methods in Enzymology, 19
4, 182-187 (1991), Processings of the National Academy of Sciences of the USA (Proc. Natl. Acad.
Sci. USA), Vol. 75, 1929 (1978). Insect cells or insects can be transformed, for example, according to the method described in Bio / Technology, 6, 47-55 (1988). In order to transform animal cells, for example, Cell Engineering Separate Volume 8, New Cell Engineering Experiment Protocol. 263-267 (1995) (published by Shujunsha), Virology, 52, 456 (1)
973). 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 used for the cultivation. 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, vitamins, growth promoting factors and the like may be added. The pH of the medium is preferably about 5 to 8.

As a medium for culturing the genus Escherichia, for example, an M9 medium containing glucose and casamino acid [Miller, Journal of Experiments in Molecular Genetics (Journa)
l of Experiments in Molecular Genetics), 431-
433, Cold Spring Harbor Laboratory, New York 1
972] is preferred. Here, in order to make the promoter work efficiently if necessary, for example, 3β-indolyl
An agent such as acrylic acid can be added. When the host is a bacterium belonging to the genus Escherichia, culturing is usually performed at about 15 to 43 ° C.
For about 3 to 24 hours, and if necessary, ventilation or stirring may be added. When the host is a bacterium belonging to the genus Bacillus, the cultivation is usually performed at about 30 to 40 ° C. for about 6 to 24 hours, and if necessary, aeration and stirring can be added. When culturing a transformant in which the host is yeast, for example, Burkholder's minimum medium [Bostian, KL
Proc. Natl. Acad. Sci. USA, Proc. Of the National Academy of Sciences of the USA, 77, 4
505 (1980)] and S containing 0.5% casamino acid.
D medium [Bitter, GA et al., "Procedures of the National Academy of Sciences of the USA (Proc. Natl. Acad. 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,
Vent or agitate as needed.

When culturing an insect cell or a transformant whose host is an insect, Grace's Insect Mediu
m (Grace, TCC, Nature, 195,788 (196
Those to which an additive such as 10% bovine serum immobilized in 2)) is appropriately added are used. The pH of the medium is about 6.2-6.
Adjustment to 4 is preferred. Culture is usually performed at about 27 ° C for about 3
５5 days, with aeration and / or agitation as needed. When culturing a transformant in which the host is an animal cell, the medium may be, for example, MEM containing about 5 to 20% fetal bovine serum.
Medium [Science, Vol. 122, 501 (19)
52)], DMEM medium [Virology,
8, 396 (1959)], RPMI 1640 medium [The Journal of the American Medica
l Association) 199, 519 (1967)], 19
9 Medium [Proceeding of the Society for the Biological Medicine (Proceeding o
fthe Society for the Biological Medicine), 73
Vol., 1 (1950)]. pH is about 6-8
It is preferred that 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 on the cell membrane of the transformant.

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, cells or cells are collected by a known method, suspended in an appropriate buffer, and subjected to sonication, lysozyme and / or freeze-thawing. After the cells or cells are destroyed by such methods as mentioned above, a method of obtaining a crude extract of the receptor protein by centrifugation or filtration is used as appropriate. The buffer may contain a protein denaturant such as urea or guanidine hydrochloride, or a surfactant such as Triton X-100 ^™ . When the receptor protein is secreted into the culture solution, after culturing, the supernatant is separated from the cells or cells by a method known per se, and the supernatant is collected. Purification of the receptor protein contained in the thus obtained culture supernatant or extract can be carried out by appropriately combining known separation and purification methods. These known separation and purification methods include methods utilizing solubility such as salting out and solvent precipitation, dialysis, ultrafiltration, gel filtration, and SDS-polyacrylamide gel electrophoresis, and the like. Method using difference in charge, method using charge difference such as ion exchange chromatography, method using specific novelty such as affinity chromatography, use of difference in hydrophobicity such as reversed-phase high-performance liquid chromatography And a method utilizing the difference in isoelectric points such as isoelectric focusing.

When the thus obtained receptor protein is obtained in a free form, it can be converted into a salt by a method known per se or a method analogous thereto, and conversely, when the receptor protein is obtained in a salt form, a known method is used. Alternatively, it can be converted into a free form or another salt by 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 thus produced receptor protein of the present invention or a salt thereof 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 a polyclonal antibody or a 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 a known antibody or antiserum using the receptor protein of the present invention as an antigen. Can be produced according to the production method of

[Preparation of Monoclonal Antibody] (a) Preparation of Monoclonal Antibody-Producing Cell The receptor protein or the like of the present invention can be itself or a carrier at a site capable of producing an antibody by administration to a mammal.
Administered with diluent. 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 2-
This is performed once every six weeks, for a total of about two to ten times. Examples of the mammal used include monkeys, rabbits, dogs, guinea pigs, mice, rats, sheep, and goats, and mice and rats are preferably used.
When producing monoclonal antibody-producing cells, a warm-blooded animal immunized with the antigen, for example, an individual having an antibody titer is selected from a mouse, and the spleen or lymph node is collected 2 to 5 days after the final immunization. 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 can be performed according to a known method, for example, the method of Kohler and Milstein [Nature, 256, 495 (1975)]. As the fusion promoter, for example, polyethylene glycol (PE
G) and Sendai virus, but PEG is preferably used. As myeloma cells, for example,
NS-1, P3U1, SP2 / 0, etc.,
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 P
EG1000-PEG6000) at a concentration of about 10-80%, and is added at about 20-40 ° C., preferably about 30-80 ° C.
By incubating at 37 ° C. for about 1 to 10 minutes, cell fusion can be carried out efficiently.

Various methods can be used to screen for monoclonal antibody-producing hybridomas. 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 labeled with a radioactive substance or an enzyme (an anti-mouse immunoglobulin antibody is used when the cells used for cell fusion are mice) or protein A 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 substances, enzymes, etc., and detect monoclonal antibody bound to solid phase And the like. The selection of the monoclonal antibody can be carried out according to a method known per se or a method analogous thereto. Usually, it can be carried out in a medium for animal cells to which HAT (hypoxanthine, aminopterin, thymidine) is added. As a selection and breeding medium, any medium can be used as long as hybridomas can grow. For example, 1-2
RP containing 0%, preferably 10-20% fetal calf serum
MI 1640 medium, G containing 1-10% fetal calf serum
IT medium (Wako Pure Chemical Industries, Ltd.) or serum-free medium for hybridoma culture (SFM-101, Nissui Pharmaceutical Co., Ltd.)
Etc. can be used. Culture temperature is usually 20-4
0 ° C., preferably about 37 ° C. Culture 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 Antibodies Separation and purification of monoclonal antibodies can be performed by the same method as that for separation and purification of normal polyclonal antibodies [eg, immunoglobulin separation and purification methods such as salting out method, alcohol precipitation method, and isoelectric point precipitation method. , Electrophoresis, adsorption / desorption with an ion exchanger (eg, DEAE), ultracentrifugation, gel filtration, antigen-bound solid phase or an active adsorbent such as protein A or protein G to collect only antibodies and bind Specific Purification Method for Obtaining Antibody by Dissociation].

[Preparation of Polyclonal Antibody] The polyclonal antibody of the present invention can be produced according to a method known per se or a method analogous thereto. For example, a complex of an immunizing antigen (an antigen such as a receptor protein) and a carrier protein is formed, a mammal is immunized in the same manner as in the above-described method for producing a monoclonal antibody, and an antibody against the receptor protein or the like of the present invention is obtained from the immunized animal. It can be produced by collecting the contents 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. Any kind may be cross-linked at any ratio. For example, bovine serum albumin, bovine thyroglobulin, keyhole limpet hemocyanin, etc. may be used in a weight ratio of about 0.1 to 20 with respect to 1 hapten. Preferably, a method of coupling at a ratio of about 1 to 5 is used. In addition, various coupling agents can be used for coupling the hapten and the carrier, but glutaraldehyde, carbodiimide, maleimide active ester, thiol group,
An active ester reagent containing a dithioviridyl group 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 the blood, ascites, etc., preferably from the 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 polyclonal antibody can be separated and purified according to the same method for separating and purifying immunoglobulins as in the above-described method for separating and purifying a monoclonal antibody.

The receptor protein of the present invention or a salt thereof,
The partial peptide or a salt thereof, and the DNA encoding the receptor protein or the partial peptide thereof,
(1) determination of a ligand (agonist) for the G protein-coupled receptor protein of the present invention, (2) preventive and / or therapeutic agent for a disease associated with dysfunction of the G protein-coupled receptor protein of the present invention, (3) Genetic diagnostics,
(4) a method for screening a compound that changes the expression level of the receptor protein or a partial peptide thereof of the present invention,
(5) a prophylactic 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; (6) a method for quantifying a ligand for the G protein-coupled receptor protein of the present invention; (7)
A 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 (8) alter the binding between the G protein-coupled receptor protein of the present invention and the ligand A preventive and / or therapeutic agent for various diseases containing a compound (agonist, antagonist), (9) quantification of the receptor protein of the present invention or a partial peptide thereof or a salt thereof, (10) a receptor protein of the present invention or a portion thereof in a cell membrane A method for screening a compound that changes the amount of a peptide, (11) a preventive and / or therapeutic agent for various diseases containing a compound that changes the amount of the receptor protein of the present invention or a partial peptide thereof in a cell membrane, and (12) a method of the present invention. Receptor protein or partial peptide Or neutralization by antibodies to salts thereof, can be used for such production of non-human animal having a DNA encoding a G protein-coupled receptor protein of the present invention (13). In particular, by using a receptor binding assay system using the expression system of the recombinant G protein-coupled receptor protein of the present invention, the binding of a ligand to a G protein-coupled receptor specific to humans or mammals is changed. Compounds (eg, agonists, antagonists, etc.) can be screened, and the agonists or antagonists can be used as agents for preventing or treating various diseases. A receptor protein or a 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 a 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) is 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 a receptor protein of the present invention, which comprises contacting a receptor compound of the present invention or a salt thereof or a partial peptide of the present invention or a salt thereof with a test compound. Test compounds include known ligands (eg,
Angiotensin, bombesin, cannabinoid, cholecystokinin, glutamine, serotonin, melatonin, neuropeptide Y, opioid, purine, vasopressin, oxytocin, PACAP, secretin, glucagon, calcitonin, adrenomedullin, somatostatin, GHRH, CRF, ACTH, ACTH PTH, V
IP (Vasoactive Intestinal and Related Polypeptide), somatostatin, dopamine, motilin, amylin, bradykinin, CGRP
(Calcitonin gene relayed peptide), leukotriene, pancreastatin, prostaglandin, thromboxane, adenosine, adrenaline, α and β-chemokines (eg, IL-
8, GROα, GROβ, GROγ, NAP-2, EN
A-78, PF4, IP10, GCP-2, MCP-
1, HC14, MCP-3, I-309, MIP1α,
MIP-1β, RANTES, etc.), endothelin, enterogastrin, histamine, neurotensin, T
In addition to RH, pancreatic polypeptide or galanin), for example, human or mammal (eg, mouse, rat, pig, cow, sheep, monkey, etc.)
Tissue extract, cell culture supernatant, and the like. For example, the tissue extract, 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 to finally obtain a single ligand.

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 receptor binding assay system, cell stimulating activity by binding to the receptor protein of the present invention (for example, arachidonic acid release, acetylcholine release, intracellular Ca ²⁺ release,
Compounds having an activity of promoting or inhibiting intracellular cAMP production, intracellular cGMP production, inositol phosphate production, cell membrane potential fluctuation, intracellular protein phosphorylation, c-fos activation, pH reduction, etc. (for example, , Peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, etc.) or salts thereof. In the ligand determination method of the present invention, when the test compound is brought into contact with the receptor protein of the present invention or a partial peptide thereof, 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 preparing 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 or a salt thereof of the present invention, 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: The labeled test compound is transformed with the DNA encoding the receptor protein of the present invention. A receptor of the present invention characterized by measuring the amount of a labeled test compound bound to the receptor protein or a salt thereof when the transformant is brought into contact with the receptor protein expressed on the cell membrane by culturing the transformant. A method for determining a ligand for a protein,

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

First, the receptor protein used in the method for determining a ligand may be any receptor protein containing the above-mentioned receptor protein of the present invention or the partial peptide of the present invention. Receptor proteins expressed in large amounts are suitable. The above-described expression method is used for producing the receptor protein of the present invention, but it is preferable to express the DNA encoding the receptor protein in mammalian cells or insect cells. Complementary DNA is usually used as the DNA fragment encoding the target protein portion, but is not necessarily limited thereto. For example, a gene fragment or a synthetic DNA may be used. In order to introduce a DNA fragment encoding the receptor protein of the present invention into a host animal cell and express them efficiently, the DNA fragment is converted into a nuclear polyhedrosis virus belonging to a baculovirus using an insect as a host. (NPV) polyhedrin promoter, SV40-derived promoter, retrovirus promoter, metallothionein promoter, human heat shock promoter, cytomegalovirus promoter, SRα promoter, etc. The amount and quality of the expressed receptor can be examined by a method known per se. For example, the literature [Nambi, P .; Et al., The Journal of Biological Chemistry (J. Biol. Chem.), 267, 1
9555-19559, 1992].

Therefore, in the method for determining a ligand of the present invention, the receptor protein of the present invention or its partial peptide or a salt thereof includes the receptor protein or its partial peptide or its salt purified according to a method known per se. Alternatively, a cell containing the receptor protein or a cell membrane fraction thereof may be used. In the method for determining a ligand of the present invention, when a cell containing the receptor protein of the present invention is used,
The cells may be fixed with glutaraldehyde, formalin, or the like. The immobilization method can be performed according to a method known per se. The cell containing the receptor protein of the present invention refers to a host cell expressing the receptor protein of the present invention. As the host cell, Escherichia coli, Bacillus subtilis, yeast, insect cells, animal cells and the like are used. The cell membrane fraction refers to a fraction abundant in cell membrane obtained by disrupting cells and then obtained by a method known per se. 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, a cell lysate is supplied at a low speed (500 rpm to 300 rpm).
0 rpm) for a short time (usually about 1 to 10 minutes),
The supernatant is further accelerated (15,000 rpm to 30,000 rpm).
m) for 30 minutes to 2 hours, and the resulting precipitate is used as a membrane fraction. The membrane fraction is rich in the expressed receptor protein 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 10 per cell.
^It is preferably ³ to 10 ⁸ molecules, and more preferably 10 ⁵ to 10 ⁷ molecules. 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 methods (1) to (3) for determining a ligand for the receptor protein of the present invention or a salt thereof, an appropriate receptor protein fraction and a labeled test compound are required. As the receptor protein fraction, a natural receptor protein fraction or a recombinant receptor fraction having an activity equivalent thereto is desirable. Here, “equivalent activity” refers to equivalent ligand binding activity, signal transduction action, and the like. As the labeled test compound, [ ³
H], [ ¹²⁵ I], [ ¹⁴ C], [ ³⁵ S] and the like, angiotensin, bombesin, cannabinoid, cholecystokinin, glutamine, serotonin, melatonin, neuropeptide Y, opioid, purine, vasopressin, oxytocin, PACAP , Secretin, glucagon, calcitonin, adrenomedullin, somatostatin, GHRH, CRF, ACTH, GRP, PTH, V
IP (Vasoactive Intestinal and Retained Polypeptide), somatostatin, dopamine, motilin, amylin, bradykinin, CGRP (calcitonin gene relayed peptide), leukotriene, pancreastatin, prostaglandin, thromboxane, adenosine, adrenaline, α And β
-Chemokines (e.g., IL-8, GR
Oα, GROβ, GROγ, NAP-2, ENA-7
8, PF4, IP10, GCP-2, MCP-1, HC
14, MCP-3, I-309, MIP1α, MIP-
1β, RANTES, etc.), endothelin, enterogastrin, histamine, neurotensin, TRH, pancreatic polypeptide, galanin and the like are suitable.

Specifically, the method for determining a ligand for the receptor protein of the present invention or a salt thereof is as follows.
First, a receptor preparation is prepared by suspending a cell or a membrane fraction of the cell containing the receptor protein of the present invention in a buffer suitable for the determination method. 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, which does not inhibit the binding between the ligand and the receptor protein. For the purpose of reducing 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, to reduce the degradation of receptors and ligands by proteases, P
Protease inhibitors such as MSF, leupeptin, E-64 (manufactured by Peptide Research Laboratories), and pepstatin can also be added. 0.01 ml to 10 ml of the receptor solution is added to a certain amount (5,000 cpm to 500,000 cps).
m) The test compound labeled with [ ³ H], [ ¹²⁵ I], [ ¹⁴ C], [ ³⁵ S], etc., is allowed to coexist. Non-specific binding (N
A reaction tube containing a large excess of an unlabeled test compound is also prepared to determine SB). The reaction is about 0 ° C to 50 ° C,
It is preferably performed at about 4 ° C. to 37 ° C. for about 20 minutes to 24 hours, preferably for 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. A test compound whose count (B-NSB) obtained by subtracting the non-specific binding amount (NSB) from the total binding amount (B) exceeds 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 (4) 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 generation, intracellular cGMP
Production, inositol phosphate production, cell membrane potential fluctuation, intracellular protein phosphorylation, activation of c-fos, activity to promote or suppress pH reduction, etc.) by a known method or a commercially available measurement kit. It can be measured using: 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. For activities such as cAMP production suppression,
It can be detected as a production inhibitory effect on cells whose basic production has been increased with forskolin or the like.

The kit for determining a ligand that binds to the receptor protein of the present invention or a salt thereof comprises a receptor protein of the present invention or a salt thereof, a partial peptide of the present invention or a salt thereof, a cell containing the receptor protein of the present invention, It contains a membrane fraction of cells containing the receptor protein of the present invention. Examples of the kit for determining a ligand of the present invention include the following. 1. Ligand determination reagent Measurement buffer and washing buffer Hanks' Balanced Salt Solution (manufactured by Gibco)
One containing 05% bovine serum albumin (manufactured by Sigma). Sterilized by filtration through a 0.45 μm filter, 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
Passage into a 12-well plate at 5 × 10 ⁵ / well, 37 ° C.
Cultured for 2 days in 5% CO ₂ and 95% air. Labeled test compound A commercially available compound labeled with [ ³ H], [ ¹²⁵ I], [ ¹⁴ C], [ ³⁵ S], or the like, or labeled by an appropriate method. Store at 0 ° C. and dilute to 1 μM with measurement buffer before use. Test compounds that are poorly soluble in water are dissolved in dimethylformamide, DMSO, methanol and the like. Unlabeled test compound The same as the labeled compound is prepared at a concentration 100 to 1000 times higher.

2. Assay Method CHO cells expressing the receptor protein of the present invention cultured on a 12-well tissue culture plate are washed twice with 1 ml of assay buffer, and then 490 μl of assay buffer is added to each well. 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, add 5 μl of an unlabeled test compound. Remove the reaction solution and wash three times with 1 ml of washing buffer. The labeled test compound bound to the cells was 0.2N NaO
Dissolve in H-1% SDS and mix with 4 ml of liquid scintillator A (Wako Pure Chemical Industries). Liquid scintillation counter (Beckman)
The radioactivity is measured using.

The ligand capable of binding to the receptor protein of the present invention or a salt thereof includes, for example, brain,
Pituitary, substances specifically present in the pancreas and the like, specifically, angiotensin, bombesin, cannabinoid, cholecystokinin, glutamine, serotonin, melatonin, neuropeptide Y, opioid, purine, vasopressin, oxytocin, PACAP, secretin, glucagon, calcitonin, adrenomedullin, somatostatin, GHRH, CRF, ACTH,
GRP, PTH, VIP (Vasoactive Intestinal and Released Polypeptide), somatostatin, dopamine, motilin, amylin, bradykinin, CGRP (calcitonin gene-related peptide), leukotriene, pancreastatin, prostaglandin, thromboxane, adenosine , Adrenaline, α and β-chemokines (eg, IL-8, GROα, GROβ, GROγ, NAP
-2, ENA-78, PF4, IP10, GCP-2,
MCP-1, HC14, MCP-3, I-309, MI
P1α, MIP-1β, RANTES, etc.), endothelin, enterogastrin, histamine, neurotensin, TRH, pancreatic polypeptide, galanin 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, Use of the receptor protein of the present invention or DNA encoding the receptor protein 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, depending on the action of the ligand. Can be. 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),
By administering the receptor protein of the present invention to the patient to supplement the amount of the receptor protein, (a) administering and expressing the DNA encoding the receptor protein of the present invention to the patient, or After the DNA encoding the receptor protein of the present invention is inserted and expressed in a cell, the amount of the receptor protein in the patient's body is increased, for example, by transplanting the cell into the patient, and the effect of the ligand is sufficiently enhanced. Can be demonstrated. That is, DN encoding the receptor protein of the present invention
A 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 has about 30% homology at the amino acid sequence level with MAS, which is a kind of G protein-coupled receptor protein. It has been reported that MAS gene-deficient mice showed changes in central functions such as increased anxiety [JBC, 273 (No. 19), 11867-11873 (199
8)], it is considered that the MAS gene has some function in central function expression. Therefore, the receptor protein of the present invention, which is homologous to MAS, can be used for diseases associated with central dysfunction (eg, anxiety, schizophrenia,
It is useful for prevention and / or treatment of manic depression, dementia, mental illness including mental retardation and movement disorder). It has been reported that the expression of MAS gene in rats shows high expression in various peripheral organs immediately after birth, and after maturation it shows high expression in testis except in the central part [FEBS Lett. 357: 27-32 (1995)]. For this reason, it is thought that there is an important role in cell proliferation, acquisition of function, and reproduction. Therefore, the receptor protein of the present invention having homology to MAS is useful for the prevention and / or treatment of respiratory diseases, cardiovascular diseases, gastrointestinal diseases, liver / bile / pancreatic diseases, and endocrine diseases. When the receptor protein of the present invention is used as the 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 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, adenovirus, or 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. The DNA of the present invention can be administered as it is or together with an auxiliary agent for promoting uptake by a gene gun or a catheter such as a hydrogel catheter. For example, the receptor protein of the present invention or the DNA encoding the receptor protein can be used as a sugar-coated tablet, capsule, elixir, microcapsule, etc., orally, or water or other pharmaceutical agent, if necessary. It can be used parenterally in the form of injections, such as sterile solutions or suspensions in liquids that are acceptable.
For example, the formulation of the receptor protein of the present invention or the DNA encoding the receptor protein with a known physiologically acceptable carrier, flavoring agent, excipient, vehicle, preservative, stabilizer, binder, etc. By mixing in the unit dose form required. The amount of the active ingredient in these preparations is such that an appropriate dose 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, gum arabic, excipients such as crystalline cellulose, corn starch, gelatin, alginic acid and the like. And a lubricating agent 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. 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 adjuvants (eg, D-sorbitol, D-mannitol, sodium chloride, etc.) and the like are used. For example, it may be used in combination with an alcohol (eg, ethanol), a polyalcohol (eg, propylene glycol, polyethylene glycol), a nonionic surfactant (eg, Polysorbate 80 ^™ , 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. Since the thus obtained preparation is safe and has low toxicity, it can be administered to, for example, humans and mammals (eg, rats, rabbits, sheep, pigs, cows, cats, dogs, monkeys, etc.). . The dose of the receptor protein of the present invention depends on the administration target, the target organ,
Although there are differences depending on symptoms, administration method, etc., in the case of oral administration, for example, in a schizophrenic patient (as 60 kg), about 0.1 mg to 100 mg, preferably about 1.0 to 50 mg per day, More preferably from about 1.0 to
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.
In a schizophrenic patient (as 60 kg), about 0.01 to 30 mg per day, preferably about 0.1 to 20 mg per day
It is convenient to administer about mg, more preferably about 0.1 to 10 mg by intravenous injection. In the case of other animals, the dose can be administered in terms of 60 kg. The dosage of the DNA of the present invention varies depending on the administration subject, target organ, condition, administration method, and the like. 0.1mg-100m
g, preferably about 1.0 to 50 mg, more preferably about 1.0 to 20 mg. If administered parenterally,
The single dose varies depending on the administration subject, target organ, condition, administration method and the like. About 30 mg, preferably about 0.3 mg.
About 1 to 20 mg, more preferably about 0.1 to 10 mg
It is convenient to administer the degree by intravenous injection. In the case of other animals, the dose can be administered in terms of 60 kg.

(3) Gene Diagnostic Agent The DNA of the present invention can be used as a probe to produce the DNA of the present invention in humans or mammals (eg, rat, rabbit, sheep, pig, cow, cat, dog, monkey, etc.). Since abnormality (gene abnormality) of DNA or mRNA encoding the receptor protein or its partial peptide can be detected, for example,
It is useful as a gene diagnostic agent for damage, mutation, or decreased expression of NA, and increased or overexpressed DNA or mRNA. 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 (Genomix)).
mics), Vol. 5, pp. 874-879 (1989), Proceedings of the National Academy.
Proceedi of Sciences of USA
ngs ofthe National Academy of Sciences of the Unit
ed States of America), Vol. 86, 2766-277
0 (1989)).

(4) Method for Screening Compounds that Alter the Expression of Receptor Protein or Partial Peptide of the Present Invention The DNA of the present invention can be used as a probe
It can be used for screening a compound that changes the expression level of the receptor protein of the present invention or its partial peptide. That is, the present invention provides, for example, the receptor protein of the present invention or a partial peptide thereof 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. M
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 amount of RNA is provided.

The measurement of the amount of mRNA of the receptor protein of the present invention or its partial peptide 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.)
Drugs (eg, anti-dementia drugs, blood pressure lowering drugs, anti-cancer drugs, anti-obesity drugs, etc.) or physical stress (eg, flooding stress, electric shock, light, dark, low temperature, etc.) Obtain a specific organ (eg, brain, liver, kidney, etc.), or tissue or cells isolated from the organ. MRN of the receptor protein of the present invention or a partial peptide thereof contained in the obtained cells
A can be quantified by, for example, extracting mRNA from cells or the like by a usual method and using, for example, a technique such as TaqMan PCR, and can also be analyzed by performing Northern blotting by a method known per se. (Ii) preparing a transformant expressing the receptor protein of the present invention or a partial peptide thereof according to the method described above;
The mRNA of the receptor protein of the present invention or its partial peptide contained in the transformant can be quantified and analyzed in the same manner.

The screening for a compound that alters the expression level of the receptor protein or its partial peptide of the present invention 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 ago or after a certain time (30 minutes to 3 days after, preferably 1 hour to 2 days after, More preferably, after 1 hour to 24 hours), or a test compound is administered simultaneously with a drug or physical stress, and after a lapse of a certain time after the 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 amount of mRNA of the receptor protein of the present invention or its partial peptide contained in the cells, and (ii) mixing the test compound in the medium when culturing the transformant according to a conventional method. After culturing for a certain period of time (1 to 7 days, preferably 1 to 3 days, more preferably 2 to 3 days), the amount of mRNA of the receptor protein of the present invention or its partial peptide contained in the transformant Can be carried out by quantification and analysis.

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 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, IntracGMP production, inositol phosphate production, cell membrane potential fluctuation, intracellular protein phosphorylation, activation of c-fos, p
Activity that promotes or suppresses the reduction of H, etc.)
And (b) a compound that reduces the cell stimulating activity by decreasing the expression level of the receptor protein of the present invention or its partial peptide. Such compounds include peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, and the like. These compounds may be novel compounds or known compounds. 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 in the above-mentioned drug containing the receptor protein of the present invention. Since the thus obtained preparation is safe and has low toxicity, it can be administered to, for example, humans and mammals (eg, rats, rabbits, sheep, pigs, cows, cats, dogs, monkeys, etc.). . The dose of the compound or a salt thereof varies depending on the administration subject, target organ, symptom, administration method and the like, but in the case of oral administration, generally, for example,
In schizophrenic patients (as 60 kg), about 0.1 to 100 mg per day, preferably about 1.0 to 50 m
g, 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 to 30 mg per day
It is convenient to administer the drug by intravenous injection, preferably of the order of about 0.1 to 20 mg, more preferably of the order of about 0.1 to 10 mg. In the case of other animals, the dose can be administered in terms of 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. Is considered to play some important role in Therefore, the compound that changes the expression level of the receptor protein of the present invention or its partial peptide can be used as a preventive and / or therapeutic agent for a disease associated with dysfunction of the receptor protein of the present invention. When the compound is used as a preventive and / or therapeutic agent for a disease associated with dysfunction of the receptor protein of the present invention, it can be formulated according to a conventional method. For example, the compound 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 may be mixed with known physiologically acceptable carriers, flavoring agents, excipients, vehicles, preservatives, stabilizers, binders, and the like, in a 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 an appropriate dose 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 acacia, excipients such as crystalline cellulose, corn starch, gelatin, alginic acid and the like. Swelling agents such as magnesium stearate, sweeteners such as sucrose, lactose or saccharin, flavoring agents such as peppermint, reddish oil or cherry, and the like. 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 adjuvants (eg, D-sorbitol, D-mannitol, sodium chloride, etc.) and the like are used. For example, it may be used in combination with an alcohol (eg, ethanol), a polyalcohol (eg, propylene glycol, polyethylene glycol), a nonionic surfactant (eg, Polysorbate 80 ^™ , 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. Since the thus obtained preparation is safe and has low toxicity, it can be administered to, for example, humans and mammals (eg, rats, 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. About 0.1 to 100 mg, preferably about 1.0 to 50 mg, more preferably about 1.0 to 20 mg
mg. In the case of parenteral administration, the single dose varies depending on the administration subject, target organ, condition, administration method, and the like. About 0.01 to 30 mg per day, preferably about 0.1 to 20 m
Conveniently, about g, more preferably about 0.1 to 10 mg, will be administered by intravenous injection. In the case of other animals, the dose can be administered in terms of 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 for ligands, the concentration of ligand in vivo can be determined with high sensitivity. can do. The quantification method of the present invention can be used, for example, by combining it with a competition method. That is, the ligand concentration in the test sample can be measured by bringing the test sample into contact with the receptor protein of the present invention. Specifically, for example, it can be used according to the method described below or the like or a method analogous thereto. Edited by Hiro Irie "Radio Immunoassay" (Kodansha, Showa 4)
(Published in 1995) Hiroshi Irie, "Continuing Radio Immunoassay" (Kodansha, published in 1979)

(7) Method for Screening Compound (Agonist, Antagonist, etc.) that Changes the Binding Property of G Protein-Coupled Receptor Protein of the Present Invention to a Ligand Using Receptor Protein of the Present Invention or Recombinant Receptor Protein By constructing an expression system such as, and using a receptor binding assay system using the expression system,
Compounds (eg, peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, etc.) or their salts that change the binding between the ligand and the receptor protein of the present invention can be efficiently screened. Such compounds include (a) cell stimulating activities (eg, arachidonic acid release, acetylcholine release, intracellular Ca ²⁺ release, intracellular cAMP production, intracellular cGMP production, inositol phosphorus via G protein-coupled receptor) A compound having an activity of promoting or suppressing acid production, fluctuation of cell membrane potential, phosphorylation of intracellular protein, activation of c-fos, reduction 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 antagonist to the receptor protein of the present invention), (c) a compound that enhances the binding force between the ligand and the G protein-coupled receptor protein of the present invention, or (d) Includes compounds that reduce the binding force between the ligand and the G protein-coupled receptor protein of the present invention. That (the compound of the above (b) is preferably screened by the ligand determination methods described above). That is, the present invention relates to (i) the 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) the 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 of the present invention or a partial peptide thereof or a salt thereof, which is characterized by comparing with a case where the test compound is brought into contact with the test compound. The screening method of the present invention is characterized in that, for example, in the cases (i) and (ii), for example, the amount of a ligand bound 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 a labeled ligand is brought into contact with the receptor protein of the present invention, and the case where a labeled ligand and a 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 of the present invention or a membrane fraction of the cell, and a method wherein the labeled ligand and the test compound are added to the cell containing the receptor protein of the present invention or the membrane fraction of the cell. The amount of the labeled ligand bound to the cell or the membrane fraction when contacted is measured and compared A method for screening a compound or a salt thereof that alters the binding property between a ligand and a receptor protein of the present invention or the like, comprising: culturing a labeled ligand on a cell membrane by culturing a transformant containing the DNA of the present invention; And the labeled ligand and test compound were brought into contact with the receptor protein of the present invention expressed on the cell membrane by culturing a transformant containing the DNA of the present invention. In this case, the amount of binding of the labeled ligand to the receptor protein or the like is measured and compared, 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 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 via the receptor (eg, arachidonic acid release, acetylcholine release, intracellular Ca ²⁺ release) when a compound that activates or the like and a test compound are brought into contact with cells containing the receptor protein or the like of the present invention. , Measurement of intracellular cAMP production, intracellular cGMP production, inositol phosphate production, cell membrane potential fluctuation, intracellular protein phosphorylation, activation of c-fos, activity of promoting or suppressing pH reduction, etc.) Change the binding between the ligand characterized by comparing with the receptor protein of the present invention. A method for screening a compound or a salt thereof, and a cell membrane obtained by 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). The compound which activates the receptor protein or the like of the present invention and the test compound when contacted with the receptor protein or the like of the present invention expressed above are treated with the DN of the present invention.
Cell-stimulating activity through the receptor (eg, arachidonic acid release, acetylcholine release, intracellular C) when the transformant containing A is brought into contact with the receptor protein of the present invention expressed on the cell membrane by culturing.
a ²⁺ release, intracellular cAMP production, intracellular cGMP production,
Inositol phosphate production, cell membrane potential fluctuation, intracellular protein phosphorylation, activation of c-fos, activity to promote or suppress pH reduction, etc.), and compare with a ligand characterized by measuring The present invention provides a method for screening a compound or a salt thereof that changes the binding to the receptor protein or the like of the present invention.

Prior to obtaining the receptor protein or the like of the present invention, when screening for a G protein-coupled receptor agonist or antagonist, cells, tissues or cell membrane fractions containing the G protein-coupled receptor protein, such as rats, are first used. To obtain candidate compounds (primary screening), and then confirm whether the candidate compounds actually inhibit the binding of human G protein-coupled receptor protein to ligand (secondary screening)
Was needed. If the cell, tissue or cell membrane fraction is used as it is, other receptor proteins will be mixed, and it has been difficult to actually screen for an agonist or antagonist for the target receptor protein. However, for example, by using the rat-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, rat-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. A complementary DNA is used as the DNA fragment encoding the target protein portion, but is not necessarily limited thereto. For example, a gene fragment or a synthetic DNA may be used. D encoding the receptor protein of the present invention
In order to introduce the NA fragment into host animal cells and express them efficiently, the DNA fragment is converted into a nuclear polyhedrosis virus (nuclear) belonging to a baculovirus using an insect as a host.
It is preferably incorporated into the downstream of polyhedrosis virus (NPV) 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 method known per se. For example, literature [Na
mbi, p. Et al., The Journal of Biological
Chemistry (J. Biol. Chem.), 267, 19555-19559
P. 1992]. Therefore, in the screening method of the present invention, the receptor protein or the like of the present invention may be a receptor protein or the like purified according to a method known per se, or a cell containing the receptor protein or the like may be used. Alternatively, a membrane fraction of cells containing the receptor protein or the like may be used.

When cells containing the receptor protein 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 method known per se. Cells containing the receptor protein or the like of the present invention refer to host cells that express the receptor protein or the like.
Bacillus subtilis, yeast, insect cells, animal cells and the like are preferred. The cell membrane fraction refers to a fraction abundant in cell membrane obtained by disrupting cells and then obtained by a method known per se. 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, a cell lysate is supplied at a low speed (500 rpm to 300 rpm).
0 rpm) for a short time (usually about 1 to 10 minutes),
The supernatant is further accelerated (15,000 rpm to 30,000 rpm).
m) for 30 minutes to 2 hours, and the resulting precipitate is used as a membrane fraction. The membrane fraction is rich in expressed receptor proteins and membrane components such as cell-derived phospholipids and membrane proteins. The amount of the receptor protein in the cell or membrane fraction containing the receptor protein or the like is 10 ³ to 10 ³ per cell.
It is preferably 10 ⁸ molecules, and more preferably 10 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 (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. As the receptor protein fraction, a natural receptor protein fraction or a recombinant receptor protein fraction having an activity equivalent thereto is desirable. 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, ligands labeled with [ ³ H], [ ¹²⁵ I], [ ¹⁴ C], [ ³⁵ S] and the like are used. Specifically, to screen for a compound that alters the binding between the 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 has a pH of 4 to 10 (preferably pH 6 to 8).
Any buffer may be used as long as it does not inhibit the binding between the ligand and the receptor protein, such as a phosphate buffer and Tris-HCl buffer. 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. Furthermore, a protease inhibitor such as PMSF, leupeptin, E-64 (manufactured by Peptide Research Laboratories), pepstatin and the like can be added for the purpose of suppressing the degradation of the receptor or ligand by the protease. A fixed amount (5,000 cpm to 500 cpm) is added to 0.01 ml to 10 ml of the receptor solution.
000 cpm) of labeled ligand and simultaneously
0 ⁻⁴ M to ^{10 −10} M test compound is allowed to coexist. 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. to 37 ° C., for about 20 minutes to 24 hours, preferably for 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. The count (B ₀ −N) obtained by subtracting the non-specific binding amount (NSB) from the count (B ₀ ) when there is no antagonistic substance
(SB) as 100%, the specific binding amount (B-NS)
For example, a test compound in which B) is 50% or less can be selected as a candidate substance having competitive inhibitory ability.

In order to carry out the above-mentioned methods (1) to (4) 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 release, intracellular cA
MP production, intracellular cGMP production, inositol phosphate production, cell membrane potential fluctuation, intracellular protein phosphorylation, cf
activity for promoting or suppressing os activation, pH reduction, etc.) can be measured using a known method or a commercially available measurement kit. Specifically, first,
Cells containing the receptor protein or the like of the present invention are cultured in a multiwell plate or the like. 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. Also, c
The activity such as AMP 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 cell stimulating activity, cells expressing an appropriate receptor protein are required. Cells expressing the receptor protein or the like of the present invention are preferably cell lines having the natural receptor protein of the present invention or the like, or cell lines expressing the above-mentioned recombinant receptor protein or the like. Test compounds include, for example, peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products,
Cell extracts, plant extracts, animal tissue extracts and the like are used, and these compounds may be novel compounds or known compounds.

A screening kit for a compound or a salt thereof that alters the binding between a ligand and the receptor protein of the present invention or the like can be obtained from cells containing the receptor protein or the like of the present invention, cells containing the receptor protein or the like of the present invention, or cells of the present invention. And those containing a membrane fraction of cells containing a receptor protein and the like. Examples of the screening kit of the present invention include the following. 1. Screening reagent Measurement buffer and washing buffer Hanks' Balanced Salt Solution (manufactured by Gibco)
One containing 05% bovine serum albumin (manufactured by Sigma). Sterilized by filtration through a 0.45 μm filter, 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
Passage into a 12-well plate at 5 × 10 ⁵ / well, 37 ° C.
Cultured for 2 days in 5% CO ₂ and 95% air. Labeled ligand Commercially available aqueous solution of ligand labeled with [ ³ H], [ ¹²⁵ I], [ ¹⁴ C], [ ³⁵ S], etc. Store at 4 ° C. or −20 ° C. Dilute the solution to 1 μM. Ligand standard solution 0.1% bovine serum albumin (Sigma)
Dissolve to 1 mM in PBS containing and store at -20 ° C.

2. Assay Method CHO cells expressing the receptor protein of the present invention cultured on a 12-well tissue culture plate are washed twice with 1 ml of assay buffer, and then 490 μl of assay buffer is added to each well. After 5 μl of a test compound solution of 10 ⁻³ to 10 ⁻¹⁰ M is added, 5 μl of a labeled ligand is added, and the mixture is reacted at room temperature for 1 hour. To determine the amount of non-specific binding, 5 μl of 10 ⁻³ M ligand is added instead of the test compound. Remove the reaction solution and wash three times with 1 ml of washing buffer. The labeled ligand bound to the cells was 0.2N NaOH
-1% SDS, 4 ml of liquid scintillator A
(Made by Wako Pure Chemical Industries). Liquid scintillation counter (Beckman)
Radioactivity was measured using Percent Maximum Binding
(PMB) is obtained by the following equation (Equation 1).

[Equation 1] PMB = [(B−NSB) / (B ₀ −NSB)] × 10
0 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 property between the 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 generation, intracellular cGMP
(A so-called receptor protein of the present invention) having activity of promoting or inhibiting the production, inositol phosphate production, fluctuation of cell membrane potential, phosphorylation of intracellular protein, activation of c-fos, reduction of pH, etc. (B) compounds having no cell-stimulating activity (so-called antagonists to the receptor protein of the present invention), (c) compounds that enhance the binding force between the ligand and the G protein-coupled receptor protein of the present invention, Alternatively, (d) a compound that decreases the binding force between the ligand and the G protein-coupled receptor protein of the present invention. Such compounds include peptides, proteins, non-peptidic compounds,
Examples thereof include synthetic compounds and fermentation products, and these compounds may be novel compounds or known compounds. 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-toxic drug for enhancing the physiological activity of the ligand for the receptor protein 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, in the same manner as the above-mentioned drug containing the receptor protein of the present invention, tablets,
Capsules, elixirs, microcapsules, sterile solutions, suspensions and the like can be used. Since the preparations obtained in this way are safe and low toxic,
It can be administered to humans and mammals (eg, rats, rabbits, 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. In the case of oral administration, generally, for example, schizophrenic patients (6
0 kg), about 0.1 to 10 per day
0 mg, 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, symptoms, administration method, etc., for example, usually in the form of an injection, for example, in schizophrenic patients (60 kg). About 0.01 to 30 mg per day, preferably about 0.1 to 20 mg, more preferably about 0.1 to 1 mg per day.
It is convenient to administer about 0 mg by intravenous injection. In the case of other animals, the dose can be administered in terms of 60 kg.

(8) Preventive 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. As described above, for example, it is considered that it plays some important role in vivo such as central function. Therefore, the compounds (agonists, antagonists) that change the binding property between the receptor protein of the present invention and the ligand can be used as agents for preventing and / or treating diseases associated with dysfunction of the receptor protein of the present invention. When the compound is used as a preventive and / or therapeutic agent for a disease associated with dysfunction of the receptor protein of the present invention, it can be formulated according to a conventional method. For example, the compound 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 may be mixed with known physiologically acceptable carriers, flavoring agents, excipients, vehicles, preservatives, stabilizers, binders, and the like, in a 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 an appropriate dose 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 acacia, excipients such as crystalline cellulose, corn starch, gelatin, alginic acid and the like. And a lubricating agent such as magnesium stearate, a sweetening agent such as sucrose, lactose or saccharin, and a flavoring agent 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 adjuvants (eg, D-sorbitol, D-mannitol, sodium chloride, etc.) and the like are used. For example, it may be used in combination with an alcohol (eg, ethanol), a polyalcohol (eg, propylene glycol, polyethylene glycol), a nonionic surfactant (eg, Polysorbate 80 ^™ , 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 above 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. Since the thus obtained preparation is safe and has low toxicity, it can be administered to, for example, humans and mammals (eg, rats, 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. About 0.1 to 100 mg, preferably about 1.0 to 50 mg, more preferably about 1.0 to 20 mg
mg. In the case of parenteral administration, the single dose varies depending on the administration subject, target organ, condition, administration method, and the like. About 0.01 to 30 mg per day, preferably about 0.1 to 20 m
Conveniently, about g, more preferably about 0.1 to 10 mg, will be administered by intravenous injection. In the case of other animals, the dose can be administered in terms of 60 kg.

(9) Quantification of the Receptor Protein of the Present Invention or Its Partial Peptide or Its Salt
Since the receptor protein or the like of the present invention can be specifically recognized, it can be used for quantification of the receptor protein or the like of the present invention in a test solution, particularly for quantification by sandwich immunoassay. That is, the present invention, for example,
(I) reacting the antibody of the present invention with a test solution and a labeled receptor protein or the like competitively, and measuring the ratio of the labeled receptor protein or the like bound to the antibody; A method for quantifying the receptor protein of the present invention,
(Ii) measuring the activity of the labeling agent on the insolubilized carrier after simultaneously or continuously reacting the test solution with the antibody of the present invention insoluble on the carrier and the labeled antibody of the present invention. A method for quantifying the receptor protein of the present invention in a test solution is provided. In the above (ii), one antibody is an antibody that recognizes the N-terminus of the receptor protein of the present invention, and the other antibody is a C-protein such as the receptor protein of the present invention.
Preferably, the antibody reacts at the end.

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, Fa
b ′ or 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 the 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 preferably used, but in terms of sensitivity and specificity, it is particularly preferable to use the sandwich method described later. 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.
Examples of radioisotopes include [ ¹²⁵ I],
[ ¹³¹ I], [ ³ H], [ ¹⁴ C] and the like are used. As the 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, luminol derivatives, 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 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, agarose,
Dextran, insoluble polysaccharides such as cellulose, polystyrene, polyacrylamide, synthetic resins such as silicon,
Alternatively, glass or the like is 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 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 kind, and a mixture of two or more kinds 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 a different binding site to the receptor protein or 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, an antibody that recognizes other than the C-terminal, for example, the N-terminal, is used.

The monoclonal antibody of the present invention can be used in a measuring system other than the sandwich method, for example, a competitive method, an immunometric method, a nephelometry and the like. In the competition method, an antigen in a test solution and a labeled antigen are allowed to react competitively with an antibody, and then the unreacted labeled antigen is reacted with the antigen.
(F) and the labeled antigen (B) bound to the antibody are separated (B
/ F separation), the labeling amount of either B or F is measured, and the amount of antigen in the test solution is quantified. In this reaction method, a soluble antibody was used as an antibody, B / F separation was performed using polyethylene glycol,
A liquid phase method using a second antibody or the like to the above antibody, and using an immobilized antibody as the first antibody, or
A solid-phase method using a soluble antibody as the first antibody and using a solid-phased 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 assay methods to the assay method of the present invention, no special conditions, operations, and other settings are required. What is necessary is just to construct the measuring system of the receptor protein of the present invention or a salt thereof by adding ordinary technical considerations of those skilled in the art to ordinary conditions and operation methods in each method. For more information on these common technical measures,
Review articles, books, etc. can be referred to [for example, Hiroe Irie, "Radio Immunoassay" (Kodansha, published in 1974), Hiroshi Irie, "Continuing Radio Immunoassay" (Kodansha, published in 1974), Eiji Ishikawa et al. "Enzyme immunoassay" (ed. Medical Shoin, published in 1978), Eiji Ishikawa et al., "Enzyme immunoassay" (second edition) (ed.), Eiji Ishikawa et. Law (3rd edition) (Medical Shoin, published in 1987), "Methods in ENZYMOLOGY" Vol. 70 (Immunoch)
emical Techniques (Part A)), ibid.Vol. 73 (Immunoch
emical Techniques (Part B)), ibid.Vol. 74 (Immunoch
emical Techniques (Part C)), ibid.Vol. 84 (Immunoch
emical Techniques (Part D: Selected Immunoassays)),
Ibid. Vol. 92 (Immunochemical Techniques (Part E: Mon
oclonal Antibodies and General Immunoassay Method
s)), ibid.Vol. 121 (Immunochemical Techniques (Part
I: Hybridoma Technology and Monoclonal Antibodie
s)) (see 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, it is possible to diagnose various diseases associated with dysfunction of the receptor protein of the present invention. Further, the antibody of the present invention can be used for specifically detecting the receptor protein of the present invention and the like 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 for a 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
For example, (i) after disrupting the blood of a non-human mammal, a specific organ, tissues or cells isolated from an organ, or the like, isolating a cell membrane fraction, and containing the receptor protein of the present invention contained in the cell membrane fraction or a receptor protein thereof. 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 partial peptide, (ii) destroying a transformant expressing the receptor protein or its partial peptide of the present invention, etc. After that, the cell membrane fraction is isolated, and the receptor protein of the present invention or its partial peptide contained in the cell membrane fraction is quantified, thereby screening for a compound that changes the amount of the receptor protein of the present invention or its partial peptide in the cell membrane The method, (iii) blood of the non-human mammal,
A specific organ, a tissue or a cell isolated from an organ is sectioned, and then, by using an immunostaining method, by quantifying the degree of staining of the receptor protein on the cell surface, the protein on the cell membrane is quantified. And a method for screening a compound that changes the amount of the receptor protein of the present invention or its partial peptide in the cell membrane by confirming the following. (Iv) Quantifying the degree of staining of the receptor protein on the cell surface by using immunostaining after sectioning a transformant or the like that expresses the receptor protein of the present invention or a partial peptide thereof. And a method for screening a compound that changes the amount of the receptor protein of the present invention or its partial peptide in a cell membrane by confirming the protein on the cell membrane.

The quantitative determination of the receptor protein of the present invention or its partial peptide contained in the cell membrane fraction is 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.)
Drugs (eg, anti-dementia drugs, blood pressure lowering drugs, anti-cancer drugs, anti-obesity drugs, etc.) or physical stress (eg, flooding stress, electric shock, light, dark, low temperature, etc.) Obtain a specific organ (eg, brain, liver, kidney, 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.),
An organ, tissue or cell is destroyed, and a cell membrane fraction is obtained by using a surfactant (eg, Triton X100 ^™ , Tween 20 ^™, etc.), and further using a method such as centrifugation, filtration, or 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 method known per se. As a method for disrupting cells, Potter-El
Examples include a method of crushing cells with a vehjem-type homogenizer, crushing with a Waring blender or a polytron (manufactured by Kinematica), crushing with ultrasonic waves, crushing by ejecting cells from a thin nozzle while applying pressure with a French press, 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 slowed (500 r
pm to 3000 rpm) for a short time (usually about 1 minute to 10 minutes).
Min) and centrifuged, and the supernatant was further spun at high speed (15000 rpm to 3
(0000 rpm) for 30 minutes to 2 hours, and the resulting precipitate is used as a membrane fraction. The membrane fraction is rich in expressed receptor proteins and membrane components such as cell-derived phospholipids and membrane proteins.

The 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 a means known per se.

(Ii) A transformant expressing the receptor protein of the present invention or its partial peptide is prepared according to the method described above, 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 ago) or after a certain time (30 minutes to 3 days later, preferably 1 hour to 2 days later) And more preferably 1 hour to 24 hours later) or a test compound is administered at the same time as the drug or physical stress, and after a certain period of time (30 minutes to 3 days, preferably 1 hour to 2 days) after administration. More preferably after 1 hour to 24
After a period of time), the amount can be determined by quantifying the amount of the receptor protein of the present invention or its partial peptide in the cell membrane. (Ii) When the transformant is cultured according to a conventional method, the test compound is mixed into the medium. , After culturing for a certain time (1
After 7 days, preferably 1 day to 3 days, more preferably 2 days to 3 days), the amount of the receptor protein of the present invention or its partial peptide in the cell membrane can be determined.

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 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 to a mouse, etc. for a certain period of time. After the passage, blood or a specific organ (eg, brain, liver, kidney, etc.), or a tissue or cell isolated from the organ is obtained. The obtained organ, tissue or cells, etc., as a tissue section according to a conventional method,
Immunostaining is performed using the antibody of the present invention. By quantifying the degree of staining of the receptor protein on the cell surface, and confirming the protein on the cell membrane, the amount of the receptor protein of the present invention or its partial peptide in the cell membrane can be quantitatively or qualitatively determined. You can check. (Iv) It can also be confirmed by using a transformant expressing the receptor protein of the present invention or a partial peptide thereof and the like, and performing the same procedure.

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 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 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, intracellular protein phosphorylation, c
(B) a compound that enhances the activity of promoting or suppressing the activation of fos, a decrease in pH, etc.), and (b) reducing the amount of the receptor protein of the present invention or its partial peptide in the cell membrane to stimulate the cell. A compound that reduces activity. Such compounds include peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, and the like. These compounds may be novel compounds or known compounds. The compound that enhances the cell stimulating activity is useful as a safe and low toxic 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 conventional means. For example,
Tablets, capsules, elixirs, microcapsules, sterile solutions, suspensions, and the like can be prepared in the same manner as in the above-mentioned drug containing the receptor protein of the present invention. Since the thus obtained preparation is safe and has low toxicity, it can be administered to, for example, humans and mammals (eg, rats, rabbits, sheep, pigs, cows, cats, dogs, monkeys, etc.). . The dose of the compound or a salt thereof varies depending on the administration subject, target organ, symptom, administration method and the like, but in the case of oral administration, generally, for example,
In schizophrenic patients (as 60 kg), about 0.1 to 100 mg per day, preferably about 1.0 to 50 m
g, 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 to 30 mg per day
It is convenient to administer the drug by intravenous injection, preferably of the order of about 0.1 to 20 mg, more preferably of the order of about 0.1 to 10 mg. In the case of other animals, the dose can be administered in terms of 60 kg.

(11) A preventive and / or therapeutic agent for various diseases containing a compound that alters the amount of the receptor protein of the present invention or its partial peptide in the cell membrane. It is thought to play some important role in the body. Therefore, a compound that alters the amount of the receptor protein of the present invention or its partial peptide 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. When the compound is used as a preventive and / or therapeutic agent for a disease associated with dysfunction of the receptor protein of the present invention, it can be formulated according to a conventional method. For example, the compound 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 may be mixed with known physiologically acceptable carriers, flavoring agents, excipients, vehicles, preservatives, stabilizers, binders, and the like, in a 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 an appropriate dose 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 and the like. Swelling agents such as magnesium stearate, sweeteners such as sucrose, lactose or saccharin, flavoring agents such as peppermint, reddish oil or cherry, and the like. 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 adjuvants (eg, D-sorbitol, D-mannitol, sodium chloride, etc.) and the like are used. For example, it may be used in combination with an alcohol (eg, ethanol), a polyalcohol (eg, propylene glycol, polyethylene glycol), a nonionic surfactant (eg, Polysorbate 80 ^™ , 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 above 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. Since the thus obtained preparation is safe and has low toxicity, it can be administered to, for example, humans and mammals (eg, rats, 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. About 0.1 to 100 mg, preferably about 1.0 to 50 mg, more preferably about 1.0 to 20 mg
mg. In the case of parenteral administration, the single dose varies depending on the administration subject, target organ, condition, administration method, and the like. About 0.01 to 30 mg per day, preferably about 0.1 to 20 m
Conveniently, about g, more preferably about 0.1 to 10 mg, will be administered by intravenous injection. In the case of other animals, the dose can be administered in terms of 60 kg.

(12) Neutralization by an antibody against the receptor protein of the present invention or its partial peptide or a salt thereof An antibody against the receptor protein of the present invention or its partial peptide or its salt neutralizes the receptor protein and the like. That is, it means an activity of inactivating a signal transduction 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, Inactivation of intracellular cAMP production, intracellular cGMP production, inositol phosphate production, cell membrane potential fluctuation, intracellular protein phosphorylation, activation of c-fos, 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.

(13) Preparation of Non-Human Animal Having DNA Encoding the G Protein-Coupled Receptor Protein of the Present Invention Using the DNA of the present invention, a transgenic non-human animal expressing the receptor protein or the like of the present invention is prepared. can do. Non-human animals include mammals (eg,
Rats, mice, rabbits, sheep, pigs, cows, cats,
Dogs, monkeys, etc.) (hereinafter abbreviated as animals), and mouse, rabbit and the like are particularly preferable. When transferring the DNA of the present invention to a target animal, the D
It is generally advantageous to use NA as a gene construct linked downstream of a promoter capable of being expressed in animal cells. For example, when the DNA of the present invention derived from a rabbit is transferred, the DNA of the present invention derived from an animal having high homology to the DNA is transferred.
A DNA transgenic animal that highly produces the receptor protein or the like of the present invention can be produced by microinjecting a gene construct in which NA is expressed downstream of various promoters capable of expressing it in animal cells, for example, into a fertilized rabbit egg. As this promoter, for example, a ubiquitous expression promoter such as a virus-derived promoter or metallothionein may be used, but an NGF gene promoter or an enolase gene promoter that is specifically expressed in the brain is preferably used.

The transfer 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 germ cells of the produced animal after DNA transfer means that all the offspring of the produced animal have the receptor protein or the like of the present invention in all of the germ cells and somatic cells. The progeny of this kind of animal that has inherited the gene has the receptor protein of the present invention in all of its germinal and somatic cells. After confirming that the DNA-transferred animal of the present invention stably retains the gene by breeding, it can be reared in a normal breeding environment as the DNA-bearing animal. Furthermore, by crossing male and female animals having the target DNA, homozygous animals having the transgene on both homologous chromosomes are obtained, and by crossing the male and female animals, all the offspring have the DNA. Breeding can be subcultured. Since the animal into which the DNA of the present invention has been transferred has high expression of the receptor protein of the present invention, it is useful as an animal for screening for an agonist or antagonist for the receptor protein of the present invention. The transgenic animal of the present invention can also be used as a cell source for tissue culture. For example, the receptor protein of the present invention can be analyzed by directly analyzing DNA or RNA in the tissue of the DNA-transferred mouse of the present invention or by analyzing the tissue in which the receptor protein of the present invention expressed by a gene is present. can do. Culturing cells of a tissue having the receptor protein or the like of the present invention by standard tissue culture techniques, and using these to study the function of cells from generally difficult-to-cultivate tissues such as those derived from brain or peripheral tissues. Can be. Also, by using the cells, for example,
It is also possible to select drugs that enhance the function of various tissues. In addition, 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 represented by abbreviations, IUPAC-IUB
It is based on the abbreviation by the Commission on Biochemical Nomenclature or the abbreviation commonly used in the relevant field. When an amino acid can have an optical isomer, the L-form is indicated unless otherwise specified. DNA: deoxyribonucleic acid cDNA: complementary deoxyribonucleic acid A: adenine T: thymine G: guanine C: cytosine RNA: ribonucleic acid mRNA: messenger ribonucleic acid dATP: deoxyadenosine triphosphate dTTP: deoxythymidine triphosphate dGTP: deoxyguanosine triphosphate Phosphate dCTP: Deoxycytidine triphosphate ATP: Adenosine triphosphate EDTA: Ethylenediaminetetraacetic acid SDS: Sodium dodecyl sulfate

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

The 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

[0111] The sequence numbers in the sequence listing in the present specification indicate the following sequences. [SEQ ID NO: 1] This shows the amino acid sequence of rCB7T084, a novel G protein-coupled receptor protein derived from rat cerebellum of the present invention. [SEQ ID NO: 2] This shows the base sequence of cDNA encoding the novel rat cerebellum-derived G protein-coupled receptor protein rCB7T084 having the amino acid sequence represented by SEQ ID NO: 2. [SEQ ID NO: 3] cDNA encoding a novel G protein-coupled receptor protein rCB7T084 derived from rat cerebellum of the present invention
Shows the nucleotide sequence of primer 1 used for cloning the DNA. [SEQ ID NO: 4] This shows the base sequence of primer 2 used for cloning cDNA encoding rCB7T084, a novel G protein-coupled receptor protein derived from rat cerebellum of the present invention.

The transformant Escherichia coli DH10B / pA obtained in Example 1 described later.
K-rCB084 was deposited on September 4, 1998 with the Ministry of International Trade and Industry, National Institute of Advanced Industrial Science and Technology (NIBH) under the deposit number FERM BP-6485.
It has been deposited with the Fermentation Research Institute (IFO) as the deposit number IFO 16199 from March 17.

[0113]

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 Derived from Rat Cerebellum and Determination of Base Sequence Using rat cerebellum cDNA 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 prepared using 1/10 of the above cDNA as a template, and using Advantage cDNA Polym.
erase Mix (CLONTECH) 1/50
Amount, primer 1 (SEQ ID NO: 3) and primer 2
(SEQ ID NO: 4) was added to each of 0.2 μM and dNTPs 20
Add 0 μM, and the attached buffer to the enzyme, and add 25 μM
1 liquid volume. The PCR reaction was performed at 95 ° C. for 30 seconds, followed by 5 cycles of 94 ° C. for 5 seconds and 70 ° C. for 5 minutes.
5 cycles of 94 ° C for 5 seconds, 68 ° C for 5 minutes,
A cycle of 94 ° C. for 5 seconds and 65 ° C. for 5 minutes was repeated 35 times, and an extension reaction was finally performed at 65 ° C. for 5 minutes. The P
The reaction product after the CR reaction is transferred to a TA cloning kit (In
The vector was subcloned into a plasmid vector pCRII (Invitrogen) according to the prescription of Vitrogen. This was introduced into Escherichia coli DH5α, and clones having the cDNA were selected on LB agar medium containing ampicillin, and the sequence of each clone was analyzed. As a result, cDNA encoding a novel G protein-coupled receptor protein was identified.
The sequence (SEQ ID NO: 2) was obtained. A novel G protein-coupled receptor protein containing an amino acid sequence (SEQ ID NO: 1) derived from this cDNA was named rCB7T084. Plasmid pAK into which cDNA (SEQ ID NO: 2) encoding the rat cerebellum-derived G protein-coupled receptor protein rCB7T084 is subcloned
-RCB084 was transformed into Escherichia coli (Esch
Escherichia coli DH10B / pAK-rCB
084. The plasmid pCRII-rCB7T084 into which cDNA (SEQ ID NO: 2) encoding the rat cerebellum-derived G protein-coupled receptor protein rCB7T084 was subcloned was introduced into Escherichia coli DH5α according to a method known per se. And transformant: Escherichia coli DH
5α / pCRII-rCB7T084 was obtained.

Example 2 Preparation of rCB7T084-Expressing CHO Cells The transformant E. coli DH5α / pC prepared in Example 1
After culturing RII-rCB7T084, pCRII-rCB7T08 was prepared using a plasmid / mid kit (Qiagen).
084 plasmid DNA was prepared. From this plasmid, the G protein-coupled receptor protein rCB7 of the present invention
The cDNA encoding T084 was cloned into a plasmid vector pcDNA3.1 / V5 / His for protein expression to construct a plasmid pcDNA3.1-rCB084 for protein expression. The thus obtained plasmid was plasmid DNA prepared in large quantities by using a plasmid Midokitto (Qiagen), CHO dhfr according to the attached protocol using this Serufekuto transfection kit (Amersham Pharmacia Biotech) ^- The cells were introduced.
That is, 10 mg of DNA was made into a coprecipitation suspension with calcium phosphate, and 24 hours before 5 × 10 ⁵ or 1 × 10 ⁶ CH
O dhfr ^- cells were added to a seeded 10 cm Petri dish, cultured in MEMα medium containing 10% fetal bovine serum for 1 day, and subcultured. Then, 0.4 mg / ml G418 (Gibco BRL) and 0.4 mg / ml selective medium were added. ME containing 10% dialyzed fetal bovine serum
The cells were cultured in Mα medium. By selecting colonies of transformed cells (CHO / rCB084) growing in the selection medium, rCB7T084 expressing CHO cells were obtained. Total RNA from the selected rCB7T084-expressing CHO cells according to a standard method
Was extracted, and then mRNA of rCB7T084 was obtained by the TaqMan method.
The amount was measured and the number of copies was calculated. The results are shown in the table below.

[Table 1]

[0116]

The G protein-coupled receptor protein of the present invention or its partial peptide or its salt, and the polynucleotide encoding it (for example, DNA, R
NA and their derivatives), determining ligands (agonists), obtaining antibodies and antisera, constructing a recombinant receptor protein expression system, developing a receptor binding assay system using the expression system, and identifying drug candidate compounds. Screening, implementation of drug design based on comparison with structurally similar ligands / receptors, reagents for preparing probes and PCR primers in gene diagnosis, preparation of transgenic animals or drugs such as gene preventive and therapeutic agents, etc. Can be used as

[0117]

[Sequence List] <110> Takeda Chemical Industries, Ltd. <120> Novel G-Protein Coupled Receptor Protein and its DNA <130> A98197 <150> JP 10-286607 <151> 1998-10-08 <160> 7 < 210> 4 <211> 319 <212> PRT <213> Rat <400> 1 Met Asn Tyr Thr Pro Tyr Ser Ser Pro Ala Pro Gly Leu Thr Ile Ser 5 10 15 Pro Thr Met Asp Pro Val Thr Trp Val Tyr Phe Ser Val Thr Phe Leu 20 25 30 Ala Met Ala Thr Cys Val Cys Gly Ile Val Gly Asn Ser Met Val Ile 35 40 45 Trp Leu Leu Ser Phe His Ser Val Gln Arg Ser Pro Phe Cys Thr Tyr 50 55 60 Val Leu Asn Leu Ala Val Ala Asp Leu Leu Phe Leu Leu Cys Met Ala 65 70 75 80 Ser Leu Leu Ser Leu Glu Thr Gly Pro Leu Leu Thr Ala Ser Thr Ser 85 90 95 Ala Arg Val Tyr Glu Gly Met Lys Arg Ile Lys Tyr Phe Ala Tyr Thr 100 105 110 Ala Gly Leu Ser Leu Leu Thr Ala Ile Ser Thr Gln Arg Cys Leu Ser 115 120 125 Val Leu Phe Pro Ile Trp Tyr Lys Cys His Arg Pro Gln His Leu Ser 130 135 140 Gly Val Val Cys Gly Val Leu Trp Ala Leu Ala Leu Leu Met Asn Phe 145 150 155 160 Leu Ala Ser Phe Phe Cys Val Gln Phe Trp His Pro Asp Lys Tyr Gln 165 170 175 Cys Phe Lys Val Asp Met Val Phe Asn Ser Leu Ile Leu Gly Ile Phe 180 185 190 Met Pro Val Met Val Leu Thrl Ser Ala Ile Ile Phe Ile Arg Met Arg 195 200 205 Lys Asn Ser Leu Leu Gln Arg Arg Gln Pro Arg Arg Leu Tyr Val Val 210 215 220 Ile Leu Thr Ser Val Leu Val Phe Leu Thr Cys Ser Leu Pro Leu Gly 225 230 235 240 Ile Asn Trp Phe Leu Leu Tyr Trp Val Glu Leu Pro Gln Ala Val Arg 245 250 255 Leu Leu Tyr Val Cys Ser Ser Arg Phe Ser Ser Ser Leu Ser Ser Ser 260 265 270 Ala Asn Pro Val Ile Tyr Phe Leu Val Gly Ser Gln Lys Ser His Arg 275 280 285 Leu Gln Glu Ser Leu Gly Ala Val Leu Gly Arg Ala Leu Gln Asp Glu 290 295 300 Pro Glu Gly Arg Glu Thr Pro Ser Thr Cys Thr Asn Asp Gly Val 305 310 315 319 <210> 2 <211> 957 <212> DNA <213> Rat <400 > 2 ATGAACTACA CTCCTTATAG CAGCCCAGCC CCAGGTCTGA CCATCAGCCC CACCATGGAC 60 CCTGTGACCT GGGTTTACTT TTCAGTGACA TTCCTGGCCA TGGCCACCTG TGTGTGTGGG 120 ATAGTGGGCA ACTCCATGGT GATTTGGCTA CTGAGTTTCC ACAGTGTGCATCGGTCCCTC CTCAA CCTGGCGGTG GCCGACCTCC TCTTCCTGCT CTGCATGGCC 240 TCCCTGCTCA GTCTGGAAAC AGGGCCCCTG CTCACAGCCA GCACCTCCGC CAGAGTCTAC 300 GAGGGGATGA AGAGAATCAA GTACTTTGCC TACACAGCAG GCCTGAGCCT GCTGACGGCC 360 ATCAGCACCC AGCGCTGTCT CTCCGTGCTT TTCCCCATCT GGTATAAGTG CCACCGGCCC 420 CAGCACCTGT CGGGGGTGGT ATGTGGTGTG CTGTGGGCAC TGGCCCTCCT GATGAACTTC 480 CTGGCTTCTT TCTTCTGTGT TCAATTCTGG CATCCCGACA AATACCAGTG CTTCAAGGTG 540 GACATGGTTT TCAACAGTCT TATCCTGGGG ATCTTCATGC CCGTCATGGT CCTGACCAGC 600 GCCATCATCT TCATCCGCAT GCGAAAGAAC AGCCTGCTGC AGAGACGGCA GCCTCGGCGG 660 CTCTACGTGG TCATCCTGAC TTCCGTCCTT GTCTTCCTTA CCTGTTCTCT GCCGTTGGGC 720 ATCAACTGGT TCTTACTCTA CTGGGTGGAA CTGCCGCAGG CCGTGAGGCT CCTGTACGTC 780 TGCTCATCAC GCTTCTCCTC GTCTTTGAGC AGCAGCGCCA ACCCAGTCAT CTACTTCCTC 840 GTGGGCAGCC AGAAGAGCCA CCGGCTGCAG GAGTCTCTGG GTGCTGTGCT GGGGCGGGCA 900 CTTCAGGACG AGCCTGAAGG CAGGGAGACG CCATCCACAT GTACTAATGA TGGGGTC 957 <210> 3 <211> 36 <212 > DNA <213> Artificial Sequence <220> <223> <400> 3 gtcgacatga actacactcc ttatagcagc cca gcc 36 <210> 4 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> <400> 4 actagttcag accccatcat tagtacatgt ggatg 35

[Brief description of the drawings]

FIG. 1 shows the nucleotide sequence of the DNA encoding the novel rat cerebellum-derived G protein-coupled receptor protein rCB7T084 obtained in Example 1 and the amino acid sequence deduced therefrom (following FIG. 2).

FIG. 2 shows rat cerebellum-derived G of the present invention obtained in Example 1.
The nucleotide sequence of the DNA encoding the protein-coupled receptor protein rCB7T084 and the amino acid sequence deduced therefrom are shown (continuation of FIG. 1).

FIG. 3 shows a hydrophobicity plot of the rat cerebellum-derived G protein-coupled receptor protein rCB7T084 prepared based on the amino acid sequences shown in FIGS. 1 and 2.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C07K 14/705 C07K 14/705 16/28 16/28 C12N 1/15 C12N 1/15 1/19 1 / 19 1/21 1/21 5/10 C12P 21/02 C C12P 21/02 21/08 21/08 G01N 33/15 Z G01N 33/15 33/50 Z 33/50 33/53 D 33/53 33 / 566 33/566 33/577 B 33/577 A61K 31/7088 // A61K 31/7088 48/00 38/00 C12N 5/00 A 48/00 B A61K 37/02

Claims (27)

[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 partial peptide of the G protein-coupled receptor protein according to claim 1, or a salt thereof. 3. A polynucleotide comprising a polynucleotide having a nucleotide sequence encoding the G protein-coupled receptor protein according to claim 1. 4. The polynucleotide according to claim 3, which is a DNA. 5. The polynucleotide according to claim 3, which has the nucleotide sequence represented by SEQ ID NO: 2. 6. A recombinant vector containing the polynucleotide according to claim 3. (7) A transformant transformed with the recombinant vector according to (6). 8. The G protein-coupled receptor protein according to claim 1, wherein the transformant according to claim 7 is cultured to produce the G protein-coupled receptor protein according to claim 1. Manufacturing method. 9. An antibody against the G protein-coupled receptor protein according to claim 1, or the partial peptide according to claim 2, or a salt thereof. 10. The antibody according to claim 9, which is a neutralizing antibody that inactivates signal transduction of the G protein-coupled receptor protein according to claim 1. (11) A diagnostic agent comprising the antibody according to (9). 12. A ligand for the G protein-coupled receptor protein of claim 1 or a salt thereof, which can be obtained by using the G protein-coupled receptor protein of claim 1 or the partial peptide of claim 2 or a salt thereof. . [13] a pharmaceutical comprising the ligand of the G protein-coupled receptor protein according to [12]; 14. 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 2 or a salt thereof is used. How to determine. 15. A ligand comprising the G protein-coupled receptor protein according to claim 1 or the partial peptide according to claim 2 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 compound. 16. A ligand comprising the G protein-coupled receptor protein according to claim 1 or the partial peptide according to claim 2 or a salt thereof, and a G protein-coupled receptor protein according to claim 1 or a salt thereof. A kit for screening a compound or a salt thereof that changes the binding property to a salt. 17. The method according to claim 15, wherein the binding between the ligand and the G protein-coupled receptor protein according to claim 1 or a salt thereof is obtained, which can be obtained by using the screening method according to claim 15 or the screening kit according to claim 16. Or a salt thereof. (18) The method according to (15), wherein the binding between the ligand and the G protein-coupled receptor protein or the salt thereof according to (1) is obtained by using the screening method according to (15) or the screening kit according to (16). A medicament comprising a compound or a salt thereof. (19) a polynucleotide which hybridizes with the polynucleotide of (3) under high stringency conditions; 20. A polynucleotide comprising a nucleotide sequence complementary to the polynucleotide according to claim 3 and a part thereof. 21. The method for quantifying mRNA of a G protein-coupled receptor protein according to claim 1, wherein the polynucleotide according to claim 3 or a part thereof is used. 22. A method for quantifying a G protein-coupled receptor protein according to claim 1, wherein the antibody according to claim 9 is used. 23. The method for diagnosing a disease associated with the function of a G protein-coupled receptor protein according to claim 1, wherein the quantification method according to claim 21 or 22 is used. 24. A method for screening a compound or a salt thereof which changes the expression level of a G protein-coupled receptor protein according to claim 1, wherein the method according to claim 21 is used. (25) A method for screening a compound or a salt thereof which alters the amount of the G protein-coupled receptor protein according to (1) in a cell membrane, which comprises using the quantification method according to (22). [26] a compound or a salt thereof, which can be obtained by using the screening method of [24], which alters the expression level of the G protein-coupled receptor protein of [1]; 27. A compound or a salt thereof, which alters the amount of the G protein-coupled receptor protein of claim 1 in a cell membrane, which can be obtained by using the screening method of claim 25.