JPH0970289A - Human crf2 recepter protein, its production and use - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new DNA for production, etc., of a human CRF2 receptor protein having a specific base sequence and useful for a primer, etc., for polymerase chain reaction of DNA coding a G protein conjugated receptor protein. SOLUTION: This new DNA has substantially same base sequence as a base sequence represented by formula I or II and is useful as a DNA primer, etc., used for polymerase chain reaction of DNA coding a G protein-conjugated receptor protein. This human CRF2 receptor protein is obtained by expressing a gene cloned by polymerase chain reaction using the DNA as a primer. The receptor protein is useful for screening an agonist such as a preventing and treating agent for dementia or obesity or a hypotensive agent and an antagonist for prevention and treatment, etc., of depression, inflammatory diseases, AIDS, Alzheimer's disease, gastrointestinal injury, reproductive failure, Cushing's disease, hypotension, etc.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel DNA useful as a DNA primer for polymerase chain reaction, a method for amplifying a DNA encoding a G protein-coupled receptor protein using the DNA, and a G protein using the DNA. D encoding a coupled receptor protein
NA screening method, DNA obtained by the screening method, and DN obtained by the screening method
The present invention relates to a G protein-coupled receptor protein encoded by A or a partial peptide thereof. Further, the present invention provides a novel CRF ₂ receptor protein, D encoding the protein.
The present invention relates to DNA containing NA, a method for producing the CRF ₂ receptor protein, and uses of the protein and DNA.

[0002]

G protein-coupled receptor proteins play a very important role as targets for molecules that regulate biological functions, such as hormones, neurotransmitters and physiologically active substances. There is a receptor protein specific to each molecule, and the specificity of the action of each physiologically active substance, that is, the target cell / organ, the pharmacological action, the action intensity, the action time, etc. are determined by the receptor protein. Therefore, if the G protein-coupled receptor gene or cDNA can be cloned, it will be useful not only for elucidating the structure, function, physiological action, etc. of the G protein-coupled receptor, but also by searching for substances that act on the receptor,
It is also thought to be useful for developing pharmaceuticals.
Although several G protein-coupled receptor genes or cDNAs have been cloned so far, it is considered that many unknown G protein-coupled receptor genes exist.

One of the characteristics of the G protein-coupled receptor proteins known so far is that seven clusters of hydrophobic amino acid residues are arranged on the primary structure and each cluster penetrates the cell membrane. To be This structure is common to all known G protein-coupled receptor proteins, and it is known that the amino acid sequence in the transmembrane region and its vicinity are often highly conserved among receptors. It is strongly suggested that when an unknown protein has such a structure, it falls into the category of G protein-coupled receptor protein. Further, some amino acid residues have a common arrangement, and these features are strongly suggested to be a G protein-coupled receptor protein.
A novel receptor gene is synthesized by the polymerase chain reaction method (hereinafter sometimes abbreviated as PCR method) by synthesizing a synthetic DNA primer based on a common amino acid sequence obtained by comparing known G protein-coupled receptor proteins. Has been reported by Libert F. et al. (Science 244: 569-572; 1989). In this report, Libert F. et al. Use a set of synthetic DNA primers corresponding to portions of the third and sixth transmembrane regions. However, the molecular species of the amplified DNA are generally defined by the design of the primer used in the PCR method. Further, based on the similarity of amino acid sequences, different codon usage affects the binding of the primer, resulting in a decrease in amplification efficiency. Therefore, although DNAs of various novel receptor proteins have been obtained using the DNA primer,
Not all receptor protein DNAs can be amplified.

An amino acid sequence common to the first to seventh transmembrane regions of 74 G protein-coupled receptor proteins has been reported by William C. Probst et al. (DNA AND CELL BIOLOGY, Vol / 11, No.1, 1992, pp
1-20). However, DN encoding these amino acid sequences
There is no suggestion of a method for screening a DNA encoding a novel G protein-coupled receptor protein by the PCR method using a DNA primer complementary to A. Therefore, by utilizing the common sequence of the G protein-coupled receptor protein or the DNA encoding the same, the DNA encoding the region closer to the full length of the novel G protein-coupled receptor protein is selectively and efficiently screened. It has been desired to develop a DNA primer for PCR for this purpose. The G protein-coupled receptor protein is very important as a target for studying a substance that regulates the function of a living body and proceeding as a pharmaceutical product. By using the G protein-coupled receptor protein and conducting receptor binding experiments and agonist / antagonist evaluation experiments using intracellular signal transduction system as an index, the discovery and development of new drug candidate compounds can be efficiently advanced. . In particular, if the existence of a novel G protein-coupled receptor protein is clarified, the existence of a specific agent for it is suggested. If the DNA encoding the novel G protein-coupled receptor protein can be efficiently screened and isolated, it is possible to efficiently proceed with the isolation of DNA having the entire coding region, construction of an expression system, and screening of an acting ligand. Is possible.

Corticotropin-Releasing Factor (hereinafter,
CRF) is a peptide consisting of 41 amino acids, which was first isolated from the hypothalamus of sheep in 1981, and has an activity of promoting ACTH / β-endorphin secretion from the pituitary gland. The mechanism of action is that CRF binds to a CRF receptor existing on the cell membrane surface of CRF target cells such as ACTH-producing cells (corticotroph) in the anterior pituitary, and then adenylate cyclase-cyclic AMP via guanine nucleotide regulatory protein (G protein). -prote
It has been clarified that it is due to activation of in kinase system [Vale, W. et al., Science 213].
Vol. 1394, 1981]. Clinically, synthetic CRF has been widely applied to the diagnosis of hypopituitarism and Cushing's syndrome. On the other hand, the CRF receptor is not only the pituitary gland,
Cerebral cortex gray matter, basal ganglia, paraventricular nucleus of hypothalamus, outer layer of central elevation, cerebral cerebral nuclei, olive nuclei, cerebellar nuclei, cerebellar cortex, olfactory cord, anterior disorders, tonsils, spinal horn Widely distributed [Vale, W., Corticotropin Releasing Fact
or), 172, 1-21 pages, 1993]. This distribution almost coincides with the distribution of CRF neuron terminals, and CRF is a neurotransmitter or neuromodu in these parts.
It is believed to act as a later. In addition, CRF receptor is also present in adrenal medulla, sympathetic ganglion, prostate, spleen, liver, kidney, testis, erythrocyte membrane, splenic macrophage, and during stress, pituitary ACTH / β
-endorphin In addition to its secretagogue action, it is speculated that it is involved in all stress adaptation reactions such as behavior, autonomic nerve or immune system reactions via these receptors.

The properties of CRF receptors present in the central nervous system have been well studied and include, for example, human CRF.
The receptor cDNA has been cloned into animal cells (COS 7 cells [Natalio Vita et al., Fabs.
Letters (FEBS Letters), Volume 335, pp. 1-5, 1993],
COS 6M cells [Ruoping Chen et al., Proc. Natl. Acad. Sci. USA],
90, 8967-8971, 1993]) has also been reported. However, it has been found that the distribution of the present CRF receptor (hereinafter referred to as CRF ₁ receptor) in the body does not always coincide with the place where CRF acts. For example, it has been suggested that CRF acts on the hypothalamus, brain stem, small intestine, stomach, testis, heart, skeletal muscle, etc.
Very few CRF ₁ receptors,
It was shown to be nonexistent [Owens, MJ, Ne
meroff, CB, Pharmacology Review (Pharma
col. Rev.) 43, 425, 1991], [Grunt, M. et a
l., American Journal of Physiology (A
M. J. Physiol.) 264, H1124, 1993], [Wei,
ET, Gao, GC, Regulatory peptides (Regul.
Pept.) 33, 93, 1991], [Lenz, HJ et a
l., American Journal of Physiology
(Am. J. Physiol.) 249, R85, 1985]. From these, it was considered that there may be a receptor having CRF as a ligand in addition to the CRF ₁ receptor.

Recently, a CRF receptor different from the CRF ₁ receptor (hereinafter referred to as CRF ₂ receptor) has been cloned from rat and mouse [Lovenberg, TW et al., Procedure of
The National Academy of Sciences USA (Proc. Natl. Acad. Sci. USA) 92, 836,
1995], [Kishimoto, T. et al., Proc. Natl. Acad. Sci. USA, Volume 92, Proc. Of the National Academy of Sciences USA.
1108, 1995], [Perrin, M. et al., Proc. Natl. Acad. Sci. USA] 9
2, p. 2969, 1995]. According to this, rat CRF ₂
The receptor cDNA encodes 411 amino acids and is a 7-transmembrane G protein-coupled receptor having about 70% homology with the CRF ₁ receptor. The mouse CRF ₂ receptor is 431 amino be composed of amino acids was found (in paper on the above mouse CRF ₂ receptors, the CRF ₂ receptor herein heart / muscle (HM) -CRF receptor or CRF
-Described in the term receptor B). On the other hand, it was not yet known whether the CRF ₂ receptor revealed in rat and mouse is also present in human.

CRF is present in many peripheral tissues (for example, placenta, adrenal medulla, pancreas, lung, stomach, duodenum, liver), and administration of CRF and CRF-like peptides soyvedin and urotensin I results in peripheral tissues. Systemic reactions have been shown to be triggered [Lenz, HJ
et al., American Journal of Physiology (Am. J. Physiol.) 249, R85, 1985]. From the rat and mouse CRF ₂ receptor studies described above,
CRF distributed in local and peripheral tissues of the central nervous system
It is strongly suggested that the receptor is the CRF ₂ receptor. From the above, it is expected that an agonist or antagonist for the CRF ₂ receptor can be a useful drug that acts on the central nervous system and peripheral tissues. When screening an agonist or an antagonist for the CRF ₂ receptor as a drug that acts on the human central nervous system or peripheral tissues, the primary screening is a tissue or cell expressing the human CRF ₂ receptor. Although it is conceivable to screen for substances that bind to the receptor by using, it is virtually impossible to obtain such human tissues because the human CRF ₂ receptor has not been confirmed. Moreover, cultured cells expressing the human CRF ₂ receptor are not known. On the other hand, although it is possible to perform screening using tissues or cells expressing the CRF ₂ receptor of rat or mouse, in this case, the difference in the characteristics of the receptor between animal species, the so-called species specificity of the receptor. Becomes a problem. In addition to the above, human CRF ₂ receptor cDNA
Since A had not been obtained, there was no way to investigate human CRF ₂ receptor signaling.

As a means for solving such a problem, if the human CRF ₂ receptor cDNA can be cloned, the human CRF ₂ can be cloned by an appropriate means.
_{Since the 2} receptor can be expressed in insect cells, animal cells, etc., it is considered that it can greatly advance the search and research of human CRF ₂ receptor agonists or antagonists. Regarding the human CRF ₂ receptor protein cDNA There was no knowledge of. That is, if the human CRF ₂ receptor protein can be used to screen for agonists / antagonists of the CRF ₂ receptor, there are drawbacks of using experimental animals (for example, compounds that cannot exert effects on humans due to different species). It is expected that the effective drug development for humans can be efficiently carried out.

[0010]

DISCLOSURE OF THE INVENTION The present invention provides a novel DNA useful as a DNA primer for PCR, and a D encoding a G protein-coupled receptor protein using the DNA.
Method for amplifying NA, method for screening DNA encoding G protein-coupled receptor protein using the DNA, DNA obtained by the screening method and G protein-coupled receptor protein encoded by the DNA obtained by the screening method, or the same A partial peptide is provided. Furthermore, the present invention provides a novel human CRF ₂ receptor protein useful for screening human CRF ₂ receptor agonists or antagonists acting on human central nervous system or peripheral tissues, DNA encoding the protein and production of the protein. Law and its uses.

[0011]

Means for Solving the Problems If the present inventors were able to efficiently collect a DNA encoding a G protein-coupled receptor protein, when the DNA encoding a novel receptor protein was contained therein. It is believed that it can be expressed by gene recombination technology and exerts a great effect on future research and drug development. As a result of intensive research, We succeeded in synthesizing a novel DNA primer based on the similarity of the nucleotide sequences encoding the seventh transmembrane region. Unexpectedly, by performing PCR using these DNA primers, G
We have succeeded in efficiently amplifying the DNA (fragment) encoding the protein-coupled receptor protein. That is, the present inventors have found that a DNA encoding a novel G protein-coupled receptor protein can be obtained by amplifying and analyzing various DNAs using the DNA primer.

More specifically, the present inventors have made known G
A DNA encoding a common amino acid sequence in the third transmembrane region by selecting a common amino acid sequence near the third transmembrane region and the seventh transmembrane region of the protein-coupled receptor protein
A primer (SEQ ID NO: 1) and a DNA primer (SEQ ID NO: 2) complementary to the base sequence encoding the common amino acid sequence near the seventh transmembrane region were designed.
These DNA primers differ in base sequence from similar DNA primers reported so far, and are novel DNA primers. In particular, a nucleotide sequence common to many receptor proteins is used at the 3'end of the primer so that the extension reaction can be efficiently performed in PCR. Taking advantage of the similarity of the base sequences also in other parts, the mixed base part was set so that the base sequences of the DNAs of as many receptor proteins as possible match. Then, the present inventors succeeded in amplifying cDNA derived from human stomach by PCR using the above-mentioned DNA primer, and proceeded with its analysis. As a result, the present inventors succeeded in isolating a human-derived cDNA encoding a novel G protein-coupled receptor protein and determining the partial structure thereof. Since this cDNA was found to have a high DNA and amino acid sequence homology with the rat and mouse CRF ₂ receptor proteins, it encodes a novel CRF ₂ receptor protein that is expressed in the human stomach. DN
I found out to be A. From these findings, this D
Using NA, cDNA having a full-length translation frame can be obtained, and the receptor protein can be produced. Furthermore, by using the receptor protein obtained by expressing the cDNA encoding the human CRF ₂ receptor protein by an appropriate means, it can be used in vivo or in the natural or natural environment by using the receptor binding experiment or the measurement of intracellular second messenger as an index. A ligand for the receptor protein can be screened from an unnatural compound, and a compound that inhibits the binding between the ligand and the receptor protein can be screened.

More specifically, the present inventors have shown in FIG.
The novel cDNA fragment derived from human stomach shown in (1) was amplified by PCR and subcloned into a plasmid vector (phs-AH1). From the analysis of the partial sequence, the c
It was revealed that the DNA encodes a novel receptor protein. When this sequence was translated into an amino acid sequence [Fig. 3], the third, fourth, fifth, sixth and seventh transmembrane regions were confirmed on the hydrophobicity plot [Fig. 4]. The size of the amplified cDNA was about 0.5 kb, which was about the same as the number of bases between the third transmembrane region and the seventh transmembrane region of the known G protein-coupled receptor protein. G protein-coupled receptor proteins show a certain degree of commonality in their amino acid sequences and form one protein family. Therefore, was subjected to homology search using the amino acid sequence encoded by the novel receptor protein DNA of the present (cDNA contained in phs-AH1), rat CRF ₂ receptor protein is a known G protein coupled receptor protein (U1623
5), mouse CRF receptor B protein (U1785
8) and 93.6% and 93.9% homology, respectively, with the novel receptor protein DNA of the present invention.
Was found to encode the human CRF ₂ receptor protein. Further, based on this, a cDNA encoding the full length of human CRF ₂ receptor protein by PCR method is used.
The fragment was obtained and the entire amino acid sequence of the human CRF ₂ receptor protein was clarified [FIG. 6 and FIG. 7].

That is, the present invention provides (1) SEQ ID NO: 1
Alternatively, a DNA having a base sequence substantially the same as the base sequence represented by SEQ ID NO: 2, (2)
The DN according to item (1), wherein the DNA is a DNA primer used for polymerase chain reaction of DNA encoding a G protein-coupled receptor protein.
A, (3) A DNA encoding a G protein-coupled receptor protein serving as a template and the DNA according to (1) are mixed to carry out a polymerase chain reaction. (4) A method for amplifying a DNA encoding DNA, wherein the DNA according to item (1) is used as a DNA primer in a polymerase chain reaction, and D containing a DNA encoding a G protein-coupled receptor protein.
Method for screening DNA encoding G protein-coupled receptor protein from NA library, (5)
DNA encoding a G protein-coupled receptor protein obtained by the screening method according to item (4), (6)
A G protein-coupled receptor protein encoded by the DNA according to item (5), a partial peptide thereof or a salt thereof,

(7) A human CRF ₂ receptor protein or a salt thereof, which contains an amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 3.
(8) A human CRF ₂ receptor protein or a salt thereof, which contains an amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 14, (9) Item (7) or (8) A partial peptide of the human CRF ₂ receptor protein or a salt thereof, (10) a DNA containing a DNA having a nucleotide sequence encoding the human CRF ₂ receptor protein of (7) or (8), 11) The DNA according to item (10), which has the base sequence represented by SEQ ID NO: 4, (12) The DNA according to item (10), which has the base sequence represented by SEQ ID NO: 15, (13) A vector containing the DNA according to item (10), (14) a transformant carrying the vector according to item (13), (15) an item (1)
The human C according to item (7) or (8), which is characterized in that the transformant according to item 4) is cultured and a human CRF ₂ receptor protein is produced on the cell membrane of the transformant.
Method for producing RF ₂ receptor protein or salt thereof, (1
6) The human CRF _{2 according to} item (7) or (8)
A receptor protein or a salt thereof, or a partial peptide or a salt thereof according to (9) is used to activate a CRF ₂ receptor or a salt thereof, or an antagonist to competitively inhibit the binding between CRF ₂ receptor and CRF Or a method for screening a salt thereof, (17) (i) item (7) or (8)
A human CRF ₂ receptor protein or a salt thereof according to item (9), a partial peptide or a salt thereof according to item (9), and a ligand, and (ii) the human according to item (7) or (8). CRF ₂ receptor protein or a salt thereof or a partial peptide or a salt thereof described in (9) is compared with a case where a ligand and a test compound are contacted with each other, and the ligand and the (7) or the (8) a method for screening a compound or its salt that inhibits the binding of human CRF ₂ receptor protein according to claim, (18) a (7) section or second (8) human CRF ₂ receptor protein according to claim or its A ligand and a partial peptide according to (9) or a salt thereof, and a ligand and (7)
Or a screening kit for a compound or a salt thereof that inhibits binding to the human CRF ₂ receptor protein according to item (8), (19) (16) or (1).
Human CR obtained using the screening method described in 7) or the screening kit described in 18)
F ₂ receptor agonist or salt thereof, human CRF ₂ receptor antagonist or salt thereof obtained by using the screening method according to (20), (16) or (17) or the screening kit according to (18) , (21) Human CR according to item (19)
A prophylactic / therapeutic agent for dementia and obesity, which contains an F ₂ receptor agonist or a salt thereof, an agent for promoting adaptation to stress, ACTH, β-endorphin,
β-lipotropin or α-MSF secretagogue, antihypertensive, mood and behavior regulator, gastrointestinal function regulator, autonomic nervous system regulator, or pituitary, cardiovascular system, digestive tract or central nervous system Depression resulting from stress characterized by containing a functional test drug, the human CRF ₂ receptor antagonist or the salt thereof according to (22) (20),
Anxiety / headache, inflammatory diseases, immunosuppression, AIDS, Alzheimer's disease, gastrointestinal disorders, loss of appetite, hemorrhagic stress, drug / alcohol withdrawal symptoms, drug dependence, reproductive disorders, Cushing's disease or hypotension Agent, and (2
3) Human CRF _{2 according to} item (7) or (8)
There is provided an antibody against the receptor protein or a salt thereof or the partial peptide according to (9) or a salt thereof.

More specifically, (24) the DNA is 1 in the nucleotide sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2 or the nucleotide sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2.
Or a nucleotide sequence in which 2 or more nucleotides are deleted, a nucleotide sequence in which 1 or 2 or more nucleotides are added to the nucleotide sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2, or SEQ ID NO: 1 or SEQ ID NO: The DNA according to item (1), which is a DNA having a base sequence in which one or more nucleotides in the base sequence represented by: 2 is replaced with another nucleotide, and the protein (25) is SEQ ID NO ::
1 or 2 or more in the amino acid sequence represented by SEQ ID NO: 3 or the amino acid sequence represented by SEQ ID NO: 3 Amino acid sequence with the addition of the amino acid of
Alternatively, the human CRF ₂ receptor protein according to item (7) or a protein thereof, which is a protein containing an amino acid sequence in which one or more amino acids in the amino acid sequence represented by SEQ ID NO: 3 are substituted with other amino acids. Salt, (26) protein, amino acid sequence represented by SEQ ID NO: 14, amino acid sequence represented by SEQ ID NO: 14 in which one or more amino acids are deleted, SEQ ID NO: 14
An amino acid sequence in which 1 or 2 or more amino acids are added to the amino acid sequence represented by, or an amino acid sequence in which 1 or 2 or more amino acids in the amino acid sequence represented by SEQ ID NO: 14 are substituted with other amino acids A human CRF ₂ receptor protein or a salt thereof according to item (8), which is a protein containing

(27) When the labeled ligand is brought into contact with the human CRF ₂ receptor protein or its salt according to the item (7) or (8) or the partial peptide or its salt according to the item (9) A label when the labeled ligand and the test compound are contacted with the human CRF ₂ receptor protein or the salt thereof according to item (7) or (8) or the partial peptide or salt thereof according to item (9) The binding amount of the ligand to the human CRF ₂ receptor protein or salt thereof according to item (7) or (8) or the partial peptide or salt thereof according to item (9) is measured and compared. ligand and second (7) sections or second (8) compounds that inhibit the binding of human CRF ₂ receptor protein according to claim or disk salt thereof to Ningu method, (28) a human C of the labeled ligand first (7) term or the (8) above, wherein
In the case of contacting with a cell containing the RF ₂ receptor protein and in the case of contacting the labeled ligand and the test compound with the cell containing the human CRF ₂ receptor protein according to the item (7) or (8) And a ligand characterized by measuring and comparing the amount of labeled ligand bound to the cell and the ligand (7) or (8)
Item 2. A method for screening a compound or a salt thereof which inhibits the binding to the human CRF ₂ receptor protein according to the item (2)
9) The case where the labeled ligand is brought into contact with the membrane fraction of cells containing the human CRF ₂ receptor protein according to the item (7) or (8), and the labeled ligand and the test compound are added to the item (7). Or the human C according to item (8)
The ligand which is characterized in that the amount of labeled ligand bound to the membrane fraction of a cell containing the RF ₂ receptor protein in contact with the membrane fraction is measured and compared. Alternatively, human C according to item (8)
Method for screening compound or salt thereof that inhibits binding to RF ₂ receptor protein, (30) Human expressing a transformant according to item (14) on the cell membrane of said transformant by culturing the transformant When contacted with the CRF ₂ receptor protein, the labeled ligand and the test compound were contacted with the human CRF ₂ receptor protein expressed on the cell membrane of the transformant by culturing the transformant described in (14). The amount of labeled ligand bound to the human CRF ₂ receptor protein is measured and compared with that of the human CRF according to item (7) or (8).
A method for screening a compound or a salt thereof which inhibits binding to an RF ₂ receptor protein, the compound which activates the human CRF ₂ receptor protein according to (31) (7) or (8) is (7). Alternatively, a compound and a test compound which activate the human CRF ₂ receptor protein according to (7) or (8) when contacted with cells containing the human CRF ₂ receptor protein according to (8) Characterized in that the cell stimulating activity mediated by the human CRF ₂ receptor protein in the case of contacting with the cells containing the human CRF ₂ receptor protein according to the item (7) or (8) is compared. And a human CR according to item (7) or (8)
A method for screening a compound or its salt that inhibits binding to F ₂ receptor protein, wherein the compound which activates human CRF ₂ receptor protein according to (32), (7) or (8) is (14). and when contacted with human CRF ₂ receptor protein expressed on the cell membrane of the transformant by culturing the transformant according, the first (7) term or the (8) human CRF ₂ receptor protein according to claim When the activating compound and the test compound are contacted with the human CRF ₂ receptor protein expressed on the cell membrane of the transformant by culturing the transformant according to (14), the human CRF ₂ receptor protein is Ligands and (7) or (8) characterized by measuring and comparing cell-mediated cell-stimulating activity
A method for screening a compound or a salt thereof, which inhibits the binding to the human CRF ₂ receptor protein according to the above item,

(33) A compound or salt thereof obtained by the screening method described in (17) or (27) to (32), or (34) a compound or salt thereof described in (33). A pharmaceutical composition comprising (3)
5) Human CRF _{2 according to} item (7) or (8)
A kit for screening according to item (18), which comprises cells containing a receptor protein,
(36) The human CR according to item (7) or (8)
A screening kit according to item (18), which comprises a membrane fraction of a cell containing an F ₂ receptor protein, (37) item (18), (35) or (36). A compound or a salt thereof obtained by using the screening kit according to Item (38), a pharmaceutical composition comprising the compound or salt thereof according to Item (37), and (39) (23). A method comprising contacting the antibody according to item (7) with the human CRF ₂ receptor protein according to item (7) or (8) or a salt thereof or the partial peptide according to item (9) or a salt thereof. A method for quantifying the human CRF ₂ receptor protein or salt thereof according to item (7) or (8) or the partial peptide or salt thereof according to item (9) is provided.

BEST MODE FOR CARRYING OUT THE INVENTION

The DNA of the present invention is a DNA having a base sequence substantially the same as the base sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2. That is, the base sequence represented by SEQ ID NO: 1 is 5'-CATTAYTKGATSGYGRCCAACT.
WCWNCTGG-3 ′ [Y represents T or C, K represents G or T, S represents C or G, R represents A or G, W represents A or T, and N represents I . ] [Fig. 1]. The base sequence represented by SEQ ID NO: 2 is: 5′-GTAGARRAYAGCCACMAMRARN
CCCTGRAA-3 ′ [R represents A or G, Y represents T or C, M represents A or C, and N represents I. ], Which is 5'-TTYCAGGGGNYTYKTKGTGGCTR
TYYTCTAC-3 ′ [Y represents T or C, N represents I, K represents G or T, and R represents A or G]. ] It is a base sequence complementary to the base sequence represented by [Fig. 2].

The DNA of the present invention includes, in addition to the DNA having the nucleotide sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2, the nucleotide sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2 Examples thereof include a DNA having a base sequence having a homology of 70 to 99.9% and having substantially the same function as the DNA having the base sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2. Examples of substantially the same function include a DNA primer binding function and amplification efficiency. "Substantially the same quality" means that the binding function and primer activity of the DNA primer are qualitatively the same. More specifically, examples of the DNA of the present invention include DNA having the nucleotide sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2. In addition, the DNA of the present invention has a nucleotide sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2 in which one or more nucleotides are deleted, SEQ ID NO: 1 or SEQ ID NO: 2. A nucleotide sequence in which 1 or 2 or more nucleotides are added to the nucleotide sequence represented, 1 or 2 or more nucleotides in the nucleotide sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2 are replaced with other nucleotides. Also included are DNAs having different base sequences. More specifically, the DNA of the present invention includes, in addition to the DNA having the nucleotide sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2, the nucleotide represented by SEQ ID NO: 1 or SEQ ID NO: 2. One or two or more (preferably 2 or more and 9 or less, more preferably 2 or more and 6 or less) nucleotides in the sequence are deleted, SEQ ID NO:
1 or 1 or 2 in the nucleotide sequence represented by SEQ ID NO: 2
One or more (preferably 2 or more and 9 or less, more preferably 2 or more and 6 or less DNA) nucleotides added, 1 or 2 in the base sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2. Examples include those in which the above nucleotides (preferably 2 or more and 9 or less, more preferably 2 or more and 6 or less DNA) are substituted with other nucleotides.

The nucleotide sequences represented by SEQ ID NO: 1 and SEQ ID NO: 2 are known G protein-coupled receptor proteins, that is, human calcitonin receptor (X824
66), human parathyroid hormone receptor (X68
596), human glucagon receptor (U0346
9), human glucagon-like polypeptide-1 receptor (U01156), rat secretin receptor (X59132), human growth hormone releasing hormone receptor (L01406), human picitary adenylate cyclase-activating polypeptide receptor (D17516), Rat-Pituitary Adenylate Cycle-Activated Polypeptide Receptor (Z23279), Human Vasoactive Intestinal Peptide Receptor (X75299), Human Vip-2 Receptor (L36566), Human Corticotropin Release Factor Receptor (L23332), and Rat Corticotropin Release Factor Amino acid sequences near the third and seventh transmembrane regions such as 2 receptor (U16253) Is a nucleotide sequence present in common to the base sequence of the DNA over de [FIGS]. The abbreviations in parentheses above are reference numbers when registered as data in GenBank / EMBL Data Bank.
It is called Number.

The DNA of the present invention is a DNA known per se.
It can be produced according to a synthetic method or a method analogous thereto. For example, either solid phase synthesis method or liquid phase synthesis method may be used. Known synthetic methods include, for example, the following methods (1) to (3). Ikehara Morio et al., Nucleic Acid Organic Chemistry, Chemistry (1979) GH Blackburn and MJ Gait, Nucleic Acids i
n Chemistry and Biology, IRL Press, Oxford (1989
Eiko Otsuka and Hideo Inoue, Jpn Jpn, 47, 87 (19)
(1989) Alternatively, a commercially available automatic DNA synthesizer can be used.
Among the DNAs of the present invention, the DNA having the nucleotide sequence represented by SEQ ID NO: 1 has a common nucleotide sequence with the DNA encoding the amino acid sequence near the third transmembrane region of the above-mentioned known G protein-coupled receptor protein. DNA having the nucleotide sequence represented by SEQ ID NO: 2 is complementary to the nucleotide sequence commonly present in the DNA encoding the amino acid sequence near the seventh transmembrane region. Since it has a nucleotide sequence, it is a DNA encoding the known G protein-coupled receptor protein (genomic DNA, c
It can bind to DNA) or RNA complementarily. Furthermore, not only the DNA / RNA encoding these known G protein-coupled receptor proteins, but also the DNA encoding other known G protein-coupled receptor proteins having a base sequence similar to the base sequence of the DNA of the present invention.
Alternatively, it can also complementarily bind to RNA or DNA or RNA encoding an unknown G protein-coupled receptor protein. Therefore, the DNA of the present invention can be used as a DNA primer for PCR. For example, by amplifying a DNA (fragment) encoding the receptor protein by mixing a small amount of DNA (fragment) encoding a G protein-coupled receptor protein as a template with the DNA primer of the present invention and performing PCR. You can Specifically, when PCR is carried out using a DNA primer having the nucleotide sequence represented by SEQ ID NO: 1, the DNA primer is a DNA (fragment) or R encoding a G protein-coupled receptor protein serving as a template.
Binds to the base sequence corresponding to the third transmembrane region of NA,
The DNA is extended from the 5'side to the 3'side. On the other hand, when the PCR method is carried out using the DNA primer having the nucleotide sequence represented by SEQ ID NO: 2, the DNA primer is used as a template for DNA (fragment) or RNA seventh membrane encoding G protein-coupled receptor protein. It binds to the base sequence corresponding to the penetrating region and extends the DNA from the 3 ′ side to the 5 ′ side. That is, by using the DNA primer having the nucleotide sequence represented by SEQ ID NO: 1 and the DNA primer having the nucleotide sequence represented by SEQ ID NO: 2 of the present invention, the third transmembrane region of the G protein-coupled receptor protein can be obtained. The DNA (fragment) encoding the seventh transmembrane region can be amplified.

This amplification method can be carried out according to the known PCR method. For example, Saiki RK et al. Sc
ience, 239: 487-491 (1988). PCR temperature, time, buffer,
Number of cycles, enzymes such as DNA polymerase, 2'-deoxy
The addition of -7-deaza-guanosine triphosphate or Inosine can be appropriately selected according to the type of target DNA. When using RNA as a template, Saiki R.
The method is described in K. et al. Science, 239: 487-491 (1988). Furthermore, the DNA of the present invention comprises the third transmembrane region or the seventh transmembrane region of the G protein-coupled receptor protein.
Since it can bind complementarily to the base sequence of DNA encoding the amino acid sequence near the transmembrane region, it can be used to screen a DNA (fragment) encoding a G protein-coupled receptor protein from a certain DNA library. It is also useful as a probe. The DNA (fragment) encoding the G protein-coupled receptor protein can be screened from a DNA library using the DNA of the present invention as a probe according to a method known per se or a method analogous thereto. In particular, when the DNA of the present invention is used as a DNA primer for the PCR method, the amplification and screening of the DNA (fragment) encoding the G protein-coupled receptor protein can be performed at once. That is, when the DNA of the present invention is used as a DNA primer for PCR, the DNA primer is a DN encoding the amino acid sequence of the third transmembrane region to the seventh transmembrane region of the G protein-coupled receptor protein.
Since the DNA primer of the present invention can bind to A (fragment) or RNA and amplify the DNA, the DNA primer of the present invention is used in the DNA library from the third transmembrane region to the seventh membrane of the G protein-coupled receptor protein. Only the DNA (fragment) encoding the amino acid sequence of the penetrating region can be selectively amplified. Then, using the DNA (fragment) encoding the amino acid sequence of the third to seventh transmembrane regions of the amplified G protein-coupled receptor protein as a probe, a DNA library was prepared according to a method known per se. DNAs that completely encode the G protein-coupled receptor protein can be screened.

That is, the present invention uses the DNA of the present invention as P
A DNA library containing a DNA (fragment) encoding a receptor protein characterized by being used as a DNA primer for CR or RNA derived from a tissue / cell
To DN encoding G protein-coupled receptor protein
A method for screening A (fragment) is provided. Specifically, the present invention provides the DNA of the present invention as a DNA for PCR.
DNA used as a primer and encoding a G protein-coupled receptor protein as a template for the DNA primer
PC with a DNA library containing (fragments)
The R method was performed, and the third of G protein-coupled receptor proteins was performed.
DNA encoding from the transmembrane region to the seventh transmembrane region
A method for amplifying and selecting (that is, screening), and using this as a probe to clone a DNA encoding a full-length G protein-coupled receptor protein from a DNA library using a method known per se To do. Analysis of cloned DNA can be performed using a DNA sequencer. D of the present invention
The DNA (fragment) or RNA encoding a G protein-coupled receptor protein to be amplified and screened by the PCR method using NA is a known DNA (fragment) or RNA encoding a G protein-coupled receptor protein. It may be DNA (fragment) or RNA encoding a novel G protein-coupled receptor protein. For example, vertebrates (eg,
Mouse, rat, cat, dog, pig, cow, horse, monkey, human, etc.) (for example, pituitary gland, brain,
G protein-coupled from pancreas, lung, adrenal gland), insects or other invertebrates (eg Drosophila, silkworm, ginger, etc.), plants (eg rice, wheat, tomato etc.) and cultured cell lines derived therefrom (Fragment) or RN encoding the receptor protein
A and the like. Specifically, for example, calcitonin, parathyroid hormone (PTH), glucagon, glucagon-like polypeptide-1, secretin, growth hormone releasing hormone (GHRH), PACAP,
Examples thereof include DNA (fragment) or RNA encoding a G protein-coupled receptor protein such as VIP, VIP2 and CRF. The DNA (fragment) used as a template for amplification by the PCR method using the DNA of the present invention may be any DNA derived from the above-mentioned tissues / cells. More specifically, it may be any of genomic DNA, genomic DNA library, tissue / cell-derived cDNA, and tissue / cell-derived cDNA library. The vector used for the library may be any of bacteriophage, plasmid, cosmid, phagemid and the like. Alternatively, it can be directly amplified by the RT-PCR method using an mRNA fraction prepared from a tissue / cell. The template DNA may be a DNA that completely encodes the G protein-coupled receptor protein,
It may be the DNA fragment. In this specification, these may be collectively referred to as DNA (fragment).

As a means for cloning DNA, a synthetic DNA primer having a partial base sequence of the DNA is used for amplification by the PCR method, or a DNA having a part or the whole region of the DNA or a synthetic DNA.
Select by hybridization with those labeled with. Hybridization methods include, for example, Molecular Cloning 2nd ed .; J. Sambrook et al.,
It is performed according to the method described in Cold Spring 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. Therefore, the DNA (fragment) obtained by the screening method using the DNA of the present invention is a DNA (fragment) encoding a G protein-coupled receptor protein contained in a DNA library or the like. That is, specifically, calcitonin, PTH, glucagon, glucagon-like polypeptide-1, secretin, GHRH, P
It is a DNA (fragment) encoding a G protein-coupled receptor protein such as ACAP, VIP, VIP2 and CRF. By using the DNA (fragment) obtained by the screening method as a probe, a DNA completely encoding a G protein-coupled receptor protein is isolated from an appropriate DNA library according to a method for screening a DNA known per se. be able to.

The DNA completely 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 has ATG as a translation initiation codon at the 5'terminal side, and TAA, TGA or TAG as a translation stop codon at the 3'terminal side.
May be provided. These translation initiation codon and translation termination codon can also be added using a suitable synthetic DNA adapter. Further, by performing Northern probing using the DNA as a probe, the tissue / cell in which the receptor protein is expressed can be determined. Further, the receptor protein can be expressed by linking a DNA having the entire coding region to an appropriate promoter and introducing it into an animal cell. Specific examples of the G protein-coupled receptor protein encoded by the DNA encoding the G protein-coupled receptor protein obtained by the screening method of the present invention include calcitonin, PTH, glucagon, glucagon-like polypeptide-1, and secretin. , GHRH, PACAP, VIP, VIP2, CR
It is a G protein-coupled receptor protein such as F. The G
The protein-coupled receptor protein can be produced from tissues or cells of warm-blooded animals by a method known per se for protein purification, or by culturing a transformant containing a DNA encoding the G protein-coupled receptor protein. Can also be manufactured by.

As the partial peptide of the G protein-coupled receptor protein, for example, a portion of the G protein-coupled receptor protein molecule that is exposed outside the cell membrane is used. Specifically, it is a partial peptide analyzed to be an extracellular region (hydrophilic site) in the hydrophobicity plot analysis of G protein-coupled receptor protein. Further, a peptide partially containing a hydrophobic (Hydtophobic) site can also be used. further,
Peptides containing individual domains may be used, but partial peptides containing multiple domains simultaneously may also be used. The partial peptide of the G protein-coupled receptor protein can be produced by publicly known methods for peptide synthesis or by cleaving the G protein-coupled receptor protein with an appropriate peptidase. 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, Interscie
nce Publishers, New York (1966) Schroeder and Luebke, The Peptide,
Academic Press, New York (1965) Nobuo Izumiya et al., Fundamentals and experiments of peptide synthesis, Maruzen Co., Ltd.
(1975) Haruaki Yajima and Shunpei Sakakibara, Laboratory for Biochemistry 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 solvent extraction, distillation, column chromatography, liquid chromatography, recrystallization and the like. When the protein obtained by the above method is a free form, it can be converted into an applicable 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. Furthermore, it can also be produced by culturing a transformant containing a DNA encoding the partial peptide. As the salt of the G protein-coupled receptor protein or its partial peptide, a physiologically acceptable acid addition salt is particularly preferable. Examples of such salts include salts with inorganic acids (eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid) 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 DNA (fragment) encoding the G protein-coupled receptor protein obtained by the aforementioned screening method using the DNA of the present invention and the G protein-coupled receptor protein or partial peptide encoded by the DNA are, for example, It can be used for determination of a ligand for the G protein-coupled receptor protein, screening for a compound that inhibits the binding between the receptor protein and the ligand, and the like. In this case, first, an expression system of DNA (fragment) encoding the G protein-coupled receptor protein is constructed. Any of animal cells, insect cells, yeast, Bacillus subtilis, Escherichia coli, etc. may be used as a host, and any promoter may be used as long as it is suitable for the host used for gene expression. The use of enhancers for expression is also effective.
The constructed expression cells can be used as they are, or a membrane fraction can be prepared therefrom by a known method and subjected to a binding experiment. The ligand to be used may be a commercially available compound labeled with a radioisotope or the like, or a culture supernatant, a tissue extract or the like directly labeled by the chloramine T method or the lactoperoxidase method. Separation of bound and free ligands may be carried out by washing as it is when cells adhering to the substrate are used, and by centrifugation or filtration in the case of floating cells or membrane fraction. Non-specific binding to a container or the like can be estimated by adding a 100-fold concentrated non-labeled ligand to the input labeled ligand.

With respect to the ligand obtained by such a binding experiment, the agonist / antagonist can be discriminated. Specifically, cells expressing the receptor protein and a natural product / compound expected to be a ligand are incubated, and then the culture supernatant is collected or cells are extracted. Changes in the components contained in these are measured, for example, with a commercially available measurement kit (cAMP, diacylglycerol, cGMP, protein kinase A, etc.). In addition, according to a known method, Fura-2, [ ³
The release of [H] arachidonic acid and [ ³ H] inositol phosphate metabolites can also be measured. The compounds and natural products obtained by such screening are agonists or antagonists to the receptor protein, and are expected to act on the tissues / cells in which the receptor is distributed. Therefore, by referring to the distribution clarified by Northern blotting or the like, it is possible to more efficiently search for its drug efficacy. In addition, compounds having new medicinal effects are desired in, for example, central nervous system, circulatory system, kidney, pancreas and the like. By selectively amplifying the DNA encoding the G protein-coupled receptor protein from the tissue, it is possible to more efficiently develop the drug by using these.

Below, D encoding the G protein-coupled receptor protein obtained by the method for screening the DNA encoding the G protein-coupled receptor protein of the present invention
Usefulness of NA (fragment) and G protein-coupled receptor protein obtained by expressing the DNA, partial peptide thereof or salts thereof (hereinafter, the salt is abbreviated as G protein-coupled receptor protein or partial peptide thereof) The sex will be described in detail below. (1) Method for determining ligand for G protein-coupled receptor protein The G protein-coupled receptor protein or its partial peptide is useful as a reagent for searching for or determining a ligand for the G protein-coupled receptor protein. That is, the present invention provides a method for determining a ligand for a G protein-coupled receptor protein, which comprises contacting a G protein-coupled receptor protein or a partial peptide thereof with a test compound. Known compounds (eg, angiotensin, bombesi,
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, prostanoid, Thromboxane, adenosine, adrenaline, α
And β-chemokine (IL-8, GROα, GRO
β, GROγ, NAP-2, ENA-78, PF4, I
P10, GCP-2, MCP-1, HC14, MCP-
3, I-309, MIP1α, MIP-1β, RANT
ES, etc.), endothelin, enterogastrin, histamine, neurotensin, TRH, pancreatic polypeptide, galanin, calcitonin, parathyroid hormone, glucagon, secretin, GHRH, P
In addition to (ACAP, CRF, etc.), for example, tissue extracts of warm-blooded animals (eg, mouse, rat, pig, cow, sheep, monkey, human, etc.), cell culture supernatant, etc. are used.
For example, the tissue extract, cell culture supernatant and the like are added to a G protein-coupled receptor protein, fractionated while measuring cell stimulating activity and the like, and finally a single ligand can be obtained.

Specifically, in the method for determining the ligand, a G protein-coupled receptor protein or a partial peptide thereof is used, or an expression system of a recombinant receptor protein is constructed, and a receptor binding assay system using the expression system is used. The use of the above-mentioned protein binds to a G protein-coupled receptor protein to stimulate cell activity (eg, arachidonic acid release, acetylcholine release, intracellular Ca ²⁺ release, intracellular c release).
Compounds (for example, peptides, proteins, non-peptide compounds, synthetic compounds) having an activity of promoting or suppressing AMP production, intracellular cGMP production, inositol phosphate production, cell membrane potential fluctuation, intracellular protein phosphorylation, etc. , Fermentation products, etc.) or salts thereof. In the method for determining a ligand, when a G protein-coupled receptor protein or partial peptide is contacted with a test compound, for example, the binding amount of the test compound to the G protein-coupled receptor protein or the partial peptide, cell stimulating activity, etc. It is characterized by measuring.

More specifically, when the labeled test compound is contacted with the G protein-coupled receptor protein or its partial peptide or its salt, the labeled test compound's protein or its salt, or its partial peptide Alternatively, a method for determining a ligand for a G protein-coupled receptor protein, which comprises measuring the amount of binding to a salt thereof, a labeled test compound is added to a cell containing the G protein-coupled receptor protein or a membrane fraction of the cell. A method for determining a ligand for a G protein-coupled receptor protein, which comprises measuring a binding amount of a labeled test compound to the cell or the membrane fraction when contacted, Cultivating Transformant Containing DNA Encoding Type I Receptor Protein G protein-coupled receptor protein is characterized by measuring the binding amount of the labeled test compound to the G protein-coupled receptor protein when it is brought into contact with the G protein-coupled receptor protein expressed on the cell membrane. Method for determining ligand for protein, cell-stimulating activity mediated by G protein-coupled receptor protein (eg, arachidonic acid release, acetylcholine release, when test compound is contacted with cells containing G protein-coupled receptor protein) Intracellular Ca ²⁺ release, intracellular cAMP production, intracellular cGMP production, inositol phosphate production, cell membrane potential fluctuation, intracellular protein phosphorylation, activity promoting or suppressing c-fos activation, etc.) A G protein-coupled receptor protein characterized by measuring A method for determining a ligand, and a test compound, which are brought into contact 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, Cell stimulating activity mediated by 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 of,
A method for determining a ligand for a G protein-coupled receptor protein, which comprises measuring the activity of promoting or suppressing the activation of c-fos).

A specific description of the ligand determination method will be given below. First, the G protein-coupled receptor protein used in the ligand determination method may be any one as long as it contains the G protein-coupled receptor protein or its partial peptide, but it is expressed in large amounts using animal cells. Suitable G protein-coupled receptor proteins are 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 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. B.
iol. Chem.), 267, 19555-19559, 1992].

Therefore, in the ligand determination method,
Those containing a G protein-coupled receptor protein or a partial peptide thereof include a G protein-coupled receptor protein purified by a method known per se or the G
It may be a partial peptide of a protein-coupled receptor protein, a cell containing the protein may be used, or a membrane fraction of cells containing the protein may be used. When cells containing a G protein-coupled receptor protein are used in the ligand determination method, the cells may be immobilized with glutaraldehyde, formalin or the like. The immobilization method can be performed according to a method known per se.
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 centrifuged at a low speed (500 rpm to 3000 rpm) for a short time (generally about 1 to 10 minutes), and the supernatant is further centrifuged at a higher speed (15,000 to 30000 rpm) for 30 minutes.
After centrifugation for 2 minutes to 2 hours, the obtained 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 the above-mentioned methods (1) to (3) for determining a ligand binding to a G protein-coupled receptor protein, an appropriate G protein-coupled receptor fraction and a labeled test compound are required. 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. The labeled test compounds include [ ³ H], [ ¹²⁵ I],
Angiotensin labeled with [ ¹⁴ C], [ ³⁵ S], etc.,
Bombesi, cannabinoids, cholecystokinin, glutamine, serotonin, melatonin, neuropeptide Y, opioids, purines, vasopressin, oxytocin, V
IP (Vasoactive Intestinal and Related Peptide), Somatostatin, Dopamine (D5 Dopamine etc.), Motilin, Amylin, Bradykinin, CGRP (Calcitonin Gene Relayed Peptide), Adrenomedullin, Leukotriene, Pancreatatin, Prostanoid, Thrombo Xanthane,
Adenosine, adrenaline, α- and β-chemokine
(IL-8, GROα, GROβ, GROγ, NAP
-2, ENA-78, PF4, IP10, GCP-2,
MCP-1, HC14, MCP-3, I-309, MI
P1α, MIP-1β, RANTES, etc.), endothelin, enterogastrin, histamine, neurotensin, TRH, pancreatic polypeptide, galanin, calcitonin, parathyroid hormone, glucagon, secretin, GHRH, PACAP, CRF and the like are preferable. is there.

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 (NSB)
Also prepare a reaction tube containing a large excess of unlabeled test compound. The reaction is carried out at 0 ° C to 50 ° C, preferably 4 ° C to 37 ° C for 20 minutes to 24 hours, preferably 3
Do from 0 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 of total binding (B) minus NSB (B-NSB) 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 methods (1) to (3) for determining a ligand that binds to a G protein-coupled receptor protein, cell stimulating activity mediated by the G protein-coupled receptor protein (eg, arachidonic acid release, acetylcholine release, cell Internal Ca ²⁺ release, intracellular cAMP production, intracellular cGMP production, inositol phosphate production, cell membrane potential fluctuation, intracellular protein phosphorylation, activity promoting or suppressing c-fos activation, etc.) It 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. Substances used as indicators of cell-stimulating activity (eg, arachidonic acid)
When it is difficult to assay the production of the enzyme by the degrading enzyme contained in the cells, an assay may be performed by adding an inhibitor to the degrading enzyme. 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 used in the method for determining a ligand that binds to a G protein-coupled receptor protein includes a G protein-coupled receptor protein or a partial peptide thereof, G
Examples thereof include those containing a membrane fraction of cells containing a protein-coupled receptor protein, or cells containing a G protein-coupled receptor protein. Examples of the ligand determination kit include the following. 1. Ligand determination reagent Measurement buffer and washing buffer Hanks' Balanced Salt Solution (manufactured by Gibco) was added with 0.
One containing 05% bovine serum albumin (manufactured by Sigma). Sterilized by filtration through a 0.45 μm filter, 4 ° C
Or may be prepared at the time of use. G protein-coupled receptor protein preparation CHO cells expressing the 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 A compound labeled with commercially available [ ³ H], [ ¹²⁵ I], [ ¹⁴ C], [ ³⁵ S], or the like, or labeled by an appropriate method. Store at 0 ° C. and dilute to 1 μM with measurement buffer before use. Test compounds that are poorly soluble in water are dissolved in dimethylformamide, DMSO, methanol and the like. Unlabeled test compound The same 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. The ligand capable of binding to the G protein-coupled receptor protein includes, for example, substances specifically present in the brain, pituitary, and the like, and specifically, angiotensin, bombesi, cannabinoids, cholecystokinin, glutamine, Serotonin, melatonin, neuropeptide Y, opioid, purine, vasopressin, oxytocin, VIP (vasoactive intestinal and related peptide), somatostatin, dopamine (such as D5 dopamine), motilin, amylin,
Bradykinin, CGRP (calcitonin gene relayed peptide), adrenomedullin, leukotriene, pancreatatin, prostanoid, thromboxane, adenosine, adrenaline, α and β-chemok
ine (IL-8, GROα, GROβ, GROγ, N
AP-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, calcitonin, parathyroid hormone,
Glucagon, secretin, GHRH, PACAP, CR
F and the like.

(2) Preventive / therapeutic agent for G protein-coupled receptor protein deficiency In the method of (1) above, when the ligand for the G protein-coupled receptor protein is clarified, G DNA encoding a protein-coupled receptor protein can be safely used as a prophylactic / therapeutic agent for G protein-coupled receptor protein deficiency with low toxicity. For example, when there is a patient in whom the physiological action of the ligand cannot be expected due to a decrease in G protein-coupled receptor protein in vivo, (a) the DNA encoding the G protein-coupled receptor protein is administered to the patient. D that encodes a G protein-coupled receptor protein by expressing or (b) brain cells
It is possible to increase the amount of G protein-coupled receptor protein in the brain cells of the patient and to sufficiently exert the action of the ligand by, for example, transplanting the brain cells into the patient after inserting and expressing NA. it can. Therefore, G
The DNA encoding the protein-coupled receptor protein is
It can be used as a safe and low-toxicity prophylactic / therapeutic agent for G protein-coupled receptor protein deficiency.

When a DNA encoding a G protein-coupled receptor protein is used as the therapeutic agent, the DN
It can be carried out by a conventional method after A is inserted alone or into an appropriate vector such as a retrovirus vector, an adenovirus vector, an adenovirus associated virus vector. 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, admixing the DNA of the present invention with physiologically acceptable carriers, flavoring agents, excipients, vehicles, preservatives, stabilizers, binders and the like in a unit dose form required for generally accepted pharmaceutical practice. 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. tablet,
Examples of additives that can be mixed with capsules include binders such as gelatin, corn starch, tragacanth, gum arabic, excipients such as crystalline cellulose, swelling agents such as corn starch, gelatin, alginic acid, and the like. And lubricating agents such as magnesium stearate, sweetening agents such as sucrose, lactose or saccharin, flavoring agents such as peppermint, reddish oil or cherry. 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), polyalcohols (eg propylene glycol, polyethylene glycol), nonionic surfactants (eg polysorbate 80 (TM), H)
CO-50) and the like 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.

Also, a buffering agent (eg, phosphate buffer,
Sodium acetate buffer), soothing agents (eg, benzalkonium chloride, procaine hydrochloride, etc.), stabilizers (eg, human serum albumin, polyethylene glycol, etc.), preservatives (eg, benzyl alcohol, phenol, etc.), antioxidants And the like. The adjusted injection solution 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 1.0 to 50 mg, more preferably 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 case of an injection, for example, in an adult (60 kg), the daily dose is about 1 day. About 0.01 to 30 mg, preferably 0.1 to 20 mg
Conveniently, about 0.1 to 10 mg is preferably administered by intravenous injection. For other animals,
The amount converted per 60 kg can be administered.

(3) Method for Quantifying Ligand for G Protein-Coupled Receptor Protein Since the G protein-coupled receptor protein or its partial peptide has a binding property to the ligand, the ligand concentration in the living body can be detected with high sensitivity. It can be quantified. This quantification method can be used, for example, in combination with a competition method. That is, the concentration of the ligand in the sample can be measured by bringing the sample into contact with the G protein-coupled receptor protein or its partial peptide. 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) Method for screening compound that inhibits binding between G protein-coupled receptor protein and ligand: G protein-coupled receptor protein or a partial peptide thereof is used, or a recombinant receptor protein expression system is constructed. A compound that inhibits the binding between a ligand and a G protein-coupled receptor protein by using a receptor binding assay system using the expression system (eg, peptide, protein, non-peptide compound, synthetic compound, fermentation product, etc.) Alternatively, the salt can be screened. Such compounds have 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-
Compounds (so-called G protein-coupled receptor agonists) having an activity that promotes or suppresses fos activation and the like and compounds that do not have the cell stimulating activity (so-called G protein-coupled receptor antagonists)
Etc. are included. That is, (i) when a G protein-coupled receptor protein or a salt thereof or a partial peptide or a salt thereof and a ligand are brought into contact with each other, and (ii)
A compound which inhibits the binding between a ligand and a G protein-coupled receptor protein, which is characterized in that a G protein-coupled receptor protein or a salt thereof or a partial peptide or a salt thereof, a ligand and a test compound are contacted with each other. Alternatively, a method for screening a salt thereof is provided. In the screening method,
(I) when a G protein-coupled receptor protein or partial peptide and a ligand are contacted, and (ii) G
Protein-coupled receptor protein or partial peptide,
When the ligand and the test compound are brought into contact with each other, the amount of ligand binding to the G protein-coupled receptor protein or the partial peptide, cell stimulating activity, etc. are measured and compared.

More specifically, the labeled ligand is brought into contact with the G protein-coupled receptor protein or its partial peptide, and the labeled ligand and the test compound are brought into contact with the G protein-coupled receptor protein or its partial peptide. The amount of labeled ligand bound to the protein or its partial peptide or salt thereof is measured and compared, and a compound or salt thereof that inhibits the binding between the ligand and the G protein-coupled receptor protein is characterized. Screening method, when a labeled ligand is brought into contact with a cell containing a G protein-coupled receptor protein or a membrane fraction of the cell, and a labeled ligand and a test compound are added to a cell containing the G protein-coupled receptor protein or Labeling when contacted with the membrane fraction of cells A method for screening a compound or its salt which inhibits the binding between a ligand and a G protein-coupled receptor protein, characterized by measuring and comparing the amount of binding of Gand to the cell or the membrane fraction, When a transformant containing a DNA encoding a protein-coupled receptor protein is brought into contact with a G protein-coupled receptor protein expressed on a cell membrane, and a labeled ligand and a test compound are added to the G protein-coupled receptor By culturing a transformant containing a protein-encoding DNA, the amount of the labeled ligand bound to the G protein-coupled receptor protein when contacted with the G protein-coupled receptor protein expressed on the cell membrane was measured. , G-protein coupled type characterized by comparing Method for screening compound or salt thereof that inhibits binding to sceptor protein, compound containing G protein-coupled receptor protein containing compound activating G protein-coupled receptor protein (eg, ligand for G protein-coupled receptor protein) Stimulation of cells mediated by G protein-coupled receptors when contacted with cells containing G protein-coupled receptors, and when a compound that activates G protein-coupled receptors and a test compound are contacted with cells containing G protein-coupled receptor proteins 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, activity promoting or suppressing c-fos activation, etc.) are measured and compared, and the ligand and G A method for screening a compound or its salt that inhibits binding to a protein-coupled receptor protein, and a compound that activates the G-protein coupled receptor (for example, a ligand for the G-protein coupled receptor protein) A transformant containing a DNA encoding a protein is brought into contact with a G protein-coupled receptor protein expressed on the cell membrane by culturing, and a compound activating the G protein-coupled receptor and a test compound are used as the G protein. Transformation containing DNA encoding a coupled receptor protein Cell-stimulating activity mediated by G protein-coupled receptor (eg, arachidonic acid release, acetylcholine release, intracellular Ca ²⁺ release) when contacted with G protein-coupled receptor protein expressed on cell membrane by culturing the body , Intracellular cAMP production, intracellular cGMP production, inositol phosphate production, cell membrane potential fluctuation, intracellular protein phosphorylation, activity promoting or suppressing c-fos activation, etc.) are measured and compared. The present invention provides a method for screening a compound or a salt thereof which inhibits the binding between a ligand and a G protein-coupled receptor protein.

A specific description of this screening method will be given below. First, the G protein-coupled receptor protein used in the screening method of the present invention may be any as long as it contains the G protein-coupled receptor protein or a partial peptide thereof. The membrane fraction of is preferred. 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 to produce the G protein-coupled receptor protein, which can be performed by expressing the DNA encoding the protein in mammalian cells or insect cells. Complementary D to the DNA fragment encoding the target portion
NA is used, but is not necessarily limited to this. For example, a gene fragment or synthetic DNA may be used. DN encoding a G protein-coupled receptor protein
In order to introduce the A fragment into host animal cells and express them efficiently, the nuclear polyhedrosis virus (nuclear porovirus) belonging to the baculovirus using the DNA fragment as an insect host (nuclear po
lyhedrosis virus (NPV) polyhedrin promoter, SV40-derived promoter, retrovirus promoter, metallothionein promoter, human heat shock promoter, cytomegalovirus 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, 19555-19559, 1992]. Therefore, in the screening method, a G protein-coupled receptor protein or a partial peptide thereof contains a G protein-coupled receptor protein purified by a method known per se or a partial peptide of the G protein-coupled receptor protein. Alternatively, 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 screening method, the cell may be fixed with glutaraldehyde, formalin or the like. The immobilization method can be performed according to a method known per se. The cell containing the 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 fed at a low speed (500 rpm to 30
Centrifuge at 00 rpm for a short time (usually about 1 minute to 10 minutes), and the supernatant is spun at a higher speed (15000 rpm to 30000).
It is usually centrifuged for 30 minutes to 2 hours at (rpm) and the obtained 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. .

Screening for a compound that inhibits the binding between the ligand and the G protein-coupled receptor
In order to carry out the procedure, an appropriate G protein-coupled receptor fraction and a labeled ligand are required. 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. As the labeled ligand, a labeled ligand, a labeled ligand analog compound, or the like is used. For example, a ligand labeled with [ ³ H], [ ¹²⁵ I], [ ¹⁴ C], [ ³⁵ 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 a pH of 6).
Any buffer may be used as long as it does not inhibit the binding between the ligand X and the receptor, such as the phosphate buffer and Tris-hydrochloric acid buffer described in 8). 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. 0.01ml-10m
l of the receptor solution, a fixed amount (5000 cpm-5
00000 cpm) of the 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). Reaction is 0 ° C
To 50 ° C, preferably 4 ° C to 37 ° C for 20 minutes to 2
It is carried out for 4 hours, preferably 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 scintillator or a γ-counter. A test compound having a specific binding amount (B-NSB) of, for example, 50% or less when the count (B ₀ -NSB) obtained by subtracting NSB from the count (B ₀ ) when there is no substance to be antagonized is defined as 100%. It 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, the cell stimulating activity via the G protein-coupled receptor protein (for example, arachidonic acid release, Acetylcholine release, intracellular Ca release, intracellular cAMP production, intracellular cGMP production, inositol phosphate production, cell membrane potential fluctuation, intracellular protein phosphorylation,
The activity of promoting or suppressing the activation of c-fos) 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. For screening, the medium was exchanged with a fresh medium or an appropriate buffer that did not show toxicity to cells in advance, and after adding a test compound or the like and incubating for a certain period of time, cells were extracted or supernatant was collected to generate The product is quantified according to the respective method. When the production of a substance that serves as an index of cell stimulating activity (for example, arachidonic acid) is difficult to assay with a degrading enzyme contained in cells, an inhibitor for the degrading enzyme may be added to the assay. Also, cA
The activity such as MP production suppression can be detected as a production suppression effect on cells whose basic production amount has been increased by forskolin or the like. To measure cell-stimulating activity and perform screening, use an appropriate G
A cell that expresses a protein-coupled receptor protein is required. Cells expressing the G protein-coupled receptor protein include cell lines having a natural G protein-coupled receptor protein (for example, mouse pancreatic β-cell line MIN6, etc.), and the recombinant G protein-coupled receptor protein expression described above. Cell lines are preferable. Examples of the test compound include peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, etc. These compounds may be novel compounds or known compounds.

The screening kit for a compound or a salt thereof that inhibits the binding between a ligand and a G protein-coupled receptor protein comprises a G protein-coupled receptor protein or a partial peptide thereof, a cell containing the G protein-coupled receptor protein, or It contains a membrane fraction of cells containing a G protein-coupled receptor protein. Examples of the screening kit include the following. 1. Screening reagent Measurement buffer and washing buffer Hanks' Balanced Salt Solution (manufactured by Gibco)
One containing 05% bovine serum albumin (manufactured by Sigma). Sterilized by filtration through a 0.45 μm filter, 4 ° C
Or may be prepared at the time of use. G protein-coupled receptor preparation CHO cells expressing the 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. Ligand standard solution 0.1% bovine serum albumin (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. Labeled ligand bound to cells is 0.2N NaOH
1% SDS, 4 ml of liquid scintillator A
(Made by Wako Pure Chemical Industries). Liquid scintillation counter (Beckman)
Radioactivity was measured using Percent Maximum Binding
(PMB) is obtained by the following equation (Equation 1).

[0052]

[Equation 1]

PMB: Percent Maximum Binding B: Value when a sample is added NSB: Non-specific Binding (non-specific binding amount) B ₀ : Maximum binding amount Compound obtained by using a screening method or screening kit, or a compound thereof A salt is a compound that inhibits the binding between a ligand and a G protein-coupled receptor,
Specifically, it is a compound having a cell stimulating activity via a 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, etc. These compounds may be novel compounds or known compounds.

Since the G protein-coupled receptor agonist has the same physiological activity as that of the ligand for the G protein-coupled receptor protein, it is a safe and low toxic pharmaceutical composition depending on the ligand activity. It is useful. On the other hand, the G protein-coupled receptor antagonist can suppress the physiological activity of the ligand for the G protein-coupled receptor protein, and is therefore useful as a safe and low-toxic pharmaceutical composition for suppressing the ligand activity. When the compound or its salt obtained by using the screening method or the screening kit is used as the above-mentioned pharmaceutical composition, it can be carried out according to a conventional method. 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.

Additives which can be mixed with tablets, capsules and the like include, for example, gelatin, corn starch, tragacanth, binders such as gum arabic, excipients such as crystalline cellulose, corn starch, gelatin, alginic acid and the like. Such as a leavening agent, a lubricant such as magnesium stearate, a sweetener such as sucrose, lactose or saccharin, and a flavoring agent such as peppermint, red oil or cherry. 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), nonionic surfactant (eg polysorbate 80 (TM), HCO-50) and the like 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. In addition, buffers (eg, phosphate buffer, sodium acetate buffer), soothing agents (eg, benzalkonium chloride, procaine hydrochloride, etc.), stabilizers (eg, human serum albumin, polyethylene glycol, etc.), preservation Agents (eg, benzyl alcohol,
Phenol, etc.), antioxidants and the like.
The adjusted injection solution 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, pigs, cows, cats, dogs, monkeys, humans, etc.). The dose of the compound or its salt varies depending on the symptoms and the like, but in the case of oral administration, it is generally about 0.1 to 100 mg, preferably 1.0 to 100 mg per day for an adult (as 60 kg). Fifty
mg, more preferably 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 case of an injection, for example, in an adult (60 kg), the daily dose is about 1 day. About 0.01 to 30 mg, preferably about 0.1 to 20 mg, more preferably 0.1 to 10 m
It is convenient to administer about g by intravenous injection. 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 or antiserum against a salt thereof or a partial peptide of the G protein-coupled receptor protein of the present invention or a salt thereof may be a G protein-coupled receptor protein of the present invention or a salt thereof or a portion of the G protein-coupled receptor protein of the present invention. It can be produced according to a method known per se for producing an antibody using a peptide or a salt thereof as an antigen. 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-coupled receptor may be abbreviated) may be administered to a warm-blooded animal at a site where antibody production can be performed by itself or together 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 producing monoclonal antibody-producing cells, warm-blooded animals immunized with an antigen, for example, an individual with an antibody titer was selected from mice, and spleens or lymph nodes were collected 2 to 5 days after the final immunization and included in them. By fusing the antibody-producing cells prepared as described above 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 (197
5)]. 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, A
P-1 and the like are mentioned, 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 selection and breeding medium, any medium can be used as long as a hybridoma can grow. For example, RPMI 1640 medium containing 1-20%, preferably 10-20% fetal calf serum, 1-10%
Medium containing fetal calf serum (Wako Pure Chemical Industries, Ltd.)
Alternatively, a serum-free medium for hybridoma culture (SFM-1
01, Nissui Pharmaceutical Co., Ltd., etc. can be used. The culturing temperature is usually 20 to 40 ° C, preferably about 37 ° C. Cultivation time is usually 5 days to 3 weeks, preferably 1 week to 2 weeks. Culturing is usually performed under 5% carbon dioxide gas. The antibody titer of the hybridoma culture supernatant can be measured in the same manner as the above-described measurement of the anti-G protein-coupled receptor antibody titer in the antiserum.

(B) Purification of monoclonal antibody The isolation and purification of the anti-G protein-coupled receptor monoclonal antibody is carried out by the same method as the isolation and purification of usual polyclonal antibodies [eg, salting-out method, alcohol precipitation method, 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]. The G protein-coupled receptor antibody of the present invention produced according to the above methods (1) and (2) can specifically recognize the G protein-coupled receptor. It can be used for quantification of a receptor, particularly by a sandwich immunoassay. That is, the present invention, for example,

(I) The antibody reactive with the G protein-coupled receptor of the present invention, the test solution and the labeled G protein-coupled receptor are competitively reacted, and the labeled G protein-coupled antibody bound to the antibody is reacted. A method for quantifying a G protein-coupled receptor in a test solution, which comprises measuring the ratio of the receptor, (ii) the test solution and the antibody insolubilized on the carrier and the labeled antibody are simultaneously or continuously In a method for quantifying a G protein-coupled receptor in a test solution, which comprises measuring the activity of a labeling agent on an insolubilized carrier after the reaction with one of the antibodies, one of the antibodies is the N-terminal portion of the G protein-coupled receptor. The present invention provides a method for quantifying a G protein-coupled receptor in a test solution, which is an antibody that recognizes G., and the other antibody reacts with the C-terminal portion of the G protein-coupled receptor. The G protein-coupled receptor can be measured using a monoclonal antibody that recognizes the G protein-coupled receptor of the present invention (hereinafter sometimes referred to as anti-G protein-coupled receptor antibody), and detection by tissue staining or the like can be performed. You can also do it. For these purposes, the antibody molecule itself may be used, or F (ab ′) ₂ , F of the antibody molecule may be used.
Ab 'or Fab fraction may be used. The assay 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 determined. 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, immunometric method and sandwich method are preferably used,
From the viewpoint of sensitivity and specificity, it is particularly preferable to use the sandwich method described later.

Examples of the labeling agent used in the measuring method using the labeling substance include radioisotopes, enzymes, fluorescent substances, luminescent substances and the like. As the radioisotope, for example, [ ¹²⁵ I], [ ¹³¹ I], [ ³ H], [ ¹⁴ C] and the like are preferable, and as the above enzyme, those having stable and large specific activity are preferable, for example, β-galactosidase, 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 usually used for insolubilizing and immobilizing proteins or enzymes may be used. 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).
After that, the amount of G protein-coupled receptor in the test solution can be quantified by measuring the activity of the labeling agent on the insolubilized carrier. 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 the immunoassay by the sandwich method, the antibody used for the solid phase antibody or the labeling antibody is not necessarily one kind, and a mixture of two or more kinds of antibodies is used for the purpose of improving measurement sensitivity and the like. You may.

In the method for measuring a G protein-coupled receptor by the sandwich method of the present invention, the anti-G protein-coupled receptor antibodies used in the primary reaction and the secondary reaction have different binding sites for the G protein-coupled receptor. Antibodies are preferably 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 can be used in a measuring system other than the sandwich method, for example, a competitive method, an immunometric method or nephrometry. In the competition method, an antigen in a test solution and a labeled antigen are allowed to competitively react with an antibody, and then the unreacted labeled antigen is separated from (F) and the labeled antigen (B) bound to the antibody. (B / F separation), the amount of any of B and F is measured, and the amount of antigen in the test solution is quantified. In this reaction method, a soluble antibody is used as an antibody, B / F separation is performed using polyethylene glycol, a liquid phase method using a second antibody against the antibody, or the like.
An immobilized antibody is used as the antibody, or an immobilization method using a soluble first antibody and an immobilized antibody as the second antibody is used. In the immunometric method,
The antigen in the test solution and the solid-phased antigen are subjected to a competitive reaction with a certain amount of the labeled antibody, and then the solid phase and the liquid phase are separated. The unreacted labeled antibody is allowed to bind 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 nephrometry 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, it is possible to refer to reviews and compendiums [for example, "Radioimmunoassay" edited by Hiro Irie (published by Kodansha, 1974), "Radioimmunoassay" edited by Hiro Irie. (Kodansha, published in 1979), Eiji Ishikawa et al., "Enzyme Immunoassay" (Medical Publishing, published in 1978), Eiji Ishikawa et al., "Enzyme Immunoassay" (second edition) (Medical Publishing,
Published in 1982), edited by Eiji Ishikawa et al. “Enzyme immunoassay”.
(3rd edition) (Medical Shoin, published in 1987), "Methods
in ENZYMOLOGY '' Vol. 70 (Immunochemical Techniques (P
art A)), ibid Vol. 73 (Immunochemical Techniques (Pa
rt B)), ibid Vol. 74 (Immunochemical Techniques (Pa
rt C)), ibid Vol. 84 (Immunochemical Techniques (Pa
rtD: Selected Immunoassays)) ibid.Vol. 92 (Immunoc)
hemical Techniques (Part E: Monoclonal Antibodies and
General Immunoassay Methods)) ibid. Vol. 121 (Imm
unochemical Techniques (Part I: Hybridoma Technology
and Monoclonal Antibodies)) (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.

The human CRF ₂ receptor protein of the present invention includes, in addition to the protein having the amino acid sequence represented by SEQ ID NO: 3 or SEQ ID NO: 14, SEQ ID NO: 3.
Alternatively, the amino acid sequence represented by SEQ ID NO: 14 and about 97
Examples include a protein having an amino acid sequence having a homology of ˜99.9% and having substantially the same activity as the protein having the amino acid sequence represented by SEQ ID NO: 3 or SEQ ID NO: 14. Examples of substantially the same activity include ligand binding activity and signal transduction. "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 human CRF ₂ receptor protein of the present invention include human stomach-derived CRF ₂ receptor protein having the amino acid sequence represented by SEQ ID NO: 3 or SEQ ID NO: 14. Further, the human CRF ₂ receptor protein of the present invention includes an amino acid sequence in which one or more amino acids are deleted from the amino acid sequence represented by SEQ ID NO: 3 or SEQ ID NO: 14, SEQ ID NO: 3 or sequence. Number: 1
A protein containing an amino acid sequence in which 1 or 2 or more amino acids are added to the amino acid sequence represented by 4 is also included. More specifically, as the human CRF ₂ receptor protein of the present invention, SEQ ID NO: 3 or SEQ ID NO:
CR derived from human stomach having the amino acid sequence represented by 14
In addition to the F ₂ receptor protein and the like, 1 or 2 in the amino acid sequence represented by SEQ ID NO: 3 or SEQ ID NO: 14
One or more (preferably 2 or more and 30 or less, more preferably 2 or more and 10 or less) amino acids deleted,
A sequence in which 1 or 2 or more (preferably 2 or more and 30 or less, more preferably 2 or more and 10 or less) amino acids are added to the amino acid sequence represented by SEQ ID NO: 3 or SEQ ID NO: 14, and a sequence 1 or 2 or more (preferably 2 or more and 30 or less, more preferably 2 or more 10 in the amino acid sequence represented by SEQ ID NO: 3 or SEQ ID NO: 14)
(Or less) amino acids are substituted with other amino acids. Furthermore, in the human CRF ₂ receptor protein of the present invention, the Met at the N-terminus is protected by a protecting group (eg, C _1-6 acyl group such as formyl group and acetyl group), and the N-terminal side of Glu is Cut in vivo,
The Glu is pyroglutamine-modified, the side chain of an amino acid in the molecule is protected by an appropriate protecting group (for example, C _1-6 acyl group such as formyl group, acetyl group, etc.),
Alternatively, a complex protein such as a so-called glycoprotein having a sugar chain bound thereto is also included.

As the salt of the human CRF ₂ receptor protein of the present invention, a physiologically acceptable acid addition salt is particularly preferable. Examples of such salts include salts with inorganic acids (eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid) 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). Human CRF of the present invention
₂ Receptor protein or its salt is a human CR described later.
It can also be produced by culturing a transformant containing DNA encoding the F ₂ receptor protein. Further, it can also be produced according to the peptide synthesis method described later. The partial peptide of human CRF ₂ receptor protein of the present invention, for example, of the human CRF ₂ receptor protein molecule of the present invention, such as site exposed to the outside of the cell membrane is used. Specifically, the portion of the human CRF ₂ receptor protein of the present invention shown in FIG. 4 or FIG. 8 which is analyzed to be an extracellular region (hydrophilic site) in the hydrophobicity plot analysis is It 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 human CRF ₂ receptor protein of the present invention, a physiologically acceptable acid addition salt is particularly preferable. Examples of such salts include salts with inorganic acids (eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid) 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 partial peptide of the human CRF ₂ receptor protein of the present invention or a salt thereof can be produced by publicly known methods for peptide synthesis, or by cleaving the human CRF ₂ receptor protein of the present invention with an appropriate peptidase. it can. 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 Peptide,
Academic Press, New York (1965) Nobuo Izumiya et al., Fundamentals and experiments of peptide synthesis, Maruzen Co., Ltd.
(1975) Haruaki Yajima and Shunpei Sakakibara, Laboratory for Biochemistry 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 solvent extraction, distillation, column chromatography, liquid chromatography, 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.

As the DNA encoding the human CRF ₂ receptor protein of the present invention, a human CRF ₂ receptor protein containing an amino acid sequence substantially the same as the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 14 of the present invention is used. Any one may be used as long as it contains the base sequence that it encodes. In addition, human genomic DNA, human genomic DNA library, cDNA derived from human tissues / cells,
Human tissue / cell-derived cDNA library, synthetic DN
Any of A may be used. The vector used for the library may be any of bacteriophage, plasmid, cosmid, phagemid and the like. In addition, reverse Tr
anscription Polymerase Chain Reaction (hereinafter RT-
It is abbreviated as PCR method. ). More specifically, RT-PCR is, as a DNA primer, a nucleotide sequence (sense sequence or antisense sequence) represented by SEQ ID NO: 4 or SEQ ID NO: 15.
Synthetic DNA having the following is used.

As a means for cloning the DNA which completely encodes the human CRF ₂ receptor protein of the present invention, it is amplified by PCR using a synthetic DNA primer having a partial base sequence of the human CRF ₂ receptor protein, or is suitable. The DNA incorporated in the vector is selected by hybridization with a DNA fragment having a part or the whole region of the human human CRF ₂ receptor protein or one labeled with a synthetic DNA. Hybridization methods include, for example, Molecul
ar Cloning 2nd (ed .; J. Sambrook et al., Cold Spr
ing 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. The cloned DNA encoding the human CRF ₂ receptor protein can be used as it is, or if desired, after digestion with a restriction enzyme or addition of a linker, depending on the purpose. The DNA may have ATG as a translation initiation codon at the 5 'end and TAA, TGA or TAG as a translation stop codon at the 3' end. These translation initiation codon and translation termination codon can also be added using a suitable synthetic DNA adapter. The human CRF ₂ receptor protein expression vector is, for example, (a) a DNA fragment of interest is excised from the DNA encoding the human CRF ₂ receptor protein of the present invention, and (b) the DNA fragment is a promoter in a suitable expression vector. Can be manufactured by connecting to the downstream side of.

As a vector, a plasmid derived from E. coli (eg, pBR322, pBR325, pUC118,
pUC119), a plasmid derived from Bacillus subtilis (eg, pUB
110, pTP5, pC194), yeast-derived plasmids (eg, pSH19, pSH15), bacteriophages such as λ phage, animal viruses such as retrovirus, vaccinia virus, baculovirus and the like. The promoter used in the present invention may be any promoter as long as it is appropriate for the host used for gene expression. If the host for transformation is Escherichia, the trp promoter, la
c promoter, recA promoter, λPL promoter, lpp promoter, etc., when the host is Bacillus, SPO1 promoter, SPO2 promoter, penP promoter, etc. When the host is yeast, PHO5 promoter, PGK promoter, GA
P promoter, 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 may be added to the human CRF ₂ receptor protein N
It can also be added to the terminal side. 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 human CRF ₂ receptor protein thus constructed. As the host, for example, Escherichia, Bacillus, yeast, insects, animal cells and the like are used. Specific examples of Escherichia and Bacillus include Escherichia coli (Escherichia coli).
hia coli) K12 ・ DH1 [Procedures of
The National Academy of Sciences
Of the USA (Proc. Natl. Acad. Sci. U
SA), 60, 160 (1968)], JM103 [Nucleic Acids Research, (Nucleic Acids Re
search), Vol. 9, 309 (1981)], JA221 [Journa of Molecular Biology (Journa
l ofMolecular Biology), 120, 517 (197)
8)], HB101 [Journal of Molecular Biology, 4
1, 459 (1969)], C600 [Genetics, 39, 440 (1954)] and the like. Bacillus spp.
Bacillus subtilis MI114 [Gene (G
ene), 24, 255 (1983)], 207-21
[Journal of Biochemistry
Biochemistry), Vol. 95, 87 (1984)] and the like. Examples of yeast include Saccharomyces
Saccaromyces cerevisiae AH22, AH
^{22R -, 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), 315, 592 (1985)]. Examples of animal cells include monkey cells COS-7, Vero cells, Chinese hamster cells CHO, DHFR gene-deficient Chinese hamster cells CHO (dhfr ^- CHO cells), CHO K-1 cells, human FL cells, 293 cells, L cells, Myeloma cells, C127 cells, Balb
/ C3T3 cells, Sp-2 / 0 cells and the like are used.

For transforming Escherichia, for example, Procedures of the National Academy of Sciences of the USA (Proc. Natl. Acad. Sci. USA), Vol. 69, 21
10 (1972) and Gene, 17, 107 (1)
982). To transform Bacillus, for example, Molecular & General Genetics (Molecular & G
Eneral Genetics), Vol. 168, 111 (1979). To transform yeast, for example, Procedures of the National Academy of Sciences of the USA (Proc. Natl. Acad. Sci. USA), Vol. 75,
1929 (1978).
Transformation of insect cells is performed according to the method described in, for example, Bio / Technology, 6, 47-55 (1988)). For transforming animal cells, for example, Virology, 52, 4
56 (1973). Among the above host cells, the host cell of the expression plasmid containing the human CRF ₂ receptor protein DNA of the present invention is particularly preferably animal cells, and examples thereof include 293 cells, CHO cells, Vero cells, L cells and myeloma cells. , C127 cells, Balb / c3T3 cells, Sp-2
/ O cells and the like, among which CHO cells or 2
93 cells and the like are preferable, and particularly CHO cells [Journal of Experiment of Medison (J. Ex.
p. Med.), 108, 945 (1958)] and the like are preferable. Furthermore, among CHO cells, CHO cells deficient in the dhfr gene (hereinafter referred to as CHO (dhf
r ^-..) there is a case to be referred to as cells) [professional sheathing's of the National Academy of Saienshiizu-of-the-USA (Proc Natl Acad.
Sci. USA), 77, 4216-4220 (198).
0)], CHO K-1 cells [Proc. Natl. Acad. Sci.
USA), 60 volumes, 1275 (1968)] and the like are preferable. Dhfr as a selectable marker in the expression plasmid
If the gene is inserted, CHO (dhfr ^-)
Cells and the like are preferred. As a method for introducing an expression plasmid into animal cells, known methods such as calcium phosphate method [Graham, FL and van der Eb, AJ Virology 52, 456-467 (1973)], electroporation [Ne
umann, E. et al. EMBO J., 1, 84
1-845 (1982)] and the like are used. As described above, a transformant transformed with the vector containing the human CRF ₂ receptor protein DNA is obtained. Also, human CR
The transformant transformed with the expression plasmid containing F ₂ receptor protein DNA can produce human CRF ₂ receptor protein.

A cell capable of highly expressing the human CRF ₂ receptor protein can be obtained by clonally selecting a cell in which the above-mentioned expression plasmid is integrated in the chromosome. Specifically, first, a transformant is selected using the above selection marker as an index. Furthermore, human CRF can be obtained by repeatedly performing clone selection on transformants obtained using the selectable marker in this manner.
It is possible to obtain a cell line that stably has a high expression ability of ₂ receptor protein. When the dhfr gene is used as a selection marker, by gradually increasing the concentration of methotrexate (MTX) and culturing to select resistant cells,
It is also possible to obtain a cell line with higher expression by amplifying the transgene intracellularly. Human CRF _{2 of the} present invention
Cells containing the receptor protein and the human CRF ₂ receptor protein of the present invention can be obtained by transforming a transformant containing a vector (particularly an expression plasmid) containing the human CRF ₂ receptor protein DNA of the present invention with the human CRF ₂ receptor protein. It can also be produced by culturing under conditions that allow expression of the encoding DNA. When culturing a transformant whose host cell is a bacterium of the genus Escherichia or Bacillus, a liquid medium is suitable as a medium used for the culture, and among them, a carbon source necessary for the growth of the transformant. , Nitrogen sources, inorganic substances, etc. are included.
As a carbon source, for example, glucose, dextrin,
Nitrogen sources such as soluble starch and sucrose include, for example, ammonium salts, nitrates, corn steep liquor,
Inorganic or organic substances such as peptone, casein, meat extract, soybean meal, potato extract, etc. Examples of the inorganic substances include calcium chloride, sodium dihydrogen phosphate, and magnesium chloride. In addition, yeast, vitamins,
A growth promoting factor or the like may be added. PH of medium is about 5
~ 8 is desirable. As a medium for culturing Escherichia, for example, M9 medium containing glucose and casamino acid [Miller, Journal of Experiments in Molecular Genetics (Jour
nal of Experiments in Molecular Genetics), 431-
433, Cold Spring Harbor Laboratory, New York
1972] is preferable. If necessary, an agent such as 3β-indolyl acrylic acid can be added in order to make the promoter work efficiently. When the host cell is Escherichia, the culture is usually about 15-4.
It is carried out at 3 ° C. for about 3 to 24 hours, and if necessary, aeration and stirring can be added. 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 added. When culturing a transformant whose host is yeast, examples of the medium include Burkholder's minimum medium [Bostian, K.
L. et al., Proc. Natl. Acad. Sci. USA, Proc. Natl. Acad. Sci. USA, 77, 4.
505 (1980)] or S containing 0.5% casamino acid.
D medium [Bitter, GA et al., Procedures of
The National Academy of Sciences
Of the USA (Proc. Natl. Acad. Sci. U
SA), 81, 5330 (1984)]. Preferably, the pH of the medium is adjusted to about 5-8. The cultivation is usually performed at about 20 ° C. to 35 ° C. for about 24 to 72 hours, and aeration and stirring are added as necessary. When culturing a transformant whose host cell is an insect, the medium is Grace's Inse
ct Medium 〔Grace, TCC, Nature, 195,
788 (1962)] to which appropriate additives such as inactivated 10% bovine serum have been added. The pH of the medium is about 6.
It is preferably adjusted to 2 to 6.4. Culture is usually about 27
It is carried out at ℃ for about 3 to 5 days, and aeration and stirring are added if necessary. When culturing a transformant whose host is an animal cell, the medium is, for example, EMEM medium containing about 0.5 to 20% fetal bovine serum [Seience, 122].
Vol. 501 (1952)], DMEM medium [Virology, Vol. 8, 396 (1959)], RPMI
1640 medium [The Jounal of the Amer
ican Medical Association) Volume 199, 519 (196
7)], 199 medium [Proceeding of the Society for the Biological Med
icine), 73, 1 (1950)], α-MEM medium and the like are used. In particular, when CHO (dhfr ⁻ ) cells and dhfr selectable marker gene are used, it is preferable to use DMEM medium containing dialyzed fetal bovine serum containing almost no thymidine. Preferably, the pH is between about 6-8. Culturing is usually carried out at about 30 ° C to 40 ° C for about 15 to 72 hours, and if necessary, medium exchange, aeration, stirring, etc. are added. Thus, vectors containing the DNA encoding human CRF ₂ receptor protein (in particular, the expression plasmid) can be produced cells containing human CRF ₂ receptor protein from transformant carrying.

The human CRF ₂ receptor protein or its partial peptide can be separated and purified from the culture of cells containing the above-mentioned human CRF ₂ receptor protein, for example, by the following method. First, human CR
When the F ₂ receptor protein is extracted from cultured bacterial cells or cells, after the culture, the bacterial cells or cells are collected by a known method, suspended in an appropriate buffer solution, and subjected to ultrasonic wave, lysozyme and / or freeze-thawing. After destroying bacterial cells or cells with human CR by centrifugation or filtration
A method for obtaining a crude extract of F ₂ receptor protein can be appropriately used. The buffer may contain a protein denaturing agent such as urea or guanidine hydrochloride, or a surfactant such as Triton X-100 (registered trademark, hereinafter sometimes abbreviated as TM). Known separation and purification methods for these are methods utilizing solubility such as salting out and solvent precipitation,
Dialysis method, ultrafiltration method, gel filtration method, SDS-polyacrylamide gel electrophoresis method and the like mainly utilizing the difference in molecular weight, ion exchange chromatography and other methods utilizing the difference in charge, affinity chromatography, etc. , A method utilizing a difference in hydrophobicity such as reversed-phase high performance liquid chromatography, a method utilizing a difference in isoelectric point such as an isoelectric focusing method, and the like. When the human CRF ₂ receptor protein thus obtained is obtained in a free form, it can be converted into a salt by a method known per se or a method analogous thereto, and conversely, when it is obtained as a salt, a method known per se. Alternatively, it can be converted into a free form or another salt by a method similar thereto. The human CRF ₂ receptor protein produced by the recombinant can be optionally modified or the polypeptide can be partially removed by acting 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 human CRF ₂ receptor protein thus produced can be measured by a binding experiment with labeled CRF or the like and an enzyme immunoassay using a specific antibody.

The cell membrane fraction of cells containing the human CRF ₂ receptor protein of the present invention contains many cell membranes obtained by a method known per se after crushing the cells containing the human CRF ₂ receptor protein. It refers to a fraction. Examples of cell disruption methods include Potter-Elve
The method includes crushing cells with an hjem homogenizer, crushing with a Waring blender or Polytron (Kinematica), crushing with ultrasonic waves, crushing by ejecting cells from a thin nozzle while applying pressure with a French press, etc. For fractionation of cell membranes, fractionation by centrifugal force, such as fractionation centrifugation and density gradient centrifugation, is mainly used. For example, use a low speed cell disruption solution (500 rpm
~ 3000 rpm) for a short time (usually about 1 to 10 minutes)
Centrifuge and the supernatant is spun at a higher speed (15000 rpm-300
It is usually centrifuged at 00 rpm) for 30 minutes to 2 hours, and the obtained precipitate is used as a membrane fraction. Expressed human CR in the membrane fraction
It contains a large amount of F ₂ receptor protein and membrane components such as cell-derived phospholipids and membrane proteins. The amount of human CRF ₂ receptor protein of a cell and the cell membrane fraction of containing human CRF ₂ receptor protein of the present invention, per cell 10 ³
It is preferably 10 ⁸ molecules, and more preferably 10 ⁵ 10 ⁷ molecules. The higher the expression level, the higher the ligand binding activity (specific activity) per membrane fraction, which not only enables the construction of a highly sensitive screening system,
A large number of samples can be measured in the same lot. DNA encoding human CRF ₂ receptor protein, the partial peptide and human CRF ₂ receptor protein of human CRF ₂ receptor protein of the present invention, to obtain the antibodies and antisera, the construction of expression systems of recombinant receptor protein,
Development of a receptor binding assay system using the same expression system, screening of drug candidate compounds, implementation of drug design based on comparison with structurally similar ligands / receptors, probes for gene diagnosis, PCR
It can be used for preparation of primers, gene therapy and the like. In particular, a CRF ₂ receptor agonist or antagonist specific to warm-blooded animals (particularly human) can be screened by a receptor binding assay system using the expression system of the human CRF ₂ receptor protein of the present invention. CRF the antagonist
It can be used as a prophylactic / therapeutic agent for various diseases caused by.

The human CRF ₂ receptor protein of the present invention, the human CRF ₂ receptor protein partial peptide,
The use of the DNA encoding the human CRF ₂ receptor protein will be specifically described. (1) Screening Method for CRF ₂ Receptor Agonist or Antagonist In the screening method for CRF ₂ receptor agonist or antagonist, it is considered that human heart or muscle is used as the human CRF ₂ receptor protein. However, since human-derived tissues are extremely difficult to obtain, they are not suitable for use in screening, and in fact, recombinants produced in large amounts in cells (especially animal cells such as CHO cells and insect cells) are used. Human CRF ₂
Receptor proteins are suitable. Furthermore, human CRF
Most preferably, a cell line that continuously and stably expresses the ₂ receptor protein is used. Thus, the human CRF ₂ receptor protein or its salt of the present invention, the partial peptide or a salt thereof of the human CRF ₂ receptor protein of the present invention or a cell or a cell membrane fraction containing human CRF ₂ receptor protein of the present invention, is, CRF ₂ It is useful as a screening reagent for receptor or antagonist. That is, the present invention provides a human CRF ₂ receptor protein or its salt of the present invention, the partial peptide or a salt thereof of the human CRF ₂ receptor protein of the present invention or a cell or a cell membrane containing human CRF ₂ receptor protein of the present invention, The present invention provides a method for screening CRF ₂ receptor agonists or antagonists, which comprises using a component.

More specifically, the present invention provides (I) (i) the human CRF ₂ receptor protein of the present invention,
Comparison between the case where CRF is contacted with the partial peptide or salts thereof and (ii) the case where CRF and a test compound are contacted with the human CRF ₂ receptor protein of the present invention, its partial peptide or salts thereof, A method for screening a CRF ₂ receptor agonist or antagonist, which comprises performing (II) (i) contacting CRF with a cell containing the human CRF ₂ receptor protein of the present invention or a cell membrane fraction thereof ( ii) A method for screening a CRF ₂ receptor agonist or antagonist, which comprises performing a comparison with a case where cells containing the human CRF ₂ receptor protein of the present invention or a cell membrane fraction thereof is contacted with CRF and a test compound. provide. Specifically, in the screening methods (I) and (II) of the present invention, in the cases of (i) and (ii), for example, the human CRF ₂ receptor protein, its partial peptide or salts thereof, or human CRF It is characterized in that the amount of CRF bound to cells containing the ₂ receptor protein or its cell membrane fraction, the cell stimulating activity, etc. are measured and compared.

More specifically, the present invention relates to (Ia) (i) labeled CRF as human CRF of the present invention.
₂ receptor protein, a partial peptide thereof or a salt thereof, and (ii) the labeled CRF and the test compound, the human CRF ₂ receptor protein of the present invention,
The amount of labeled CRF bound to the receptor protein, the partial peptide or salts thereof when contacted with the partial peptide or salts thereof is measured,
Method for Screening Human CRF ₂ Receptor Agonist or Antagonist Comparing (IIa) (i) Labeled CRF with Human CRF of the Present Invention
_When the cells containing the _2- receptor protein or the cell membrane fraction thereof are brought into contact, (ii) the labeled CRF and the test compound are brought into contact with the cells containing the human CRF ₂ receptor protein of the present invention or the cell membrane fraction thereof. In the above case, the amount of labeled CRF bound to the cell or its cell membrane fraction is measured and compared, and a method for screening a human CRF ₂ receptor agonist or antagonist, and (IIb) (i) CRF, When a cell containing the human CRF ₂ receptor protein of the present invention or a cell membrane fraction thereof is contacted, (ii) CRF and a test compound are added to the cell containing the human CRF ₂ receptor protein of the present invention or a cell membrane fraction thereof. CR when contacted with
Cell stimulating activity via F ₂ receptor (for example, opening of K ⁺ channel, closing of N-type Ca ²⁺ channel, arachidonic acid release, acetylcholine release, fluctuation of intracellular Ca ²⁺ concentration, promotion of intracellular cAMP production or Suppression, inositol phosphate production, cell membrane potential fluctuation, intracellular protein phosphorylation, c-fos activation, pH decrease, cell migration activity, hormone secretion, G protein activation, cell proliferation, etc. The present invention provides a method for screening a human CRF ₂ receptor agonist or antagonist, which comprises measuring and comparing the activity or suppressing activity).

[0076] In the screening method of the above (Ia) or (IIa), binds to human CRF ₂ receptor protein, a compound is human CRF ₂ receptor agonist or antagonist that inhibits the binding of CRF and human CRF ₂ receptor protein You can choose. further,
In the screening method of (IIb) above, human CR
Binding to F ₂ receptor and cell stimulating activity via the receptor (for example, opening of K ⁺ channel, closing of N-type Ca ²⁺ channel, arachidonic acid release, acetylcholine release, fluctuation of intracellular Ca ²⁺ concentration, cell Intracellular cAMP production, promotion or inhibition of intracellular cGMP production, inositol phosphate production, cell membrane potential fluctuation, intracellular protein phosphorylation, c
-Fos activation, pH decrease, cell migration activity, hormone secretion, G protein activation, activity promoting or suppressing cell proliferation, etc.)
It can be selected as an RF ₂ receptor agonist. On the other hand, in the above-mentioned screening method (Ia) or (IIa), among the test compounds which have been found to have an activity of inhibiting the binding of CRF to the human CRF ₂ receptor protein, a compound having no cell stimulating activity was selected as CRF ₂
It can be selected as a receptor antagonist. Since cells containing cDNA and human CRF ₂ receptor protein encoding human CRF ₂ receptor protein of the present invention is previously obtained, there is no cell that can highly express human CRF ₂ receptor protein, human CRF ₂
It was not possible to efficiently screen for receptor agonists or antagonists.

A detailed description of the screening method of the present invention is given below. As the CRF used as a ligand, a commercially available product or the like can be used. Also, CR
Instead of F, a known CRF receptor agonist or antagonist can also be used. For example, soyvedin or urotensin I can be used. Examples of the labeled CRF include [ ³ H] and [ ¹²⁵
CRF or the like labeled with I] or [ ¹⁴ C] can be used, and further, known CRF agonists or antagonists labeled with [ ³ H], [ ¹²⁵ I], [ ¹⁴ C] or the like can be used. You can also For example, sheep CRF labeled with [ ¹²⁵ I] (NEN Research Products, NEX
-217) and human CRF (NEN Research Products,
NEX-216) and the like are preferable. Examples of test compounds include peptides, proteins, non-peptidic compounds,
Examples thereof include synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, etc. These compounds may be novel compounds or known compounds.

Specifically, the above (Ia) or (II
To carry out the screening method of a), first, a cell containing the human CRF ₂ receptor protein of the present invention or a cell membrane fraction thereof, or a human CRF ₂ receptor protein or a partial peptide thereof is added to a reaction buffer suitable for screening. A receptor preparation is prepared by suspending. The reaction buffer may be any buffer which does not inhibit the binding between CRF and the receptor, such as a phosphate buffer having a pH of about 4 to 10 (desirably, a pH of about 6 to 8) and a Tris-hydrochloric acid buffer. Moreover, in order to reduce non-specific binding, CHAPS, T
ween-80 ^™ (Kao-Atlas), digitonin,
A detergent such as deoxycholate can also be added to the buffer. Furthermore, for the purpose of suppressing the degradation of the receptor or ligand by the protease, a protease inhibitor such as PMSF, leupeptin, bacitracin, aprotinin, E-64 (manufactured by Peptide Research Institute), pepstatin and the like can be added. Further, as a protein component, about 0.01-5% (preferably 0.1-1%) of B is used.
SA, about 0.01-5% (preferably 0.05-0.5)
%) Gelatin etc. can also be added. Conventionally, CR
Since F is a peptide consisting of 41 amino acids and having a relatively large molecular weight, it is easily adsorbed to a reaction vessel or a pipette in a ligand binding experiment, and accurate and highly sensitive measurement has not been easy. Therefore, in the screening methods (Ia) and (IIa) of the present invention, the reaction conditions for measuring the specific binding amount of CRF include the human CRF ₂ receptor protein of the present invention or its partial peptide, or the present invention. It is desirable to appropriately set the composition of the reaction buffer, the reaction time, the reaction temperature and the like when the CRF is brought into contact with the cell containing the human CRF ₂ receptor protein or the cell membrane fraction thereof.

For the purpose of reducing non-specific binding in the composition of the reaction buffer, it is effective to add a surfactant or a protein. Particularly, as the surfactant, for example, about 0.0
It is desirable to add 1-0.05% CHAPS, about 0.05% digitonin. Further, as the protein component, for example, it is desirable to add about 0.2% BSA or about 0.3% gelatin. The reaction temperature and the reaction time are preferably about 25 ° C. and about 2 hours, respectively. On the other hand, human CR
When CHO cells containing the F ₂ receptor protein are used, CRF and CR can be used while being attached to the incubator, that is, in the state of growing CHO cells or using cells fixed with glutaraldehyde or paraformaldehyde.
The F ₂ receptor protein can be bound. In this case, as the reaction buffer, for example, a medium or Hank's solution is used. And 0.01ml-10m
l of the receptor solution, a fixed amount (eg, 2200C)
In the case of i / mmol, about 10,000 cpm to 10,000
00 cpm labeled CRF (eg, [ ¹²⁵ I] C
RF) is added, and at the same time, 10 ⁻⁴ M to ^{10 −10} M of the test compound is allowed to coexist. A reaction tube containing a large excess of unlabeled CRF is also prepared in order to know the amount of nonspecific binding (NSB). The reaction is carried out at 0 ° C to 50 ° C, preferably at 4 ° C to 3 ° C.
The reaction is carried out at 7 ° C. for 20 minutes to 24 hours, preferably 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 a washing buffer, and the radioactivity (for example, the amount of [ ¹²⁵ I]) remaining on the glass fiber filter paper is measured with a liquid scintillation counter or a γ-counter. . Although a manifold or cell harvester can be used for filtration, the use of a cell harvester is desirable for treating large amounts of test compound. The count (B ₀ −NSB) obtained by subtracting the non-specific binding amount (NSB) from the count (B ₀ ) when there is no antagonistic substance is 100.
%, The specific binding amount when the test compound was added (B
-NSB) is, for example, 50 of the count (B ₀ -NSB).
Test compounds that are less than or equal to% can be selected as agonist or antagonist candidate compounds.

Further, in order to carry out the screening method of (IIb), cell stimulating activity mediated by human CRF ₂ receptor protein (eg, arachidonic acid release, acetylcholine release, fluctuation of intracellular Ca ²⁺ concentration, intracellular cA
MP production, inositol phosphate production, cell membrane potential fluctuation,
Phosphorylation of intracellular proteins, c-fos activation, pH decrease, hormone release, activity promoting or suppressing cell migration activity, etc.) is measured using a known method or a commercially available measurement kit. can do. Specifically, first, C containing human CRF ₂ receptor protein
HO cells 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 (eg, cAMP, arachidonic acid, etc.) is difficult to assay by the degrading enzyme contained in the cell, an inhibitor may be added to the degrading enzyme to perform the assay. .

The screening kit of the present invention comprises a CHO cell containing the human CRF ₂ receptor protein of the present invention or a cell membrane fraction thereof, or the human CRF of the present invention.
_{(2) It} contains a receptor protein, a partial peptide thereof or a salt thereof. Examples of the screening kit of the present invention include the following. [Screening reagent] Measurement buffer and washing buffer Hanks' Balanced Salt Solution (manufactured by Gibco)
One containing 05% bovine serum albumin (manufactured by Sigma). Sterilized by filtration through a 0.45 μm filter, 4 ° C
Or may be prepared at the time of use. Human CRF ₂ Receptor Protein Preparation CHO cells containing human CRF ₂ receptor protein were subcultured on a 12-well plate at 5 × 10 ⁵ cells / well, and 37
Cultivated at 5 ° C., 5% CO ₂ , 95% air for 2 days. Labeled CRF Commercially available CRF solution labeled with [ ³ H], [ ¹²⁵ I], [ ¹⁴ C], [ ³⁵ S] or the like is stored at 4 ° C. or -20 ° C.,
When using, dilute to 5 nM with the measurement buffer. CRF standard solution CRF was dissolved in PBS containing 0.1% bovine serum albumin (manufactured by Sigma) to a concentration of 0.1 mM, and the solution was -20 ° C.
Save with.

[Measurement Method] Human CRF ₂ cultured in a 12-well tissue culture plate
The CHO cells containing the receptor protein are washed twice with 1 ml of the measurement buffer, and then 490 μl of the measurement 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 5 nM labeled CRF is added and reacted at room temperature for 1 hour. In order to know the amount of non-specific binding, 5 μl of 10 ⁻⁴ M CRF was added in place of the test compound. Remove the reaction solution and wash 3 times with 1 ml of washing buffer. Labeled CRF bound to cells was added to 0.5 ml of 0.2N.
It is dissolved in NaOH-1% SDS and mixed with 4 ml of liquid scintillator A (manufactured by Wako Pure Chemical Industries). Liquid scintillation counter (Beckman)
Radioactivity was measured using Percent Maximum Binding
(PMB) is calculated by the following equation [Equation 2]. In addition,
When labeled with [ ¹²⁵ I], it can be measured directly with a gamma counter without mixing with a liquid scintillator.

[0083]

[Equation 2]

PMB: Percent Maximum Binding B: Value when a sample is added NSB: Non-specific Binding (non-specific binding amount) B ₀ : Maximum binding amount Obtained by using the screening method or screening kit of the present invention. The compound or a salt thereof is a compound that inhibits the binding between CRF and the human CRF ₂ receptor protein of the present invention, and specifically, a compound or a salt thereof having a cell stimulating activity via the human CRF ₂ receptor (so-called, Human CRF ₂ receptor agonist), or a compound or salt thereof having no such cell stimulating activity (so-called human CRF ₂ receptor antagonist)
It is.

The human CRF ₂ receptor agonist is C
Since it has all or part of the physiological activity possessed by RF, it is useful as a safe and low-toxic pharmaceutical composition depending on the physiological activity. For example, in addition to being useful as a prophylactic / therapeutic agent for dementia and obesity, for example, an adaptation promoter for stress, a secretagogue for ACTH, β-endorphin, β-lipotropin or α-MSF, a blood pressure lowering agent, mood It is useful as a behavior regulator, gastrointestinal function regulator, autonomic nervous system regulator, pituitary, cardiovascular system, digestive tract, or central nervous system function test agent. On the other hand, since the human CRF ₂ receptor antagonist can suppress all or part of the physiological activity of CRF, it is useful as a safe and low-toxic pharmaceutical composition for suppressing the physiological activity. For example, depression / anxiety / headache caused by stress, inflammatory diseases, immunosuppression, AIDS, Alzheimer's disease, gastrointestinal disorders, anorexia, bleeding stress, withdrawal symptoms such as drugs / alcohol, drug addiction, reproductive disorders, Cushing's disease It is also useful as a prophylactic / therapeutic agent for hypotension. further,
Chemically modified or substituted structural formula of the human CRF ₂ receptor agonist or antagonist obtained by using the screening method or screening kit of the present invention, and a compound designed based on the structural formula of the compound Human CRF obtained using the screening method or screening kit of the present invention
₂ Included in receptor agonists or antagonists. As the salt of the human CRF ₂ receptor agonist or antagonist, a physiologically acceptable acid addition salt is particularly preferable. Examples of such salts include salts with inorganic salts (eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid), or organic acids (eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, succinic acid). , Tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid) and the like.

When the agonist or antagonist obtained by using the screening method or the screening kit of the present invention is used as the above-mentioned pharmaceutical composition,
It has low toxicity and can be used safely according to a conventional method. 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 mixed with tablets, capsules and the like include binders such as gelatin, corn starch, tragacanth, and gum arabic.
Excipients such as crystalline cellulose, swelling agents such as corn starch, gelatin, alginic acid, lubricants such as magnesium stearate, sweeteners such as sucrose, lactose or saccharin, peppermint, red oil or cherry. Such a flavoring agent is 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. .

As the aqueous solution for injection, for example, physiological saline, isotonic solution containing glucose and other auxiliary agents (for example,
D-sorbitol, D-mannitol, sodium chloride, etc., and suitable solubilizing agents such as alcohols (eg ethanol), polyalcohols (eg propylene glycol, polyethylene glycol), nonionic surfactants (eg polysorbate). You may use together with 80 (TM), HCO-50, etc.
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), soothing agents (eg, benzalkonium chloride, procaine hydrochloride, etc.), stabilizers (eg, human serum albumin, polyethylene glycol, etc.), preservation You may mix | blend with agents (for example, benzyl alcohol, phenol, etc.), antioxidants, etc. The adjusted injection solution is usually filled in a suitable ampoule. Since the formulation thus obtained is safe and has low toxicity,
For example, it can be administered to a warm-blooded mammal (eg, rat, rabbit, sheep, pig, cow, cat, dog, monkey, human, etc., particularly human). The dose of the compound or its salt varies depending on the symptoms and the like, but in the case of oral administration, it is generally about 0.1 to 100 mg, preferably about 1.0 mg per day for an adult (as 60 kg). ~ 50 mg, more preferably about 1.0-20 mg
It is. 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). It is convenient to administer about 0.01 to 30 mg, preferably about 0.1 to 20 mg, more preferably about 0.1 to 10 mg by intravenous injection. In the case of other animals, the dose converted per 60 kg can be administered.

(2) Preventive / therapeutic agent for CRF ₂ receptor protein deficiency D encoding the human CRF ₂ receptor protein of the present invention
NA can be used as a prophylactic / therapeutic agent for CRF ₂ receptor protein deficiency. For example, when the human CRF ₂ receptor protein of the present invention is reduced in vivo and there are patients who cannot expect the physiological action of CRF, (a) the DNA encoding the human CRF ₂ receptor protein of the present invention Administered to a patient, the human C in vivo
By expressing the RF ₂ receptor protein or (b) inserting the DNA encoding the human CRF ₂ receptor protein of the present invention into the cell and expressing the human CRF ₂ receptor protein, the cell is transferred to the patient. By transplanting or the like, the amount of human CRF ₂ receptor protein in the patient can be increased and the action of CRF can be sufficiently exerted. Therefore, the human CRF _{2 of the} present invention
The DNA encoding the receptor protein is a safe and low toxic CRF ₂ receptor protein deficiency (eg, hypertension,
It can be used as a prophylactic / therapeutic agent for autonomic imbalance or stress. When the DNA of the present invention is used as the above-mentioned prophylactic / therapeutic agent, the DNA is inserted alone or into an appropriate vector such as a retrovirus vector, an adenovirus vector or an adenovirus associated virus vector, and then the procedure is carried out according to a conventional method. can do. 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 is a physiologically acceptable carrier, flavoring agent,
It can be prepared by mixing with excipients, vehicles, preservatives, stabilizers, binders and the like in a unit dosage form generally required for accepted pharmaceutical practice. The amount of the active ingredient in these preparations is such that an appropriate dose in the specified range can be obtained.

Examples of additives that can be mixed with tablets, capsules and the like include binders such as gelatin, corn starch, tragacanth and gum arabic, excipients such as crystalline cellulose, corn starch, gelatin, alginic acid and the like. Such as a leavening agent, a lubricant such as magnesium stearate, a sweetener such as sucrose, lactose or saccharin, and a flavoring agent such as peppermint, red oil or cherry. 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), nonionic surfactant (eg polysorbate 80 (TM), HCO-50) and the like 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. In addition, buffers (eg, phosphate buffer, sodium acetate buffer), soothing agents (eg, benzalkonium chloride, procaine hydrochloride, etc.), stabilizers (eg, human serum albumin, polyethylene glycol, etc.), preservation Agents (eg, benzyl alcohol,
Phenol, etc.), antioxidants and the like.
The adjusted injection solution 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,
It can be administered to sheep, pigs, cows, cats, dogs, monkeys, humans, etc., especially humans). Although the dose of the DNA varies depending on the symptoms and the like, in the case of oral administration, it is generally about 0.1 mg to 100 mg, preferably about 1.0 mg per day for an adult (60 kg).
50 mg, more preferably about 1.0-20 mg.
In the case of parenteral administration, the single dose varies depending on the administration subject, target organ, symptoms, administration method, etc., but in the form of an injection, it is usually about 1 day in an adult (as 60 kg). It is convenient to administer about 0.01 to 30 mg, preferably about 0.1 to 20 mg, more preferably about 0.1 to 10 mg by intravenous injection. In the case of other animals, the dose converted per 60 kg can be administered. The D of the present invention
As a method for inserting NA into cells to express the human CRF ₂ receptor protein and transplanting the cells into a patient, a method known per se or a method analogous thereto can be used.

(3) Method for Quantifying CRF The human CRF ₂ receptor protein of the present invention or a salt thereof, or the partial peptide of the present invention or a salt thereof is CR.
Since it has a binding property to F, it can bind to C in vivo.
It can be used as a reagent when quantifying the RF concentration with high sensitivity. That is, the present invention relates to the human CRF _{2 of the} present invention.
Provided is a method for quantifying CRF concentration in a subject, which comprises contacting the subject with a receptor protein or a salt thereof, or a partial peptide of the present invention or a salt thereof.
The quantification method of the present invention can be used, for example, by combining it with a competition method. 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, "Continued Radioimmunoassay" (Kodansha, published in 1979) Furthermore, the CRF quantification method of the present invention is a disease caused by an increase or decrease in CRF concentration (for example, stress, inflammation, Alzheimer's disease, gastrointestinal It can also be used as a method for diagnosing problems such as disorders.

[0091] (4) human CRF ₂ receptor protein or human CRF ₂ receptor protein partial peptide or human CRF ₂ receptor protein or its salt of the preparation the present invention an antibody or antiserum against a salt of salt thereof of the present invention or Alternatively, the antibody against the partial peptide of the human CRF ₂ receptor protein of the present invention or a salt thereof (eg, polyclonal antibody, monoclonal antibody) or antiserum is the human CRF ₂ receptor protein of the present invention or its salt or the human CRF ₂ receptor of the present invention. It can be produced according to a method for producing an antibody or antiserum known per se using a partial peptide of a protein or a salt thereof as an antigen.
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 Human CRF ₂ receptor protein of the present invention or a salt thereof, or partial peptide of human CRF ₂ receptor protein of the present invention or a salt thereof (hereinafter referred to as human CRF) (Sometimes abbreviated as " ₂ 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 producing monoclonal antibody-producing cells, warm-blooded animals immunized with an antigen, for example, an individual with an antibody titer was selected from mice, and spleens or lymph nodes were collected 2 to 5 days after the final immunization and included in them. By fusing the antibody-producing cells prepared as described above with myeloma cells, a monoclonal antibody-producing hybridoma can be prepared. The antibody titer in antiserum can be measured by, for example, the labeled human CRF described below.
₂ After reacting the receptor with antiserum, the activity of the labeling agent bound to the antibody is measured. The fusion operation can be carried out according to a known method, for example, the method of Kohler and Milstein [Nature, 256, 495 (1975)]. Examples of the fusion promoter include polyethylene glycol (PEG) and Sendai virus.
Preferably PEG is used. Examples of myeloma cells include NS-1, P3U1, SP2 / 0, 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.
(Preferably PEG1000 to PEG6000) is 10
The cell fusion can be efficiently carried out by adding at a concentration of about 80% and incubating at 20 to 40 ° C., preferably 30 to 37 ° C. for 1 to 10 minutes. Anti-human CRF
_Although various methods can be used for screening a hybridoma producing a _2- receptor antibody, for example, human CRF ₂
Hybridoma culture supernatant is added to a solid phase (eg, microplate) on which the receptor antigen is directly or adsorbed with a carrier, and then an anti-immunoglobulin antibody labeled with a radioactive substance or enzyme (cells used for cell fusion are mouse In the case of, an anti-mouse immunoglobulin antibody is used) or protein A is added, and the anti-human CRF bound to a solid phase is added.
Method for detecting ₂ receptor monoclonal antibody, hybridoma culture supernatant is added to a solid phase on which anti-immunoglobulin antibody or protein A is adsorbed, and human CRF ₂ receptor labeled with a radioactive substance or enzyme is added to bind to the solid phase And a method for detecting the anti-human CRF ₂ receptor monoclonal antibody. The anti-human CRF ₂ receptor monoclonal antibody can be selected according to a method known per se or a method analogous thereto. Normal H
It is carried out in a medium for animal cells supplemented with AT (hypoxanthine, aminopterin, thymidine). As a selection and breeding medium, any medium can be used as long as hybridomas can grow. For example, 1-20
%, Preferably 10-20% fetal calf serum
I 1640 medium, GI containing 1-10% fetal bovine serum
T medium (Wako Pure Chemical Industries, Ltd.) or serum-free medium for hybridoma culture (SFM-101, Nissui Pharmaceutical Co., Ltd.)
Etc. can be used. The culture temperature is usually 20 to 4
It is 0 ° C, preferably about 37 ° C. Cultivation time is usually 5
Day to 3 weeks, preferably 1 to 2 weeks. Culturing is usually performed under 5% carbon dioxide gas. The antibody titer of the hybridoma culture supernatant was determined by the anti-human CRF ₂
It can be measured in the same manner as the measurement of the receptor antibody titer.

(B) Purification of monoclonal antibody The isolation and purification of the anti-human CRF ₂ receptor monoclonal antibody is carried out in the same manner as the isolation and purification of a usual polyclonal antibody [eg, salting out method, alcohol precipitation method, etc.]. Focusing point precipitation method, electrophoresis method, ion exchanger (eg, D
Specific purification method to obtain antibody by adsorption / desorption method by EAE, ultracentrifugation method, gel filtration method, antigen-binding solid phase or active adsorbent such as protein A or protein G, and dissociating the bond to obtain antibody] Is done according to. Since the human CRF ₂ receptor antibody of the present invention produced according to the above methods (1) and (2) can specifically recognize the human CRF ₂ receptor, it is possible to quantify the human CRF ₂ receptor in a test solution. In particular, it can be used for quantification by sandwich immunoassay. That is, the present invention provides, for example, (i) an antibody that reacts with the human CRF ₂ receptor of the present invention, a test solution and labeled human C
A method for quantifying human CRF ₂ receptor in a test solution, which comprises reacting competitively with RF ₂ receptor to measure the ratio of labeled human CRF ₂ receptor bound to the antibody, (ii) test liquid and After the carrier has been insolubilized antibody and labeled on the antibody is simultaneously or sequentially reacting the human CRF ₂ of the test fluid, which comprises measuring the activity of the labeling agent on the insoluble carrier In the assay method for receptors, one antibody is an antibody that recognizes the N-terminal portion of the human CRF ₂ receptor, and the other antibody is an antibody that reacts with the C-terminal portion of the human CRF ₂ receptor. A method for quantifying the human CRF ₂ receptor therein is provided. Human CRF ₂ receptor of the present invention a monoclonal antibody recognizing (hereinafter, anti-human CRF ₂ may be referred to as a receptor antibody) other enables to measure the human CRF ₂ receptor using, also be detected by tissue staining etc. it can. For these purposes, the antibody molecule itself may be used, or F (ab ′) ₂ , Fab ′, or Fab fraction of the antibody molecule may be used. The assay 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 (for example, the amount of human CRF ₂ receptor) in the solution to be measured is chemically determined. Any measuring method may be used as long as it is detected by physical or physical means and is calculated from a standard curve prepared using a standard solution containing a known amount of antigen. For example, nephelometry, a competitive method, an immunometric method, and a sandwich method are suitably used, but in terms of sensitivity and specificity, it is particularly preferable to use a sandwich method described later.

Examples of the labeling agent used in the measuring method using a labeling substance include radioisotopes, enzymes, fluorescent substances, luminescent substances and the like. As the radioisotope, for example, [ ¹²⁵ I], [ ¹³¹ I], [ ³ H], [ ¹⁴ C] and the like are preferable, and as the above enzyme, those having stable and large specific activity are preferable, for example, β-galactosidase, 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 usually used for insolubilizing and immobilizing proteins or enzymes may be used. 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-human CRF ₂ receptor antibody is reacted with a test solution (first reaction), and further, a labeled anti-human CRF ₂ receptor antibody is reacted (secondary reaction), followed by labeling on an insolubilized carrier. The amount of human CRF ₂ receptor in the test solution can be quantified by measuring the activity of the 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 the immunoassay by the sandwich method, the antibody used for the solid phase antibody or the labeling antibody is not necessarily one kind, and a mixture of two or more kinds of antibodies is used for the purpose of improving measurement sensitivity and the like. You may.

Human CRF by the sandwich method of the present invention
_{In the} method for measuring ₂ receptors, the anti-human CRF ₂ receptor antibody used in the primary and secondary reactions is human CR.
Antibodies having different F ₂ receptor binding sites are preferably used. That is, when the antibody used in the secondary reaction recognizes the C-terminal part of the human CRF ₂ receptor, the antibody used in the primary reaction is preferably an antibody used in the primary reaction. An antibody that recognizes an N-terminal other than the C-terminal is used. Human CR of the present invention
The F ₂ receptor antibody can be used in a measuring system other than the sandwich method, for example, a competitive method, an immunometric method or nephrometry. In the competition method, an antigen in a test solution and a labeled antigen are allowed to competitively react with an antibody, and then the unreacted labeled antigen is separated from (F) and the labeled antigen (B) bound to the antibody. (B / F separation), the amount of any of B and F is measured, and the amount of antigen in the test solution is quantified. In this reaction method, a soluble antibody is used as an antibody, and B /
F separation is performed by a liquid phase method using polyethylene glycol, a second antibody against the above antibody, and a solid phase antibody is used as the first antibody, or alternatively, a soluble antibody is used as the first antibody and a solid phase is used as the second antibody. A solid phase immobilization method using a conjugated antibody is used. In the immunometric method, an antigen in a test solution and a solid-phased antigen are subjected to a competitive reaction with a fixed amount of a labeled antibody, and then the solid phase and the liquid phase are separated. Is reacted with an excess amount of the labeled antibody, and then the immobilized antigen is added to bind the unreacted labeled antibody to the solid phase, and then the solid phase and the liquid phase are separated. Next, the 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 assay methods to the assay method of the present invention, it is not necessary to set special conditions, operations and the like. The measurement system for human CRF ₂ 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 reviews, books, etc. [for example, Hiro Irie's "Radioimmunoassay" (Kodansha, published in 1974), Irie
Hiroshi "Sequel Radioimmunoassay" (Kodansha, published in 1979), Eiji Ishikawa et al. "Enzyme Immunoassay" (Medical Shoin,
"Published in 1978)" Eiji Ishikawa et al. "Enzyme Immunoassay"
(2nd edition) (Medical Shoin, published in 1982), Eiji Ishikawa et al. “Enzyme Immunoassay” (3rd edition) (Medical Shoin, 1987)
Yearly), "Methods in ENZYMOLOGY" Vol. 70 (Immunoc
hemical Techniques (Part A)), ibid Vol. 73 (Immunoc
hemical Techniques (Part B)), ibid Vol. 74 (Immunoc
hemical Techniques (Part C)), Vol. 84 (Immunoc)
hemical Techniques (Part D: Selected Immunoassay
s)), 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)) (above, issued by Academic Press) etc.).
As described above, the human CRF ₂ receptor can be quantified with high sensitivity by using the human CRF ₂ receptor antibody of the present invention.

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 Phosphoric acid dCTP: Deoxycytidine triphosphate ATP: Adenosine triphosphate EDTA: Ethylenediaminetetraacetic acid SDS: Sodium dodecyl sulfate EIA: Enzyme immunoassay Gly: Glycine Ala: Alanine Val: Valine Leu: Leucine Ileh: Isoleucine: Isoleucine. Cys: Cysteine Met: Methionine Glu: Glutamic acid Asp: Aspartic acid Lys: Lysine Arg: Ar Nin His: Histidine Phe: Phenylalanine Tyr: Tyrosine Trp: Tryptophan Pro: Proline Asn: Asparagine Gln: Glutamine pGlu: Pyroglutamic acid Me: Methyl group Et: Ethyl group Bu: Butyl group PhC: Phyl group: T: Phenyl: Ph- -Carboxamide group

The sequence numbers in the sequence listing in this specification show the following sequences. [SEQ ID NO: 1] This shows the base sequence of synthetic DNA used for cloning the cDNA encoding the human CRF ₂ receptor protein of the present invention. [SEQ ID NO: 2] This shows the base sequence of synthetic DNA used for cloning the cDNA encoding the human CRF ₂ receptor protein of the present invention. [SEQ ID NO: 3] This shows the amino acid sequence of the human CRF ₂ receptor protein of the present invention. [SEQ ID NO: 4] This shows the base sequence of cDNA fragment encoding human CRF ₂ receptor protein of the present invention. [SEQ ID NO: 5] This shows the base sequence of synthetic DNA used for cloning cDNA encoding human CRF ₂ receptor protein of the present invention. [SEQ ID NO: 6] This shows the base sequence of synthetic DNA used for cloning cDNA encoding human CRF ₂ receptor protein of the present invention. [SEQ ID NO: 7] This shows the base sequence of synthetic DNA used for cloning cDNA encoding human CRF ₂ receptor protein of the present invention. [SEQ ID NO: 8] This shows the base sequence of synthetic DNA used for cloning cDNA encoding human CRF ₂ receptor protein of the present invention. [SEQ ID NO: 9] This shows the base sequence of synthetic DNA used for cloning cDNA encoding human CRF ₂ receptor protein of the present invention. [SEQ ID NO: 10] This shows the base sequence of synthetic DNA used for cloning cDNA encoding human CRF ₂ receptor protein of the present invention. [SEQ ID NO: 11] This shows the base sequence of synthetic DNA used for cloning cDNA encoding human CRF ₂ receptor protein of the present invention. [SEQ ID NO: 12] This shows the base sequence of synthetic DNA used for cloning cDNA encoding human CRF ₂ receptor protein of the present invention. [SEQ ID NO: 13] This shows the base sequence of synthetic DNA used for cloning cDNA encoding human CRF ₂ receptor protein of the present invention. [SEQ ID NO: 14] This shows the entire amino acid sequence of human CRF ₂ receptor protein of the present invention. [SEQ ID NO: 15] This shows the base sequence of cDNA fragment encoding the full length of human CRF ₂ receptor protein of the present invention. The transformant Escherichia coli JM109 / phs-AH1 obtained in Example 4 described later.
Has been depositing with the deposit number FERM from the Ministry of International Trade and Industry, Institute of Industrial Science and Technology, Institute of Biotechnology (NIBH) since June 15, 1995.
It has been deposited as BP-5136, and also in June 1995.
IFO to Fermentation Research Institute (IFO) from 12th of March
Deposited as 15827. In addition, the transformant Escherichia coli (Escheric
hia coli) JM109 / pCRF2-10 has been deposited with the deposit number FERM BP-5226 at the Institute of Biotechnology, National Institute of Advanced Industrial Science and Technology (NIBH) from September 5, 1995.
Has been deposited as IFO 15868 from the Institute of Fermentation (IFO) since August 29, 1995.

[0099]

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.

Example 1 Production of Synthetic DNA Primer for Amplifying DNA Encoding G Protein-Coupled Receptor Protein Known human calcitonin receptor (X82466),
Human parathyroid hormone receptor (X6859
6), human glucagon receptor (U03469), human glucagon-like polypeptide-1 receptor (U
01156), rat secretin receptor (X591
32), human growth hormone-releasing hormone receptor (L01406), human picitary adenylate cyclase-activated polypeptide receptor (D17516), rat picitary adenylate cyclase-activated polypeptide receptor (Z23279), human vasoactive Intestinal peptide receptor (X75299), human VIP 2
Receptor (L36566), Human Corticotropin Release Factor Receptor (L23332), and Rat Corticotropin Release Factor 2 Receptor (U1)
6253), the nucleotide sequences of the cDNAs encoding the amino acid sequences near the third and seventh transmembrane regions were compared with each other, and highly similar portions were found in the respective regions [FIGS. 1 and 2]. Abbreviations in () above are DNASIS G
ene / Protein sequence database (CD019,
Hitachi Software Engineering)
This is the reference number shown when you search EMBL Data Bank, and is usually called the Accession number. 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. A synthetic DN having a base sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2 which is complementary to a common base sequence based on this sequence
Two A sheets were produced. 5'-CATTAYTKGATSGYGRCCAACT
WCWNCTGG-3 ′ [Y represents T or C, K represents G or T, S represents C or G, R represents A or G, W represents A or T, and N represents I . ] (SEQ ID NO: 1) 5'-GTAGARRAYAGCCACMAMRARN
CCCTGRAA-3 ′ [R represents A or G, Y represents T or C, M represents A or C, and N represents I. ] (SEQ ID NO: 2) Y, K, S, R, W and M are synthesized by mixing with a plurality of bases at the time of synthesis.

[0100]

[Example 2] c from poly (A) ⁺ RNA fraction derived from human stomach
Synthesis of DNA Random DN was used as a primer for 5 μg of human stomach poly (A) ⁺ RNA fraction (Clontech, Catalog No. 6548-1).
A hexamer (BRL) was added, and complementary DNA was synthesized using Moronii mouse leukemia virus reverse transcriptase (BRL) using the attached buffer. The product after the reaction was precipitated with ethanol and then dissolved in 30 μl of TE.

[0101]

[Example 3] Amplification of receptor cDNA by PCR using human stomach-derived cDNA 1 µl of cDNA prepared from human stomach in Example 2 was used as a template, and DNA primers synthesized in Example 1 were used for PCR. Amplification was performed. The composition of the reaction solution is
Synthetic DNA primers (sequence: 5'primer sequence and 3'primer sequence) 100 pM each, 0.25 mM dN
TPs (Deoxyribonucleoside triphosphate), Taq
The total reaction solution volume was 100 μl with 1 μl of DNA polymerase and 10 μl of buffer attached to the enzyme. The cycle for amplification is the thermal cycler (Perkin
Elmer), 96 ° C for 30 seconds, 45 ° C for 1 minute,
The cycle of 60 ° C. for 3 minutes was repeated 25 times. The amplification product was confirmed by 1.2% agarose gel electrophoresis and ethidium bromide staining.

[0102]

Example 4 Subcloning of PCR Product into Plasmid Vector and Selection of Human CRF ₂ Receptor Candidate Clones by Decoding the Nucleotide Sequence of Inserted cDNA Part The reaction product after PCR performed in Example 3 was 1.5% agarose gel. Was separated, and the band portion was cut out with a razor, followed by electroelution, phenol extraction, and ethanol precipitation to recover DNA. T
The recovered DNA was subcloned into the plasmid vector pCR ^™ II according to the prescription of A cloning kit (Invitrogen). E. coli JM109 compe
After being introduced into a tent cell (Takara Shuzo Co., Ltd.) for transformation, the clone having the cDNA insert fragment was treated with ampicillin, IPTG (isopropylthio-β-D-galactoside) and X-gal (5-Bromo-4-chloro-3-). indolyl-β-D-galacto
side) and selected in LB agar medium, and only white clones were separated using a sterilized toothpick to obtain 100 transformants. Individual clones were cultured overnight in LB medium containing ampicillin, and plasmid DNA was prepared using an automatic plasmid extractor PI-100 (Kurabo).
Was prepared. EcoRI using a part of the prepared DNA
Then, the size of the inserted cDNA fragment was confirmed. A part of the remaining DNA is further purified with RNase.
Treatment, extraction with phenol / chloroform, and concentration by ethanol precipitation. The reaction for determining the nucleotide sequence is
DyeDeoxy Terminator Cycle Sequencing Kit (ABI
Company) and decoded using a fluorescent automatic sequencer. Based on the obtained nucleotide sequence, a homology search was performed using DNASIS (Hitachi System Engineering Co., Ltd.). As a result, the transformant Escherichia coli (Esch.
erichiacoli) JM109 / phs-AH1 was found to have a cDNA fragment inserted into a plasmid that encodes a novel G protein-coupled receptor protein. The nucleotide sequence of the cDNA fragment is shown in FIG. To further confirm, after using DNASIS (Hitachi System Engineering Co., Ltd.) to convert the nucleotide sequence into an amino acid sequence [FIG. 3], a hydrophobicity plot [FIG. 4] was performed.
It was confirmed that there is a hydrophobic domain indicating that it is a G protein-coupled receptor protein. In addition, as a result of conducting a homology search based on the amino acid sequence, it was found that the rat CRF2 receptor protein (U16235) and 96.6
%, 93.8% with the mouse CRF ₂ (CRF-RB) receptor protein (U17858), and 84.5% with the human CRF receptor protein (L23332). This indicates that the cDNA fragment of the present invention encodes a human CRF ₂ receptor protein which has not been reported so far. The abbreviations in parentheses above are reference numbers when registered as data in GenBank, and are usually called Accession numbers.

[0103]

Example 5 Human CRF ₂ receptor c by PCR method
Amplification of 5'side and 3'side sequences of DNA The 5'side and 3'side sequences of human CRF ₂ receptor cDNA were prepared using human gastric poly (A) ⁺ RNA fraction (Clontech, Catalog No. 6548-1) as a material. PC to amplify
R was performed. Introduction Marathon cDNA amplification
Human CRF ₂ receptor cD obtained in Example 4 using a kit (Clontech, Catalog No. K1802-1)
PCR was carried out to amplify a cDNA having a 5'side sequence of the NA fragment. SEQ ID NO: 5 or SEQ ID NO: which is complementary to the sequence (SEQ ID NO: 4) of the human CRF ₂ receptor cDNA fragment obtained in Example 4 as a primer.
Two synthetic DNAs having a base sequence represented by 6 were prepared. N0 5'-ATGAAGAGGAAGAGGCACTTGCGCA-3 '(SEQ ID NO: 5) N1 5'- CAGTGGAGTAGGTCATGACAATGGC-3' (SEQ ID NO: 6) Next, using human stomach poly (A) ⁺ RNA fraction 1 μg, Mara.
Double-stranded cDNA for use as a template was synthesized according to the method of thon cDNA amplification kit. Subsequently, the first PCR was performed using this as a material. The composition of the reaction solution was 5 μl of double-stranded cDNA and 200 n each of synthetic DNA primer (N0 and AP1 attached to the kit).
M, 200 μM dNTP, ExTaq DNA polymerase
(Takara Shuzo) 0.5 μl and 10 × ExTaq reaction buffer 5 μl, and the total reaction volume was adjusted to 50 μl by adding water. The reaction for amplification uses a thermal cycler (Perkin-Elmer) at 94 ° C for 30 seconds and 60 ° C for 30 seconds.
The cycle of 68 ° C. for 3 minutes was repeated 30 times. Next, a second PCR was performed using 0.1 μl of the reaction solution as a template. The composition of the reaction solution is 200 nM, 200 μM for each synthetic primer (N1 and AP2 attached to the kit).
dNTP, ExTaq DNA polymerase 0.5 μl, 10
× ExTaq reaction buffer was 5 μl, and the total reaction solution volume was adjusted to 50 μl by adding water. Reaction conditions: 94 ° C, 30 seconds, 6
8 ° C. and 3 minutes were repeated 25 times. Confirmation of amplified product is 1.
After 2% agarose gel electrophoresis, staining was performed with ethidium bromide. About 350 has been amplified specifically
A bp long DNA fragment was recovered from the gel by electroelution. After cloning this into the plasmid vector pCRII by the method described in Example 4, the nucleotide sequence was determined. As a result, the recovered DNA fragment was human CR
It was found that the F ₂ receptor cDNA fragment contained about 350 bp from the position of primer N1 toward the 5 ′ side. Based on the information on the nucleotide sequence of human CRF ₂ receptor cDNA obtained by the above operation, further 5'and 3 '
The DNA fragment on the side was amplified. For amplification of the 5'-side DNA fragment, 5'-AmpliFINDER RACE Kit (Clontech, catalog number K1800-1) was used as follows. Two new synthetic DNAs having the nucleotide sequence represented by SEQ ID NO: 7 or SEQ ID NO: 8 were newly prepared as primers for amplifying the 5 ′ side. N11 5'-ATGATGGGGCCTTGGTAGATGTTAGTCCA CC-3 '(SEQ ID NO: 7) N12 5'-GCTCCTTGCCAAACAGCACTGTTTCATT CT-3' (SEQ ID NO: 8) Next, 5'-A of human stomach poly (A) ⁺ RNA fraction was used.
cDNA synthesis and PCR were performed according to the method of mpliFINDER RACE Kit. N11 was used as a primer for cDNA synthesis. For the first PCR, N12 was used as a primer and the anchor primer attached to the kit, and for the second PCR, the previously synthesized N12 was used.
0 and anchor primer were used. The composition of the PCR reaction was 200 nM for each synthetic primer and 200 μM d
NTP, ExTaq DNA polymerase 0.5 μl, 10 × Ex
The reaction volume was 5 μl for Taq, and the total reaction volume was 50 μl by adding water. The first PCR is 94 ℃ ・ 45
Second, 55 ° C., 45 seconds, 68 ° C., 3 minutes were repeated 30 times. The second PCR was 94 ° C for 45 seconds, 60 ° C for
45 seconds, 68 ° C. and 3 minutes were repeated 25 times. As a result of confirming the amplified product by 1.5% agarose gel electrophoresis, about 7
Since the 00 bp DNA fragment was amplified, the DNA
The fragment was recovered from the gel by electroelution, cloned into the plasmid vector pCRII by the method described in Example 4, and the nucleotide sequence was determined. As a result, the recovered DNA fragment was found to be approximately 700 bp in the 5'direction from the position of the primer N0 of the human CRF ₂ receptor cDNA fragment.
Was found to contain. For amplification of 3'side DNA fragment, 3'RACE System (GIBCO BRL: Catalog No. 837
3SA) was performed as follows. As a primer for amplifying the 3'side, three synthetic DNAs having a nucleotide sequence represented by SEQ ID NO: 9, SEQ ID NO: 10 or SEQ ID NO: 11 were newly prepared. C1 5′-GAGGACGACCTGTCACAGATCATGT-3 ′ (SEQ ID NO: 9) C11 5′-ATTCGCTGTCTGCCGGAATGTGATTCACT
GG-3 ′ (SEQ ID NO: 10) C12 5′-CTGGAACCTCATCACCACCTTTATCCTG CG-3 ′ (SEQ ID NO: 11) Next, using human stomach poly (A) ⁺ RNA fraction 1 μg, 3 ′ R
After synthesizing the cDNA according to the method of ACE System, the first PCR was performed using C11 and the anchor primer attached to the kit. The composition of the reaction solution and the reaction conditions were the same as those for the amplification of the 5′-side DNA fragment described above. Next, the second PCR is similarly performed using C12 and the anchor primer as the primers, and the third PCR is performed.
Was similarly performed using C1 and the anchor primer as primers. As a result of confirming the amplified product by 1.5% agarose gel electrophoresis, a DNA fragment of about 1200 bp was amplified. Therefore, the nucleotide sequence of the DNA fragment was analyzed by the above method. As a result, it was revealed that the recovered DNA fragment contained about 1200 bp in the 3'direction from the position of the primer C1 of the human CRF ₂ receptor cDNA fragment. Human CR represented by SEQ ID NO: 15 by the above operation
The entire coding region of the F ₂ receptor cDNA was obtained. By this, the nucleotide sequence of the entire coding region of human CRF ₂ receptor cDNA was clarified, and the entire amino acid sequence of human CRF ₂ receptor protein represented by SEQ ID NO: 14 was clarified for the first time (FIGS. 6 and 7).

[0104]

Example 6 Human CRF ₂ receptor c by PCR method
Amplification of a DNA fragment containing the entire coding region of DNA Using the human stomach cDNA prepared in Example 2 as a template, human C
A cDNA fragment encoding the entire length of the RF ₂ receptor protein was amplified. First, the human CR found in Example 5
Based on the sequence of F ₂ receptor cDNA, SEQ ID NO: 12
Alternatively, two synthetic DNAs having the base sequence represented by SEQ ID NO: 13 were prepared. C2-F 5'-GTCGACACGCCGCTGCCGGACGCGAGTG GA-3 '(SEQ ID NO: 12) C2-R 5'-GTCGACTCTTGCAGGTGGGCGACCGAGG GGTCA-3' (SEQ ID NO: 13) C2-F contains the start codon of human CRF ₂ receptor cDNA. C2-R with a sense sequence corresponding to 18 to +5 (A of start codon ATG is +1)
Is an antisense sequence corresponding to +1258 to +1283 including the stop codon of human CRF ₂ receptor cDNA. In addition, a restriction enzyme site (SalI) was added to the 5'end of both primers. The composition of the PCR reaction solution was as follows: human stomach cDNA 1 μl, synthetic DNA primers (C2-F and C2-R) 200 nM and 200 μM, respectively.
dNTP, 0.5 μl of ExTaq DNA polymerase, and 5 μl of 10 × ExTaq reaction buffer, and the total reaction volume was adjusted to 50 μl by adding water. The reaction conditions are 95 ° C and 3
0 seconds, 70 ° C. and 4 minutes were repeated 35 times. 1% of reaction solution
As a result of confirmation by agarose gel electrophoresis, a DNA fragment of the desired size (about 1.3 kb) was specifically amplified. The DNA fragment was recovered from the gel by electroelution and the plasmid vector pCR was prepared by the method described above.
After cloning into II, automatic base sequence analyzer 370A
The base sequence of the inserted DNA fragment was analyzed by (Applied Biosystems). As a result, it was confirmed that the DNA fragment was a DNA fragment containing the entire coding region of human CRF ₂ receptor cDNA found in Example 5.
Among the clones obtained here, pCRF2-10
To be used in the subsequent experiments. pCRF2-10
Since the entire coding region of human CRF ₂ receptor cDNA can be excised with restriction enzymes SalI or EcoRI, it is suitable for construction of human CRF ₂ receptor expression plasmid, preparation of DNA probe and the like.

[0105]

EFFECT OF THE INVENTION SEQ ID NO: 1 or SEQ ID NO: of the present invention
By using a DNA having a nucleotide sequence substantially the same as the nucleotide sequence represented by 2, the DNA encoding the G protein-coupled receptor protein can be efficiently amplified. Thereby, the DNA encoding the G protein-coupled receptor protein can be efficiently screened and cloned. The human CRF ₂ receptor protein of the present invention is a known human C
It is a novel protein having an amino acid sequence different from that of the RF receptor protein. Human CR of the present invention
DNA encoding F ₂ receptor protein and the DN
A human CRF ₂ receptor protein encoded by A, a partial peptide thereof, or a cell containing the human CRF ₂ receptor protein or a cell membrane fraction thereof can be efficiently screened for a human CRF ₂ receptor agonist or antagonist. According to the above screening method, an agonist or an antagonist can be advantageously selected, so that a drug can be developed at an early stage. Agonists include, for example, prophylactic / therapeutic agents for dementia and obesity, agents for promoting adaptation to stress, ACT
H, β-endorphin, β-lipotropin or α
-MSF secretagogues, antihypertensive agents, mood and behavior regulators, gastrointestinal function regulators, autonomic nervous system regulators, and further pituitary, cardiovascular, gastrointestinal or central nervous system function test agents, etc. Is useful as On the other hand, antagonists include, for example, depression / anxiety / headache caused by stress, inflammatory diseases, immunosuppression, AIDS, Alzheimer's disease, gastrointestinal disorders, anorexia, bleeding stress, drug / alcohol withdrawal symptoms, and drug dependence. It is also useful as a prophylactic / therapeutic agent for reproductive disorders, Cushing's disease or hypotension.

[0106]

[Sequence list]

[SEQ ID NO: 1] Sequence length: 30 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence features: N indicates I. Sequence CATTAYTKGA TSGYGRCCAA CTWCWNCTGG 30

[0107]

[SEQ ID NO: 2] Sequence length: 30 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence characteristics: N represents I. Array GTAGARRAYA GCCACMAMRA RNCCCTGRAA 30

[0108]

[SEQ ID NO: 3] Sequence length: 148 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Met Phe Val Glu Gly Cys Tyr Leu His Thr Ala Ile Val Met Thr Tyr 1 5 10 15 Ser Thr Glu Arg Leu Arg Lys Cys Leu Phe Leu Phe Ile Gly Trp Cys 20 25 30 Ile Pro Phe Pro Ile Ile Val Ala Trp Ala Ile Gly Lys Leu Tyr Tyr 35 40 45 Glu Asn Glu Gln Cys Trp Phe Gly Lys Glu Pro Gly Asp Leu Val Asp 50 55 60 Tyr Ile Tyr Gln Gly Pro Ile Ile Leu Val Leu Leu Ile Asn Phe Val 65 70 75 80 Phe Leu Phe Asn Ile Val Arg Ile Leu Met Thr Lys Leu Arg Ala Ser 85 90 95 Thr Thr Ser Glu Thr Ile Gln Tyr Arg Lys Ala Val Lys Ala Thr Leu 100 105 110 Val Leu Leu Pro Leu Leu Gly Ile Thr Tyr Met Leu Phe Phe Val Asn 115 120 125 Pro Gly Glu Asp Asp Leu Ser Gln Ile Met Phe Ile Tyr Phe Asn Ser 130 135 140 Phe Leu Gln Ser 145

[0109]

[SEQ ID NO: 4] Sequence length: 444 Sequence type: Nucleic acid Number of strands: Double stranded Topology: Linear Sequence type: cDNA Characterization method: S sequence ATGTTTGTGG AAGGCTGCTA CCTGCACACG GCCATTGTCA TGACCTACTC CACTGAGCGC 60 CTGCGCAAGT GCCTCTTCCT CTTCATCGGA TGGTGCATCC CCTTCCCCAT CATCGTCGCC 120 TGGGCCATCG GCAAGCTCTA CTATGAGAAT GAACAGTGCT GGTTTGGCAA GGAGCCTGGC 180 GACCTGGTGG ACTACATCTA CCAAGGCCCC ATCATTCTCG TGCTCCTGAT CAATTTCGTA 240 TTTCTGTTCA ACATCGTCAG GATCCTAATG ACAAAGTTAC GCGCGTCCAC CACATCCGAG 300 ACAATCCAGT ACAGGAAGGC AGTGAAGGCC ACCCTGGTGC TCCTGCCCCT CCTGGGCATC 360 ACCTACATGC TCTTCTTCGT CAATCCCGGG GAGGACGACC TGTCACAGAT CATGTTCATC 420 TATTTCAACT CCTTCCTGCA GTCG 444

[0110]

[SEQ ID NO: 5] Sequence length: 25 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence ATGAAGAGGA AGAGGCACTT GCG
CA 25

[0111]

[SEQ ID NO: 6] Sequence length: 25 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence CATGGGAGTA GGTCATGACA ATG
GC 25

[0112]

[SEQ ID NO: 7] Sequence length: 30 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence ATGATGGGGC CTTGGTAGAT GTAGTCCACC 30

[0113]

[SEQ ID NO: 8] Sequence length: 30 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence GCTCCTTGCC AAACCAGCAC TGT
TCATTTCT 30

[0114]

[SEQ ID NO: 9] Sequence length: 25 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence GAGGACGACC TGTCACAGAT CAT
GT 25

[0115]

[SEQ ID NO: 10] Sequence length: 30 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence ATTCGCTGTC TGCGGAATGT GATTCACTGG 30

[0116]

[SEQ ID NO: 11] Sequence length: 30 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence CTGGAACCTC ATCACCACCT TTA
TCCT GCG 30

[0117]

[SEQ ID NO: 12] Sequence length: 29 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence GTCGACACGC GGCTGCGGGA CGC
GATGGA 29

[0118]

[SEQ ID NO: 13] Sequence length: 32 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence GTCGACTGTG CAGGTGGGCG ACCGAGGGGT CA 32

[0119]

[SEQ ID NO: 14] Sequence length: 411 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Met Asp Ala Ala Leu Leu His Ser Leu Leu Glu Ala Asn Cys Ser Leu 1 5 10 15 Ala Leu Ala Glu Glu Leu Leu Leu Asp Gly Trp Gly Pro Pro Leu Asp 20 25 30 Pro Glu Gly Pro Tyr Ser Tyr Cys Asn Thr Thr Leu Asp Gln Ile Gly 35 40 45 Thr Cys Trp Pro Arg Ser Ala Ala Gly Ala Leu Val Glu Arg Pro Cys 50 55 60 Pro Glu Tyr Phe Asn Gly Val Lys Tyr Asn Thr Thr Arg Asn Ala Tyr 65 70 75 80 Arg Glu Cys Leu Glu Asn Gly Thr Trp Ala Ser Lys Ile Asn Tyr Ser 85 90 95 Gln Cys Glu Pro Ile Leu Asp Asp Lys Gln Arg Lys Tyr Asp Leu His 100 105 110 Tyr Arg Ile Ala Leu Val Val Asn Tyr Leu Gly His Cys Val Ser Val 115 120 125 Ala Ala Leu Val Ala Ala Phe Leu Leu Phe Leu Ala Leu Arg Ser Ile 130 135 140 Arg Cys Leu Arg Asn Val Ile His Trp Asn Leu Ile Thr Thr Phe Ile 145 150 155 160 Leu Arg Asn Val Met Trp Phe Leu Leu Gln Leu Val Asp His Glu Val 165 170 175 His Glu Ser Asn Glu Val Trp Cys Arg Cys Ile Thr Thr Ile Phe Asn 180 185 190 Tyr Phe Val Val Thr Asn Phe Phe Trp Met Phe Val Glu Gly Cys Tyr 195 200 205 Leu His Thr Ala Ile Val Met Thr Tyr Ser Thr Glu Arg Leu Arg Lys 210 215 220 Cys Leu Phe Leu Phe Ile Gly Trp Cys Ile Pro Phe Pro Ile Ile Val 225 230 235 240 Ala Trp Ala Ile Gly Lys Leu Tyr Tyr Glu Asn Glu Gln Cys Trp Phe 245 250 255 Gly Lys Glu Pro Gly Asp Leu Val Asp Tyr Ile Tyr Gln Gly Pro Ile 260 265 270 Ile Leu Val Leu Leu Ile Asn Phe Val Phe Leu Phe Asn Ile Val Arg 275 280 285 Ile Leu Met Thr Lys Leu Arg Ala Ser Thr Thr Ser Glu Thr Ile Gln 290 295 300 Tyr Arg Lys Ala Val Lys Ala Thr Leu Val Leu Leu Pro Leu Leu Gly 305 310 315 320 Ile Thr Tyr Met Leu Phe Phe Val Asn Pro Gly Glu Asp Asp Leu Ser 325 330 335 Gln Ile Met Phe Ile Tyr Phe Asn Ser Phe Leu Gln Ser Phe Gln Gly 340 345 350 Phe Phe Val Ser Val Phe Tyr Cys Phe Phe Asn Gly Glu Val Arg Ser 355 360 365 Ala Val Arg Lys Arg Trp His Arg Trp Gln Asp His His Ser Leu Arg 370 375 380 Val Pro Met Ala Arg Ala Met Ser Ile Pro Thr Ser Pro Thr Arg Ile 385 390 395 400 Ser Phe His Ser Ile Lys Gln Thr Ala Ala Val 405 410

[0120]

[SEQ ID NO: 15] Sequence length: 1277 Sequence type: Nucleic acid Number of strands: Double-stranded topology: Linear Sequence type: cDNA Characterization method: S sequence ACGCGGCTGC GGGACGCGAT GGACGCGGCA CTGCTCCACA GCCTGCTGGA GGCCAACTGC 60 AGCCTGGCGC TGGCTGAAGA GCTGCTCTTG GACGGCTGGG GGCCACCCCT GGACCCCGAG 120 GGTCCCTACT CCTACTGCAA CACGACCTTG GACCAGATCG GAACGTGCTG GCCCCGCAGC 180 GCTGCCGGAG CCCTCGTGGA GAGGCCGTGC CCCGAGTACT TCAACGGCGT CAAGTACAAC 240 ACGACCCGGA ATGCCTATCG AGAATGCTTG GAGAATGGGA CGTGGGCCTC AAAGATCAAC 300 TACTCACAGT GTGAGCCCAT TTTGGATGAC AAGCAGAGGA AGTATGACCT GCACTACCGC 360 ATCGCCCTTG TCGTCAACTA CCTGGGCCAC TGCGTATCTG TGGCAGCCCT GGTGGCCGCC 420 TTCCTGCTTT TCCTGGCCCT GCGGAGCATT CGCTGTCTGC GGAATGTGAT TCACTGGAAC 480 CTCATCACCA CCTTTATCCT GCGAAATGTC ATGTGGTTCC TGCTGCAGCT CGTTGACCAT 540 GAAGTGCACG AGAGCAATGA GGTCTGGTGC CGCTGCATCA CCACCATCTT CAACTACTTC 600 GTGGTGACCA ACTTCTTCTG GATGTTTGTG GAAGGCTGCT ACCTGCACAC GGCCATTGTC 660 ATGACCTACT CCCGGAGCGTG CAAG TGCCTCTTCC TCTTCATCGG ATGGTGCATC 720 CCCTTCCCCA TCATCGTCGC CTGGGCCATC GGCAAGCTCT ACTATGAGAA TGAACAGTGC 780 TGGTTTGGCA AGGAGCCTGG CGACCTGGTG GACTACATCT ACCAAGGCCC CATCATTCTC 840 GTGCTCCTGA TCAATTTCGT ATTTCTGTTC AACATCGTCA GGATCCTAAT GACAAAGTTA 900 CGCGCGTCCA CCACATCCGA GACAATCCAG TACAGGAAGG CAGTGAAGGC CACCCTGGTG 960 CTCCTGCCCC TCCTGGGCAT CACCTACATG CTCTTCTTCG TCAATCCCGG GGAGGACGAC 1020 CTGTCACAGA TCATGTTCAT CTATTTCAAC TCCTTCCTGC AGTCGTTCCA GGGTTTCTTC 1080 GTGTCTGTCT TCTACTGCTT CTTCAATGGA GAGGTGCGCT CAGCCGTGAG GAAGAGGTGG 1140 CACCGCTGGC AGGACCATCA CTCCCTTCGA GTCCCCATGG CCCGGGCCAT GTCCATCCCT 1200 ACATCACCCA CACGGATCAG CTTCCACAGC ATCAAGCAGA CGGCCGCTGT GTGACCCCTC 1260 GGTCGCCCAC CTGCACA 1277

[0121]

[Brief description of drawings]

FIG. 1 is a 5 ′ having a nucleotide sequence represented by SEQ ID NO: 1.
Of the synthetic DNA primer (TM3-C) on the side and cDNA encoding another G protein-coupled receptor protein
It is the figure which showed the commonality with A or the base sequence of a gene.

FIG. 2 is 3 ′ having the nucleotide sequence represented by SEQ ID NO: 2.
FIG. 3 is a diagram showing the commonality between the nucleotide sequence complementary to the synthetic DNA primer (TM7-D) on the side and the nucleotide sequence of a cDNA or gene encoding another G protein-coupled receptor protein.

FIG. 3 shows the nucleotide sequence of the human CRF ₂ receptor cDNA fragment of the present invention and the amino acid sequence encoded thereby. The underlined parts at the 5'end and 3'end of the base sequence correspond to the synthetic primers used for PCR amplification.

FIG. 4 was prepared based on the amino acid sequence shown in FIG.
1 shows a hydrophobicity plot of a protein encoded by a human CRF ₂ receptor protein cDNA fragment. From this figure TM3
The presence of the hydrophobic domain shown by ~ TM7 is clear.

FIG. 5: Human CRF ₂ receptor cDNA shown in FIG.
Partial amino acid sequence of the protein encoded by the fragment (phs
-AH1) is a diagram comparing the known rat CRF ₂ receptor protein (RNU16253). The shaded areas indicate the matching amino acid residues. The underlined part is
The primer portion used in PCR is shown.

FIG. 6 shows the nucleotide sequence of the human CRF ₂ receptor cDNA fragment of the present invention (-18 to +630 when A of the start codon ATG is +1) and the amino acid sequence encoded thereby.

FIG. 7 shows the nucleotide sequence of the human CRF ₂ receptor cDNA fragment of the present invention (+631 to +1259 when A of the start codon ATG is +1) and the amino acid sequence encoded thereby.

FIG. 8 shows a hydrophobicity plot of human CRF ₂ receptor protein prepared based on the amino acid sequences shown in FIGS. 6 and 7. From this figure, the existence of the hydrophobic domains TM1 to TM7 is clear.

FIG. 9 shows the amino acid sequences shown in FIG. 6 and FIG. 7 in the known rat CRF ₂ receptor protein (RNU1625).
The figure compared with 3) is shown. The black portions indicate that the amino acid residues are the same in both.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification number Internal reference number FI Technical indication C12N 1/21 C12N 1/21 C12P 21/02 C12P 21/02 C C12Q 1/68 9453-4B C12Q 1/68 Z G01N 33/566 G01N 33/566 // A61K 38/00 AAM A61K 37/02 AAM ABU ABU ADD ADD 38/04 AAT 37/43 AAT ACJ ACJ (C12N 1/21 C12R 1:19) (C12P 21/02 C12R 1:19)

Claims (23)

[Claims] 1. A DNA having a base sequence substantially the same as the base sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2. 2. The DNA according to claim 1, which is a DNA primer used for polymerase chain reaction of DNA encoding a G protein-coupled receptor protein. 3. A DNA encoding a G protein-coupled receptor protein as a template is mixed with the DNA according to claim 1 to carry out a polymerase chain reaction, which encodes the G protein-coupled receptor protein. A method for amplifying DNA. 4. A G protein-coupled receptor protein from a DNA library containing a DNA encoding a G protein-coupled receptor protein, characterized in that the DNA according to claim 1 is used as a DNA primer in a polymerase chain reaction. A method for screening a DNA encoding the protein. 5. A DN encoding a G protein-coupled receptor protein obtained by the screening method according to claim 4.
A. 6. A G protein-coupled receptor protein encoded by the DNA according to claim 5, a partial peptide thereof or a salt thereof. 7. A human CRF ₂ receptor protein or a salt thereof, which contains an amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 3. 8. A human CRF ₂ receptor protein or a salt thereof, which contains an amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 14. 9. The human CR according to claim 7 or 8.
A partial peptide of F ₂ receptor protein or a salt thereof. 10. The human C according to claim 7 or claim 8.
A DNA containing a DNA having a nucleotide sequence encoding an RF ₂ receptor protein. 11. The DNA according to claim 10, which has the base sequence represented by SEQ ID NO: 4. 12. The DNA according to claim 10, which has the nucleotide sequence represented by SEQ ID NO: 15. 13. A vector containing the DNA according to claim 10. 14. A transformant carrying the vector according to claim 13. 15. The transformant according to claim 14 is cultured,
Claim 7 or claim 8 human CRF ₂ receptor protein or a salt thereof as claimed, characterized in that allowed to produce a human CRF ₂ receptor protein in the cell membrane of the transformant. 16. The human C according to claim 7 or 8.
An RF ₂ receptor protein or a salt thereof or claim 9.
A method for screening an agonist or a salt thereof that activates a CRF ₂ receptor, or an antagonist or a salt thereof that competitively inhibits the binding between the CRF ₂ receptor and CRF, characterized by using the partial peptide or the salt thereof. (17) A case in which a ligand is brought into contact with the human CRF ₂ receptor protein or a salt thereof according to claim 7 or 8, or a partial peptide or a salt thereof according to claim 9, and (ii). 7. A ligand characterized in that the human CRF ₂ receptor protein or salt thereof according to claim 8 or the salt thereof or the partial peptide or salt thereof according to claim 9 is contacted with a ligand and a test compound. A method for screening a compound or a salt thereof which inhibits the binding to the human CRF ₂ receptor protein according to claim 7 or 8. 18. The human C according to claim 7 or claim 8.
An RF ₂ receptor protein or a salt thereof or claim 9.
A kit for screening a compound or a salt thereof which inhibits the binding between the ligand and the human CRF ₂ receptor protein according to claim 7 or 8, characterized by containing the partial peptide or the salt thereof. 19. A human CRF ₂ receptor agonist or a salt thereof obtained by using the screening method according to claim 16 or 17, or the screening kit according to claim 18. 20. A human CRF ₂ receptor antagonist or a salt thereof obtained by using the screening method according to claim 16 or 17, or the screening kit according to claim 18. 21. A preventive / therapeutic agent for dementia and obesity, which comprises the human CRF ₂ receptor agonist or a salt thereof according to claim 19, an agent for promoting adaptation to stress, ACTH, β-endorphin, β -Lipotropin or α-MSF secretagogues, antihypertensive agents, mood and behavior regulators, gastrointestinal function regulators, autonomic nervous system regulators, or pituitary, cardiovascular, gastrointestinal or central nervous system functions Test drug. 22. Depression / anxiety / headache caused by stress, inflammatory disease, which comprises the human CRF ₂ receptor antagonist or the salt thereof according to claim 20.
A prophylactic / therapeutic agent for immunosuppression, AIDS, Alzheimer's disease, gastrointestinal disorders, anorexia, bleeding stress, drug / alcohol withdrawal symptoms, drug dependence, reproductive disorders, Cushing's disease or hypotension. 23. Human C according to claim 7 or claim 8.
An RF ₂ receptor protein or a salt thereof or claim 9.
An antibody against the described partial peptide or a salt thereof.