JPH09121865A - New g-protein-coupled type receptor protein, its production and use thereof - Google Patents

Abstract

PROBLEM TO BE SOLVED: To produce the subject protein derived from a human brain and human lungs and useful for development, etc., of medicines containing a receptor agonist or antagonist. SOLUTION: This guanine nucleotide-binding protein (G-protein)-coupled type receptor protein derived from a human brain and human lungs or its salt or further a partial peptide of the G-protein coupled type receptor protein or its salt comprises the same or the substantially same amino acid sequence as that of formula I or II. The G-protein coupled type receptor protein is obtained from a tissue of a warm-blooded animal or its cell according to a well-known method for purification.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention encodes a novel G protein-coupled receptor protein derived from human brain and human lung, a salt thereof or a partial peptide thereof, a salt thereof, the G protein-coupled receptor protein or a partial peptide thereof. DNA containing DNA, method for producing G protein-coupled receptor protein or salt thereof or partial peptide or salt thereof, G protein coupled receptor protein (or partial peptide thereof) and DNA thereof
And a method for measuring a ligand using the G protein-coupled receptor protein or cells expressing the same, and a property of changing the binding property to the ligand characterized by using the G protein-coupled receptor protein or cells expressing the same. The present invention relates to a method for screening a receptor agonist or antagonist having, a kit for the screening, an agonist or antagonist obtained by the screening method, and a medicine containing the agonist or antagonist.

[0002]

2. Description of the Related Art Many hormones and neurotransmitters regulate the function of the living body through specific receptor proteins present in the cell membrane. Many of these receptor proteins are guanine nucleotide-binding proteins
(Hereinafter sometimes abbreviated as G protein) to signal intracellularly through activation and to have a common structure having seven transmembrane regions. Collectively referred to as transmembrane receptor proteins. G protein-coupled receptor proteins are present on the surface of various functional cells in living cells and organs,
They play a very important role as targets for molecules that regulate the functions of cells and organs of the living body, such as hormones, neurotransmitters, and physiologically active substances.

To clarify the relationship between substances that regulate complex functions in cells and organs of various organisms and their specific receptors, particularly the specific G protein-coupled receptor proteins expressed by them. , Elucidate the functions of cells and organs of various living organisms, and provide a very important means for drug development closely related to those functions. For example, in central nervous system organs such as the brain, physiological functions of the brain are regulated under the control of many hormones, hormone-like substances, neurotransmitters or physiologically active substances. In particular, neurotransmitters are present in various parts of the brain, and regulate their physiological functions through their corresponding receptors. There are many unknown neurotransmitters in the brain, and regarding the structure of its receptor protein cDNA,
It is considered that there are many that have not been reported so far. Furthermore, it was not known whether there was a known receptor protein subtype. Furthermore, in the respiratory organs such as the lungs, physiological functions of the respiratory system are regulated under the control of many hormones, hormone-like substances, neurotransmitters or physiologically active substances. Particularly in the lungs, various kinds of these physiologically active substances are present, and they fulfill their regulatory functions through receptors corresponding to blood pressure, respiratory activity and the like. In addition, many unknown physiologically active substances are considered to exist in the lungs, and it is considered that many of them have not been reported so far regarding the structure of the receptor protein cDNA. In addition, it is possible that there are known receptor protein subtypes.

Substances that regulate complex functions in the brain,
To clarify the relationship with its specific receptor,
It is a very important tool for drug development. In addition, in order to efficiently screen for agonists and antagonists to receptor proteins and to develop pharmaceuticals, it is necessary to elucidate the functions of receptor protein genes expressed in the brain and to express them in an appropriate expression system. Was needed. Furthermore, clarifying the relationship between substances that regulate complex functions in the respiratory system such as lungs and their specific receptors is a very important means for drug development. In particular, in order to efficiently screen for agonists and antagonists to receptor proteins for regulating the functions of the respiratory system such as lungs, and to develop pharmaceuticals, elucidate the function of the gene for the receptor protein expressed in lungs, It was necessary to express them in an appropriate expression system.

[0005]

DISCLOSURE OF THE INVENTION The present invention provides a novel G protein-coupled receptor protein derived from human brain and human lung, DNA containing a DNA encoding the protein, a method for producing the protein, and the protein and the DNA. The purpose of use is to provide The present invention provides a novel G protein-coupled receptor protein or a salt thereof or a partial peptide or a salt thereof, a DNA containing a DNA encoding the G protein-coupled receptor protein or a partial peptide thereof, a vector containing the DNA, Host transformed with vector, cell membrane fraction obtained from the transformant, method for producing G protein-coupled receptor protein or salt thereof or partial peptide or salt thereof, G protein-coupled receptor protein or salt thereof Method for measuring a ligand for a G protein-coupled receptor using expression cells (including the transformant), method for screening a G protein-coupled receptor agonist or antagonist using the G protein-coupled receptor protein or cells expressing the same, said screening Kit An agonist or antagonist obtained by the screening, a medicine containing an agonist or antagonist to the G protein-coupled receptor protein, an antibody against the G protein-coupled receptor protein, immunization using the G protein-coupled receptor protein or the antibody The present invention provides assay technology, uses of the G protein-coupled receptor protein and its DNA, and the like.

[0006]

[Means for Solving the Problems] In recent years, by utilizing the fact that the G protein-coupled receptor protein shows similarity in amino acid sequence to a part of its structure, polymerase chain
Reaction (Polymerase Chain Reaction: Below, P
The method of searching for a novel receptor protein has come to be carried out by the method (abbreviated as CR). The present inventors
A synthetic DNA primer for more efficient isolation of a DNA encoding a protein-coupled receptor protein was synthesized, and cDNA derived from human brain and cDNA derived from human lung were amplified using the primer, and the analysis was advanced. It was As a result, the present inventors succeeded in isolating a cDNA encoding a novel G protein-coupled receptor protein and determining the partial structure thereof. These G protein-coupled receptor protein cDNAs encode homologous DNAs and amino acids with known G protein-coupled receptors and further encode a novel G protein-coupled receptor protein that is functionally expressed in humans. Thought to be Since the present inventors have isolated these G protein-coupled receptor proteins from human brain and human lung, these G protein-coupled receptor proteins are expressed in human brain and human lung, respectively, and have some function. It is suggested that they are responsible for. From these facts, these receptor proteins are considered to be expressed and function in human brain and human lung, respectively. Using this DNA, cDNA having a full-length translation frame can be obtained, and the receptor protein can be produced.
Alternatively, a human-derived cDNA can be cloned from a human tissue or cell using the cDNA as a primer or a probe. Furthermore, when the receptor protein in which the cDNA encoding the G protein-coupled receptor protein is expressed by an appropriate means is used, it can be used in vivo or in a natural or non-natural state by using a receptor binding experiment or measurement of intracellular second messenger as an index. From the compounds described above, a ligand for the receptor protein can be screened. Furthermore, it is also possible to screen a compound that changes the binding property between the ligand and the receptor protein, for example, a compound that inhibits the binding between the ligand and the receptor protein, or a compound that promotes the binding between the ligand and the receptor protein. I found it.

[0007] More specifically, the present inventors have constructed a novel cDNA fragment derived from human brain as shown in FIG.
Amplification by the CR method, subcloning, and analysis of the sequence thus obtained revealed that the cDNA encoded a novel receptor protein. When this sequence was translated into an amino acid sequence (SEQ ID NO: 1) [FIG. 1], the second, third, fourth, fifth, sixth and seventh transmembrane regions were confirmed on the hydrophobicity plot [ Figure 2]. Also, the size of the amplified cDNA is about 700 bp, which is about the same as that of the known G protein-coupled receptor protein.
Met. When the present inventors conducted a database search using the nucleotide sequence of this DNA as a template, they found that human somatostatin receptor subtype 1 (P30872), which is a known G protein-coupled receptor protein, human somatostatin receptor subtype. Type 2 (P3087
4) and human somatostatin receptor subtype 3 (P32745) with 36 amino acid sequences, respectively.
%, 37% and 41% homology was observed [Fig. 3]. Abbreviations in () above are NBRF-PIR / Swiss-PROT
It is a reference number when it is registered as data in
It is called a cession number.

Furthermore, the inventors of the present invention amplified the human lung-derived novel cDNA fragment shown in FIG. 4 by the PCR method, subcloned it, and analyzed its sequence. It turned out to be coded. When this sequence was translated into an amino acid sequence (SEQ ID NO: 2) [Fig. 4], second, third,
The fourth, fifth, sixth and seventh transmembrane regions were confirmed on the hydrophobicity plot [Fig. 5]. Also, the size of the amplified cDNA was about 700 bp, which is comparable to that of the known G protein-coupled receptor protein. We have:
When a database search was performed using the nucleotide sequence of this DNA as a template, human somatostatin receptor subtype 2 (P30874), human somatostatin receptor subtype 3 (P32745) and 38%, 41% and 39% homology was observed in the amino acid sequences of human somatostatin receptor subtype 1 (P30872), respectively (Fig. 6). The abbreviations in parentheses above are reference numbers when registered as data in NBRF-PIR / Swiss-PROT, and are usually called Accession Numbers.

That is, the present invention provides (1) SEQ ID NO: 1
Or a G protein-coupled receptor protein or a salt thereof, which contains the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 2, or a partial peptide of the G protein-coupled receptor protein, or a portion thereof A salt, (2) a DNA containing a DNA having a nucleotide sequence encoding the G protein-coupled receptor protein or the partial peptide thereof described in (1), (3) represented by SEQ ID NO: 3 or SEQ ID NO: 4 A DNA according to item (2) having a nucleotide sequence, a vector containing the DNA according to (4) item (2) or (3), and a vector according to item (5) (4). Culturing the transformant to be retained, (6) the transformant according to (5),
The G according to item (1), which produces a G protein-coupled receptor protein or a partial peptide thereof.
Method for producing protein-coupled receptor protein or salt thereof or partial peptide or salt thereof, (7) G protein-coupled receptor protein or salt thereof or partial peptide or salt thereof according to item (1), and test sample A method for determining a ligand for a G protein-coupled receptor protein or a salt thereof or a partial peptide thereof or a salt thereof according to (1),

(8) (i) When the ligand is brought into contact with the G protein-coupled receptor protein or salt thereof or the partial peptide or salt thereof according to item (1), and (ii) according to item (1). And a G protein-coupled receptor protein or a salt thereof or a partial peptide or a salt thereof are compared with a case where a ligand and a test compound are contacted with each other, and the G protein-coupled according to item (1). Type receptor protein or a salt thereof or a partial peptide or a method of screening a compound or a salt thereof that changes the binding property to a salt thereof,
(9) A ligand comprising the G protein-coupled receptor protein or salt thereof according to item (1) or a partial peptide or salt thereof, and the G protein-coupled receptor protein according to item (1) or A kit for screening a compound or a salt thereof, which changes the binding property to the salt or a partial peptide thereof or a salt thereof,
(10) An antibody against the G protein-coupled receptor protein or a salt thereof or a partial peptide or a salt thereof according to (1), (11) the screening method according to (8) or the screening according to (9). G protein-coupled receptor agonist or antagonist obtained by using the kit for use, (12) having a nucleotide sequence cloned using the DNA according to item (2) as a primer or probe and encoding a G protein-coupled receptor protein DNA containing DNA, and G protein-coupled receptor protein encoded by DNA cloned by using the DNA described in (13) (2) above as a primer or probe, or a salt thereof, or the G protein-coupled receptor protein Partial peptides of A salt thereof.

More specifically, (14) the protein is the amino acid sequence represented by SEQ ID NO: 1, or 1 or 2 or more amino acids in the amino acid sequence represented by SEQ ID NO: 1 (preferably 1 or more 30 1 or less, more preferably 1
1 or more and 20 or less, more preferably 1 or more and 10 or less amino acids are deleted, and 1 or 2 or more amino acids (preferably 1 or more and 30 amino acids) in the amino acid sequence represented by SEQ ID NO: 1. Or less, more preferably 1
1 or more and 20 or less, more preferably 1 or more and 10 or less amino acids), or 1 or 2 or more amino acids (preferably 1 or more and 30) in the amino acid sequence represented by SEQ ID NO: 1. G or less, more preferably 1 or more and 20 or less, and more preferably 1 or more and 10 or less amino acid), which is a protein containing an amino acid sequence substituted with another amino acid.
Protein-coupled receptor protein or salt thereof, (15)
The protein is the amino acid sequence represented by SEQ ID NO: 2, or 1 or 2 or more amino acids in the amino acid sequence represented by SEQ ID NO: 2 (preferably 1 or more and 30 or less, more preferably 1 or more and 20 or less) , More preferably 1 to 10 amino acids) deleted amino acid sequence,
The amino acid sequence represented by SEQ ID NO: 2 contains 1 or 2 or more amino acids (preferably 1 or more and 30 or less, more preferably 1 or more and 20 or less, more preferably 1 or more and 10 or less amino acids). The added amino acid sequence,
Alternatively, 1 in the amino acid sequence represented by SEQ ID NO: 2
Or an amino acid sequence in which two or more amino acids (preferably 1 or more and 30 or less, more preferably 1 or more and 20 or less, more preferably 1 or more and 10 or less amino acid) are substituted with other amino acids G protein-coupled receptor protein or a salt thereof according to item (1), which is a protein to be used, (16) wherein the ligand is angiotensin, montbesin, bombesin, cannabinoid, cholecystokinin,
Glutamine, serotonin, melatonin, neuropeptide Y, opioid, purine, vasopressin, oxytocin, VIP (vasoactive intestinal and related peptide), somatostatin, dopamine, motilin, amylin, bradykinin, CGR.
P (calcitonin gene relayed peptide), adrenomedullin, leukotriene, pancreatatin, prostaglandin, thromboxane, adenosine, adrenaline, α and β-chemokine (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
RH, pancreatic polypeptide, galanin,
A method for determining a ligand according to the item (6), which is a member of the family,

(17) The case where the labeled ligand is brought into contact with the G protein-coupled receptor protein or its salt or its partial peptide or its salt described in (1), and the labeled ligand and the test compound are treated with ) G protein-coupled receptor protein or a salt thereof or a partial peptide or salt thereof, the labeled ligand described in (1), the G protein-coupled receptor protein or a salt thereof or a portion thereof. The amount of binding to a peptide or a salt thereof is measured and compared with the ligand described in (1) above.
A method for screening a compound or a salt thereof which changes the binding property to a protein-coupled receptor protein or a salt thereof or a partial peptide or a salt thereof, (18) a G protein-coupled receptor protein according to item (1) for labeled ligand Alternatively, when a cell containing a salt thereof or a partial peptide thereof or a salt thereof is brought into contact, the labeled ligand and the test compound are added to the G protein-coupled receptor protein or a salt thereof or a partial peptide thereof or the partial peptide thereof. The G protein-coupled receptor protein or salt thereof according to item (1), which is characterized in that the amount of labeled ligand bound to the cells is measured and compared when the cells are contacted with the salt-containing cells. Or, change the binding property with its partial peptide or its salt Compound or screening method of a compound represented by the formula, (19) G of the labeled ligand subsection (1), wherein
The G protein-coupled receptor protein according to item (1), wherein the labeled ligand and the test compound are brought into contact with a membrane fraction of a cell containing the protein-coupled receptor protein or its salt or its partial peptide or its salt Alternatively, a ligand characterized by measuring the amount of binding of a labeled ligand to a membrane fraction of a cell containing the salt or a partial peptide thereof or a cell membrane fraction containing the salt, and comparing the amount. And a method for screening a compound or its salt, which changes the binding property to the G protein-coupled receptor protein or its salt or its partial peptide or its salt according to (1),

(20) A case where a labeled ligand is brought into contact with a G protein-coupled receptor protein expressed on the cell membrane of the transformant or a partial peptide thereof by culturing the transformant according to (5). Labeling when a labeled ligand and a test compound are contacted with a G protein-coupled receptor protein expressed on the cell membrane of the transformant or a partial peptide thereof by culturing the transformant according to (5) The amount of the bound ligand to the G protein-coupled receptor protein or its partial peptide is measured and compared, and the ligand is bound to the G protein-coupled receptor protein or its partial peptide according to item (1). A method for screening a compound or its salt that alters sex, (2
1) The change in the binding property between the ligand and the G protein-coupled receptor protein or the salt thereof or the partial peptide or the salt thereof according to the item (1) is caused by the ligand and the G protein-coupled type according to the item (1). Any of (8) and (17) to (20), which inhibits or promotes binding to a receptor protein or a salt thereof or a partial peptide thereof or a salt thereof. The method for screening according to any one of (1) to (22), wherein the compound that activates the G protein-coupled receptor protein or salt thereof according to item (1) is a G protein-coupled receptor protein or salt thereof according to item (1) or a salt thereof. When the cells containing the partial peptide or its salt are contacted, the G protein-coupled receptor protein or its salt described in (1) is activated. G protein-coupled receptor protein or a salt thereof when the compound to be converted and a test compound are contacted with cells containing the G protein-coupled receptor protein or a salt thereof or a partial peptide thereof or a salt thereof according to (1) Or measuring the cell stimulating activity via its partial peptide or its salt,
(23) A method for screening a compound or a salt thereof which inhibits binding between a ligand and a G protein-coupled receptor protein or a salt thereof or a partial peptide thereof or a salt thereof according to (1), A G protein-coupled receptor expressed on the cell membrane of the transformant by culturing the transformant according to (5) with a compound that activates the G protein-coupled receptor protein or salt thereof according to item (1). When the protein or its partial peptide is contacted, the compound activating the G protein-coupled receptor protein or its salt described in (1) and the test compound are cultured in the transformant described in (5). By contacting it with the G protein-coupled receptor protein expressed on the cell membrane of the transformant or a partial peptide thereof. In this case, the G protein-coupled receptor protein or a salt thereof or a portion thereof is characterized by measuring and comparing the cell stimulating activity via a G protein-coupled receptor protein or a partial peptide thereof. A method for screening a compound or a salt thereof that inhibits binding to a peptide or a salt thereof,

(24) The compound that activates the G protein-coupled receptor protein or salt thereof according to item (1) is angiotensin, montbesin, bombesin, cannabinoid, cholecystokinin, glutamine, serotonin, melatonin, neuropeptide Y, Opioid, pudding,
Vasopressin, oxytocin, VIP (vasoactive intestinal and related peptide), somatostatin, dopamine, motilin, amylin, bradykinin, CGRP (calcitonin gene relayed peptide), adrenomedullin, leukotriene, pancreatatin, prostaglandin, thrombosis. Xanthine, adenosine, adrenaline, α and β
-Chemokine (IL-8, GROα, GROβ, GRO
γ, NAP-2, ENA-78, PF4, IP10, G
CP-2, MCP-1, HC14, MCP-3, I-3
09, MIP1α, MIP-1β, RANTES, etc.), endothelin, enterogastrin, histamine, neurotensin, TRH, pancreatic polypeptide, galanin, and family members thereof (17) to (23). Any of the screening methods described in (25), (8), and (17)
(26) A compound or salt thereof obtained by the screening method according to any one of (23) to (23), (26) A pharmaceutical composition comprising the compound or salt thereof according to (11), (27) ) A pharmaceutical composition comprising the compound according to item (1) or (13) or a salt thereof, (28) the DNA according to item (2) or (12) or a salt thereof. (29) A pharmaceutical composition comprising the compound according to (25) or a salt thereof,

(30) For screening according to item (9), which comprises cells containing the G protein-coupled receptor protein according to item (1) or a salt thereof, or a partial peptide thereof or a salt thereof. Kit, (3
1) For screening according to item (9), which comprises a G protein-coupled receptor protein according to item (1) or a salt thereof, or a partial peptide thereof or a membrane fraction of a cell containing the salt. Kit, (32) Item (9), (30) or (31) Compound obtained by using the screening kit or salt thereof, (33) Item (32) or its salt A pharmaceutical composition comprising a salt, (34) No. (1)
The antibody according to item (0) is contacted with the G protein-coupled receptor protein according to item (1) or a salt thereof, or a partial peptide thereof or a salt thereof, and the G protein-coupled according to item (1). -Type receptor protein or a salt thereof or a partial peptide or a salt thereof, which comprises the G protein-coupled receptor protein or a salt thereof or a partial peptide or a salt thereof according to (35) (1). Or a cell membrane fraction thereof, wherein the DNA according to (36) (2) is used as a primer or a probe.
(37) A method for screening a DNA having a nucleotide sequence encoding a G protein-coupled receptor protein from an A library,
A method for screening DNA according to the above (36), which is a cell-derived cDNA library, and (38) an example of human tissue is an adrenal gland,
Umbilical cord, brain, tongue, liver, lymph gland, lung, thymus, placenta, peritoneum, retina, spleen, heart, smooth muscle, intestine, blood vessel, bone, kidney,
Skin, fetus, mammary gland, ovary, testis, pituitary gland, pancreas, submandibular gland, spine, prostate, stomach, thyroid, trachea, skeletal muscle, uterus,
Examples include adipose tissue, bladder, cornea, olfactory bulb, bone marrow, amniotic membrane, and examples of human cells include nerve cells, epithelial cells, leukocytes, lymphocytes, glial cells, fibroblasts, keratinocytes,
Provided is a method for screening DNA according to the above (37), which is an osteoblast, an osteoclast, an astrocyte, a melanocyte, various cancer cells, or a cell derived from the human tissue. To do.

Further, the present invention (39) is capable of hybridizing to the DNA described in the above (2), wherein a nucleotide sequence complementary to at least a part of the DNA described in the above (2) is used. Antisense DNA and RNA characterized by containing G (40) (1)
Monoclonal antibody against protein-coupled receptor protein or salt thereof or partial peptide or salt thereof, (41) Purified polyclonal against G protein-coupled receptor protein or salt thereof or partial peptide or salt thereof according to (1) Antibody preparation, (42) (i) Incubating the antibody according to the above (10), (40) or (41) with a sample to be measured to form an antigen-antibody complex,
(Ii) An immunoassay method for detecting a G protein-coupled receptor protein or a partial peptide thereof, which comprises detecting the antigen-antibody complex formed in the above step (i), (43) ( i) G described in the above item (1)
The protein-coupled receptor protein or its salt or its partial peptide or its salt is incubated with the sample to be measured to form an antigen-antibody complex,
(Ii) An immunoassay method for detecting an antibody against a G protein-coupled receptor protein or a partial peptide thereof, which comprises detecting the antigen-antibody complex formed in the above step (i), (44) ) Antisense DNA and RNA according to the above (39), which is in a pharmaceutically acceptable carrier, (45) 2-5
Characterized in that it contains a base sequence of 0.
9) the antisense DNA and RNA described in (4)
6) A G protein-coupled receptor protein or a G protein-coupled receptor protein characterized by bringing the antisense DNA or RNA according to the above (39) into contact with a cell expressing the G protein-coupled receptor protein or a partial peptide thereof Method for controlling / regulating activity of partial peptide, (47)
The antisense DNA or RN according to the above (39).
Controlling the functional expression of a G protein-coupled receptor protein or its partial peptide, which comprises contacting A with a DNA containing a DNA having a nucleotide sequence encoding the G protein-coupled receptor protein or its partial peptide (48) The method according to (48) above, wherein the G protein-coupled receptor protein according to item (28) above or a DNA encoding the partial peptide thereof or a salt thereof is administered to an individual. Method for improving or treating deficiency of protein-coupled receptor protein or salt thereof or partial peptide or salt thereof, and (49) (i) G protein-coupled receptor protein or salt thereof or portion thereof according to (1) above Immunizing an individual with a peptide or salt thereof, (ii) obtained from the immunized individual The cells permanence can be cultured if tighten, (i
ii) selecting a subcultureable cell producing an antibody that reacts with a G protein-coupled receptor protein, and (i
v) A method for producing a hybridoma for producing a monoclonal antibody against the G protein-coupled receptor protein or a salt thereof or a partial peptide thereof or a salt thereof according to item (1), wherein the cells that can be continuously cultured are grown. provide.

[0017]

BEST MODE FOR CARRYING OUT THE INVENTION In the present specification, "substantially the same" means the activity of a protein, for example, the binding activity of a ligand,
It means that physiological properties and the like are substantially the same. Amino acid substitutions, deletions or insertions often do not result in a significant change in the physiological or chemical properties of the polypeptide, in which case the polypeptide with the substitutions, deletions or insertions, may have such substitutions, It would be substantially identical to the one without the deletion or insertion. Substantially identical substitutions of amino acids in the amino acid sequence can be selected from other amino acids of the class to which the amino acid belongs. Non-polar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, methionine and the like. Examples of polar (neutral) amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, glutamine and the like. Examples of positively charged (basic) amino acids include arginine, lysine, and histidine. Examples of negatively charged (acidic) amino acids include aspartic acid and glutamic acid.

The G protein-coupled receptor protein of the present invention includes all tissues of warm-blooded animals (eg, guinea pig, rat, mouse, pig, sheep, cow, horse, rabbit, cat, dog, monkey, human). For example, pituitary, pancreas, brain, kidney, liver, gonad, thyroid, gallbladder,
G protein-coupled receptor protein derived from bone marrow, adrenal gland, skin, muscle, lung, digestive tract, blood vessel, heart, etc.) or cell, which is represented by the amino acid sequence represented by SEQ ID NO: 1 or represented by SEQ ID NO: 2. Any amino acid sequence may be used as long as it contains the same or substantially the same amino acid sequence as the amino acid sequence described above. That is, as the G protein-coupled receptor protein of the present invention, in addition to the protein containing the amino acid sequence represented by SEQ ID NO: 1, the protein containing the amino acid sequence represented by SEQ ID NO: 2, etc., SEQ ID NO: 1 A protein containing an amino acid sequence having a homology of about 90 to 99.9% with the amino acid sequence represented by SEQ ID NO: 2 and having substantially the same activity as the protein containing SEQ ID NO: 1, represented by SEQ ID NO: 2. Examples thereof include a protein containing an amino acid sequence having about 90 to 99.9% homology with the amino acid sequence and having substantially the same activity as the protein containing SEQ ID NO: 2. Examples of substantially the same activity include ligand binding activity,
Examples include signal transmission. "Substantially the same quality" means that the ligand binding activity and the like are qualitatively the same. Therefore, strength and weakness such as strength of ligand binding activity and quantitative factors such as molecular weight of receptor protein may be different.

More specifically, examples of the G protein-coupled receptor protein of the present invention include human brain-derived G protein-coupled receptor protein containing the amino acid sequence represented by SEQ ID NO: 1. The G protein-coupled receptor protein of the present invention includes 1 or 2 or more amino acids (preferably 1 or more and 30 or less, more preferably 1 or 20 or more) in the amino acid sequence represented by SEQ ID NO: 1. Hereinafter, more preferably, an amino acid sequence in which 1 to 10 amino acids are deleted, SEQ ID NO: 1
An amino acid sequence in which 1 or 2 or more amino acids (preferably 1 or more and 30 or less, more preferably 1 or more and 20 or less, more preferably 1 or more and 10 or less amino acid) are added to the amino acid sequence represented by , SEQ ID NO: 1
1 or 2 or more amino acids (preferably 1 or more and 30 or less, more preferably 1 or more and 20 or less, more preferably 1 or more and 10 or less amino acid) in the amino acid sequence represented by Also included are proteins containing the amino acid sequence substituted with. Examples of the G protein-coupled receptor protein of the present invention include human lung-derived G protein-coupled receptor protein containing the amino acid sequence represented by SEQ ID NO: 2.
The G protein-coupled receptor protein of the present invention includes 1 or 2 or more amino acids (preferably 1 or more and 30 or less, more preferably 1 or more and 20) in the amino acid sequence represented by SEQ ID NO: 2. In the following, more preferably, 1 or more and 10 or less amino acids are deleted, or 1 or 2 or more amino acids (preferably 1 or more and 30 or less, further preferably in the amino acid sequence represented by SEQ ID NO: 2). Is 1 or more and 20 or less, more preferably 1 or more and 10 or less amino acid), 1 in the amino acid sequence represented by SEQ ID NO: 2.
Alternatively, it contains an amino acid sequence in which two or more amino acids (preferably 1 or more and 30 or less, more preferably 1 or more and 20 or less, more preferably 1 or more and 10 or less) are substituted with other amino acids. Also included are proteins that do. Further, in the G protein-coupled receptor protein of the present invention, Met at the N-terminus is protected by a protecting group (for example, C _1-6 acyl group such as formyl group and acetyl group), N-terminal of Glu. The side is cut in vivo and the G
A compound in which lu is pyroglutaminated, a compound in which the side chain of an amino acid in the molecule is protected with an appropriate protecting group (eg, C _1-6 acyl group such as formyl group, acetyl group, etc.), or a sugar chain is bound It also includes complex proteins such as the so-called glycoproteins. Furthermore, in the G protein-coupled receptor protein of the present invention, DN containing a nucleotide sequence encoding the same or substantially the same amino acid sequence as the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 of the present invention.
It will also be understood that it may be a G protein-coupled receptor protein or a salt thereof encoded by DNA cloned using A as a primer or a probe, or a partial peptide of the G protein-coupled receptor protein or a salt thereof. More specifically, DN cloned by using the DNA itself of SEQ ID NO: 3 or SEQ ID NO: 4 of the present invention or a partial sequence thereof or a DNA based on the information thereof as a primer or a probe.
It will also be understood that it may be a G protein-coupled receptor protein encoded by A or a salt thereof or a partial peptide of the G protein-coupled receptor protein or a salt thereof.

The salt of the G protein-coupled receptor protein of the present invention is preferably a physiologically acceptable acid addition salt. 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) And tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid and benzenesulfonic acid). The G protein-coupled receptor protein of the present invention or a salt thereof can be produced from a tissue or cell of a warm-blooded animal by a method known per se for protein purification. It can also be produced by culturing the transformant containing it. Also,
It can also be produced according to the peptide synthesis method described below.
As the partial peptide of the G protein-coupled receptor protein of the present invention, for example, a portion of the G protein-coupled receptor protein molecule of the present invention that is exposed outside the cell membrane is used. Specifically, the portion of the G protein-coupled receptor protein of the present invention shown in FIG. 2 or FIG. 4 which is analyzed to be an extracellular region (hydrophilic region) in the hydrophobicity plot analysis. Is a peptide containing. 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. As the salt of the partial peptide of the G protein-coupled receptor protein of the present invention, a physiologically acceptable acid addition salt is particularly preferable. Such salts include, for example, inorganic acids (for example,
Salts with hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid) or organic acids (eg acetic acid, formic acid, propionic acid, fumaric acid,
Maleic acid, succinic acid, tartaric acid, citric acid, malic acid,
Salts with oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid, etc. are used.

The partial peptide of the G protein-coupled receptor protein of the present invention or a salt thereof is produced according to a peptide synthesis method known per se, or by cleaving the G protein-coupled receptor protein of the present invention with an appropriate peptidase. be able to. Furthermore, it can also be produced by culturing a transformant containing a DNA encoding the partial peptide. The peptide synthesis method may be either a solid phase synthesis method or a liquid phase synthesis method. That is, the target peptide can be produced by condensing a partial peptide or amino acid 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. Examples of known condensation methods and elimination of protective groups include the methods described in the following (1) to (3). M. Bodanszky and MA Ondetti, Peptide Synthesis, Interscience Publisher
s, New York (1966) Schroeder and Luebke, The Peptid
e), Academic Press, N York (1965) Nobuo Izumiya et al., Fundamentals and experiments of peptide synthesis, Maruzen Co., Ltd.
(1975) Haruaki Yajima and Shunpei Sakakibara, Biochemistry Laboratory 1, Protein Chemistry IV, 205, (1977) Supervised by Haruaki Yajima, Development of Pharmaceuticals Volume 14 Peptide Synthesis Hirokawa Shoten The protein of the present invention can be purified and isolated by a combination of purification methods such as salting out, solvent extraction, distillation, column chromatography, liquid chromatography, electrophoresis, recrystallization and the like. When the protein obtained by the above method is a free form, it can be converted into an appropriate salt by a known method, and conversely, when it is obtained with a salt, it can be converted into a free form by a known method. it can.

The DNA encoding the G protein-coupled receptor protein of the present invention encodes a G protein-coupled receptor protein containing the same or substantially the same amino acid sequence as the amino acid sequence of SEQ ID NO: 1 of the present invention. That contains a base sequence that encodes a G protein-coupled receptor protein that contains the same or substantially the same amino acid sequence as the amino acid sequence of SEQ ID NO: 2. It may be. Further, it may be any of human genomic DNA, human genomic DNA library, human tissue / cell-derived cDNA, human tissue / cell-derived cDNA library, and synthetic DNA. The vector used for the library may be any of bacteriophage, plasmid, cosmid, phagemid and the like. In addition, from tissue / cell, mRN
Direct reverse transcrip using the prepared A fraction
tase Polymerase Chain Reaction (hereinafter RT-PCR
Abbreviated as law. ). More specifically, as the DNA encoding the human brain-derived G protein-coupled receptor protein containing the amino acid sequence of SEQ ID NO: 1, DNA having the nucleotide sequence represented by SEQ ID NO: 3, SEQ ID NO: 2 As the DNA encoding the human lung-derived G protein-coupled receptor protein containing the amino acid sequence of, the DNA having the nucleotide sequence represented by SEQ ID NO: 4 is used. Furthermore, DN encoding the G protein-coupled receptor protein of the present invention
As A, SEQ ID NO: 1 of the present invention or SEQ ID NO:
2 was cloned by using as a primer or a probe a DNA containing a nucleotide sequence encoding a G protein-coupled receptor protein containing the same or substantially the same amino acid sequence as the amino acid sequence of 2 and encodes a G protein-coupled receptor protein. D having a base sequence that
It will also be understood that NA is good. More specifically, it is cloned using the DNA of SEQ ID NO: 3 or SEQ ID NO: 4 of the present invention itself or a partial sequence thereof or a DNA based on the information as a primer or a probe, and encodes a G protein-coupled receptor protein. It will also be understood that it may be DNA containing DNA having a base sequence.

As a means for cloning the DNA which completely encodes the G protein-coupled receptor protein of the present invention, a synthetic DNA primer having a partial base sequence of the G protein-coupled receptor protein is used for amplification by the PCR method, Alternatively, the DNA incorporated into an appropriate vector
Are selected by hybridization with a DNA fragment having a part or whole region of human G protein-coupled receptor protein or labeled with a synthetic DNA. Hybridization methods are described, for example, in Molec
ular Cloning 2nd (ed .; J. Sambrook et al., Cold S
pring Harbor Lab. Press, 1989). When a commercially available library is used, the procedure is performed according to the method described in the attached instruction manual. This PCR amplification method can be carried out according to a known PCR method. For example, Saiki RK et al. Science, 23.
9: 487-491 (1988). PCR temperature, time, buffer, cycle number, enzymes such as DNA polymerase, 2'-deoxy-7-deaza
The addition of -guanosine triphosphate or Inosine can be appropriately selected according to the type of target DNA.
When RNA is used as a template, Saiki RK et
al. Science, 239: 487-491 (1988). The cloned DNA encoding the G protein-coupled receptor protein can be used as it is, or if desired after digestion with a restriction enzyme or addition of a linker, depending on the purpose. The DNA is the 5 '
It has ATG as a translation initiation codon on the terminal side, and TAA and TGA as translation termination codons on the 3'terminal side.
Alternatively, it may have a TAG. These translation initiation codon and translation termination codon can also be added using a suitable synthetic DNA adapter. The G protein-coupled receptor protein expression vector is, for example, (a) a DNA fragment of interest is excised from the DNA encoding the G protein-coupled receptor protein of the present invention, and (b) the DNA fragment is used in an appropriate expression vector. It can be produced by ligating to the downstream of the promoter. As the vector, a plasmid derived from E. coli (eg, pBR322, pB
R325, pUC12, pUC13, etc.), plasmids derived from Bacillus subtilis (eg, pUB110, pTP5, pC19)
4, etc.), yeast-derived plasmid (eg, pSH19, pS
H15, etc.), bacteriophages such as λ phage,
Animal viruses such as retrovirus, vaccinia virus, and baculovirus 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.

When the host for transformation is a bacterium of the genus Escherichia, the trp promoter, the lac promoter,
rec A promoter, λPL promoter, lpp promoter, etc., when the host is Bacillus, SP
When the host is yeast, such as O1 promoter, SPO2 promoter and pen P promoter, PHO5 promoter, PGK promoter, GAP promoter,
The ADH promoter and the like are preferable. When the host is an animal cell, an SV40-derived promoter, a retrovirus promoter, a metallothionein promoter, a heat shock promoter, a cytomegalovirus promoter, an SRα promoter, and the like can be used. The use of enhancers for expression is also effective. In addition, if necessary, a signal sequence suitable for the host is added to the N-terminal side of the G protein-coupled receptor protein. When the host is a bacterium of the genus Escherichia, alkaline phosphatase signal sequence, OmpA signal sequence, etc., when the host is a bacterium of the genus Bacillus, α-amylase signal sequence, subtilisin signal sequence, etc., the host is yeast When the host is an animal cell, for example, an insulin signal sequence, an α-interferon signal sequence, an antibody molecule / signal sequence, etc., respectively. Available. A transformant is produced using the vector containing the DNA encoding the G protein-coupled receptor protein thus constructed.

As the host, for example, Escherichia genus, Bacillus genus, yeast, insect, animal cell and the like are used. Specific examples of the genus Escherichia and the genus Bacillus include Escherichia coli K12.
DH1 [Proc. Natl. Acad. Sci. USA], 60, 16
0 (1968)], JM103 [Nucleic Acids Research], Volume 9, 3.
09 (1981)], JA221 [Journal of
Molecular Biology (Journal of Molecular Bi
ology), 120 volumes, 517 (1978)], HB10
1 [Journal of Molecular Biology,
41, 459 (1969)], C600 [Genetics, 39, 440 (1954)] and the like. Examples of the genus Bacillus include Bacillus subtilis MI114 [Gene, 24, 255 (1983)], 207-21.
[Journal of Biochemistry
Biochemistry), Vol. 95, 87 (1984)] and the like.

Examples of yeast include Saccaromyces cerevisiae AH22, A
^{H22R -, NA87-11A, DKD-5D} , 20B
-12 or the like is used. As insects, for example, silkworm larvae are used [Maeda et al., Nature (Natur
e), Volume 315, 592 (1985)]. Examples of animal cells include monkey cells COS-7, Vero, Chinese hamster cells CHO, DHFR gene-deficient Chinese hamster cells CHO (dhfr ^- CHO cells), mouse L cells, mouse myeloma cells, human FL.
Cells and the like are used. For transformation of Escherichia, for example, Proceedings of the National Academy of Sciences of the
USA (Proc. Natl. Acad. Sci. USA), 69
Volume, 2110 (1972) and Gene, Volume 17,
107 (1982) and the like. To transform Bacillus, for example, Molecular and General Genetics (Mole
cular & General Genetics), 168, 111 (1
979) and the like. For transforming yeast, for example, Proceeds of the National Academy of Sciences of the USA (Proc. Natl. Acad. Sci. US
A), Vol. 75, 1929 (1978).

For transforming insect cells, for example, Bio / Technology, 6, 47-55 (198)
8) It is performed according to the method described in). To transform animal cells, for example, Virolog
y), 52, 456 (1973). Thus, a transformant transformed with the expression vector containing the DNA encoding the G protein-coupled receptor protein is obtained. When culturing a transformant whose host is a genus Escherichia or Bacillus, a liquid medium is suitable as a medium used for the culturing, and a carbon source necessary for growth of the transformant is used therein. Nitrogen sources, inorganic substances, etc. are contained. Examples of the carbon source include glucose, dextrin, soluble starch,
Examples of nitrogen sources such as sucrose include inorganic or organic substances such as ammonium salts, nitrates, corn steep liquor, peptone, casein, meat extract, soybean meal and potato extract, and examples of inorganic substances include calcium chloride and phosphoric acid. Examples thereof include sodium dihydrogen and magnesium chloride. In addition, yeast, vitamins, growth promoting factors and the like may be added. The pH of the medium is preferably about 5-8.

As a medium for culturing Escherichia, for example, M9 medium containing glucose and casamino acid [Miller, Journal of Experiments in Molecular Genetics (Journa
l of Experiments in Molecular Genetics), 431-
433, Cold Spring Harbor Laboratory, New York 1
972] is preferred. If necessary, in order to make the promoter work efficiently, for example, 3β-indolyl is used.
An agent such as acrylic acid can be added. When the host is a bacterium belonging to the genus Escherichia, the cultivation is usually carried out at about 15 to 43 ° C. for about 3 to 24 hours, and if necessary, aeration and stirring can be applied. When the host is a bacterium belonging to the genus Bacillus, the cultivation is usually carried out at about 30 to 40 ° C. for about 6 to 24 hours, and if necessary, aeration and stirring can be applied. When culturing a transformant whose host is yeast, examples of the medium include Burkholder minimal medium [Bostian, KL et al.
Procedures of the National Academy of Sciences of the USA (Pr
oc. Natl. Acad. Sci. USA), 77, 4505 (1
980)] or an SD medium containing 0.5% casamino acid [B
itter, GA et al., Proc. Natl. Acad. Sci. USA, Proc. of the National Academy of Sciences of the USA, 8
1, 5330 (1984)]. Medium p
H is preferably adjusted to about 5-8. Culture is usually about 2
It is performed at 0 ° C to 35 ° C for about 24 to 72 hours, and aeration and stirring are added if necessary.

When culturing a transformant whose host is an insect, Grace's Insect Medium (Grace,
TCC, Nature, 195, 788 (1962)) to which an additive such as immobilized 10% bovine serum is appropriately added may be used. The pH of the medium is preferably adjusted to about 6.2 to 6.4. Culturing is usually performed at about 27 ° C. for about 3 to 5 days, and aeration and agitation are added if necessary. When culturing a transformant whose host is an animal cell, the medium is, for example, MEM medium containing about 5 to 20% fetal bovine serum [Science, Volume 122, 501 (1952)],
DMEM medium [Virology, 8 volumes, 39
6 (1959)], RPMI1640 medium [The Jounal of the American Medical Associatio (Journal of the American Medical Association
n) 199, 519 (1967)], 199 medium [Procedure of the Society for the.
Biological Medicine (Proceeding of the Soci
ety for the Biological Medicine), 73, 1 (1
950)] and the like are used. Preferably, the pH is between about 6-8. Culturing is usually performed at about 30 to 40 ° C for about 15 to 6
Perform for 0 hours and add aeration and agitation as needed.

The G protein-coupled receptor protein can be separated and purified from the culture described above, for example, by the following method. When the G protein-coupled receptor protein is extracted from cultured cells or cells, the cells or cells are collected by a known method after culturing, and the cells or cells are suspended in an appropriate buffer solution and subjected to ultrasonic wave, lysozyme and / or freezing. A method of obtaining a crude extract of G protein-coupled receptor protein by disrupting cells or cells by thawing and then centrifuging or filtering can be appropriately used. In the buffer solution, protein denaturants such as urea and guanidine hydrochloride,
A surfactant such as Triton X-100 (registered trademark; hereinafter, sometimes abbreviated as TM) may be included. When the G protein-coupled receptor protein is secreted into the culture medium, after completion of the culture, the cells or cells are separated from the supernatant by a method known per se, and the supernatant is collected. Purification of the G protein-coupled receptor protein contained in the thus obtained culture supernatant or the extract can be carried out by appropriately combining separation / purification methods known per se. 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 G protein-coupled receptor protein thus obtained is obtained as a free form, it can be converted into a salt by a method known per se or a method analogous thereto, and conversely, when it is obtained as a salt. The free form or other salt can be converted by a method known per se or a method analogous thereto. The G protein-coupled receptor protein produced by the recombinant can be optionally modified or partially removed by reacting it with an appropriate protein-modifying enzyme before or after purification. . Examples of the protein-modifying enzyme include trypsin, chymotrypsin, arginyl endopeptidase, protein kinase, glycosidase and the like. The activity of the G protein-coupled receptor protein thus produced can be measured by a binding experiment with a labeled ligand, an enzyme immunoassay using a specific antibody, or the like. The DNA encoding the G protein-coupled receptor protein of the present invention and the G protein-coupled receptor protein are
Method for determining ligand for G protein-coupled receptor protein of the present invention, acquisition of antibody and antiserum, construction of recombinant receptor protein expression system, development of receptor binding assay system using the same expression system, and drug candidate compound It can be used for screening, implementation of drug design based on comparison with structurally similar ligands / receptors, preparation of probes in gene diagnosis, preparation of PCR primers, gene therapy and the like. Especially,
By the receptor binding assay system using the expression system of the recombinant G protein-coupled receptor protein of the present invention, a G protein-coupled receptor agonist or antagonist specific to warm-blooded animals such as humans can be screened. An agonist or an antagonist can be used as a preventive / therapeutic agent for various diseases. G protein-coupled receptor protein of the present invention, partial peptide, DN encoding G protein-coupled receptor protein
The use of A and the antibody will be specifically described below. A DNA encoding the G protein-coupled receptor protein of the present invention, a G protein-coupled receptor protein encoded by the DNA, a partial peptide thereof or a salt thereof (hereinafter, referred to as a salt and simply referred to as "G protein-coupled receptor protein") Alternatively, it may be abbreviated as "G protein-coupled receptor protein or its partial peptide"), and the utility of cells containing recombinant G protein-coupled receptor protein or cell membrane fractions thereof will be described in more detail. To do.

(1) Method for determining ligand for G protein-coupled receptor protein of the present invention The G protein-coupled receptor protein of the present invention or a salt thereof or the partial peptide of the present invention or a salt thereof is a G protein-coupled type of the present invention. It is useful as a reagent for searching or determining a ligand for a receptor protein. That is, the present invention provides the G protein-coupled receptor protein of the present invention or a salt thereof, or the partial peptide of the present invention or a salt thereof,
Provided is a method for determining a ligand for a G protein-coupled receptor protein of the present invention, which comprises contacting with a test sample such as a test compound. As the test sample, known ligands (for example, angiotensin, montbesin, bombesin, cannabinoid, cholecystokinin, glutamine, serotonin, melatonin, neuropeptide Y, opioid, purine, vasopressin, oxytocin, VIP (vasoactive intestinal and related). Peptide), somatostatin, dopamine, motilin, amylin, bradykinin, CGRP
(Calcitonin gene relayed peptide), adrenomedullin, leukotriene, pancreatatin, prostaglandin, thromboxane, adenosine, adrenaline, α- and β-chemokine (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
RH, pancreatic polypeptide, galanin,
In addition to their modified derivatives, their analogs, their family members, etc.) or their new modified derivatives, their analogs, their family members, etc., for example, warm blood Animals (eg,
Mouse, rat, pig, cow, sheep, monkey, human etc.) tissue extracts, cell culture supernatants and the like are used. For example, the tissue extract, cell culture supernatant, etc. may be added to the G protein-coupled receptor protein of the present invention and fractionated while measuring the cell stimulating activity and the like to finally obtain a single ligand. . In particular, those which can be detected by biological activity or biological response reaction can be preferably measured.

Specifically, the method for determining a ligand of the present invention uses the G protein-coupled receptor protein of the present invention or a salt thereof, or the partial peptide of the present invention or a salt thereof, or expresses a recombinant receptor protein. By constructing a system and using a receptor binding assay system using the expression system, a cell stimulating activity (eg, arachidonic acid release, binding to a G protein-coupled receptor protein,
Acetylcholine release, intracellular Ca ²⁺ release, intracellular cAM
P production, intracellular cGMP production, inositol phosphate production, cell membrane potential fluctuation, intracellular protein phosphorylation, cf
os activation, a sample having a compound having an activity of promoting or suppressing a decrease in pH, etc. (for example, a peptide, a protein, a non-peptidic compound, a synthetic compound, a fermentation product, etc.) or a salt of the compound, etc. It is a way to decide. In the ligand determination method of the present invention, when the G protein-coupled receptor protein of the present invention or the partial peptide of the present invention is contacted with a sample such as a test compound,
For example, the binding amount of a sample such as a test compound to the G protein-coupled receptor protein or the partial peptide, and the cell stimulating activity are measured.

More specifically, in the present invention, the labeled test compound or the like is labeled when it is brought into contact with the G protein-coupled receptor protein of the present invention or a salt thereof or the partial peptide of the present invention or a salt thereof. A method for determining a ligand for a G protein-coupled receptor protein, which comprises measuring the binding amount of a test compound or the like to the protein or a salt thereof, or the partial peptide or a salt thereof, a labeled test compound, etc. A G protein characterized by measuring the amount of a labeled test compound or the like bound to a cell containing the G protein-coupled receptor protein or a membrane fraction of the cell and the membrane fraction of the cell. A method for determining a ligand for a coupled receptor protein, a labeled test compound, etc. can be used as the G of the present invention. In the case of contacting the G protein coupled receptor protein expressed on the cell membrane by culturing a transformant containing DNA encoding the white matter coupled receptor protein,
G characterized by measuring the binding amount of the labeled test compound or the like to the G protein-coupled receptor protein
A method for determining a ligand for a protein-coupled receptor protein,

When a test compound or the like is brought into contact with cells containing the G protein-coupled receptor protein of the present invention, cell stimulating activity mediated by the G protein-coupled 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-fos activation,
A method for determining a ligand for a G protein-coupled receptor protein, which comprises the activity of promoting or suppressing a decrease in pH, etc., and a test compound, etc. Cell-stimulating activity mediated by a G protein-coupled receptor protein (for example, arachidonic acid release, when contacted with a G protein-coupled receptor protein expressed on a cell membrane by culturing a transformant containing the encoding DNA) Acetylcholine release, intracellular C
a ²⁺ release, intracellular cAMP production, intracellular cGMP production,
G protein-coupled receptor characterized by measuring inositol phosphate production, cell membrane potential fluctuation, intracellular protein phosphorylation, c-fos activation, activity promoting or suppressing pH decrease, etc. A method for determining a ligand for a protein is provided.

A specific description of the ligand determination method of the present invention will be given below. First, any G protein-coupled receptor protein used in the method for determining a ligand may be used as long as it contains the G protein-coupled receptor protein of the present invention or a partial peptide of the G protein-coupled receptor protein of the present invention. However, a G protein-coupled receptor protein expressed in large quantities using animal cells is suitable. To produce a G protein-coupled receptor protein,
The above-mentioned method can be used, but DN encoding the protein is used.
It can be carried out by expressing A in mammalian cells or insect cells. Although complementary DNA is used as the DNA fragment encoding the target portion, it is not necessarily limited thereto. For example, a gene fragment or a synthetic DNA may be used. To introduce DNA fragments encoding G protein-coupled receptor proteins into host animal cells and to express them efficiently, in order to efficiently express them, a nuclear polyhedrosis virus (nuclea) belonging to a baculovirus using insects as a host (nuclea
r polyhedrosis virus (NPV) polyhedrin promoter, SV40-derived promoter, retrovirus promoter, metallothionein promoter, human heat shock promoter, cytomegalovirus promoter, SRα promoter and the like are preferably incorporated downstream. The amount and quality of the expressed receptor can be examined by a method known per se. For example, the literature [Nambi, P. Et al., The Journal of Biological Chemistry (J. Biol. Chem.), Vol. 267, 19555-.
19559, 1992].

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

The amount of the G protein-coupled receptor protein in the cell or the membrane fraction containing the G protein-coupled receptor protein is preferably 10 ³ to 10 ⁸ molecules per cell, and 10 ⁵ to 10 ⁷ It is preferably a molecule. The higher the expression level, the higher the ligand binding activity (specific activity) per membrane fraction, 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 determine the ligand binding to the G protein-coupled receptor protein, the above-mentioned method (1) requires an appropriate G protein-coupled receptor fraction and a labeled test compound. As the G protein-coupled receptor fraction, a natural G protein-coupled receptor fraction or a recombinant G protein-coupled receptor fraction having an activity equivalent to that is desirable.
Here, the equivalent activity refers to equivalent ligand binding activity and the like. Labeled test compounds include [ ³ H], [
¹²⁵ I], [ ¹⁴ C], [ ³⁵ S] etc. labeled angiotensin, monbesin, bombesin, cannabinoid, cholecystokinin, glutamine, serotonin, melatonin, neuropeptide Y, opioid, purine, vasopressin, oxytocin, VIP (vaso Active intestinal and related peptide), somatostatin, dopamine, motilin, amylin, bradykinin, CGRP (calcitonin gene relayed peptide), adrenomedullin, leukotriene,
Pancreatatin, prostaglandins, thromboxane, adenosine, adrenaline, α and β-chemok
ine (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, galanin, and analogs thereof are preferable.

Specifically, in order to carry out a method for determining a ligand that binds to a G protein-coupled receptor protein, first, G
A receptor preparation is prepared by suspending cells or a membrane fraction of cells containing a protein-coupled receptor protein in a buffer suitable for the determination method. Any buffer may be used as long as it does not inhibit the binding between the ligand and the receptor, such as a phosphate buffer having a pH of 4 to 10 (preferably a pH of 6 to 8) and a Tris-hydrochloric acid buffer. In addition, for the purpose of reducing non-specific binding,
Surfactants such as CHAPS, Tween-80 ^™ (Kao-Atlas), digitonin and deoxycholate, and various proteins such as bovine serum albumin and gelatin may be added to the buffer. In addition, PM is used to suppress the decomposition of receptors and ligands by protease.
SF, leupeptin, E-64 (made by Peptide Institute),
Protease inhibitors such as pepstatin can also be added. A fixed amount (5000 cpm to 500,000 cpm) of a test compound labeled with [ ³ H], [ ¹²⁵ I], [ ¹⁴ C], [ ³⁵ S] or the like is allowed to coexist with 0.01 ml to 10 ml of the receptor solution. Non-specific binding (N
To know SB), a reaction tube containing a large excess of unlabeled test compound and the like is also prepared. The reaction is performed at 0 ° C to 50
C., preferably 4 to 37.degree. C. for 20 minutes to 24 hours,
Desirably, it is carried out for 30 minutes to 3 hours. After the reaction, the mixture is filtered through a glass fiber filter paper or the like, washed with an appropriate amount of the same buffer, and the radioactivity remaining on the glass fiber filter paper is measured with a liquid scintillation counter or a γ-counter. A test compound having a count (B-NSB) obtained by subtracting the nonspecific binding amount (NSB) from the total binding amount (B) exceeding 0 cpm can be selected as a ligand for the G protein-coupled receptor protein of the present invention.

In order to carry out the above-mentioned methods (1) to (3) for determining a ligand that binds to a G protein-coupled receptor protein, a cell stimulating activity mediated by the G protein-coupled receptor protein (eg, arachidonic acid release, acetylcholine release, cell Intracellular Ca ²⁺ release, intracellular cAMP production, intracellular cGMP production, inositol phosphate production, cell membrane potential fluctuation, intracellular protein phosphorylation, c-fos activation, p
Activity that promotes or suppresses reduction of H, etc.)
Can be measured using a known method or a commercially available measurement kit. Specifically, first, cells containing the G protein-coupled receptor protein are cultured in a multiwell plate or the like. Prior to ligand determination, replace the medium with a fresh medium or an appropriate buffer that is not toxic to cells, add test compounds, etc., incubate for a certain period of time, and then extract cells or collect supernatant to generate The quantified product is quantified according to each method. When the production of a substance that serves as an index of cell stimulating activity (for example, arachidonic acid) is difficult to assay with a degrading enzyme contained in cells, an inhibitor for the degrading enzyme may be added to the assay. Further, the activity of suppressing cAMP production and the like can be detected as a production suppressing action on cells in which the basic production amount of cells has been increased by forskolin or the like.

The kit for determining a ligand that binds to the G protein-coupled receptor protein of the present invention comprises a G protein-coupled receptor protein of the present invention or a salt thereof, a partial peptide of the G protein-coupled receptor protein of the present invention or a salt thereof, It contains a membrane fraction of cells containing the G protein-coupled receptor protein of the present invention or cells containing the G protein-coupled receptor protein of the present invention. Examples of the ligand determination kit of the present invention include the following. 1. Reagent for ligand determination Assay buffer and wash buffer Hanks' Balanced Salt Solution (manufactured by Gibco)
To which 0.05% bovine serum albumin (manufactured by Sigma) was added. Sterilize by filtration with a 0.45μm filter,
It may be stored at 4 ° C or may be prepared at the time of use. G protein-coupled receptor protein preparation CHO cells expressing the G protein-coupled receptor protein were subcultured on a 12-well plate at 5 × 10 ⁵ cells / well and 3
One cultured for 2 days at 7 ° C. and 5% CO ₂ 95% air. Labeled test compound Compound labeled with commercially available [ ³ H], [ ¹²⁵ I], [ ¹⁴ C], [ ³⁵ S], etc. 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 labeled compound is prepared at a concentration of 100 to 1000 times higher.

2. Assay method G-protein coupled receptor protein-expressing CHO cells cultured in 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 the mixture is reacted at room temperature for 1 hour. To know the amount of non-specific binding, 5 μl of unlabeled test compound is added. Remove the reaction solution and wash 3 times with 1 ml of washing buffer. The labeled test compound bound to the cells was treated with 0.2 N NaO.
It is dissolved in H-1% SDS and mixed with 4 ml of liquid scintillator A (manufactured by Wako Pure Chemical Industries). Liquid scintillation counter (Beckman)
Is used to measure the radioactivity. Examples of the ligand capable of binding to the G protein-coupled receptor protein of the present invention include brain, lung, pituitary gland,
Examples thereof include substances specifically present in the pancreas and the like, and specifically, angiotensin, monbesin, bombesin, cannabinoid, cholecystokinin, glutamine, serotonin, melatonin, neuropeptide Y, opioid, purine, vasopressin, oxytocin, VIP ( Vasoactive intestinal and related peptide), somatostatin, dopamine, motilin, amylin, bradykinin, CGRP (calcitonin gene relayed peptide), adrenomedullin, leukotriene, pancreatatin, prostaglandin, thromboxane, adenosine, adrenaline, α and β-chemokine (IL-8, GROα, GROβ,
GROγ, NAP-2, ENA-78, PF4, IP1
0, GCP-2, MCP-1, HC14, MCP-3,
I-309, MIP1α, MIP-1β, RANTES
Etc.), endothelin, enterogastrin, histamine, neurotensin, TRH, pancreatic polypeptide, galanin, modified derivatives thereof, related derivatives thereof, family members thereof, and the like.

(2) Preventive / therapeutic agent for G protein-coupled receptor protein deficiency of the present invention If the ligand for the G protein-coupled receptor protein of the present invention is identified in the method of (1) above, the ligand has the ligand. Depending on the action, the DNA encoding the G protein-coupled receptor protein of the present invention can be used as a prophylactic / therapeutic agent for G protein-coupled receptor protein deficiency. For example, when there is a patient in whom the physiological action of the ligand cannot be expected because the G protein-coupled receptor protein of the present invention is reduced in vivo,
(A) by administering the DNA encoding the G protein-coupled receptor protein of the present invention to the patient and expressing it, or (b) encoding the G protein-coupled receptor protein of the present invention in brain cells, lung cells, etc. DNA
By inserting and expressing the brain cells or lung cells into the patient, the amount of G protein-coupled receptor protein in the brain cells or lung cells of the patient is increased, and the action of the ligand is sufficiently increased. Can be demonstrated. Therefore, the DNA encoding the G protein-coupled receptor protein of the present invention can be used as a safe and low toxic preventive / therapeutic agent for the G protein-coupled receptor protein deficiency of the present invention. When the DNA of the present invention is used as the above-mentioned therapeutic agent, the DNA may be used alone or after inserting it into an appropriate vector such as a retrovirus vector, an adenovirus vector, an adenovirus associated virus vector, etc., and then performing it according to a conventional method. it can. The agent can be used in various pharmaceutical compositions or preparations. For example, orally as tablets, capsules, elixirs, microcapsules, etc., optionally sugar-coated, or aseptic solution with water or other pharmaceutically acceptable liquid, or suspension It can be used parenterally in the form of injections such as. For example, the DNA of the present invention together with physiologically acceptable carriers, flavors, excipients, adjuvants, diluents, vehicles, preservatives, stabilizers, binders, etc., generally accepted unit dosage required for the implementation of the formulation. It can be manufactured by mixing in the form. 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 incorporated into 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. 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, a liquid carrier such as oil and fat can be further contained in the above-mentioned type of material. Sterile compositions for injection can be formulated according to conventional pharmaceutical practices such as dissolving or suspending the active substance in a vehicle such as water for injection, naturally occurring vegetable oils such as sesame oil, coconut oil, etc. . Examples of the aqueous solution for injection include physiological saline, isotonic solution containing glucose and other adjuvants (eg, D-sorbitol, D-mannitol, sodium chloride, etc.), and a suitable solubilizing agent such as alcohol. (Eg ethanol), polyalcohol (eg propylene glycol, polyethylene glycol etc.), nonionic surfactant (eg polysorbate 80 (TM), HCO-50 etc.) etc. may be used in combination. Examples of the oily liquid include sesame oil and soybean oil, which may be used in combination with solubilizing agents such as benzyl benzoate and benzyl alcohol.

Further, a buffering agent (eg, phosphate buffer,
Sodium acetate buffer etc.), soothing agent (eg benzalkonium chloride, procaine hydrochloride etc.), stabilizer (eg human serum albumin, polyethylene glycol etc.), preservative (eg benzyl alcohol, phenol etc.), oxidation You may mix | blend with an inhibitor etc. 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, for example, to warm-blooded mammals (eg, rats, rabbits, sheep, pigs, cows, cats, dogs, monkeys, humans, etc.). it can. Although the dose of the DNA varies depending on the symptoms and the like, when administered orally, it is generally adult (60
(as kg), about 0.1 mg to 1 per day
00 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., but in the form of an injection, it is usually about 1 day in an adult (as 60 kg). About 0.01 to 30 mg, preferably about 0.1 to 20
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 converted per 60 kg can be administered.

(3) Method for quantifying ligand for G protein-coupled receptor protein of the present invention The G protein-coupled receptor protein of the present invention or a salt thereof, or the partial peptide of the present invention or a salt thereof has a binding property to a ligand. Since it has, the ligand concentration in the living body can be quantified with high sensitivity. The quantification method of the present invention can be used, for example, by combining it with a competition method. That is, the subject is a G protein-coupled receptor protein of the present invention or a salt thereof, or G
The ligand concentration in the test sample can be measured by bringing it into contact with a partial peptide of the protein-coupled receptor protein or a salt thereof. 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 1997) edited by Hiroshi Irie, "Radio Immunoassay" (Kodansha, published in 1979)

(4) Screening method for a compound that changes the binding property between a G protein-coupled receptor protein of the present invention and a ligand: The G protein-coupled receptor protein of the present invention or a salt thereof, or a partial peptide of the present invention or a salt thereof A compound that alters the binding property between a ligand and a G protein-coupled receptor protein by using or by constructing an expression system of a recombinant receptor protein and using a receptor binding assay system using the expression system (for example, A compound that inhibits the binding between the ligand and the G protein-coupled receptor protein, a compound that promotes the binding between the ligand and the G protein-coupled receptor protein, or a salt thereof can be screened. Especially ligand and G
A compound that inhibits binding to a protein-coupled receptor protein (for example, peptide, protein, non-peptide compound,
Synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, etc.) or salts thereof, G protein-coupled receptor proteins of the present invention or salts thereof, or partial peptides of the present invention or salts thereof Or by constructing a recombinant receptor protein expression system and using a receptor binding assay system using the expression system,
Can be screened. Such compounds include cell stimulating activity (eg, arachidonic acid release, acetylcholine release, intracellular Ca ²⁺ release, intracellular cAMP production, intracellular cGMP) via G protein-coupled receptors.
Production, inositol phosphate production, cell membrane potential fluctuation, intracellular protein phosphorylation, c-fos activation, activity promoting or suppressing pH decrease, etc. (so-called "G protein of the present invention" And a compound having no such cell stimulating activity (so-called “G protein-coupled receptor antagonist” of the present invention) and the like.

That is, the present invention includes (i) a case where a ligand is contacted with the G protein-coupled receptor protein of the present invention or a salt thereof, or a partial peptide of the present invention or a salt thereof, and (ii) the G protein of the present invention. Comparison of a ligand and a G protein-coupled receptor protein of the present invention, which is characterized in that a ligand and a test compound are contacted with the coupled receptor protein or a salt thereof or a partial peptide of the present invention or a salt thereof. A compound that changes the binding property (for example, a compound that inhibits the binding between the ligand and the G protein-coupled receptor protein of the present invention or a salt thereof, or a compound that promotes the binding between the ligand and the G protein-coupled receptor protein of the present invention, or A salt thereof). In the screening method of the present invention, (i) a case where a ligand is contacted with the G protein-coupled receptor protein of the present invention or a partial peptide of the present invention, and (ii) a G protein-coupled receptor protein of the present invention or the present invention When the ligand and the test compound are contacted with the partial peptide of, the binding amount of the ligand to the G protein-coupled receptor protein or the partial peptide, the cell stimulating activity, etc. are measured and compared. . In particular, those which can be detected by biological activity or biological response reaction can be preferably measured.

Hereinafter, the method for screening a compound or a salt thereof that inhibits the binding between the ligand and the G protein-coupled receptor protein of the present invention will be described more specifically, but it is understood that the present invention is not limited thereto. Should be. More specifically, the present invention relates to a case where a labeled ligand is brought into contact with the G protein-coupled receptor protein of the present invention or a salt thereof or a partial peptide of the G protein coupled receptor protein of the present invention or a salt thereof, When the labeled ligand and the test compound are contacted with the G protein-coupled receptor protein of the present invention or a salt thereof or a partial peptide of the G protein coupled receptor protein of the present invention or a salt thereof, the labeled ligand protein or the protein thereof A method for screening a compound or a salt thereof, which inhibits the binding between a ligand and a G protein-coupled receptor protein of the present invention, characterized in that the amount of binding to a salt or the partial peptide or a salt thereof is measured and compared. The ligand includes the G protein-coupled receptor protein of the present invention. And a membrane fraction of the cell, and a labeled ligand and a test compound are brought into contact with a cell containing the G protein-coupled receptor protein of the present invention or a membrane fraction of the cell, A method for screening a compound or a salt thereof which inhibits the binding between the ligand and the G protein-coupled receptor protein of the present invention, which comprises measuring and comparing the amount of the labeled ligand bound to the cells or the membrane fraction, When the labeled ligand was brought into contact with the G protein-coupled receptor protein expressed on the cell membrane by culturing a transformant containing the DNA encoding the G protein-coupled receptor protein of the present invention, and the labeled ligand And a test compound containing a DNA encoding a G protein-coupled receptor protein of the present invention. A ligand characterized by measuring and comparing the amount of labeled ligand bound to the G protein-coupled receptor protein when the G protein-coupled receptor protein expressed on the cell membrane is contacted by culturing the body And a method for screening a compound or a salt thereof which inhibits the binding of the G protein-coupled receptor protein of the present invention,

A compound that activates the G protein-coupled receptor protein of the present invention (eg, a ligand for the G protein-coupled receptor protein of the present invention) is brought into contact with cells containing the G protein-coupled receptor protein of the present invention. And a compound that activates the G protein-coupled receptor of the present invention and a test compound are contacted with cells containing the G protein-coupled receptor protein of the present invention, cells mediated by the G protein-coupled receptor Stimulatory activity (eg arachidonic acid release, acetylcholine release, intracellular Ca ²⁺ release, intracellular cAMP production, intracellular c
GMP production, inositol phosphate production, cell membrane potential fluctuation, intracellular protein phosphorylation, activation of c-fos, p
Activity that promotes or suppresses reduction of H, etc.)
And a method for screening a compound or a salt thereof that inhibits the binding between the ligand and the G protein-coupled receptor protein of the present invention, and a compound that activates the G protein-coupled receptor of the present invention A G protein-coupled type expressed on a cell membrane by culturing a transformant containing a DNA encoding the G protein-coupled receptor protein of the present invention (for example, a ligand for the G protein-coupled receptor protein of the present invention) When contacted with a receptor protein, G of the present invention
A compound that activates a protein-coupled receptor and a test compound are contacted with a G protein-coupled receptor protein expressed on a cell membrane by culturing a transformant containing a DNA encoding the G protein-coupled receptor protein of the present invention. G protein-coupled receptor-mediated cell stimulating activity (eg, arachidonic acid release, acetylcholine release, intracellular Ca ²⁺ release, intracellular c
AMP production, intracellular cGMP production, inositol phosphate production, cell membrane potential fluctuation, intracellular protein phosphorylation, c-
and a compound which inhibits the binding between the ligand and the G protein-coupled receptor protein of the present invention, characterized by measuring and comparing the activity of promoting fos, the activity of promoting fos, or suppressing the decrease of pH) Provided is a salt screening method.

Before the G protein-coupled receptor protein of the present invention was obtained, when a G protein-coupled receptor agonist or antagonist was screened, cells, tissues, or cell membranes thereof containing the G protein-coupled receptor protein, such as rat, were first prepared. To obtain a candidate compound (primary screening), and then confirm whether or not the candidate compound actually inhibits the binding of the human G protein-coupled receptor protein to the 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, by using the human-derived G protein-coupled receptor protein of the present invention, the need for primary screening is eliminated, and a compound that inhibits the binding between the ligand and the G protein-coupled receptor can be efficiently screened. Furthermore, it is possible to evaluate whether the screened compound is a G protein coupled receptor agonist or a G protein coupled receptor antagonist. The specific description of the screening method of the present invention is as follows. First, any G protein-coupled receptor protein used in the screening method of the present invention may be used as long as it contains the G protein-coupled receptor protein of the present invention or a partial peptide of the G protein-coupled receptor protein. However, a membrane fraction of an organ of a warm-blooded animal is preferable. However, since human-derived organs are extremely difficult to obtain, a G protein-coupled receptor protein expressed in large amounts using a recombinant is suitable for use in screening.

The above-mentioned method is used for producing the G protein-coupled receptor protein, which can be carried out by expressing the DNA encoding the protein in mammalian cells or insect cells. Although complementary DNA is used as the DNA fragment encoding the target portion, it is not necessarily limited thereto. For example, a gene fragment or synthetic DNA may be used. To introduce a DNA fragment encoding a G protein-coupled receptor protein into a host animal cell and express them efficiently, in order to efficiently express them, a nuclear polyhedrosis virus (nuclear polyhedrosis virus) belonging to a baculovirus in which an insect is used as a host is used. virus; NPV) polyhedrin promoter, SV40-derived promoter, retrovirus promoter, metallothionein promoter, human heat shock promoter, cytomegalovirus promoter, SRα promoter and the like are preferably incorporated downstream. The amount and quality of the expressed receptor can be examined by a method known per se. For example, the literature [Nambi, P. Et al., The Journal of Biological Chemistry (J. Biol. Chem.), 267.
Vol., 19555-19559, 1992]. Therefore, in the screening method of the present invention, a G protein-coupled receptor protein or a partial peptide of a G protein-coupled receptor protein is a G protein-coupled receptor protein purified by a method known per se or the G protein. It may be a partial peptide of the coupled receptor protein, a cell containing the protein may be used, or a membrane fraction of cells containing the protein may be used.

In the screening method of the present invention, G
When a cell containing a protein-coupled receptor protein is used, the cell may be immobilized with glutaraldehyde, formalin or the like. The immobilization method can be carried out according to a method known per se or a modification method substantially similar thereto. The cell containing the G protein-coupled receptor protein refers to a host cell expressing the G protein-coupled receptor protein, and examples of the host cell include Escherichia coli, Bacillus subtilis, yeast, insect cells, animal cells and the like. . The membrane fraction refers to a fraction containing a large amount of cell membrane obtained by a method known per se after crushing cells. As a method for crushing cells, a method of crushing cells with a Potter-Elvehjem type homogenizer, crushing with a Waring blender or Polytron (Kinematica), crushing with ultrasonic waves, and jetting the cells from a thin nozzle while pressing with a French press etc. Examples include crushing by things. For fractionation of cell membranes, fractionation by centrifugal force, such as fractionation centrifugation and density gradient centrifugation, is mainly used. For example, the cell lysate is fed at a low speed (500 rpm to 3000 rpm).
m) for a short time (generally about 1 minute to 10 minutes), and the supernatant is further centrifuged at a high speed (15,000 to 30000 rpm) for usually 30 minutes to 2 hours, and the resulting precipitate is used as a membrane fraction. The membrane fraction is rich in expressed G protein-coupled receptor protein and membrane components such as cell-derived phospholipids and membrane proteins. The amount of G protein-coupled receptor protein in cells or membrane fractions containing the G protein-coupled receptor protein is preferably 10 ³ to 10 ⁸ molecules per cell, and is 10 ⁵ to 10 ⁷ molecules. Is preferred. 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 (1) to (3) above for screening a compound that inhibits the binding between the ligand and the G protein-coupled receptor of the present invention, an appropriate G protein-coupled receptor fraction and a labeled ligand are required. is there. G
As the protein-coupled receptor fraction, a natural G protein-coupled receptor fraction or a recombinant G protein-coupled receptor fraction having an activity equivalent to that is desirable. Here, the equivalent activity refers to equivalent ligand binding activity and the like. As the labeled ligand, a labeled ligand, a labeled ligand analog compound, or the like is used. For example, [ ³ H], [ ¹²⁵ I], [ ¹⁴ C], [ ³⁵
For example, a ligand labeled with S] or the like can be used. Specifically, to screen a compound that inhibits the binding between a ligand and a G protein-coupled receptor protein, first, a cell containing the G protein-coupled receptor protein or a membrane fraction of the cell is suitable for screening. Prepare the receptor preparation by suspending it in a buffer. The buffer should have a pH of 4 to 10 (preferably p
Any buffer that does not inhibit the binding between the ligand X and the receptor, such as a phosphate buffer of H6 to 8) or a Tris-hydrochloric acid buffer, may be used. Further, for the purpose of reducing non-specific binding, CHAPS, Tween-80
Surfactants such as ^TM (Kao-Atlas), digitonin and deoxycholate may be added to the buffer. Furthermore, PMSF, leupeptin, E-6 for the purpose of suppressing the degradation of the receptor and ligand by protease.
4 (manufactured by Peptide Institute), a protease inhibitor such as pepstatin can also be added. 0.01 ml-10
ml of the receptor solution, a fixed amount (5000 cpm-
(500,000 cpm) of labeled ligand is added, and 10 ⁻⁴ M to ^{10 −10} M of the test compound is simultaneously added.
A reaction tube containing a large excess of unlabeled ligand is also prepared in order to know the amount of non-specific binding (NSB). The reaction is 0
C. to 50.degree. C., preferably 4.degree. C. to 37.degree. C., for 20 minutes to 24 hours, preferably 30 minutes to 3 hours. After the reaction,
After filtering with glass fiber filter paper and the like and washing with an appropriate amount of the same buffer, the radioactivity remaining on the glass fiber filter paper is measured with a liquid scintillation counter or a γ-counter. Count (B ₀ -NS) obtained by subtracting the amount of non-specific binding (NSB) from the count (B ₀ ) in the absence of an antagonist.
Specific binding amount (B-NSB) when B) is 100%
A test compound having a ratio of 50% or less can be selected as a candidate substance capable of competitive inhibition.

In order to carry out the above-mentioned methods (1) to (3) for screening a compound which inhibits the binding between the ligand and the G protein-coupled receptor protein of the present invention, the cell stimulating activity mediated by the G protein-coupled receptor protein (eg,
Arachidonic acid release, acetylcholine release, intracellular Ca release, intracellular cAMP production, intracellular cGMP production, inositol phosphate production, cell membrane potential fluctuation, intracellular protein phosphorylation, c-fos activation, pH decrease, etc. The promoting activity or the suppressing activity) can be measured using a known method or a commercially available measurement kit. Specifically, first, cells containing the G protein-coupled receptor protein are cultured in a multiwell plate or the like. Prior to screening, exchange with fresh medium or an appropriate buffer that does not show toxicity to cells,
After adding a test compound or the like and incubating for a certain period of time, cells are extracted or the supernatant is collected, and the produced product is quantified according to each method. When the production of a substance that serves as an index of cell stimulating activity (for example, arachidonic acid) is difficult to assay with a degrading enzyme contained in cells, an inhibitor for the degrading enzyme may be added to the assay. Further, the activity of suppressing cAMP production and the like can be detected as a production suppressing action on cells in which the basic production amount of cells has been increased by forskolin or the like. In order to perform screening by measuring cell stimulating activity, cells expressing an appropriate G protein-coupled receptor protein are required. The cells expressing the G protein-coupled receptor protein of the present invention include cell lines having a natural G protein-coupled receptor protein of the present invention, the above-mentioned recombinant G protein-coupled receptor protein-expressing cell line (for example, CHO cells, COS cells, etc.) are preferable. Examples of test compounds include peptides, proteins, non-peptide compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, serum,
Examples thereof include blood and body fluid, and these compounds may be novel compounds or known compounds.

The screening kit for a compound or a salt thereof that inhibits the binding between the ligand and the G protein-coupled receptor protein of the present invention is a G protein-coupled receptor protein of the present invention or a salt thereof, and a G protein-coupled type of the present invention. It comprises a partial peptide of a receptor protein or a salt thereof, a cell containing the G protein-coupled receptor protein of the present invention, or a membrane fraction of a cell containing the G protein-coupled receptor protein of the present invention. 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)
To which 0.05% bovine serum albumin (manufactured by Sigma) was added. Sterilize by filtration with a 0.45μm filter,
It may be stored at 4 ° C or may be prepared at the time of use. G protein-coupled receptor preparation CHO cells expressing the G protein-coupled receptor protein were subcultured on a 12-well plate at 5 × 10 ⁵ cells / well and 3
One cultured at 7 ° C., 5% CO ₂ , 95% air for 2 days. Labeled ligand Commercially available ligand labeled with [ ³ H], [ ¹²⁵ I], [ ¹⁴ C], [ ³⁵ S], etc. Aqueous solution is stored at 4 ° C or -20 ° C and used as a measurement buffer. Dilute to 1 μM with solution. Standard ligand solution 0.1% bovine serum albumin (manufactured by Sigma)
And dissolved at 1 mM in PBS, and stored at -20 ° C.

2. Assay method G-protein coupled receptor protein-expressing CHO cells cultured in 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 adding 5 μl of a test compound solution of 10 ⁻³ to 10 ⁻¹⁰ M, 5 μl of a labeled ligand is added, and the mixture is reacted at room temperature for 1 hour. In order to know the amount of non-specific binding, 5 μl of 10 ⁻³ M ligand was added in place of the test compound. Remove the reaction solution and wash 3 times with 1 ml of washing buffer. The labeled ligand bound to the cells is treated with 0.2 N 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).

[0058]

[Number 1] PMB = [(B-NSB)] / (B ₀ -NS
B)] × 100 PMB: Percent Maximum Binding B: Value when a sample is added NSB: Non-specific Binding (non-specific binding amount) B ₀ : Maximum binding amount

The compound or its salt obtained by using the screening method or the screening kit of the present invention is a compound or its salt that changes the binding property between the ligand and the G protein-coupled receptor of the present invention. Particularly preferably, the compound or its salt obtained by using the screening method or screening kit of the present invention,
A compound which inhibits the binding between a ligand and the G protein-coupled receptor of the present invention, and specifically, a compound having a cell stimulating activity via the G protein-coupled receptor or a salt thereof (so-called “G protein-coupled receptor agonist”). )) Or a compound having no such stimulating activity (so-called “G protein-coupled receptor antagonist”). Examples of the compound include peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products,
These compounds may be new compounds or known compounds. The G protein-coupled receptor agonist has the same physiological activity as that of the ligand for the G protein-coupled receptor protein of the present invention, and therefore, as a safe and low-toxic pharmaceutical composition depending on the ligand activity. It is useful. On the contrary, since the G protein-coupled receptor antagonist can suppress the physiological activity of the ligand for the G protein-coupled receptor protein of the present invention, it is useful as a safe and low-toxic pharmaceutical composition for suppressing the ligand activity. Is. For example, when the ligand for the G protein-coupled receptor protein of the present invention is somatostatin, the somatostatin receptor agonist has all or part of the physiological activity of somatostatin, and is safe and low depending on the physiological activity. It is useful as a toxic pharmaceutical composition. For example, it is useful as a production inhibitor of hormones such as growth hormone and pituitary hormone (eg, gastrin, insulin, etc.), and further, endocrine disease, endocrine tumor, hormone-producing tumor, acromegaly, giant disease, dementia It is useful as a preventive and / or therapeutic agent for diabetes, gastric ulcer, etc. On the other hand, the somatostatin receptor antagonist can suppress all or part of the physiological activity of somatostatin, and is therefore useful as a safe and low-toxic pharmaceutical composition for suppressing the physiological activity. For example,
It is useful as a production inhibitor of hormones such as growth hormone, pituitary hormone (eg, thyroid stimulating hormone, prolactin, etc.), digestive tract hormone (eg, gastrin, insulin, etc.), dwarfism, and elderly people. Prophylactic or (and) therapeutic agent for wounds, osteoporosis, insomnia, liver function disease, lactation deficiency, diabetes, etc., or functional regulator of gastrointestinal organs (eg, stomach, small intestine, pancreas, liver, etc. It is useful as a function regulator).

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. The composition can be used in various pharmaceutical formulations or preparations. For example, orally as tablets, capsules, elixirs, microcapsules, etc., optionally sugar-coated, or aseptic solution with water or other pharmaceutically acceptable liquid, or suspension It can be used parenterally in the form of injections such as. For example, the compound or salt thereof is admixed with a physiologically acceptable carrier, flavoring agent, excipient, vehicle, preservative, stabilizer, binder and the like in a unit dosage form generally required for pharmaceutical practice. It 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 incorporated into 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. Swelling agents, lubricants such as magnesium stearate, sweeteners such as sucrose, lactose or saccharin, flavoring agents such as peppermint, reddish oil or cherry are used. When the preparation unit form is a capsule, a liquid carrier such as oil and fat can be further contained in the above-mentioned type of material. Sterile compositions for injection can be formulated according to conventional pharmaceutical practices such as dissolving or suspending the active substance in a vehicle such as water for injection, naturally occurring vegetable oils such as sesame oil, coconut oil, etc. .

Examples of the aqueous solution for injection include physiological saline, isotonic solution containing glucose and other adjuvants (for example, D-sorbitol, D-mannitol, sodium chloride etc.) and the like. It may be used together with a solubilizing agent such as alcohol (eg ethanol), polyalcohol (eg propylene glycol, polyethylene glycol etc.), nonionic surfactant (eg Polysorbate 80 (TM), HCO-50 etc.) and the like. . Examples of the oily liquid include sesame oil and soybean oil, which may be used in combination with solubilizing agents such as benzyl benzoate and benzyl alcohol. In addition, buffers (eg, phosphate buffer, sodium acetate buffer, etc.), soothing agents (eg, benzalkonium chloride, procaine hydrochloride, etc.), stabilizers (eg, human serum albumin, polyethylene glycol, etc.), You may mix | blend with a preservative (for example, benzyl alcohol, phenol, etc.), an antioxidant, etc. The prepared injection is usually filled in a suitable ampoule. Since the preparation thus obtained is safe and has low toxicity, for example, warm-blooded mammals (for example, rat, rabbit, sheep, pig, cow, cat, dog, monkey,
Human). The dose of the compound or its salt may vary depending on the symptoms,
In the case of oral administration, generally in an adult (as 60 kg), about 0.1 to 100 mg, preferably about 1.0 to 50 mg, more preferably about 1.0 to 20 m per day.
g. When administered parenterally, the single dose varies depending on the administration subject, target organs, symptoms, administration method, etc., but for example in the form of an injection, it is usually for adults (60 kg
As) in about 0.01 to 30 mg per day
It is convenient to administer intravenously, preferably about 0.1 to 20 mg, more preferably about 0.1 to 10 mg. In the case of other animals, the dose converted per 60 kg can be administered.

(5) Production of antibody or antiserum against the G protein-coupled receptor protein of the present invention or a salt thereof or a partial peptide of the G protein-coupled receptor protein of the present invention or a salt thereof The G protein-coupled receptor protein of the present invention Alternatively, an antibody (for example, a polyclonal antibody, a monoclonal antibody) or an antiserum against a salt thereof, a partial peptide of the G protein-coupled receptor protein of the present invention or a salt thereof is a G protein-coupled receptor protein of the present invention or a salt thereof or the present invention. Using the partial peptide of the G protein-coupled receptor protein or the salt thereof as an antigen, it can be produced according to a method for producing an antibody or antiserum known per se. For example, a monoclonal antibody can be produced according to the method described below. [Preparation of Monoclonal Antibody] (a) Preparation of Monoclonal Antibody-Producing Cell G protein-coupled receptor protein of the present invention or a salt thereof, or partial peptide of G protein-coupled receptor protein of the present invention or a salt thereof (hereinafter referred to as G protein conjugate) (Sometimes abbreviated as "type receptor") is administered to warm-blooded animals at a site where antibody can be produced by administration, or itself or with a carrier or diluent. Complete Freund's adjuvant or incomplete Freund's adjuvant may be administered in order to enhance the antibody-producing ability upon administration. The administration is usually performed once every 2 to 6 weeks, about 2 to 10 times in total. Examples of warm-blooded animals used include monkeys, rabbits, dogs, guinea pigs, mice, rats, sheep, goats, and chickens, and mice and rats are preferably used.

In the preparation of monoclonal antibody-producing cells, warm-blooded animals immunized with an antigen, for example, mice having an antibody titer were selected and the spleen or lymph nodes were collected 2 to 5 days after the final immunization. By fusing the antibody-producing cells contained therein with myeloma cells, a monoclonal antibody-producing hybridoma can be prepared. The antibody titer in the antiserum can be measured by, for example, labeled G described below.
After reacting the protein-coupled receptor with the antiserum,
This is done by measuring the activity of the labeling agent bound to the antibody. The fusion operation is a known method, for example, the method of Koehler and Milstein [Nature, 256, 495 (1
975)]. Examples of the fusion promoter include polyethylene glycol (PEG) and Sendai virus, and PEG is preferably used. Examples of myeloma cells include NS-1, P3U1, SP2 / 0,
Examples include AP-1 and the like, but P3U1 is preferably used. The preferred ratio of the number of antibody-producing cells (spleen cells) to the number of myeloma cells used is about 1: 1 to 20: 1, and PEG (preferably PEG1000 to PEG600) is used.
0) is added at a concentration of about 10 to 80%, and 20 to 40
Cell fusion can be efficiently carried out by incubating at 1 ° C., preferably 30 to 37 ° C. for 1 to 10 minutes. Although various methods can be used for screening anti-G protein-coupled receptor antibody-producing hybridomas, for example, the hybridoma culture supernatant is applied to a solid phase (eg, a microplate) on which the G protein-coupled receptor antigen is directly or adsorbed with a carrier. Then, anti-immunoglobulin antibody labeled with a radioactive substance or enzyme (anti-mouse immunoglobulin antibody is used when the cells used for cell fusion are mice) or protein A is added, and the anti-immunoglobulin antibody bound to the solid phase is added. Method for detecting G protein-coupled receptor monoclonal antibody, anti-immunoglobulin antibody or protein A
Hybridoma culture supernatant was added to the solid phase on which
Examples include a method of adding a G protein-coupled receptor labeled with a radioactive substance or an enzyme, and detecting an anti-G protein-coupled receptor monoclonal antibody bound to a solid phase.

The anti-G protein coupled receptor monoclonal antibody can be selected by a method known per se or a method analogous thereto. It is usually carried out in a medium for animal cells supplemented with HAT (hypoxanthine, aminopterin, thymidine). As a medium for selection, cloning and breeding, any medium can be used as long as it can grow a hybridoma. For example, RPMI16 containing 1-20%, preferably 10-20% fetal calf serum.
40 medium (Dainippon Pharmaceutical Co., Ltd.), GIT medium containing 1 to 10% fetal calf serum (Wako Pure Chemical Industries, Ltd.) or serum-free medium for hybridoma culture (SFM-101, Nissui Pharmaceutical Co., Ltd.) ) Or the like can be used. The culture temperature is
It is usually 20 to 40 ° C, preferably about 37 ° C. Cultivation time is usually 5 days to 3 weeks, preferably 1 week to 2 weeks. Culturing is usually performed under 5% carbon dioxide gas. The antibody titer of the hybridoma culture supernatant is the anti-G in the above antiserum.
It can be measured in the same manner as the protein-coupled receptor antibody titer. Cloning can be performed by a method known per se such as a semi-solid agger method or a limiting dilution method, and the cloned hybridoma is preferably cultured in a serum-free medium and an optimal amount of the antibody is added to the supernatant. give. The target monoclonal antibody can also be obtained preferably by ascites.

(B) Purification of monoclonal antibody The isolation and purification of the anti-G protein-coupled receptor monoclonal antibody is carried out in the same manner as the usual isolation and purification of a polyclonal antibody [eg, salting out method, alcohol precipitation method, etc.]. Isoelectric precipitation, electrophoresis, ion exchanger (eg,
DEAE) adsorption / desorption method, ultracentrifugation method, gel filtration method, antigen-binding solid phase or protein A or protein G
Specific purification method in which only the antibody is collected by an active adsorbent such as, and the bond is dissociated to obtain the antibody]. Since the G protein-coupled receptor antibody of the present invention produced according to the above methods (a) and (b) can specifically recognize the G protein-coupled receptor, the G protein-coupled receptor in the test solution is coupled. It can be used for the quantification of type receptors, especially for the quantification by sandwich immunoassay. That is, the present invention provides, for example, (i) a label bound to the antibody that reacts with the G protein-coupled receptor of the present invention, the test solution and the labeled G protein-coupled receptor in a competitive manner. Method for quantifying a G protein-coupled receptor in a test solution, which comprises measuring the ratio of a modified G protein-coupled receptor, (ii) a test solution, an antibody insolubilized on a carrier, and a labeled antibody In the method for quantifying a G protein-coupled receptor in a test solution, the activity of the labeling agent on the insolubilized carrier is measured after reacting simultaneously or successively with one antibody Provided is a method for quantifying a G protein-coupled receptor in a test solution, which is an antibody that recognizes the N-terminal portion of the receptor and the other antibody reacts with the C-terminal portion of the G protein-coupled receptor. You .

The G protein-coupled receptor can be measured using the monoclonal antibody of the present invention which recognizes the G protein-coupled receptor (hereinafter sometimes referred to as anti-G protein-coupled receptor antibody), and tissue staining and the like. Can also be detected. The antibody molecule itself may be used for these purposes, and the F (a
b ′) ₂ , Fab ′, or Fab fraction may be used. The measuring method using the antibody of the present invention is not particularly limited, and the amount of the antibody, the antigen or the antibody-antigen complex corresponding to the amount of the antigen (eg, the amount of G protein-coupled receptor) in the liquid to be measured can be adjusted. Any measuring method may be used as long as it is detected by chemical or physical means and is calculated from a standard curve prepared using a standard solution containing a known amount of antigen. For example, nephrometry, competitive method,
Although the immunometric method and the sandwich method are preferably used, the sandwich method described below is particularly preferably used in terms of sensitivity and specificity. The labeling agent used in the measurement method using a labeling substance includes radioisotopes, enzymes,
Examples thereof include fluorescent substances and luminescent substances. Examples of radioisotopes include [ ¹²⁵ I], [ ¹³¹ I], [ ³ H],
[ ¹⁴ C] and the like are preferably those which are stable and have a large specific activity, such as β-galactosidase and β
-Glucosidase, alkaline phosphatase, peroxidase, malate dehydrogenase, etc., fluorescent substances such as fluorescamine, fluorescein isothiocyanate, and luminescent substances such as luminol, luminol derivatives, luciferin, lucigenin, etc. To be Further, a biotin-avidin system can be used for binding the antibody or antigen to the labeling agent.

For the insolubilization of the antigen or antibody, physical adsorption may be used, or a method using a chemical bond which is usually used for insolubilizing or immobilizing proteins or enzymes. Examples of the carrier include insoluble polysaccharides such as agarose, dextran, and cellulose; synthetic resins such as polystyrene, polyacrylamide, and silicon; and glass. In the sandwich method, an insolubilized anti-G protein-coupled receptor antibody is reacted with a test solution (first reaction), and further a labeled anti-G protein-coupled receptor antibody is reacted (second reaction), and then on an insolubilized carrier. The amount of the G protein-coupled receptor in the test solution can be quantified by measuring the activity of the labeling agent. 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 in accordance with those described above. In addition, 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 may be used for the purpose of improving measurement sensitivity and the like. In the method for measuring a G protein-coupled receptor according to the sandwich method of the present invention, the anti-G protein-coupled receptor antibodies used in the primary reaction and the secondary reaction are preferably antibodies having different binding sites for the G protein-coupled receptor. Used. That is, when the antibody used in the secondary reaction recognizes the C-terminal portion of the G protein-coupled receptor, the antibody used in the primary reaction is preferably the antibody used in the primary reaction. An antibody that recognizes an N-terminal other than the C-terminal is used.

The G protein-coupled receptor antibody of the present invention may be assayed by a measuring system other than the sandwich method, for example, the competitive method,
It can be used for immunometric method or nephrometry. In the competitive method, the antigen in the test solution and the labeled antigen are reacted competitively with the antibody, and then the unreacted labeled antigen (F) and the labeled antigen (B) bound to the antibody are separated. (B / F separation), the labeled amount of either B or F is measured, and the amount of antigen in the test liquid is quantified. In this reaction method, a soluble antibody is used as the antibody, a liquid phase method using polyethylene glycol, a second antibody against the antibody is used for B / F separation, and a solid phase antibody is used as the first antibody, or The first antibody is soluble and the second antibody is a solid-phased antibody. 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 labeled amount of either phase is measured to quantify the amount of antigen in the test liquid. In nephelometry, the amount of insoluble precipitates generated as a result of an antigen-antibody reaction in a gel or in a solution is measured. Even when the amount of antigen in the test solution is small and only a small amount of sediment is obtained, laser nephelometry utilizing laser scattering is preferably used.

In applying these individual immunological measurement methods to the measurement method of the present invention, no special conditions, operations and the like need to be set. The measurement system for the G protein-coupled receptor may be constructed by adding ordinary technical consideration to those skilled in the art to ordinary conditions and operating methods in each method. For details of these general technical means, reference can be made to review articles, books, etc. [for example, Hiro Irie's "Radioimmunoassay" (Kodansha, 1974), Hiroshi Irie's "Continued Radioimmunoassay". (Kodansha, Showa 54
Issue), Eiji Ishikawa et al. “Enzyme Immunoassay” (medical booklet, published in 1978), Eiji Ishikawa et al. “Enzyme Immunoassay” (second edition) (medical booklet, published in 1982) Eiji et al., “Enzyme Immunoassay” (3rd edition) (Medical Shoin, published in 1987), “Methods in ENZYMOLOGY” Vol. 70 (Im
munochemical Techniques (Part A)), ibid Vol. 73 (Imm
unochemical Techniques (Part B)), ibid Vol. 74 (Imm
unochemical Techniques (Part C)), ibid Vol. 84 (Imm
unochemical Techniques (PartD: Selected Immunoassay
s)), ibid.Vol. 92 (Immunochemical Techniques (Part
E: Monoclonal Antibodies and General Immunoassay Me
thods)), ibid Vol. 121 (Immunochemical Techniques
(Part I: Hybridoma Technology and Monoclonal Antibo
dies)) (see above, issued by Academic Press) etc.). As described above, by using the G protein-coupled receptor antibody of the present invention, the G protein-coupled receptor can be quantified with high sensitivity. (6) Preparation of Animal Having DNA Encoding G Protein-Coupled Receptor Protein of the Present Invention Transgenic animals expressing G protein-coupled receptor protein are prepared using DNA encoding G protein-coupled receptor protein be able to. Examples of animals include warm-blooded mammals (eg, rat, rabbit, sheep, pig, cow, cat, dog, monkey, etc.). DN encoding a G protein-coupled receptor protein
In transferring A into a target animal, it is generally advantageous to bind the DNA downstream of a promoter that can be expressed in animal cells and use it. For example, when a G protein-coupled receptor protein DNA is introduced into a rabbit, a gene construct in which the G protein-coupled receptor protein DNA derived from an animal having high homology with this is linked downstream of various promoters capable of expressing in animal cells, By microinjecting a fertilized egg of a target animal, for example, a fertilized egg of a rabbit, a transgenic animal that produces a high G protein-coupled receptor protein can be produced.

As the promoter, for example, a virus-derived promoter or a ubiquitous expression promoter such as metallothionein can be used, but preferably an NGF gene promoter or an enolase gene promoter which is specifically expressed in brain or lung is used. The transfer of G protein-coupled receptor protein DNA at the fertilized egg cell stage is ensured to be present in all embryonic cells and somatic cells of the target animal. The presence of the G protein-coupled receptor protein in the embryonic cells of the producing animal after DNA transfer means that all the progeny of the producing animal have the G protein-coupled receptor protein in all of the embryonic cells and somatic cells. The offspring of this type of animal that inherits the gene has the G protein-coupled receptor protein in all of its germ cells and somatic cells. After confirming that the G protein-coupled receptor protein DNA-introduced animal stably retains the gene by mating, the animal can be passaged in a normal breeding environment as the DNA-carrying animal. Furthermore, a male and female animal carrying the target DNA is mated to obtain a homozygous animal having the transgene on both homologous chromosomes. By mating the male and female animals, all offspring are made to have the DNA. Can be bred and passaged. G protein-coupled receptor protein DNA
Since the G protein-coupled receptor protein is highly expressed in the animal into which is introduced, it is useful as an animal for screening an agonist or antagonist for the G protein-coupled receptor protein.

This transgenic animal can also be used as a cell source for tissue culture. For example, the G protein-coupled receptor protein can be analyzed by directly analyzing the DNA or RNA in the tissue of the transgenic mouse or by analyzing the protein tissue expressed by the gene. Cells of a tissue having a G protein-coupled receptor protein are cultured by a standard tissue culture technique, and by using these, cells from a tissue which is generally difficult to culture, such as those derived from brain or peripheral tissues, and further lung cells and lungs are used. It is possible to study the respective function of cells from tissues that are generally difficult to culture, such as those of tissue origin. Further, by using the cells, it is possible to select a drug that enhances the functions of various tissues, for example. If a highly expressing cell line is available, it is possible to isolate and purify the G protein-coupled receptor protein from the cell line.

(7) Antisense DNA capable of inhibiting the replication of G protein-coupled receptor protein gene
(Or an oligonucleotide) According to the present invention, an antisense oligonucleotide (nucleic acid) capable of inhibiting the replication or expression of a G protein-coupled receptor protein gene is a cloned or determined G protein-coupled receptor. It can be designed and synthesized based on the nucleotide sequence information of the DNA encoding the protein. Such oligonucleotide (nucleic acid) is DNA or RNA of G protein-coupled receptor protein gene.
Etc., and can inhibit the synthesis, expression or function of the DNA or RNA, or the G protein-coupled receptor protein through interaction with the G protein-coupled receptor protein-related nucleic acid. It can regulate and control the expression of genes. An oligonucleotide complementary to a selected sequence of a G protein-coupled receptor protein-related nucleic acid and an oligonucleotide capable of specifically hybridizing with a G protein-coupled receptor protein-related RNA are It is useful for regulating and controlling the expression of protein-coupled receptor protein gene, and also useful for treatment or diagnosis of diseases and the like. The term "corresponding" means having homology or being complementary to a specific sequence of nucleotides, base sequences or nucleic acids including genes. “Corresponding” between a nucleotide, a nucleotide sequence or a nucleic acid and a peptide (protein) usually means an amino acid of a peptide (protein) in a command derived from the nucleotide (nucleic acid) sequence or its complement. . 5'end hairpin loop of G protein-coupled receptor protein gene, 5'end 6-base pair repeat, 5'end untranslated region, polypeptide translation initiation codon, protein coding region, ORF translation initiation codon, 3'end non The translation region, the 3'-end palindromic region, and the 3'-end hairpin loop can be selected as preferred regions of interest, but any region within the G protein-coupled receptor protein gene can be selected as a region of interest.

An oligonucleotide capable of complementing or hybridizing with the target nucleic acid and at least a part of the target region can be said to be "antisense" with the target. Antisense oligonucleotides are polydeoxynucleotides containing 2'-deoxy-D-ribose, polydeoxynucleotides containing D-ribose, purines or other types of N-glycosides of pyrimidine bases. Polynucleotides, or other polymers with non-nucleotide backbones (eg, commercially available protein nucleic acids and synthetic sequence-specific nucleic acid polymers) or other polymers containing special linkages, provided that the polymers are found in DNA or RNA. Containing a nucleotide having a configuration that allows pairing of bases and attachment of bases). They are double-stranded DNA, single-stranded DNA, double-stranded RNA,
Single-stranded RNA, which may be further a DNA: RNA hybrid, and further an unmodified polynucleotide or an unmodified oligonucleotide, and further with a known modification such as one with a label known in the art, Capped, methylated, substituted with one or more natural nucleotides by analogs, modified with an intramolecular nucleotide, such as an uncharged bond (eg, methylphosphonate, phosphotriester, Those having a charged bond or a sulfur-containing bond (eg, phosphorothioate, phosphorodithioate, etc.), such as a protein (nuclease, nuclease inhibitor,
Toxins, antibodies, signal peptides, poly-L-lysine, etc.) and those having side chain groups such as sugars (eg, monosaccharides), those having an intercurrent compound (eg, acridine, psoralen, etc.), Those containing chelating compounds (eg, metals, radioactive metals, boron, oxidizing metals, etc.), those containing alkylating agents, those having modified bonds (eg,
α anomeric nucleic acid). Here, "nucleoside", "nucleotide" and "nucleic acid" not only contain known purine and pyrimidine bases,
Those having other modified heterocyclic bases may be included. Such modifications may include methylated purines and pyrimidines, acylated purines and pyrimidines, or other heterocycles.
The modified nucleosides and modified nucleotides may also be modified at the sugar moiety, e.g., one or more hydroxyl groups are replaced with halogens, aliphatic groups, etc., or converted to functional groups such as ethers, amines, etc. May have been.

The antisense oligonucleotide of the present invention is RNA, DNA, or a modified nucleic acid. 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 oligonucleotide of the present invention can be preferably designed according to the following policy. That is, it makes the antisense oligonucleotide more stable in the cell, increases the cell permeability of the antisense nucleic acid, increases the affinity for the target sense strand, and is toxic. Then make the antisense oligonucleotide less toxic. Many such modifications are known in the art, for example J. Kawakam.
i et al., Pharm Tech Japan, Vol. 8, pp.247, 1992; V
ol. 8, pp.395, 1992; ST Crooke et al. ed., Antis
ense Research and Applications, CRC Press, 1993 etc. The antisense oligonucleotides of the present invention include altered or modified sugars, bases,
It may contain a bond and the like, and may be provided in a special form such as liposome, microsphere, applied in a form suitable for gene therapy, or provided in an added form. Such additional forms include polycations such as polylysine, which acts to neutralize the charge of the phosphate backbone, and lipids, which enhance the interaction with cell membranes and increase the uptake of nucleic acids ( For example, crude water-based substances such as phospholipid and cholesterol can be used. 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 capping groups specifically arranged at the 3'-end or 5'-end of nucleic acid, such as exonuclease and RNas.
Examples thereof include those for preventing decomposition by nuclease such as e. 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 inhibitory activity of antisense oligonucleotides is
It 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 G protein-coupled receptor protein. The oligonucleotide can be applied to cells by various methods known per se.

In the present specification and the drawings, when an abbreviation is used for a base or amino acid, IUPAC-IUB is used.
Abbreviations based on Commision on Biochemical Nomenclature or common abbreviations in the field,
An example is given below. 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 dCTP: Deoxycytidine triphosphate ATP: Adenosine triphosphate EDTA: Ethylenediaminetetraacetic acid SDS: Sodium dodecyl sulfate EIA: Enzyme immunoassay Gly: Glycine Ala: Alanine Val: Valine Leu: Leucine IleS: leucine IleS

Thr: Threonine Cys: Cysteine Met: Methionine Glu: Glutamic acid Asp: Aspartic acid Lys: Lysine Arg: Arginine His: Histidine Phe: Phenylalanine Tyr: Tyrosine Trp: Tryptophan: parasulin: Pron: Asparagine: Pyrosine: Asparagine: Pyrosine: Asparagine: Pyrosine: Pyrosine: Asparagine: Pyrosine: Pyrosine: Glyn: Pyrosine: Pyrosine: Pyrosine: Pyrosine: Pyrosine: Pyrosine: Pyrosine: Pyrosine: Pyrosine: Pyrosine: Pyrosine: Pyrosine: Pyrosine: Pyrosine: Pyrosine: Pyrosine: Pol: Asparaginine: Pyrosine: Pyrosine: Pyrosine: Pyrosine: Pyrosine: Pyrosin: Glytin: Pyrosine: Pyrosine: Pyrosine: Pyrosine: Pyrosine: Pyrosine: Pyrosine: Pyrosine: Pyrosine: Pyrosine: Pyrosine: Glytin: Pyrosine: Pyrosine. Me: Methyl group Et: Ethyl group Bu: Butyl group Ph: Phenyl group TC: Thiazolidine-4 (R) -carboxamide group The transformant Escherichia coli JM109 / pPBBS002 obtained in Example 3 described later.
Has been depositing with the deposit number FERM on September 29, 1995 at the Institute of Biotechnology, National Institute of Advanced Industrial Science and Technology (NIBH), Ministry of International Trade and Industry.
It has been deposited as BP-5253, and also on September 1995.
IFO to the Fermentation Research Institute (IFO) from 26th of March
Deposited as 15878. The transformant Escherichia coli obtained in Example 6 described below was used.
li) JM109 / pPLBS003 is 2 September 1995
It has been deposited at the Institute of Biotechnology and Industrial Technology (NIBH) of the Ministry of International Trade and Industry since the 9th under the deposit number FERM BP-5254, and from September 26, 1995 to the Fermentation Research Institute (IFO). Deposited as 15879.

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. Various specific embodiments will be apparent to those skilled in the art from the disclosure of the present invention within the scope of the claims.

[0078]

[Reference Example] Production of Synthetic DNA Primer for Amplifying DNA Encoding G Protein-Coupled Receptor Protein Known G protein-coupled receptor, namely human-derived galanin receptor (HUMGALAREC), rat-derived α-1B-adrenergic receptor (RATADR
1B), human-derived β-1-adrenergic receptor (HUMADRB1), rabbit-derived IL-8 receptor (RABIL8RSB), human-derived opioid receptor (HUMOPIODRE), bovine-derived substance K
Receptor (BTSR), human somatostatin receptor-2 (HUMSRI2A), human somatostatin receptor-3 (HUMSSTR3Y), human gastrin receptor (HUMGARE), human cholecystokinin A receptor (HUMCCKAR),
Human-derived dopamine receptor-D5 (HUMD1B),
Human-derived serotonin receptor 5HT1E (HUM5H
T1E), human-derived dopamine receptor D4 (HUMD
4C), mouse-derived serotonin receptor-2 (MMS
ERO), rat-derived α-1A-adrenergic receptor (RATADRA1A), rat-derived histamine H
The nucleotide sequences of the DNAs encoding the amino acid sequences near the second transmembrane region of the 2 receptor (S57565) and the like were compared to find a highly similar portion. The abbreviations in parentheses above are the reference numbers shown when searching the GenBank / EMBL Data Bank using the DNASIS Gene / Protein Sequence Database (CD019, Hitachi Software Engineering), usually the Accession Number and the Entry Name, respectively. It is called.

Furthermore, a known G protein-coupled receptor, that is, human-derived galanin receptor (HUMGA)
LAREC), rat-derived A1 adenosine receptor (RAT1ADREC), porcine-derived angiotensin receptor (PIGA2R), rat-derived serotonin receptor (RAT5HTRTC), human-derived dopamine receptor (S58541), human-derived gastrin releasing peptide receptor (HUMGRPR), mouse-derived GRP / Bombesin Receptor (MUSGRPBO
M), rat-derived vascular type 1 angiotensin receptor (RRVT1AIIR), human-derived musculinic acetylcholine receptor (HSHM4), human-derived β-1 adrenergic receptor (HUMDRB)
1), human-derived gastrin receptor (HUMGAR
E), rat-derived cholecystokinin receptor (RAT
CCKAR), rat-derived ligand unknown receptor (S
59748), human somatostatin receptor (H
UMSST28A), rat-derived ligand unknown receptor (RNGPROCR), mouse-derived somatostatin receptor 1 (MUSSRI1A), human-derived α-A1-
Adrenergic receptor (HUMA1AADR), mouse-derived delta opioid receptor (S6618)
1), human somatostatin receptor-3 (HUM
The base sequences of the DNAs encoding the amino acid sequences near the seventh transmembrane region (SSTR3Y) and the like were compared and a highly similar portion was found. Abbreviations in () above are DNASIS G
ene / Protein sequence database (CD019,
Hitachi Software Engineering) using GenBank
/ EMBL This is the reference number displayed when you search Data Bank, which are usually called Accession Number and Entry Name, respectively. In particular, the introduction of mixed bases was planned in order to increase the conformity of the sequence with as many receptor cDNAs as possible in the other portions, with the base portion corresponding to the cDNAs encoding many receptor proteins as a reference. Based on this sequence, SEQ ID NO: 5 (T2A) or SEQ ID NO: 6 complementary to the common nucleotide sequence
A synthetic DNA having a base sequence represented by (T7A) was prepared.

The base sequence represented by SEQ ID NO: 5 (T2A primer) is 5'-GYCACCAACN ₂ W.
STTCATCCCTSWN ₂ HCTG-3 ′ [S represents G or C, Y represents C or T, W represents A or T
, H represents A, C or T, and N ₂ represents I.
Items in parentheses are mixed with multiple bases during synthesis. However, I is inosine. ].

The base sequence represented by SEQ ID NO: 6 (T7A primer) is 5'-ASN ₂ SAN ₂ RAAG.
SARTAGAN ₂ GAN ₂ RGGRTT-3 ′ [R represents A or G, S represents G or C, and N ₂ represents I. Items in parentheses are mixed with multiple bases during synthesis. However, I is inosine. ].

[0082]

Example 1 Synthesis of cDNA from human brain-derived poly (A) ⁺ RNA fraction Human brain-derived poly (A) ⁺ RNA was purchased from Wako Pure Chemical Industries, Ltd. Random DNA hexamer (BRL) was added to 5 μg of the purchased poly (A) ⁺ RNA fraction as a primer.
), And complemented with Moronii murine leukemia virus reverse transcriptase (BRL) using the attached buffer.
NA was synthesized. After the reaction, the product was subjected to ethanol precipitation and then 30 μl of TE (Tris-EDTA solution; 10 mM Tri).
It was dissolved in s-HCl (pH 8.0), 1 mM EDTA (pH 8.0)).

[0083]

[Example 2] Amplification of receptor cDNA by PCR using human brain-derived cDNA Using 3 µl of cDNA prepared from human brain in Example 1 as a template, D of T2A and T7A synthesized in the above Reference Example
Amplification by the PCR method was performed using NA primers.
The composition of the reaction solution is 100 p for each synthetic DNA primer.
M, 0.25 mM dNTPs (Deoxyribonucleotide tr)
iphosphates), Taq DNA polymerase (Takara Shuzo)
1 μl and 10 × Taq buffer 10 attached to the enzyme
In μl, the total reaction solution volume was 100 μl. The cycle for amplification uses a thermal cycler (Perkin Elmer), 96 ° C for 30 seconds, 45 ° C for 1 minute, 60 ° C.
The 3 minute cycle was repeated 25 times. Taq DNA p
Before adding olymerase, mix the remaining reaction mixture and
Heating was performed at 5 ° C for 5 minutes. Confirmation of amplification products is 1.2%
It was performed by agarose gel electrophoresis and ethidium bromide staining.

[0084]

Example 3 Subcloning of PCR Product into Plasmid Vector and Selection of Novel Candidate Receptor Protein Clone by Decoding Nucleotide Sequence of Inserted cDNA Part The reaction product after PCR performed in Example 2 was 1.2% electroeluted. Separate using agarose gel, cut out the band part with a razor, heat melt,
DNA was recovered by phenol extraction and ethanol precipitation. According to the prescription of TA cloning kit (Invitrogen), the recovered DNA was used as plasmid vector pC.
Subcloned into R ^™ II (TM stands for Trademark). This was introduced into E. coli JM109 competent cell (Takara Shuzo Co., Ltd.) for transformation, and then cDNA
The clone having the insert fragment was cloned into an LB agar medium (Luria- containing amphicillin, isopropylthio-β-D-galactoside (IPTG) and 5-bromo-4-chloro-3-indolyl-β-D-galactoside (X-gal). Bertan
(Iagar medium), only white clones were isolated using a sterilized toothpick, and transformed into Escherichia coli JM109 / pPB.
BS002 was obtained. Individual clones were cultured overnight in LB medium containing ampicillin, and plasmid DNA was prepared using an automatic plasmid extractor (Kurabo). A part of the prepared DNA was digested with EcoRI to confirm the size of the inserted cDNA fragment.
A part of the remaining DNA was further treated with RNase, extracted with phenol / chloroform, and concentrated by ethanol precipitation. The reaction for determining the nucleotide sequence is DyeDeoxy Term
Inator Cycle Sequencing Kit (Applied Biosystems: ABI) was performed, and it was decoded using a fluorescent automatic sequencer, and the obtained nucleotide sequence was analyzed by D.
It was performed using NASIS (Hitachi System Engineering Co., Ltd.). The underlined portion in FIG. 1 corresponds to the synthetic primer used in PCR. As a result of homology search based on the determined nucleotide sequence [SEQ ID NO: 3 (nucleotide sequence of FIG. 1)], the transformant Escherichia coli JM109 / pPBBS0 was obtained.
It was found that the cDNA fragment inserted in the plasmid contained in 02 encodes a novel G protein-coupled receptor protein. To further confirm that, DNASI
The base sequence was converted into an amino acid sequence using S (Hitachi System Engineering Co., Ltd.) [SEQ ID NO: 1 (amino acid sequence of FIG. 1)]. A homology search based on the hydrophobicity plot [FIG. 2] and the amino acid sequence was performed, and human somatostatin receptor subtype 1 (P30872),
Human somatostatin receptor subtype 2 (P3
0874) and human somatostatin receptor subtype 3 (P32745) were found to be homologous [Fig. 3]. The abbreviations in parentheses above are NBRF-PIR / Swiss-PRO
It is a reference number when it is registered as data in T, usually
It is called Accession Number or Entry Name.

[0085]

[Example 4] Synthesis of cDNA from human lung-derived poly (A) ⁺ RNA fraction Human lung-derived poly (A) ⁺ RNA was produced by Clontech.
(Clontech Laboratories, USA). Random DNA hexamer (BRL) was added as a primer to 5 μg of the purchased poly (A) ⁺ RNA fraction, and complementary DNA was synthesized using the attached buffer with Moroni mouse leukemia virus reverse transcriptase (BRL). After the reaction, the product was precipitated with ethanol and then treated with 30 μl of T
E (Tris-EDTA solution; 10mM Tris-HCl (pH 8.0), 1m
It was dissolved in M EDTA (pH 8.0)).

[0086]

Example 5 Amplification of Receptor cDNA by PCR Using Human Lung-Derived cDNA T2A and T7A synthesized in the above Reference Example using 3 μl of cDNA prepared from human lung in Example 4 as a template
Amplification was carried out by the PCR method using the DNA primer of 1. The composition of the reaction solution is 100 for each of the synthetic DNA primers.
pM, 0.25 mM dNTPs (Deoxyribonucleotid)
e triphosphates), Taq DNA polymerase (Takara Shuzo) 1 μl and 10 × Taq buffer 10 μl attached to the enzyme, and the total reaction solution volume was 100 μl. The cycle for amplification uses a thermal cycler (Perkin Elmer), 96 ° C for 30 seconds, 45 ° C for 1 minute, 60
The cycle of 3 minutes at ° C was repeated 25 times. Taq DN
Before adding A polymerase, the remaining reaction solution was mixed and heated at 95 ° C for 5 minutes and 65 ° C for 5 minutes. The amplification product was confirmed by 1.2% agarose gel electrophoresis and ethidium bromide staining.

[0087]

Example 6 Subcloning of PCR Product into Plasmid Vector and Selection of Novel Receptor Protein Candidate Clones by Decoding the Nucleotide Sequence of Inserted cDNA Part The reaction product after PCR performed in Example 5 was 1.2% electroeluted. Separate using agarose gel, cut out the band part with a razor, heat melt,
DNA was recovered by phenol extraction and ethanol precipitation. According to the prescription of TA cloning kit (Invitrogen), the recovered DNA was used as plasmid vector pC.
Subcloned into R ^™ II (TM stands for Trademark). This was introduced into E. coli JM109 competent cell (Takara Shuzo Co., Ltd.) for transformation, and then cDNA
The clone having the insert fragment was cloned into an LB agar medium (Luria- containing amphicillin, isopropylthio-β-D-galactoside (IPTG) and 5-bromo-4-chloro-3-indolyl-β-D-galactoside (X-gal). Bertan
(Iagar medium), only white clones were isolated using a sterilized toothpick, and transformed into Escherichia coli JM109 / pPL.
BS003 was obtained. Individual clones were cultured overnight in LB medium containing ampicillin, and plasmid DNA was prepared using an automatic plasmid extractor (Kurabo). A part of the prepared DNA was digested with EcoRI to confirm the size of the inserted cDNA fragment.
A part of the remaining DNA was further treated with RNase, extracted with phenol / chloroform, and concentrated by ethanol precipitation. The reaction for determining the nucleotide sequence is DyeDeoxy Term
Inator Cycle Sequencing Kit (Applied Biosystems: ABI) was performed, and it was decoded using a fluorescent automatic sequencer, and the obtained nucleotide sequence was analyzed by D.
It was performed using NASIS (Hitachi System Engineering Co., Ltd.). The underlined portion in FIG. 4 corresponds to the synthetic primer used in PCR. As a result of homology search based on the determined nucleotide sequence [SEQ ID NO: 4 (nucleotide sequence of FIG. 4)], the transformant Escherichia coli JM109 / pPLBS0 was obtained.
It was found that the cDNA fragment inserted into the plasmid possessed by 03 codes for a novel G protein-coupled receptor protein. To further confirm that, DNASI
The base sequence was converted into an amino acid sequence using S (Hitachi System Engineering Co., Ltd.) [SEQ ID NO: 2 (amino acid sequence in FIG. 4)]. A homology search based on the hydrophobicity plot [FIG. 5] and the amino acid sequence was performed, and human somatostatin receptor subtype 2 (P30874),
Human somatostatin receptor subtype 3 (P3
2745) and human somatostatin receptor subtype 1 (P30872) were found to be homologous [Fig. 6]. Abbreviations in () above are NBRF-PIR / Swiss-P
This is a reference number when it is registered as data in the ROT, and is usually called an Accession Number or entry name.

[0088]

The G protein-coupled receptor protein of the present invention and the DNA encoding the protein are the G protein of the present invention.
Determination of ligand for protein-coupled receptor protein, acquisition of antibody and antiserum, construction of recombinant receptor protein expression system, development of receptor binding assay system using the expression system and screening of drug candidate compounds, structurally It can be used for drug design based on comparison with similar ligands / receptors, probes for gene diagnosis, preparation of PCR primers, gene therapy and the like. In particular, elucidation of the structure and properties of G protein-coupled receptors will lead to the development of unique drugs that act on these systems.

[0089]

[Sequence list]

[SEQ ID NO: 1] Sequence length: 223 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Ala Ile Ala Asp Glu Leu Phe Thr Leu Val Leu Pro Ile Asn Ile Ala 1 5 10 15 Asp Phe Leu Leu Arg Gln Trp Pro Phe Gly Glu Leu Met Cys Lys Leu 20 25 30 Ile Val Ala Ile Asp Gln Tyr Asn Thr Phe Ser Ser Leu Tyr Phe Leu 35 40 45 Thr Val Met Ser Ala Asp Arg Tyr Leu Val Val Leu Ala Thr Ala Glu 50 55 60 Ser Arg Arg Val Val Gly Arg Thr Tyr Ser Ala Ala Arg Ala Val Ser 65 70 75 80 Leu Ala Val Trp Gly Ile Val Thr Leu Val Val Leu Ser Phe Ala Val 85 90 95 Phe Ala Arg Leu Asp Asp Glu Gln Gly Arg Arg Gln Cys Val Leu Val 100 105 110 Phe Pro Gln Pro Glu Ala Phe Trp Trp Arg Ala Ser Arg Leu Tyr Thr 115 120 125 Leu Val Leu Gly Phe Ala Ile Pro Val Ser Thr Ile Cys Val Leu Tyr 130 135 140 Thr Thr Leu Leu Cys Arg Leu His Ala Met Gly Leu Asp Ser His Ala 145 150 155 160 Lys Ala Leu Glu Arg Ala Lys Lys Arg Val Thr Phe Leu Val Val Ala 165 170 175 Ile Leu Ala Val Cys Leu Le u Cys Trp Thr Pro Tyr His Leu Ser Thr 180 185 190 Val Val Ala Leu Thr Thr Asp Leu Pro Gln Thr Pro Leu Val Ile Ala 195 200 205 Ile Ser Tyr Phe Ile Thr Ser Leu Ser Tyr Ala Asn Ser Cys Leu 210 215 220 223

[0090]

[SEQ ID NO: 2] Sequence length: 224 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Ala Val Ala Asp Gly Leu Phe Thr Leu Val Leu Pro Val Asn Ile Ala 1 5 10 15 Glu His Leu Leu Gln Tyr Trp Pro Phe Gly Glu Leu Leu Cys Lys Leu 20 25 30 Val Leu Ala Val Asp His Tyr Asn Ile Phe Ser Ser Ile Tyr Phe Leu 35 40 45 Ala Val Met Ser Val Asp Arg Tyr Leu Val Val Leu Ala Thr Val Arg 50 55 60 Ser Arg His Met Pro Trp Arg Thr Tyr Arg Gly Ala Lys Val Ala Ser 65 70 75 80 Leu Cys Val Trp Leu Gly Val Thr Val Leu Val Leu Pro Phe Phe Ser 85 90 95 Phe Ala Gly Val Tyr Ser Asn Glu Leu Gln Val Pro Ser Cys Gly Leu 100 105 110 Ser Phe Pro Trp Pro Glu Gln Val Trp Phe Lys Ala Ser Arg Val Tyr 115 120 125 Thr Leu Val Leu Gly Phe Val Leu Pro Val Cys Thr Ile Cys Val Leu 130 135 140 Tyr Thr Asp Leu Leu Arg Arg Leu Arg Ala Val Arg Leu Arg Ser Gly 145 150 155 160 Ala Lys Ala Leu Gly Lys Ala Arg Arg Lys Val Thr Val Leu Val Leu 165 170 175 Val Val Leu Ala Val Cys Leu Leu Cys Trp Thr Pro Phe His Leu Ala 180 185 190 Ser Val Val Ala Leu Thr Thr Asp Leu Pro Gln Thr Pro Leu Val Ile 195 200 205 Ser Met Ser Tyr Val Ile Thr Ser Leu Ser Tyr Ala Asn Ser Cys Leu 210 215 220 224

[0091]

[SEQ ID NO: 3] Sequence length: 669 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: cDNA Characterization method: S sequence GCCATCGCCG ACGAGCTCTT CACGCTGGTG CTGCCCATCA ACATCGCCGA CTTCCTGCTG 60 CGGCAGTGGC CCTTCGGGGA GCTCATGTGC AAGCTCATCG TGGCTATCGA CCAGTACAAC 120 ACCTTCTCCA GTCTCTACTT CCTCACCGTC ATGAGCGCCG ACCGCTACCT GGTGGTGTTG 180 GCCACTGCGG AGTCGCGCCG GGTGGTCGGT CGTACCTACA GTGCCGCGCG CGCGGTGAGC 240 CTGGCCGTGT GGGGGATCGT CACACTCGTC GTGCTGTCCT TCGCAGTCTT CGCCCGGCTA 300 GACGACGAGC AGGGCCGGCG CCAGTGCGTG CTAGTCTTTC CGCAGCCCGA GGCCTTCTGG 360 TGGCGCGCGA GCCGCCTCTA CACGCTCGTG CTGGGCTTCG CCATCCCCGT GTCCACCATC 420 TGTGTCCTCT ATACCACCCT GCTGTGCCGG CTGCATGCAA TGGGGCTGGA CAGCCACGCC 480 AAGGCCCTGG AGCGCGCCAA GAAGCGGGTG ACCTTCCTGG TGGTGGCAAT CCTGGCGGTG 540 TGCCTCCTCT GCTGGACGCC CTACCACCTG AGCACCGTGG TGGCGCTCAC CACCGACCTC 600 CCGCAGACGC CGCTGGTCAT CGCTATCTCC TACTTCATCA CCAGCCTGAG CTACGCCAAC 660 AGCTGCCTC 669

[0092]

[SEQ ID NO: 4] Sequence length: 672 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: cDNA Characterization method: S sequence GCCGTCGCCG ACGGGCTCTT CACGCTGGTA CTGCCCGTCA ACATCGCGGA GCACCTGCTG 60 CAGTACTGGC CCTTCGGGGA GCTGCTCTGC AAGCTGGTGC TGGCCGTCGA CCACTACAAC 120 ATCTTCTCCA GCATCTACTT CCTAGCCGTG ATGAGCGTGG ACCGATACCT GGTGGTGCTG 180 GCCACCGTGA GGTCCCGCCA CATGCCCTGG CGCACCTACC GGGGGGCGAA GGTCGCCAGC 240 CTGTGTGTCT GGCTGGGCGT CACGGTCCTG GTTCTGCCCT TCTTCTCTTT CGCTGGCGTC 300 TACAGCAACG AGCTGCAGGT CCCAAGCTGT GGGCTGAGCT TCCCGTGGCC CGAGCAGGTC 360 TGGTTCAAGG CCAGCCGTGT CTACACGTTG GTCCTGGGCT TCGTGCTGCC CGTGTGCACC 420 ATCTGTGTGC TCTACACAGA CCTCCTGCGC AGGCTGCGGG CCGTGCGGCT CCGCTCTGGA 480 GCCAAGGCTC TAGGCAAGGC CAGGCGGAAG GTGACCGTCC TGGTCCTCGT CGTGCTGGCC 540 GTGTGCCTCC TCTGCTGGAC GCCCTTCCAC CTGGCCTCTG TCGTGGCCCT GACCACGGAC 600 CTGCCCCAGA CCCCACTGGT CATCAGTATG TCCTACGTCA TCACCAGCCT CAGCTACGCC 660 AACTCGTGCC TG672

[0093]

[SEQ ID NO: 5] Sequence length: 27 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence characteristics: N ₂ represents I. Sequence GYCACCAACN ₂ WSTTCATCCT SWN ₂ HCTG 27

[0094]

[SEQ ID NO: 6] Sequence length: 27 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence features: N ₂ represents I. Array ASN ₂ SAN ₂ RAAG SARTAGAN ₂ GA N ₂ RGGRTT 27

[Brief description of the drawings]

FIG. 1 shows the nucleotide sequence of a receptor protein cDNA fragment contained in a novel receptor protein cDNA clone pPBBS002 obtained from human brain by PCR and the amino acid sequence encoded thereby. The underlined portion corresponds to the synthetic primer used for PCR amplification.

FIG. 2 was prepared based on the amino acid sequence shown in FIG.
A hydrophobicity plot of the receptor protein is shown. This figure suggests the presence of the hydrophobic domains shown in 2-7.

FIG. 3 is a partial amino acid sequence of a novel receptor protein cDNA encoded by pPBBS002 (pPBBS00
2, AA) for human somatostatin receptor subtype 1 (P30872), human somatostatin receptor subtype 2 (P30874) and human somatostatin receptor subtype 3 (P3274)
The figure which compared with the partial amino acid sequence of 5) is shown. The shaded areas indicate the matching amino acid residues.

FIG. 4 shows the nucleotide sequence of the receptor protein cDNA fragment contained in the novel receptor protein cDNA clone pPLBS003 obtained from human lungs by the PCR method and the amino acid sequence encoded thereby. The underlined portion corresponds to the synthetic primer used for PCR amplification.

FIG. 5 is prepared based on the amino acid sequence shown in FIG.
A hydrophobicity plot of the receptor protein is shown. This figure suggests the presence of the hydrophobic domains shown in 2-7.

FIG. 6 is a partial amino acid sequence of a novel receptor protein cDNA encoded by pPLBS003 (pPLBS00
3, AA) for human somatostatin receptor subtype 2 (P30874), human somatostatin receptor subtype 3 (P32745) and human somatostatin receptor subtype 1 (P3087)
The figure which compared with the partial amino acid sequence of 2) is shown. The shaded areas indicate the matching amino acid residues.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI Technical display location G01N 33/566 G01N 33/566 // A61K 38/00 A61K 39/395 D 39/395 N 48 / 00 48/00 9453-4B C12Q 1/68 A C12Q 1/68 A61K 37/02 (C12N 1/21 C12R 1:19) (72) Inventor Ryo Fujii 9 Takeda, 1-7 Kasuga, Tsukuba, Ibaraki Kasuga Heights No. 303

Claims (13)

[Claims] 1. A G protein-coupled receptor protein or a salt thereof or the G protein, which contains the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2. A partial peptide of a coupled receptor protein or a salt thereof. 2. A DNA containing a DNA having a nucleotide sequence encoding the G protein-coupled receptor protein or the partial peptide thereof according to claim 1. 3. The DNA according to claim 2, which has the nucleotide sequence represented by SEQ ID NO: 3 or SEQ ID NO: 4. 4. A vector containing the DNA according to claim 2 or 3. 5. A transformant carrying the vector according to claim 4. 6. The transformant according to claim 5 is cultured, and the transformant is caused to produce a G protein-coupled receptor protein or a partial peptide thereof.
A method for producing the G protein-coupled receptor protein or salt thereof or partial peptide thereof or salt thereof as described above. 7. The G protein-coupled receptor protein according to claim 1, wherein the G protein-coupled receptor protein according to claim 1 or a salt thereof or a partial peptide thereof or a salt thereof is contacted with a test sample. Alternatively, a method for determining a ligand for a salt thereof or a partial peptide thereof or a salt thereof. 8. (i) A case in which a ligand is contacted with the G protein-coupled receptor protein or its salt or its partial peptide or its salt according to claim 1, and (ii)
The G protein-coupled receptor protein according to claim 1 or a salt thereof or a partial peptide thereof or a salt thereof is compared with a case where a ligand and a test compound are contacted with each other, and the ligand according to claim 1 A method for screening a compound or a salt thereof that changes the binding property to a protein-coupled receptor protein or a salt thereof or a partial peptide thereof or a salt thereof. 9. A ligand and a G protein-coupled receptor protein or a salt thereof according to claim 1, which comprises the G protein-coupled receptor protein or a salt thereof or a partial peptide or salt thereof according to claim 1. A kit for screening a salt or a partial peptide thereof or a compound or a salt thereof that changes the binding property to the salt. 10. An antibody against the G protein-coupled receptor protein according to claim 1 or a salt thereof or a partial peptide thereof or a salt thereof. 11. A G protein-coupled receptor agonist or antagonist obtained by using the screening method according to claim 8 or the screening kit according to claim 9. 12. A DNA comprising a DNA cloned using the DNA according to claim 2 as a primer or a probe and having a nucleotide sequence encoding a G protein-coupled receptor protein. 13. A DNA cloned using the DNA according to claim 2 as a primer or a probe.
A G protein-coupled receptor protein or a salt thereof encoded by: or a partial peptide of the G protein-coupled receptor protein or a salt thereof.