JP2001211885A - New polypeptide - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new G protein-conjugated receptor polypeptide, and to provide a method of screening ligand, agonist, antagonist or a function-modifying substance against the polypeptide. SOLUTION: A DNA encoding a new polypeptide having a high homology to a known G protein-conjugated receptor can be obtained by randomly sequencing a cDNA clone selected from a cDNA library derived from the human hypophysis. A system or kit for screening ligand, agonist, antagonist or function- modifying substance against the polypeptide, a compound obtained by using the system or kit for screening, and an antibody against the polypeptide can be obtained by using (a part of) a polypeptide encoded by the DNA. A non-human animal from which the DNA is deleted can be obtained by using the DNA.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a novel G-protein-receptor polypeptide derived from human pituitary gland, a partial peptide of the polypeptide, a DNA encoding the polypeptide or the partial peptide, and a DNA incorporating the same. Recombinant vector, transformant carrying the recombinant vector, a method for producing the polypeptide or the partial peptide, an antibody recognizing the polypeptide, and a method for producing the polypeptide using the polypeptide or the partial peptide A method for screening a ligand, agonist, antagonist or function-modifying substance, a kit for screening a ligand, agonist, antagonist or function-modifying substance of the polypeptide containing the polypeptide or the partial peptide, and a method for screening or a kit for screening Compound Or a pharmaceutically acceptable salt thereof, deficiency or to some modified animals and their usage a gene encoding the polypeptide.

[0002]

2. Description of the Related Art Many hormones and neurotransmitters regulate the functions of living organisms through specific receptors present on cell membranes. Many of these receptors are collectively referred to as G protein-coupled receptors (hereinafter sometimes abbreviated as GPCRs). GPCR is a heterotrimeric G protein (guan
conjugated with ine nucleotide-binding protein) and transmits signals into cells through activation of G protein. GPC
Since R has seven transmembrane regions, it is also called a seven transmembrane receptor.

[0003] GPCRs are present on the surface of various functional cells of living cells and organs, and function as receptors for molecules that regulate the functions of living cells and organs, such as hormones, neurotransmitters and physiologically active substances. Plays a very important role physiologically or pathologically. GPCRs are
It also functions as a receptor for light, taste substances, and odor substances. As specific ligands for GPCRs, a wide variety of proteins, peptides, biogenic amines, lipid mediators, photons, calcium, sugars, nucleic acids and the like are known.

[0004] GPCRs are very good targets for drug discovery, and natural ligands, agonists or antagonists of GPCRs have been used as drugs so far. Approximately 60% of currently marketed drugs target GPCRs. GPCRs also cause genetic diseases and are important targets in the diagnosis and treatment of genetic diseases [Trends in Pharmacological Science, 18 , 430
(1984)].

[0005] Therefore, obtaining a new GPCR and analyzing its function provides a very useful means for developing a drug closely related to its function. For example, in the central nervous system organs such as the brain, the physiological functions of the brain are regulated by many hormones, hormone-like substances, neurotransmitters or biologically active substances, but unknown in the brain. It is thought that hormones, hormone-like substances, neurotransmitters or bioactive substances are present. It is known that the physiological functions of organs such as the stomach, intestine, and pancreas are also regulated by many hormones, hormone-like substances, neurotransmitters or bioactive substances,
It is thought that unknown hormones, hormone-like substances, neurotransmitters or biologically active substances exist in organs such as the stomach, intestine and pancreas. In organs such as the brain, stomach, intestine, and pancreas, receptors (eg, GPC) corresponding to the above hormones, hormone-like substances, neurotransmitters or bioactive substances
R) is also known to be present, but it is likely that unknown receptors (eg, GPCRs) also exist in these organs. New subtypes may also exist for known GPCRs.

If a novel GPCR gene expressed in the brain, stomach, intestine, and pancreas can be obtained, the amino acid sequence of the GPCR can be compared with the amino acid sequence of a known GPCR, and the expression distribution of the transcript of the GPCR gene can be determined. By investigating, the function of the GPCR can be estimated and useful information for drug development can be obtained. In addition, if a new GPCR gene can be obtained, a natural ligand, agonist or antagonist for the GPCR can be efficiently screened. The natural ligand, agonist or antagonist is expected as a pharmaceutical.

[0007] The anterior pituitary gland secretes various hormones that regulate the functions of living organisms. Secretion of these hormones
It is well known that various ligands released from the hypothalamus are controlled by acting on corresponding GPCRs present on anterior pituitary cells. Therefore, the pituitary gland may have an unknown GPCR involved in the regulation of secretion of a known or novel bioactive substance.

On the other hand, in recent years, as a means for analyzing genes expressed in a living body, the sequence of each cDNA clone in a cDNA library has been randomly analyzed to obtain a novel sequence which has not been known until now. Methods for searching for DNA have been implemented.

[0009]

If a G protein-coupled receptor polypeptide and a DNA encoding the polypeptide, which have not been known, can be obtained, a ligand, an agonist, an antagonist or a function modifying substance of the polypeptide may be obtained. Screening becomes possible, and substances obtained by the screening are useful as pharmaceuticals.

[0010] The present invention provides a novel G protein-coupled receptor polypeptide derived from human pituitary gland or a partial peptide thereof,
DN encoding the polypeptide or the partial peptide
A, a recombinant vector containing the DNA, a transformant harboring the recombinant vector, a method for producing the polypeptide or the partial peptide, a method for screening a ligand, agonist, antagonist or functional modifier for the polypeptide and Screening kit, screening method, and ligand, agonist, antagonist or function-modifying substance obtained by using the screening kit, medicine containing the ligand, agonist, antagonist or function-modifying substance, and polypeptide or partial peptide thereof The purpose is to provide antibodies and the like.

[0011]

Means for Solving the Problems As a result of intensive studies, the present inventors have isolated a cDNA encoding a novel G protein-coupled receptor polypeptide from a human pituitary-derived cDNA library. Successful analysis of the entire nucleotide sequence has led to the completion of the present invention.

The present invention relates to the following (1) to (31). (1) A G protein-coupled receptor polypeptide having the amino acid sequence of SEQ ID NO: 1. (2) a polypeptide having an amino acid sequence in which one or more amino acids have been deleted, substituted or added in the amino acid sequence of the polypeptide described in SEQ ID NO: 1, and substantially the same as the polypeptide described in (1) A polypeptide having the same activity as (3) A partial peptide which is a partial peptide of the G protein-coupled receptor polypeptide according to (1) or (2), and has a binding ability to a ligand, agonist, antagonist or functional modifier of the polypeptide. (4) A DNA encoding the G protein-coupled receptor polypeptide according to (1) or (2). (5) A DNA encoding a partial peptide of the G protein-coupled receptor polypeptide according to (3). (6) a DNA having a base sequence represented by base numbers 58 to 1140 in the DNA of SEQ ID NO: 2
A. (7) The DNA according to any one of (4) to (6)
A DNA that hybridizes with a DNA selected from the group consisting of the above under stringent conditions and encodes a polypeptide having substantially the same activity as the G protein-coupled receptor polypeptide according to (1). (8) The DNA according to any one of (4) to (7)
A recombinant DNA obtained by incorporating a DNA selected from the above into a vector. (9) A transformed cell, transformed plant or transformed non-human animal having the recombinant DNA according to (8). (10) Using the transformed cell, transformed plant or transformed non-human animal according to (9), [1] culturing the transformed cell in a medium, and [2] performing the transformation in the culture. Cultivating a plant and [3] breeding the transformed non-human animal to produce the polypeptide according to (1) or (2) or the partial peptide according to (3) in the animal; Collecting the polypeptide or the partial peptide from the culture, the plant or the animal, and collecting the polypeptide or the peptide according to (1) or (2). Production method. (11) the polypeptide according to (1) or (2),
Or an antibody that recognizes the partial peptide according to (3). (12) A method for immunologically quantifying the polypeptide according to (1) or (2) or the partial peptide according to (3), wherein the antibody according to (11) is used. (13) A method for diagnosing cancer or pituitary dysfunction using the quantification method according to (12). (14) A method for diagnosing cancer or pituitary dysfunction by measuring the amount of mRNA encoding the polypeptide according to (1) or (2). (15) A method for diagnosing cancer or pituitary dysfunction by detecting deletion, substitution or insertion of the gene encoding the polypeptide according to (1) or (2). (1
6) The polypeptide according to (1) or (2) is brought into contact with the polypeptide according to (1) or (2), or the partial peptide according to (3), and the test sample. (1) or (2), wherein an agonist, an antagonist or a function modifier is selected.
A method for screening an antagonist or a functional modifier. (17) [i] When the polypeptide according to (1) or (2) or the partial peptide according to (3) is contacted with a ligand, and [ii] according to (1) or (2). Or the partial peptide described in (3) is brought into contact with the ligand and the test sample, and the test sample is compared with the agonist, antagonist, or antagonist of the polypeptide described in (1) or (2). A method for screening a polypeptide agonist, antagonist or function-modifying substance according to (1) or (2), which comprises selecting a function-modifying substance. (18) the polypeptide according to (1) or (2),
Or a kit for screening a ligand, agonist, antagonist or function-modifying substance of the polypeptide according to (1) or (2), which comprises the partial peptide according to (3). (19) The ligand, agonist, or antagonist of the polypeptide according to (1) or (2), which is obtained by using the screening method according to (16) or (17), or the screening kit according to (18). Or a function modifier, or a pharmacologically acceptable salt thereof. (20) A cancer or pituitary dysfunction containing a substance selected from the antibody according to (11), the ligand, agonist, antagonist and function-modifying substance according to (19) or a pharmacologically acceptable salt thereof. Remedies for the disease. (21) [i] A cell that expresses the polypeptide according to (1) or (2) is brought into contact with a cell that expresses the polypeptide according to (ii) or (2) and a test sample. (1) or (2) from the test sample
A method for screening a compound that changes the expression level of a DNA encoding the polypeptide according to (1) or (2), which comprises selecting a compound that alters the expression of the gene encoding the polypeptide according to (1). . (22) The method according to (22), wherein the variation in the expression level is measured by the method described in (12) or the method for quantifying the amount of mRNA encoding the polypeptide according to (1) or (2). ). (23) A test sample is contacted with a transformant containing a reporter gene-linked DNA downstream of a region that controls transcription of the gene encoding the polypeptide according to (1) or (2). Selecting from the sample a compound that changes the expression level of the DNA encoding the polypeptide according to (1) or (2),
A method for screening a compound that changes the expression level of a gene encoding the polypeptide according to (1) or (2). (24) A compound obtained by a method selected from the screening method according to any one of (21) to (23) or a pharmacologically acceptable salt thereof. (25) An agent for treating cancer or pituitary abnormality, comprising the compound according to (24) or a pharmacologically acceptable salt thereof. (26) an oligonucleotide having the same sequence as 5 to 60 consecutive bases in the base sequence of a DNA selected from the DNA of (4) and (6) and the DNA of SEQ ID NO: 2, complementary to the oligonucleotide; Selected from oligonucleotides having specific sequences and derivative oligonucleotides of these oligonucleotides. (27) Any one of (4) to (6) and (26)
A method for suppressing the transcription or translation of mRNA encoding the polypeptide of (1) or (2), using a DNA selected from the DNAs described in (1). (28) All or part of a gene containing a DNA encoding the polypeptide according to (1) or (2) is deleted or replaced, and the expression level of the polypeptide according to (1) or (2) is changed A non-human animal with a deleted or substituted gene. (29) A cancer characterized by contacting the animal according to (28) or an organ, tissue or cell of the animal with a test sample and selecting a therapeutic agent for cancer or pituitary disorder from the test sample. Or a method for screening or evaluating a therapeutic drug for pituitary disorders. (30) A compound or a pharmacologically acceptable salt thereof obtained by the screening method according to (29). (31) An agent for treating cancer or pituitary disorder, comprising the compound according to (30) or a pharmacologically acceptable salt thereof.

[0013]

BEST MODE FOR CARRYING OUT THE INVENTION (1) G protein-coupled receptor polypeptide of the present invention or a partial peptide thereof The polypeptide of the present invention is a G protein-coupled receptor polypeptide, for example, represented by SEQ ID NO: 1. Or a polypeptide having an amino acid sequence in which one or more amino acids have been deleted, substituted or inserted in the amino acid sequence of the polypeptide, and having the amino acid sequence represented by SEQ ID NO: 1. A polypeptide having substantially the same activity as the polypeptide can be mentioned.

The polypeptide of the present invention can be used, for example, in all cells (eg, spleen cells, neurons, etc.) of humans and mammals (eg, guinea pig, rat, mouse, chicken, rabbit, pig, sheep, cow, monkey, etc.). Glial cells, pancreatic β cells, bone marrow cells, mesangial cells, Langerhans cells, epidermal cells, epithelial cells, endothelial cells, fibroblasts,
Fiber cells, muscle cells, fat cells, immune cells (eg, macrophages, T cells, B cells, natural killer cells, mast cells, neutrophils, basophils, eosinophils, monocytes), megakaryocytes, synovium Cells, chondrocytes, bone cells, osteoblasts, osteoclasts, mammary cells, hepatocytes or stromal cells, or precursors of these cells, stem cells or cancer cells), or any tissue in which those cells are present, For example, the brain, various parts of the brain (eg, olfactory bulb, squamous nucleus, basal sphere, hippocampus, thalamus, hypothalamus, hypothalamus, cerebral cortex, medulla, cerebellum, occipital lobe, frontal lobe, temporal lobe, putamen , Caudate nucleus, brain stain, substantia nigra), spinal cord, pituitary, stomach, pancreas, kidney, liver, gonad, thyroid, gall bladder,
Bone marrow, adrenal gland, skin, muscle, lung, digestive tract, blood vessels, heart, thymus, spleen, submandibular gland, peripheral blood, peripheral blood cells, intestinal tract, prostate,
It may be a polypeptide derived from the testis, testis, ovary, placenta, uterus, bone, joint, small intestine, large intestine, skeletal muscle and the like (particularly, brain and various parts of the brain). Further, the polypeptide of the present invention may be a polypeptide synthesized by chemical synthesis.

The above-mentioned deletion, substitution or addition of one or more amino acids means molecular cloning, A laboratory ma
nual, Second Edition. (1989) (hereinafter molecular
Cloning 2nd edition), Current Protocols in M
olecular Biology, John and Wily & Sons (1987-1997)
(Hereinafter abbreviated as Current Protocols in Molecular Biology), Nucleic Acids Research,
10 , 6487 (1982), Proc. Natl. Acad. Sci. USA, 79 , 6
409 (1982), Gene, 34 , 315 (1985), NucleicAcids Res
earch, 13 , 4431 (1985), Proc. Natl. Acad. Sci. US
A, D, which has a base sequence in which a sufficient number of bases have been deleted, substituted or added to DNA by the site-directed mutagenesis method described in A, 82 , 488 (1985), etc.
It means deletion, substitution or addition of amino acids of the polypeptide encoded by NA, for example, deletion of any number of amino acids of about 1 to 20, preferably about 1 to 15, more preferably 1 to 5. Lost, substituted or added amino acid sequences can be mentioned. In addition, a polypeptide having an amino acid sequence in which the amino acid has been deleted, substituted or added has substantially the same activity as the polypeptide described in SEQ ID NO: 1 in BLAST [J. Mol. Biol.,
215 , 403 (1990)), FASTA (Method in Enzymology, 18
3 , 63 (1990)], when calculated using analysis software such as the FrameSearch method (manufactured by Compugen), the amino acid sequence represented by SEQ ID NO: 1 is about 70% or more, preferably about 80% or more.
% Or more, more preferably about 90% or more.

The above-mentioned substantially identical activities include, for example, a ligand binding activity and a signal information transmitting activity of the peptide represented by the amino acid sequence of SEQ ID NO: 1. Substantially identical indicates that their activities are identical in nature. Therefore, the quantitative factors such as the ligand binding activity, the degree of signal transduction, and the molecular weight of the protein may be different.

More specifically, examples of the polypeptide of the present invention include a human pituitary-derived G protein-coupled receptor polypeptide having the amino acid sequence represented by SEQ ID NO: 1.

Further, in the polypeptide of the present invention, the amino group of the N-terminal methionine residue in the polypeptide may be a protecting group (for example, C-formyl group, acetyl group, etc.).
1-6 acyl group), a glutamyl group formed by cleavage of the N-terminal side in vivo and a pyroglutamate, a substituent on the side chain of an amino acid in the molecule (for example, -OH, -COOH, amino group, imidazole group,
Those in which an indole group, a guanidino group, etc.) are protected with a suitable protecting group (eg, a C1-6 acyl group such as a formyl group, an acetyl group, etc.), or complex proteins such as so-called glycoproteins to which sugar chains are bonded. Is also included. Furthermore, the polypeptide of the present invention has an amide (-CO
NH ₂ ) or an ester (—COOR). Here, R of the ester group is methyl, ethyl,
C1-6 alkyl group such as n-propyl, isopropyl or n-butyl, for example, C3-8 cycloalkyl group such as cyclopentyl, cyclohexyl, for example, C6-12 aryl group such as phenyl, α-naphthyl, etc. Benzyl, C7-14 aralkyl group such as phenyl-C1-2 alkyl group such as phenethyl or α-naphthyl-C1-2 alkyl group such as α-naphthylmethyl,
Pivaloyloxymethyl ester commonly used as an oral ester may be used. When the polypeptide of the present invention has a carboxyl group (or carboxylate) other than at the C-terminus, those in which the carboxyl group is amidated or esterified are also included in the polypeptide of the present invention. As the ester at this time, for example, the above C
Examples include terminal esters.

As the salt of the polypeptide of the present invention, a physiologically acceptable acid addition salt is particularly preferable. Such salts include, for example, inorganic acids (eg, hydrochloric acid, phosphoric acid,
Salts with hydrobromic acid, sulfuric acid, or organic acids (for example,
Examples thereof include salts with acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, succinic acid, tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, and benzenesulfonic acid.

The partial peptide of the present invention is a partial peptide of the polypeptide of the present invention and a partial peptide capable of binding to the ligand, agonist, antagonist or functional modifier of the polypeptide of the present invention. G
It is known that the ligand binding region of PCR is an extracellular region, a transmembrane region, or both an extracellular region and a transmembrane region (Current Opinion of Cell Biology,
6 , 191 (1994), EMBO J., 18 , 1723 (1999)]. Therefore, as a partial peptide capable of binding to the ligand, agonist, antagonist or functional modifier of the polypeptide, for example, in a cell expressing the polypeptide, the partial peptide is exposed outside the cell membrane. Portion (extracellular region) or a partial peptide containing a membrane-bound region. Further, the partial peptide of the present invention may be a peptide individually containing each domain or a partial peptide containing a plurality of domains simultaneously. The membrane binding region of any GPCR may be a known GPCR
EMBO J.,
12 , 1693, (1993)]. Therefore, the extracellular region and intracellular region of any GPCR can be predicted based on the membrane-bound region predicted by the method. In addition, it is also possible to perform hydropathy analysis (using the analysis software MacMolly 3.5 purchased from Aloka) or membrane binding region prediction analysis (using the analysis software SOSUI system ver1.0 / 10 purchased from Mitsui Information Development). Can predict the membrane-bound, extracellular, and intracellular regions of GPCRs. Therefore, by performing the above analysis on the polypeptide having the amino acid sequence represented by SEQ ID NO: 1, specific extracellular regions (hydrophilic regions), membrane-bound regions (hydrophobic regions), and intracellular regions (hydrophilic regions) Region) can be predicted. Specifically, for example, as the partial peptide containing the extracellular region, the first to 40th and 98th to 111th amino acids of the amino acid sequence represented by SEQ ID NO: 1
And partial peptides having the 179th to 205th amino acids or the 288th to 300th amino acid sequences. In addition, the partial peptide containing the membrane-binding region portion includes the 41st to 66th, 72nd to 97th, 2112th to 137th, and 153rd to 153rd amino acids of the amino acid sequence represented by SEQ ID NO: 1. Partial peptides having the 178th, 206th to 231rd, 262nd to 287th, or 301st to 326th amino acid sequences can be mentioned.

Furthermore, in the partial peptide of the present invention, the amino group of the N-terminal methionine residue in the above partial peptide is protected with a protecting group (for example, a C1-6 acyl group such as a formyl group or an acetyl group). Those in which the glutamyl group formed by cleavage of the N-terminal side in vivo is pyroglutaminated; substituents on the side chains of amino acids in the molecule (for example, -OH, -COOH, amino groups, imidazole groups, Those in which an indole group, a guanidino group, etc.) are protected with a suitable protecting group (eg, a C1-6 acyl group such as a formyl group, an acetyl group, etc.), or a complex peptide such as a so-called glycopeptide having a sugar chain bonded thereto, etc. Is also included. Furthermore, the partial peptide of the present invention has a carboxyl group (-COOH) or a carboxylate at the C-terminal.
(—COO—), and the C-terminus may be an amide or an ester as in the polypeptide of the present invention described above. When the partial peptide of the present invention has a carboxyl group (or carboxylate) other than the C-terminus, those in which the carboxyl group is amidated or esterified are also included in the partial peptide of the present invention. Examples of the ester at this time include the above-mentioned C-terminal ester and the like. As the salt of the partial peptide of the present invention, a physiologically acceptable acid addition salt is particularly preferable. Such salts include, for example, inorganic acids (eg, hydrochloric acid, phosphoric acid,
Salts with hydrobromic acid, sulfuric acid, or organic acids (for example,
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. (2) DNA encoding the G protein-coupled receptor polypeptide of the present invention or a partial peptide thereof The DNA encoding the polypeptide of the present invention may be the DNA encoding the polypeptide of the present invention described in (1). Any DNA may be used, but specifically, for example,
(A) base number 5 in the base sequence of SEQ ID NO: 2
DNA having a nucleotide sequence represented by Nos. 8 to 1140,
(B) a DNA having a nucleotide sequence in which one or more bases have been deleted, substituted or added in the nucleotide sequence of the DNA described in (a), and substantially the same as the polypeptide having the amino acid sequence of SEQ ID NO: 1 (C) a DNA encoding the polypeptide having the same activity, or a DNA having the amino acid sequence of SEQ ID NO: 1 which hybridizes under stringent conditions with the DNA of (a) or (b). DNA encoding a polypeptide having substantially the same activity as the peptide.

In the above description, a DNA having a base sequence in which one or more bases have been deleted, substituted or added means a DNA in which a sufficient number of bases have been deleted, substituted or added by a known method.

The DNA which hybridizes under the above-mentioned stringent conditions and encodes a polypeptide having substantially the same activity as the polypeptide having the amino acid sequence of SEQ ID NO: 1 D described in
A DNA obtained by using a colony hybridization method, a plaque hybridization method, a Southern blot hybridization method, or the like with NA as a probe. After hybridization at 42-65 ° C. in the presence of 0.7-1.0 mol / L NaCl, and then 0.1- to 2-fold concentration of SSC (saline-sodium citrat).
e) Solution (The composition of a 1-fold concentration SSC solution is 150 mmol
/ L sodium chloride and 15 mmol / L sodium citrate) and washing the filter under the conditions of 42 to 65 ° C. Hybridization is performed using Molecular Cloning 2nd Edition, Current Protocols in
Molecular biology, DNA Cloning 1: Core Te
chniques, A Practical Approach, Second Edition, Oxf
It can be carried out according to the method described in an experimental book such as ord University Press (1995). Specifically, the hybridizable DNA has at least 60% homology with the DNA described in (a) or (b) above when calculated using analysis software such as BLAST or FASTA. DNAs, preferably DNAs having a homology of 80% or more, more preferably DNAs having a homology of 95% or more.

DNA encoding the partial peptide of the present invention
Any DNA may be used as long as it has a base sequence encoding the partial peptide of the present invention described in (1) above, and specifically, for example, (a) described in SEQ ID NO: 2 Nucleotide numbers 58 to 11 in the nucleotide sequence of
DNA having a partial nucleotide sequence selected from the nucleotide sequence represented by No. 40, (b) a nucleotide sequence represented by SEQ ID NO: 2,
A DNA having a base sequence in which one or more bases are deleted, substituted or added in the base sequence represented by base numbers 58 to 1140, and substantially the same as the polypeptide having the amino acid sequence of SEQ ID NO: 1 Hybridizes under stringent conditions with a DNA having a partial nucleotide sequence selected from the nucleotide sequence of a DNA encoding a polypeptide having the same activity as described in (c), (a) or (b), and And a DNA having a partial nucleotide sequence selected from the nucleotide sequence of a DNA encoding a polypeptide having substantially the same activity as the polypeptide having the amino acid sequence of SEQ ID NO: 1.

DNA encoding the partial peptide of the present invention
Is a ligand, an agonist of the polypeptide of the present invention,
DNA encoding a partial peptide of the polypeptide of the present invention, which is capable of binding to an antagonist or a function-modifying substance, for example, a portion containing an extracellular region or a membrane-bound region predicted by the method described in (1) above. Examples include DNA encoding a peptide. Specifically, the 1st to 40th amino acids of the amino acid sequence represented by SEQ ID NO: 1
Th, 98th to 111th, 179th to 2nd
DNA encoding a partial peptide having an amino acid sequence at position 05 or at position 288 to position 300;
Nos. 58 to 17 of the nucleotide sequence represented by SEQ ID NO: 2
7th, 349th to 390th, 592th to
DNAs having the 672nd or 919th to 957th nucleotide sequences can be mentioned. In addition, the 41st to 66th amino acids of the amino acid sequence represented by SEQ ID NO: 1
, 72nd to 97th, 2112th to 1st
A sequence encoding a partial peptide having an amino acid sequence at position 37, position 153 to position 178, position 206 to position 231, position 262 to position 287, or position 301 to position 326; Nos. 178-255 of the nucleotide sequence represented by No. 2,
71st to 348th, 391st to 468th, 514th to 591st, 673th to 7th
50th, 841th to 918th or 958th
And DNA having the base sequence from the 1st to the 1035th. The DNA encoding the partial peptide of the present invention may be a DNA individually encoding each partial peptide, or may be a DNA containing a plurality of DNAs encoding each partial peptide at the same time.

(3) Obtaining the DNA encoding the polypeptide of the present invention, and producing the DNA and the oligonucleotide The DNA encoding the polypeptide of the present invention is, for example,
All cells of humans and mammals (eg, guinea pig, rat, mouse, chicken, rabbit, pig, sheep, cow, monkey, etc.) (eg, spleen cells, nerve cells, glial cells, pancreatic β cells, bone marrow cells, mesangial cells) , Langerhans cells, epidermal cells, epithelial cells, endothelial cells, fibroblasts, fibrocytes, muscle cells, adipocytes, immune cells (eg, macrophages, T cells, B cells, natural killer cells, mast cells, neutrophils, Basophils, eosinophils, monocytes), megakaryocytes, synovial cells, chondrocytes, osteocytes, osteoblasts, osteoclasts, mammary cells, hepatocytes or stromal cells, or precursor cells of these cells, Stem cells or cancer cells), or any tissue in which these cells are present, for example, the brain, various parts of the brain (eg, olfactory bulb, nucleus pulposus, basal sphere, hippocampus, thalamus, hypothalamus, thalamus Nucleus, cerebral cortex, medulla, cerebellum, occipital lobe, frontal lobe, temporal lobe, putamen, caudate nucleus, brain stain, substantia nigra), spinal cord, pituitary, stomach, pancreas, kidney, liver, gonad, thyroid, gall bladder , Bone marrow, adrenal gland, skin, muscle, lungs,
Gastrointestinal tract, blood vessels, heart, thymus, spleen, submandibular gland, peripheral blood, peripheral blood cells, intestinal tract, prostate, testes, testis, ovaries, placenta, uterus, bones, joints, small intestine, large intestine, skeletal muscle, etc. Genomic DNA, genomic DNA library, cDNA derived from the above cells or tissues, or c
Each genomic DNA selected from a DNA library etc.
Alternatively, it can be obtained by randomly sequencing cDNA.

The preparation of a genomic DNA and the preparation of a genomic DNA library can be carried out, for example, using the above-mentioned various cells, organs or tissues according to a conventional method. Specific methods include Molecular Cloning 2nd Edition and Current Cloning
Examples include the methods described in Protocols in Molecular Biology.

The cDNA can be prepared by a conventional method using, for example, mRNA derived from the above various cells, organs or tissues. Specifically, Molecular Cloning 2nd Edition, Current Protocols in Molecular Biology, DNA Cloning 1: Core Techniq
ues, A Practical Approach, Second Edition, OxfordU
niversity Press (1995) etc., full-length c
DNA preparation method [Methods in Enzymology, 303 , 19 (199
9), Gene, 138 , 171 (1994)] or a commercially available kit, for example, SuperScript Plasmid System for cDNA Synthesis and Plasmid Cloning [SuperScript Plasmid System for cDN
A Synthesis and Plasmid Cloning; Gibco BRL (Gib
co BRL)] and Zap-cDNA synthesis kit [ZAP-cDNA Synthesis Kit, manufactured by Stratagene]
It can be manufactured using.

As a cloning vector for preparing a library, any phage vector, plasmid vector, or the like can be used as long as it can replicate autonomously in E. coli K12 strain. Specifically, ZAP Express
(Strategies, 5 , 58 (1992), manufactured by Stratagene)
pBluescript II SK (+) (Nucleic Acids Research, 17 , 9
494 (1989)], λzap II (manufactured by Stratagene),
λgt10, λgt11 [DNA Cloning, A Practical
Approach, 1 , 49 (1985)], λTriplEx (Clontech), λExCell (Pharmacia), pT7T
318U (Pharmacia), pcD2 [Mol. Cell. Bi
ol., 3 , 280 (1983)], pUC18 [Gene, 33 , 103
(1985)], pAMo [J. Biol. Chem., 268 , 22782 (19
93), also known as pAMoPRC3Sc (JP-A-05-336963), pAMo
-D (see Example 1) and the like.

[0030] As the host microorganism used for preparing the library, any microorganism belonging to Escherichia coli can be used. Specifically, Escherichia coli XL1-B
lueMRF '[Stratagies, 5 , 81 (19
92)), Escherichia coli C600 [Genetics, 39 , 440 (1
954)), Escherichia coli Y1088 (Science, 222 , 778
(1983)), Escherichia coli Y1090 (Science, 222 , 7
78 (1983)), Escherichia coli NM522 (J. Mol. Bio
l., 166 , 1 (1983)], Escherichia coli K802 (J. Mo.
l. Biol., 16 , 118 (1966)], Escherichia coli JM10
5 (Gene, 38 , 275 (1985)), Escherichia coli SOLR
^TM Strain (commercially available from Stratagene) and E. coli
LE392 (Molecular Cloning 2nd Edition) or the like can be used.

A more specific method for preparing a cDNA library is as follows.

From mRNA derived from human pituitary gland (Clontech), a cDNA synthesis system (cDNA Synthes
is System, manufactured by GIBCO BRL), and Eco RI- Not I- Sal I adapt at both ends.
or (SuperScript Choice System for cDNA Synthesis;
(GIBCO BRL), and the cloning vector λ
ZAP II (λZAP II / EcoRI / CIAP Cloning Kit, STRATAGENE
EcoRI site of Stratagene Gigap
in vitro pac using the ack III Gold Packaging Extract
By performing kaging and infecting Escherichia coli, a human pituitary-derived cDNA library can be prepared. Alternatively, a commercially available cDNA library can be purchased.

For the nucleotide sequence of the cDNA, each E. coli clone constituting the cDNA library is randomly selected, a plasmid DNA is prepared from the clone, and the nucleotide sequences at both ends of the cDNA contained in the plasmid are usually used. Nucleotide sequence analysis method, for example, the dideoxy method of Sanger et al. [Proc. Natl. Acad. Sci. USA, 74 , 546].
3 (1977)] or a nucleotide sequence analyzer such as a 373A DNA sequencer (manufactured by Perkin Elmer).

The presence of the thus obtained gene having homology to the base sequence or a protein having homology to the amino acid sequence encoded by the base sequence is examined by database search. For the search, a program such as the Blast or FrameSearch method can be used. As the database, a public database such as GenBank can be used, or a private database can also be used. According to the search, a known GP at the base level or the amino acid level
For the cDNA showing homology to CR, the entire nucleotide sequence is determined, and the entire amino acid sequence of the polypeptide encoded by the cDNA is determined.

When the cDNA does not encode a full-length polypeptide, a cDNA encoding the full-length polypeptide can be obtained as follows.

Using a single-stranded cDNA library prepared from various organs or various cells or a cDNA library prepared by the above-described method as a template, and using a primer set specific to the cDNA, polymerase chain reaction ( Polymerase Chain Reaction; hereinafter abbreviated as PCR) [Molecular Cloning Second Edition and PCR Protocols Academic Press (199
0)], an organ or cell that expresses the gene corresponding to the cDNA is specified, and a cDNA library derived from the specified organ or cell is used as a probe for the colony hybridization method (molecular -Cloning 2nd edition) can newly select a cDNA containing the full length of the cDNA from the cDNA library.

Further, using a single-stranded cDNA library or cDNA library derived from an organ or a cell expressing the gene corresponding to the cDNA as a template, 5 ′ RAC
E method and 3 'RACE method (Proc. Natl. Acad. Sci. USA, 85 , 89
98 (1988)], a 5′-end fragment and a 3′-end fragment of the cDNA are obtained, and by linking both fragments, a full-length cDNA can be obtained. In addition, commercially available kits (for example, Boehringer's 5 '/ 3' RA
The 5 'RACE method and the 3' RACE method can also be performed using a CE kit.

Single-chain cD derived from various organs or cells
The NA library can be prepared by a conventional method or a commercially available kit. For example, the NA library can be prepared by the following method.

A guanidium thiocyanate phenol-chloroform method [Anal.
Biochem., 162 , 156 (1987)]. If necessary, the total RNA is treated with deoxyribonuclease I (manufactured by Life Technologies) to degrade chromosomal DNA that may be contaminated. Total RNA obtained
For each, SUPERSCRIPT ^™ Preamplification using oligo (dT) or random primers
System for First Strand cDNA System (LifeTechnolo
gies) to produce a single-stranded cDNA library. The single-stranded cDNA library prepared as described above includes, for example, a single-stranded cDNA library prepared from human pituitary gland.

The entire nucleotide sequence of the cDNA encoding the full-length polypeptide obtained as described above and the amino acid sequence of the polypeptide were again subjected to a database search in the same manner as described above, and the homology with the known GPCR was determined. You can find out. Further, a hydrophobicity plot analysis is performed using the amino acid sequence of the polypeptide, and it is determined whether the polypeptide has a seven-transmembrane region common to GPCRs. The polypeptide has a seven-transmembrane region, And if it shows homology to a known GPCR, the polypeptide is GP
It can be considered a CR. If the polypeptide is different from the known GPCR, the polypeptide
It can be considered a CR.

Further, based on the determined amino acid sequence of the novel polypeptide, the DN encoding the polypeptide is
The target DNA can also be prepared by chemically synthesizing A. Chemical synthesis of DNA can be performed using a DNA synthesizer manufactured by Shimadzu Corporation using a thiophosphite method, a DNA synthesizer model 392 manufactured by Perkin-Elmer Inc. using a phosphoramidite method, or the like.

Using the oligonucleotides described below as sense primers and antisense primers, the mRN of cells expressing mRNA complementary to these DNAs was used.
The target DNA can also be prepared by performing PCR using the cDNA prepared from A as a template.

As the DNA encoding the novel G protein-coupled receptor polypeptide obtained by the above method,
For example, a DNA encoding the polypeptide represented by SEQ ID NO: 1 and the like can be mentioned. Specifically, it has a nucleotide sequence represented by nucleotide numbers from 58 to 1140 in the nucleotide sequence represented by SEQ ID NO: 2 DNA and the like can be mentioned. Nucleotide number 5 in the nucleotide sequence of SEQ ID NO: 2
The polypeptide encoded by the DNA having the nucleotide sequence represented by Nos. 8 to 1140 is expected to have the following structure by the above analysis method (see FIGS. 1 and 2).

The N-terminal extracellular region (40 amino acids), the first transmembrane region (26 amino acids), the first intracellular loop (5 amino acids)
Amino acid), second transmembrane region (26 amino acids), first extracellular loop (14 amino acids), third transmembrane region (26 amino acids), second intracellular loop (15 amino acids), fourth transmembrane region (26 amino acids) Amino acid), second extracellular loop (27 amino acids), fifth transmembrane region (26 amino acids), third intracellular loop (30 amino acids), sixth transmembrane region (26 amino acids), third extracellular loop (13 amino acids) Amino acid), seventh transmembrane region (26 amino acids), C-terminal intracellular region (35 amino acids) Hydropathy analysis of the amino acid sequence suggests that the polypeptide does not have a signal peptide (see FIG. 1). .

DN having the nucleotide sequence of SEQ ID NO: 2
As a plasmid containing A, for example, pBS-PGM
O334. pBS-PGMO33
Escherichia coli DH5α / pBS carrying No. 4
-PGMO334 has been deposited as FERMBP-6966 on December 8, 1999 at the Institute of Biotechnology and Industrial Technology, Institute of Industrial Science and Technology, 1-3-1 Higashi, Tsukuba, Ibaraki, Japan (zip code 305-8566). .

Using the DNA and DNA fragment of the present invention obtained by the above-mentioned method, a standard method described in Molecular Cloning, 2nd edition, etc., or a DNA synthesizer using the base sequence information of the DNA is used. Oligonucleotides such as antisense oligonucleotides and sense oligonucleotides having a partial sequence of the DNA of the present invention can be prepared.

Examples of the oligonucleotide include a DNA having the same base sequence as 5 to 60 consecutive bases in the base sequence of the DNA of the present invention or a DNA having a sequence complementary to the DNA. Examples of the DNA include a DNA having the same sequence as 5 to 60 consecutive nucleotides in the base sequence of the DNA described in SEQ ID NO: 2 or a DNA having a sequence complementary to the DNA. When used as a sense primer and an antisense primer, it is preferable to select an oligonucleotide whose melting temperature (Tm) and the number of bases of both do not extremely change. Specifically, for example, a primer set of an oligonucleotide having a base sequence represented by SEQ ID NO: 15 and SEQ ID NO: 16, or a primer set of an oligonucleotide having a base sequence represented by SEQ ID NO: 16 and SEQ ID NO: 17 can be mentioned. be able to.

Further, derivatives of these oligonucleotides (hereinafter referred to as oligonucleotide derivatives) can also be used as the oligonucleotide of the present invention. Examples of the oligonucleotide derivative include an oligonucleotide derivative in which a phosphodiester bond in an oligonucleotide is converted to a phosphorothioate bond, and a phosphodiester bond in an oligonucleotide converted to an N3′-P5 ′ phosphoramidate bond. Oligonucleotide derivative, the ribose and the phosphodiester bond in the oligonucleotide are converted to a peptide nucleic acid bond, the uracil in the oligonucleotide is C-
Oligonucleotide derivatives substituted with 5-propynyluracil, oligonucleotide derivatives in which uracil in the oligonucleotide is substituted with C-5 thiazoleuracil, oligonucleotide derivatives in which cytosine in the oligonucleotide is substituted with C-5 propynylcytosine, oligo Oligonucleotide derivative in which cytosine in nucleotide is replaced with phenoxazine-modified cytosine, oligonucleotide derivative in which ribose in oligonucleotide is replaced by 2′-O-propyl ribose, or ribose in oligonucleotide Can be exemplified by oligonucleotide derivatives substituted with 2'-methoxyethoxyribose [Cell Engineering, 16 , 1463 (1997)]. (4) Method for Producing Polypeptide and Partial Peptide of the Present Invention The polypeptide of the present invention or a salt thereof can be produced from the above-mentioned method for purifying a protein from human or mammalian cells or tissues, and will be described later. It can also be produced using a transformant containing a DNA encoding the polypeptide of the present invention. Further, it can also be produced according to the peptide synthesis method described later. When producing from human or mammalian tissues or cells, for example, after homogenizing human or mammalian tissues or cells, extraction is performed with an acid or the like, and the extract is subjected to chromatography such as reverse phase chromatography or ion exchange chromatography. Purification and isolation can be achieved by combining the chromatography.

The partial peptide of the present invention or a salt thereof can be synthesized according to a known peptide synthesis method, or can be produced by cleaving the G protein-coupled receptor polypeptide of the present invention with an appropriate peptidase. can do. As a method for synthesizing a peptide, for example, any of a solid phase synthesis method and a liquid phase synthesis method may be used.
That is, the target peptide can be produced by condensing a partial peptide or amino acid capable of constituting the partial peptide of the present invention with the remaining portion, and removing the protective group when the product has a protective group. Known condensation methods and elimination of protecting groups include, for example, the following (a) to
The method described in (e) is mentioned. (A) M. Bodanszky and MA Ondetti, peptide
Synthesis (Peptide Synthesis), Interscience Publi
shers, New York (1966) (b) Schroeder and Luebke, The Pepti
de), Academic Press, New York (1965) (c) Nobuo Izumiya et al., Fundamentals and experiments of peptide synthesis, Maruzen
(1975) (d) Haruaki Yajima and Shunpei Sakakibara, Laboratory of Biochemical Experiments 1,
Protein Chemistry IV, 205, (1977) (e) Supervised by Haruaki Yajima, Development of Continuing Drugs Volume 14 Peptide Synthesis Hirokawa Shoten Also, after the reaction, conventional purification methods such as solvent extraction, distillation, and column chromatography -The partial peptide of the present invention can be purified and isolated by a combination of liquid chromatography, recrystallization and the like. When the partial peptide obtained by the above method is a free form, it can be converted to an appropriate salt by a known method, and conversely, when it is obtained by a salt, it can be converted to a free form by a known method. Can be.

In addition to the above method, the polypeptide or partial peptide of the present invention can be produced by expressing the DNA of the present invention obtained in the above (3) in a host cell.

That is, a recombinant DNA in which the DNA of the present invention is inserted downstream of a promoter of an appropriate expression vector is constructed, and the recombinant DNA is introduced into a host cell to obtain a polypeptide or a partial peptide of the present invention. A polypeptide or a partial peptide of the present invention can be produced by obtaining a transformant that expresses and then culturing the transformant.

As the host cell, any prokaryotic cell, yeast, animal cell, insect cell, plant cell, or the like can be used as long as it can express the gene of interest. Moreover, an animal individual or a plant individual can also be used.

As the expression vector, those which can replicate autonomously in the above-mentioned host cells or can be integrated into the chromosome, and which contain a promoter at a position suitable for transcription of the DNA of the present invention can be used. .

When a prokaryote such as a bacterium is used as a host cell, the DNA expression vector of the present invention is capable of autonomous replication in a prokaryote and has a promoter, a ribosome binding sequence, a novel receptor gene, a transcription termination sequence, and the like. , It is preferable to be constituted. A gene that controls the promoter may be included.

As an expression vector, for example, pBTr
p2, pBTac1, pBTac2 (all commercially available from Boehringer Mannheim), pSE280 (Invitrogen), pGEMEX-1 (Promega),
pQE-8 (manufactured by QIAGEN), pKYP10 (JP-A-58
-110600), pKYP200 [Agric. Biol. Che
m., 48 , 669 (1984)], pLSA1 [Agric. Biol. Che.
m., 53 , 277 (1989)], pGEL1 [Proc. Natl. Aca
d. Sci., USA, 82 , 4306 (1985)], pBluescript II SK
(-) (STRATAGENE), pTrs30 (FERM BP)
-5407), pTrs32 (FERM BP-540)
8), pGHA2 (FERM BP-400), pGK
A2 (FERM B-6798), pTerm2 (JP-A-3-22979, US4686191, US4939)
094, US5160735), pKK233-2 (Ph
armacia), pGEX (Pharmacia), pET system (Novagen), pSuex, pUB11
0, pTP5, pC194, pTrxFus (Invitrog
en company), pMAL-c2 (New England Biolabs) and the like.

The promoter may be any promoter as long as it can be expressed in a host cell such as E. coli.
For example, trp promoter (P trp), lac promoter (P lac), P _L promoter, such as P _R promoter,
Promoters derived from E. coli, phage, etc., SPO1
Promoter, SPO2 promoter, penP promoter and the like can be mentioned. In addition, artificially designed and modified promoters such as a promoter in which two Ptrps are connected in series (P trp x2), a tac promoter, a lacT7 promoter, and a let I promoter can also be used.

As the ribosome binding sequence, Shine-
It is preferable to use a plasmid in which the distance between the Shine-Dalgarno sequence and the initiation codon is adjusted to an appropriate distance (for example, 6 to 18 bases).

Although a transcription termination sequence is not always necessary for expression of the DNA of the present invention, it is preferable to arrange a transcription termination sequence immediately below a structural gene.

As the host cell, microorganisms belonging to the genus Escherichia, Serratia, Bacillus, Brevibacterium, Corynebacterium, Microbacterium, Pseudomonas, etc., for example, Escherichia coli XL1-B
lue, Escherichia coli XL2-Blue, Escherichia coli D
H1, Escherichia coli MC1000, Escherichia coli KY32
76, Escherichia coli W1485, Escherichia coli JM10
9, Escherichia coli HB101, Escherichia coli No.4
9, Escherichia coli W3110, Escherichia coli NY49,
Escherichia coli BL21 (DE3), Escherichia coli BL21
(DE3) pLysS, Escherichia coli HMS174 (DE3), Escheric
hia coli HMS174 (DE3) pLysS, Serratia ficaria , Serra
tia fonticola , Serratia liquefaciens , Serratia mar
cescens , Bacillus subtilis , Bacillus amyloliquefac
iens , Corynebacterium ammoniagenes , Brevibacterium
immariophilum ATCC14068, Brevibacterium saccharol
yticum ATCC14066, Corynebacterium glutamicum ATCC1
3032, Corynebacterium glutamicum ATCC14067, Coryne
bacterium glutamicum ATCC13869, Corynebacterium ac
etoacidophilum ATCC13870, Microbacterium ammoniaph
ilum ATCC15354, Pseudomonas sp. D-0110 and the like.

As a method for introducing a recombinant vector, any method for introducing DNA into the above host cells can be used. For example, electroporation [Nu
cleic Acids Res., 16 , 6127 (1988)], a method using calcium ions [Proc. Natl. Acad. Sci. USA, 69 ,
2110 (1972)], the protoplast method (JP-A-63-24839).
4), Gene, 17 , 107 (1982) and Molecular & General Gen
etics, 168 , 111 (1979).

When a yeast strain is used as a host cell, as an expression vector, for example, YEp13 (ATCC37)
115), YEp24 (ATCC37051), YCp50 (ATCC37
419), pHS19, pHS15 and the like. Any promoter can be used as long as it can be expressed in yeast strains.
Promoter, PGK promoter, GAP promoter, ADH promoter, gal 1 promoter, g
Examples of the promoter include an al10 promoter, a heat shock protein promoter, a MFα1 promoter, and a CUP1 promoter.

As the host cell, Saccharomyces sp.
Yeast strains belonging to the genus Schizosaccharomyces, Kluyveromyces, Trichosporone, Siwaniomyces and the like can be mentioned, and specifically, Saccharomyces cerevisi
ae , Schizosaccharomyces pombe , Kluyveromyces lacti
s , Trichosporon pullulans , Schwanniomyces alluvius
Etc. can be given. Alternatively, a mutant suitable for GPCR expression can be used [Trends in Biotechnolog
y, 15 , 487 (1997), Mol.Cell.Biol., 15 , 6188 (199
5), Mol. Cell. Biol., 16 , 4700 (1996)].

As a method for introducing a recombinant vector, any method for introducing DNA into yeast can be used. For example, an electroporation method [Methods.
nzymol., 194 , 182 (1990)), spheroplast method (Pr
Natl. Acad. Sci. USA, 84 , 1929 (1978)], lithium acetate method (J. Bacteriol., 153 , 163 (1983)), Proc.
Natl. Acad. Sci. USA, 75 , 1929 (1978).

When an animal cell is used as a host cell, pcDNAI / Am
p, pcDNAI, pCDM8, pAGE107, pR
EP4, pAGE103, pAMo, pAMoA, pA
Mod-d (see Example 1), pAS3-3 and the like can be exemplified.

Any promoter can be used as long as it can be expressed in animal cells. For example, the IE (imme) of cytomegalovirus (human CMV) can be used.
diateearly) promoter, early promoter of SV40, long terminal repeat promoter of Moloney Murine Leukemia Virus
at promoter, a retrovirus promoter, a heat shock promoter, an SRα promoter, a metallothionein promoter, and the like. Further, an enhancer of the IE gene of human CMV may be used together with the promoter.

Examples of host cells include mouse myeloma cells, rat myeloma cells, mouse hybridoma cells, CHO cells, BHK cells, African green monkey kidney cells, Namalwa cells, Namalwa KJM-1 cells, human fetal kidney cells, and human leukemia. Cell, HBT5637 (JP-A-63-299), human colon cancer cell line, frog oocyte, frog melanocyte and the like.

Further, as mouse / myeloma cells,
YB2 / 0 for rat myeloma cells such as SP2 / 0 and NSO
HEK293 etc. as human fetal kidney cells, BALL-1 etc. as human leukemia cells, COS-1 and COS-7 as African green monkey kidney cells, HCT-15 etc. as human colon cancer cell line. it can.

As a method for introducing a recombinant vector, any method for introducing DNA into animal cells can be used. For example, electroporation [Cytote
chnology, 3 , 133 (1990)], calcium phosphate method (Japanese Patent Laid-Open No. 2-227075), lipofection method [Proc. Natl. Ac
ad. Sci. USA, 84 , 7413 (1987)], Virology, 52 , 456.
(1973). Obtaining and culturing the transformant can be performed according to the method described in JP-A-2-227075 or JP-A-2-25791.

When an insect cell is used as a host, for example, baculovirus expression vectors, a laboratory manual [Baculovirus Exp.
ression Vectors, A Laboratory Manual, WH Freema
n and Company, New York (1992)], Molecular Biology A Laboratory Manual (Molecular
Biology, A Laboratory Manual), Current Protocols in Molecular Biology, Bio / T
The polypeptide can be expressed by the method described in Echinology, 6 , 47 (1988) and the like.

That is, after a recombinant gene transfer vector and a baculovirus are co-transfected into insect cells to obtain a recombinant virus in the culture supernatant of insect cells, the recombinant virus is further infected into insect cells, and the polypeptide is transcribed. Can be expressed.

The gene transfer vector used in the method includes, for example, pVL1392, pVL1393, pBlueBacII
I (all manufactured by Invitrogen) and the like.

Examples of the baculovirus include, for example, Autographa californica nuclea polyhedrosis virus, which is a virus that infects night moth insects
(Autographa californica nuclear polyhedrosis viru
s) etc. can be used.

As insect cells, Spodoptera frugiperd
a ovarian cells, ovarian cells of Trichoplusia ni, can be used cultured cells derived from silkworm ovary. Spodoptera f
Rugi perda ovary cells include Sf9 and Sf21 (baculovirus expression vectors, a laboratory manual), and Trichoplusia ni ovary cells are High 5, BTI-TN-5B1-4 (manufactured by Invitrogen).
Bombyx mori N4 is a cultured cell derived from silkworm ovary.
Etc. can be given.

The method for co-transferring the above-described recombinant gene transfer vector and the above baculovirus into insect cells for preparing a recombinant virus includes, for example, the calcium phosphate method (Japanese Patent Laid-Open No. 2-227075), the lipofection method [Proc.
Natl. Acad. Sci. USA, 84 , 7413 (1987)].

Further, DNA can be introduced into insect cells using the same method as that for introducing DNA into animal cells. For example, electroporation [Cytotech.
nology, 3 , 133 (1990)], calcium phosphate method (Japanese Patent Laid-Open No. 2-227075), lipofection method [Proc. Natl. Aca
d. Sci. USA, 84 , 7413 (1987)]. When a plant cell or a plant individual is used as a host, a known method [tissue culture, 20 (1994), tissue culture, 21
(1995), Trends in Biotechnology, 15 , 45 (1997)].

As the promoter used for gene expression, any promoter can be used as long as it can be expressed in plant cells, and examples thereof include the cauliflower mosaic virus (CaMV) 35S promoter and rice actin 1 promoter. Further, by inserting intron 1 and the like of a maize alcohol dehydrogenase gene between the promoter and the gene to be expressed,
Gene expression efficiency can also be increased.

The host cells include potato, tobacco,
Corn, rice, rape, soy, tomato, wheat,
Plant cells such as barley, rye, alfalfa, flax and the like can be mentioned.

As a method for introducing a recombinant vector, any method for introducing DNA into plant cells can be used. For example, Agrobacterium ( Agrobacterium) can be used.
m ) (JP-A-59-140885, JP-A-60-70080, WO94 / 0097)
7), electroporation [Cytotechnology, 3 ,
133 (1990), JP-A-60-251887], a method using a particle gun (gene gun) (Patent Nos. 2606856, 251
7813).

The cells or organs of the plant into which the gene has been introduced can be cultured in large quantities using a jar fermenter. Further, a plant individual (transgenic plant) into which the gene has been introduced can also be created by redifferentiating the plant cell into which the gene has been introduced.

The polypeptide of the present invention can be produced using an animal individual. For example, known methods [American
Journal of Clinical Nutrition, 63 , 639S (1996), A
American Journal of Clinical Nutrition, 63 , 627S (1
996), Bio / Technology, 9 , 830 (1991)], and the polypeptide of the present invention can be produced in an animal into which a gene has been introduced.

As the promoter, any promoter that can be expressed in animals can be used.
Mammary cell-specific promoters such as α-casein promoter, β-casein promoter, β-lactoglobulin promoter, whey acidic protein promoter and the like are preferably used.

A transformant derived from a microorganism, animal cell, or plant cell having a recombinant DNA into which DNA encoding the polypeptide or partial peptide of the present invention has been incorporated is cultured according to a conventional culture method, and The polypeptide can be produced by producing and accumulating the peptide and collecting the polypeptide from the culture.

When the transformant is an animal or plant individual, the transformant is bred or cultivated according to a conventional method to produce and accumulate the polypeptide, and the polypeptide is collected from the animal or plant individual. The polypeptide can be produced.

That is, in the case of an animal individual, for example, a non-human transgenic animal carrying the DNA of the present invention is bred and the polypeptide or partial peptide of the present invention encoded by the recombinant DNA is produced in the animal. -The polypeptide or partial peptide of the present invention can be produced by accumulating and collecting the polypeptide or partial peptide from the animal. Examples of the production / accumulation site in the animal include cell membrane fractions of various cells, milk, eggs, and the like of the animal.

In the case of a plant individual, for example, the DNA of the present invention
Cultivating a transgenic plant carrying the recombinant DNA, producing and accumulating the polypeptide or partial peptide of the present invention encoded by the recombinant DNA in the plant, and collecting the polypeptide or partial peptide from the plant. ,
The polypeptide or partial peptide of the present invention can be produced.

When the transformant for producing the polypeptide or the partial peptide of the present invention is a prokaryote such as Escherichia coli or a eukaryote such as yeast, the medium for culturing these organisms may be a carbon-containing medium capable of assimilating the organism. Any of a natural medium and a synthetic medium may be used as long as the medium contains a source, a nitrogen source, inorganic salts, and the like and can efficiently culture the transformant.

Any carbon source may be used as long as the transformant can be assimilated, such as glucose, fructose, sucrose, molasses containing these, carbohydrates such as starch or starch hydrolyzate, acetic acid, propionic acid and the like. Alcohols such as organic acids, ethanol, and propanol are used.

Examples of the nitrogen source include ammonium, ammonium chloride, ammonium sulfate, ammonium acetate, ammonium phosphate and the like, ammonium salts of various inorganic and organic acids, other nitrogen-containing compounds, peptone, meat extract, yeast extract, and corn steep. Liquor, casein hydrolyzate, soybean meal and soybean meal hydrolyzate, various fermented cells and digested products thereof can be used.

As the inorganic substance, potassium (I) phosphate, potassium (II) phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, calcium carbonate and the like can be used.

The culture is performed under aerobic conditions such as shaking culture or deep aeration stirring culture. The culturing temperature is preferably 15 to 40 ° C, and the culturing time is usually 16 to 96 hours. During culture
H is kept between 3.0 and 9.0. The pH is adjusted using an inorganic or organic acid, an alkali solution, urea, calcium carbonate, ammonia, or the like.

[0091] If necessary, an antibiotic such as ampicillin or tetracycline may be added to the medium during the culture.

When a microorganism transformed with an expression vector using an inducible promoter is cultured, an inducer may be added to the medium, if necessary. For example, when culturing a microorganism transformed with an expression vector using a lac promoter, culturing a microorganism transformed with an expression vector using a trp promoter such as isopropyl-β-D-thiogalactopyranoside (IPTG). At times, indole acrylic acid (IAA) or the like may be added to the medium.

When the transformant for producing the polypeptide or the partial peptide of the present invention is an animal cell, the medium for culturing the cell may be a commonly used RPMI1640 medium.
Medium (The Journal of the American Medical Associat
ion, 199 , 519 (1967)], Eagle's MEM medium [Sc
ience, 122 , 501 (1952)), DMEM medium (Virology,
8 , 396 (1959)), 199 medium (Proceeding of the Soc
Society for the Biological Medicine, 73 , 1 (1950)], or a medium obtained by adding fetal bovine serum or the like to such a medium.

The cultivation is usually carried out at pH 6-8, 30-40 ° C.
This is performed for 1 to 7 days under conditions such as the presence of 5% CO ₂ .

During the culture, kanamycin,
An antibiotic such as penicillin may be added to the medium.

As a medium for culturing a transformant obtained by using an insect cell as a host cell, a commonly used medium is used.
NM-FH medium (Pharmingen), Sf-900 II SFM medium (Gibco BRL), ExCell400, Ex
Cell 405 (manufactured by JRH Biosciences), Grace's Ins
ect Medium [Nature, 195 , 788 (1962)] and the like can be used.

The culture conditions are pH 6-7, and the culture temperature is 25-
Preferably, the cultivation time is usually 1 to 5 days at 30 ° C. Also,
If necessary, an antibiotic such as gentamicin may be added to the medium during the culture.

The polypeptide or partial peptide of the present invention can be expressed as a secretory protein or a fusion protein in accordance with the method described in Molecular Cloning, 2nd edition, etc., in addition to direct expression. Examples of proteins to be fused include β-galactosidase, protein A, an IgG binding region of protein A,
Chloramphenicol acetyltransferase,
Poly (Arg), poly (Glu), protein G, maltose binding protein, glutathione S-transferase, polyhistidine chain (His-tag), S peptide,
DNA binding protein domain, Tac antigen, thioredoxin, green fluorescent protein, F
LAG peptide and epitopes of any antibodies [Akio Yamakawa, Experimental Medicine, 13 , 469-474 (199)
Five)〕.

The polypeptide or the partial peptide of the present invention may be produced in a host cell, secreted out of the host cell, or produced on the host cell membrane. The method can be selected by changing the structure of the polypeptide to be produced.

In the case of secretory expression outside the host cell or expression on the host cell membrane, a signal sequence suitable for the host is added to the N-terminal side of the polypeptide or partial peptide of the present invention, if necessary. In some cases, it is better to add the above-mentioned secretion signal after partially deleting the N-terminus of the polypeptide or partial peptide. When the host is a bacterium belonging to the genus Escherichia, an alkaline phosphatase signal sequence, an OmpA signal sequence, or the like is used. When the host is a bacterium belonging to the genus Bacillus, an α-amylase signal sequence, a subtilisin signal sequence, or the like is used. If the host is an animal cell, an insulin signal sequence, an α signal sequence, an invertase signal sequence, etc.
-Interferon signal sequence, antibody molecule / signal sequence, etc. can be used.

The polypeptide or partial peptide of the present invention can be expressed by adding a tag for purification or detection. Tags can be added anywhere,
When expressed in mammalian cells, it is preferable to add the gene to the extracellular region or intracellular region described above.

Examples of tags for purification and detection include β-galactosidase, protein A, an IgG binding region of protein A, chloramphenicol acetyltransferase, poly (Arg), poly (Glu), protein G, and maltose binding protein. , Glutathione S-transferase, polyhistidine chain (His-tag), S peptide, DNA binding protein domain, Tac antigen,
Examples include thioredoxin, green fluorescent protein, FLAG peptide, and an epitope of any antibody [Akio Yamakawa, Experimental Medicine, 13 , 469-47.
4 (1995)].

Further, according to the method described in Japanese Patent Application Laid-Open No. 2-227075, the production amount can be increased using a gene amplification system using a dihydrofolate reductase gene or the like.

In order to isolate and purify the polypeptide of the present invention from the culture of the transformant for producing the polypeptide or the partial peptide of the present invention, the usual isolation and purification of the polypeptide can be used.
Purification methods can be used.

For example, when the polypeptide or partial peptide of the present invention accumulates in a lysed state in the cells of the transformant for producing the polypeptide or partial peptide of the present invention, the culture is centrifuged. After collecting the cells in the culture and washing the cells, the cells are crushed with an ultrasonic crusher, a French press, a Mantongaulin homogenizer, a Dynomill or the like to obtain a cell-free extract.

From the supernatant obtained by centrifuging the cell-free extract, a solvent extraction method, a salting-out method using ammonium sulfate, a desalting method, a precipitation method using an organic solvent, diethylaminoethyl (DEAE) -Sepharose, DIAION Anion exchange chromatography using a resin such as HPA-75 (Mitsubishi Kasei), cation exchange chromatography using a resin such as S-Sepharose FF (Pharmacia), butyl sepharose, phenyl sepharose, etc. Purified samples can be obtained using techniques such as hydrophobic chromatography using resins, gel filtration using molecular sieves, affinity chromatography, chromatofocusing, and electrophoresis such as isoelectric focusing. it can.

When the polypeptide or partial peptide of the present invention accumulates on the cell membrane of the transformant for producing the polypeptide or partial peptide of the present invention, the cells are similarly recovered, crushed, centrifuged or filtered. , A polypeptide such as Triton X-100 is used to solubilize the polypeptide or the partial peptide from the membrane, and a purified sample is obtained by the same isolation and purification method as described above. be able to.

When the polypeptide or the partial peptide is expressed by forming an insoluble substance in the cells, the cells are similarly collected, crushed, and centrifuged to obtain a precipitate fraction. After recovering the polypeptide or the peptide by an ordinary method, the insoluble form of the polypeptide or the partial peptide is solubilized with a denaturing agent. The lysate is
After the denaturant is not contained or the concentration of the denaturant is diluted to a dilute solution such that the polypeptide or the partial peptide is not denatured, or dialyzed to form the polypeptide or the partial peptide into a normal three-dimensional structure. A purified sample can be obtained by the same isolation and purification method as described above.

When the polypeptide or the partial peptide is secreted extracellularly, the culture is treated by a technique such as centrifugation to obtain a soluble fraction. From the soluble fraction, a purified sample of the polypeptide or the partial peptide can be obtained by the same method as in the above-mentioned isolation and purification method from the cell-free extract supernatant.

Further, the polypeptide of the present invention or the partial peptide may be produced as a fusion protein with another protein, and purified by affinity chromatography using a substance having affinity for the fused protein. [Akio Yamakawa, Experimental Medicine, 13 , 469-474 (199
Five)〕. For example, the method of Lowe et al. [Proc. Natl. Acad. Sci.
USA, 86 , 8227 (1989), Genes Develop., 4 , 1288 (1
990)], the polypeptide or partial peptide of the present invention is produced as a fusion protein with protein A, and purified by affinity chromatography using immunoglobulin G according to the method described in JP-A-05-336963 and WO94 / 23201. can do. Further, the polypeptide or partial peptide of the present invention can be produced as a fusion protein with a FLAG peptide and purified by affinity chromatography using an anti-FLAG antibody [Proc. Natl. Acad. Sci. USA, 86 , 8227 ( 1989), G
enes Develop., 4 , 1288 (1990)].

Further, the polypeptide or partial peptide of the present invention can be purified by affinity chromatography using an antibody against itself.

Further, a known method [J. Biomolecular NMR,
6 , 129-134, Science, 242 , 1162-1164, J. Biochem.,
110 , 166-168 (1991)], and the polypeptide or partial peptide of the present invention can be produced using an in vitro transcription / translation system.

Further, the polypeptide or the partial peptide of the present invention can also be produced by a chemical synthesis method such as the Fmoc method (fluorenylmethyloxycarbonyl method) and the tBoc method (t-butyloxycarbonyl method).
Advanced ChemTech, Perkin-Elmer,
Pharmacia Biotech, Protein Technology Instr
Chemical synthesis can also be carried out using a peptide synthesizer such as ument, Synthecell-Vega, PerSeptive, Shimadzu Corporation.

The structural analysis of the purified polypeptide of the present invention is performed by a method usually used in protein chemistry, for example, the method described in Protein Structural Analysis for Gene Cloning (Hisashi Hirano, Tokyo Chemical Dojin, 1993). It is feasible.

When the polypeptide or partial peptide of the present invention obtained by the above method is obtained in a free form, it can be converted into a salt by a known method or a method analogous thereto, and conversely, it can be obtained by a salt. In this case, it can be converted to a free form or another salt by a known method or a method analogous thereto. The polypeptide or partial peptide of the present invention can be arbitrarily modified or partially removed by the action of an appropriate protein modifying enzyme. As protein modifying enzymes,
For example, trypsin, chymotrypsin, arginyl endopeptidase, protein kinase, glycosidase and the like are used.

The activity of the polypeptide or partial peptide of the present invention or the salt thereof which can be produced by the above method is as follows.
It can be measured by a binding experiment with a labeled ligand, an enzyme immunoassay using a specific antibody, or the like. (5) Use of Polypeptide or Partial Peptide of the Present Invention, and DNA Encoding the Same The polypeptide or its partial peptide of the present invention, or a salt thereof, and the DNA encoding the polypeptide or its partial peptide are as follows: Can be used for the purpose. (A) Production of polypeptide or partial peptide of the present invention (b) Preparation of antibody and antiserum to polypeptide of the present invention (c) Determination of ligand for polypeptide of the present invention (d) Ligand for polypeptide of the present invention Of screening system for agonists, antagonists, and functional modifiers and screening of drug candidate compounds using the screening system (e) Implementation of drug design based on comparison with structurally similar ligands / receptors (f) Reagents for the preparation of probes and PCR primers in gene diagnosis, etc. (g) Pharmaceuticals such as gene preventive agents and gene therapeutic agents (h) Preparation of knockout animals Polypeptides of the present invention or partial peptides thereof, or salts thereof, polypeptides Or a DN encoding a partial peptide thereof For applications will be specifically described below. (6) Method for Determining Ligands for G Protein-Coupled Receptor Polypeptides of the Present Invention Methods for searching for or determining ligands for the polypeptides of the present invention include, for example, polypeptides of the present invention, partial peptides or salts thereof, A method for selecting a ligand for the polypeptide of the present invention from a test substance by contacting the test substance with a test substance, or contacting a cell expressing the polypeptide or partial peptide of the present invention or a membrane fraction of the cell with the test substance. And a method for selecting a ligand for the polypeptide of the present invention from a test substance.

As test substances, known ligands (for example, angiotensin, bombesin, cannabinoid, cholecystokinin, glutamine, serotonin, melatonin, neuropeptide Y, opioid, purine, vasopressin, oxytocin, PACAP (Pichuitari)
Adenylate cyclase activating protein), secretin, glucagon, calcitonin, adrenomedullin, somatostatin, GHRH (growth hormone-releasing hormone), CRF (corticotropin-releasing factor), ACTH (adrenocorticotropic hormone), melanin stimulant Ration hormone, GRP (gastrin-releasing peptide), PTH (parathyroid hormone), VIP (vasoactive intestinal and reduced polypeptide), dopamine, motilin, amylin, bradykinin, CGRP (calcitonin gene-related peptide), leukotriene,
Pancreatastatin, prostaglandin, thromboxane, adenosine, adrenaline, α and β-chemokines [eg, IL-8 (interleukin-8), G
ROα (Growth Relayed Gene α), GRO
β, GROγ, NAP-2 (neural calmodulin binding protein-2), ENA-78
(Epserial Cell-Derived Neutrofil-Activating Protein-78), PF4 (Platelet Factor 4), IP10 (Interferon-γ Inducible Protein of 10k)
d), GCP-2 (granulosite chemotactic protein-2), MCP-1 (monosite chemoretractant protein-1), HC14, MCP-
3, I-309, MIP1α (macrophage inflammatri protein 1α), MIP-1β, RANT
ES (regulated on activation, normal T-cell Expressed and Secreted, etc.), endothelin, enterogastrin, histamine, neurotensin, TRH, pancreatic polypeptide, galanin, urotensin I and II,
Neuropeptide FF, orexin, melanin concentrating hormone, etc.], as well as, for example, tissue extracts of humans or mammals (eg, mice, rats, pigs, cows, sheep, monkeys, etc.), cell culture supernatants and the like. Can be

As a specific method for searching for or determining a ligand for the polypeptide of the present invention, a method for searching for a polypeptide or a partial peptide of the present invention or a salt thereof, or a cell expressing the polypeptide or partial peptide of the present invention may be used. Examples of the method include a method of measuring the amount of a test substance bound to a polypeptide or a partial peptide of the present invention or a salt thereof, or detecting a response of a cell expressing the polypeptide of the present invention.

More specifically, (a) when the labeled test substance is brought into contact with the polypeptide or partial peptide of the present invention or a salt thereof, the polypeptide or partial peptide of the labeled test substance or their salts (B) a method for determining a ligand for a polypeptide of the present invention, which comprises measuring a binding amount of the compound to a salt and selecting a ligand for the polypeptide of the present invention or a salt thereof from the test substance; Is contacted with a cell containing the polypeptide or partial peptide of the present invention or a membrane fraction of the cell, a binding amount of the labeled test substance to the cell or the membrane fraction is measured, and the test substance is measured. Selecting a ligand for the polypeptide of the present invention from a ligand for the polypeptide of the present invention. Determination method, (c) a test substance, when in contact with the membrane fraction of the cell or the cell containing the polypeptide of the present invention, the labeled GTPyS G
A method for determining a ligand for the polypeptide of the present invention, which comprises measuring the amount of binding to the α protein and selecting a ligand for the polypeptide of the present invention from the test substance. A ligand for the polypeptide of the present invention, which comprises measuring the GTPase activity when the cell is contacted with a cell containing the polypeptide or a membrane fraction of the cell, and selecting a ligand for the peptide of the present invention from a test substance. How to determine
(E) polypeptide-mediated cell stimulating activity (eg, arachidonic acid release, acetylcholine release, intracellular Ca ²⁺ release, intracellular) when a test substance is brought into contact with a cell containing the polypeptide of the present invention. cAMP production, intracellular cGMP production, inositol phosphate production, cell membrane potential fluctuation, intracellular protein phosphorylation, c-fos activation, p
H decrease, cell proliferation activity, aggregation or diffusion of melanin pigment, or activity to promote or suppress reporter gene expression, etc.) and select a ligand for the polypeptide of the present invention from the test substance. And a method for determining a ligand for the polypeptide of the present invention.

Hereinafter, the method for determining a ligand of the present invention will be described in detail. (I) Method for Measuring the Amount of Test Substance to be Bound As shown in (a) and (b) above, the polypeptide or partial peptide of the present invention or a salt thereof, or the polypeptide or partial peptide of the present invention is expressed. The labeled test substance is allowed to act on the cells, the amount of the test substance bound to the polypeptide of the present invention is measured, and the ligand for the polypeptide of the present invention is selected from the test substance, whereby the ligand for the polypeptide of the present invention is obtained. Can be searched or determined.

As test substances, for example,^ThreeH,¹⁴C,
¹²⁵I,³⁵S,³²Known labeling with radioactive isotopes such as P
GPCR ligands [angiotensin, Bombesi
, Cannabinoids, cholecystokinin, glutamine,
Rotonin, melatonin, neuropeptide Y, opioid
Do, pudding, vasopressin, oxytocin, PACA
P, secretin, glucagon, calcitonin, adreno
Medulin, somatostatin, GHRH, CRF, AC
TH, melanin stimulation hormone, GR
P, PTH, VIP, dopamine, motilin, amiri
, Bradykinin, CGRP, leukotriene, punk
Reastatin, prostaglandin, thromboxane,
Adenosine, adrenaline, α and β-chemokines
(For example, IL-8, GROα, GROβ, GROγ,
NAP-2, ENA-78, PF4, IP10, GCP
-2, MCP-1, HC14, MCP-3, I-30
9, MIP1α, MIP-1β, RANTES, etc.),
Endothelin, enterogastrine, histamine,
-Rotensin, TRH, pancreatic polypeptide
Id, galanin, urotensin I and II, neuro
Peptide FF, orexin and melanin concentrate
And other hormones). Ma
Was ^ThreeH,¹⁴C,¹²⁵I,³⁵S,³²Radioactive isotopes such as P
Using any protein, peptide or compound labeled with
You can also.

The polypeptide or partial peptide of the present invention used in the above method may be any one containing a polypeptide or partial peptide having ligand binding ability, and may be a cell or a cell containing the polypeptide or partial peptide. The purified polypeptide or partial peptide may be used, or the cell itself or the cell membrane fraction containing the polypeptide or partial peptide may be used. Examples of the partial peptide used in the above method include a partial peptide having both a hydrophilic site having ligand binding ability and a hydrophobic site that is a cell membrane transmembrane region, and the partial peptide is, like the polypeptide of the present invention, It is thought that it exists on the cell membrane of a cell containing the partial peptide, and can be used in the above method. When cells containing the polypeptide or partial peptide are used, the cells may be immobilized with glutaraldehyde, formalin, or the like.

The polypeptide or partial peptide of the present invention may be a naturally occurring polypeptide or partial peptide, or a recombinant polypeptide or a recombinant partial peptide prepared by using a gene recombination technique. Was constitutively active in the polypeptide encoded by the mutated DNA obtained by introducing a mutation into the DNA having the base sequence represented by SEQ ID NO: 2 by the method described in (3). Variant polypeptides are particularly useful.

Among G protein-coupled receptors (GPCRs), there are ones which, when the GPCR polypeptide is overexpressed in cells, signal even in the absence of ligand, and which are active components. It is called a type GPCR. Also,
It is known that even GPCRs that are not originally constitutively active become constitutively active by introducing mutations such as amino acid substitutions and deletions. Since it is known that a mutant GPCR changed to a constitutively active form may have an increased affinity for an agonist, a constitutively active mutant GPCR is considered to be useful in the search for a ligand [Journal of Pharmacology and Experimental Ther
apeutics, 275 , 1274 (1995), Endocrinology, 137 , 39
36 (1996), J. Biol. Chem., 272 , 1822 (1997)].

The constitutively active GPCR can be obtained according to a known method [J. Biol. Chem., 271 , 1857].
(1996), Science, 268 , 98 (1995), Journal of Pharm
acology and Experimental Therapeutics, 275 , 1274
(1995), J. Biol. Chem., 272 , 1822 (1997), Journal
of Receptor and Signal Transduction Research, 17 , 5
7 (1997), WO98 / 46995] The above-mentioned cell membrane fraction refers to a fraction containing a large amount of cell membrane obtained by a method known per se after cell disruption. As a method for crushing cells, a method of crushing cells with a Potter-Elvehjem type homogenizer, crushing with a Waring blender or a polytron (manufactured by Kinematica), crushing with ultrasonic waves, spouting cells from a thin nozzle while applying pressure with a French press, etc. Crushing and the like. For the fractionation of cell membranes, fractionation by centrifugal force such as fractionation centrifugation or density gradient centrifugation is mainly used. For example, the cell lysate is supplied at a low speed (500 rpm to 30 rpm).
(1 min. To 10 min.) And centrifugation at a higher speed (15000 rpm to 3000 rpm).
(0 rpm) for 30 minutes to 2 hours, and the resulting precipitate is used as a membrane fraction. The membrane fraction is rich in the expressed receptor protein and membrane components such as cell-derived phospholipids and membrane proteins.

As the cells expressing the polypeptide of the present invention, as described in (5) above, a trait obtained by introducing a recombinant DNA containing a DNA encoding the polypeptide into an appropriate host cell. Cells expressing the polypeptide in large amounts, such as transformed cells, can also be used.
In addition to microorganisms such as Escherichia coli, Bacillus subtilis, and yeast, insect cells, frog oocytes, frog melanocytes, animal cells, plant cells, and the like can be mentioned, and the polypeptide of the present invention produced by the transformant can be used. In order to maintain the higher-order structure and maintain the binding property to the ligand, it is preferable to express in yeast, insect cells, frog oocytes, frog melanocytes, animal cells, plant cells and the like.

The amount of the polypeptide or the partial peptide in the cell containing the polypeptide or the partial peptide of the present invention or the cell membrane fraction thereof is preferably, for example, 10 ³ to 10 ⁸ molecules per cell, and preferably 10 ⁵ to 10 ⁵ molecules. Preferably it is 10 ⁷ molecules. The higher the expression level, the higher the ligand binding activity (specific activity) per membrane fraction, which makes it possible not only to construct a highly sensitive screening system, but also to measure a large number of samples in the same lot. .

Hereinafter, specific examples will be described.

A sample of the polypeptide of the present invention is prepared by suspending a cell containing the polypeptide or the partial peptide of the present invention or a cell membrane fraction of the cell in an appropriate buffer. The buffer may be any buffer as long as it does not inhibit the binding between the ligand and the polypeptide of the present invention, for example, pH 4 to 10 (preferably pH 6 to 8).
Phosphate buffer or Tris-HCl buffer. In addition, CHA was used for the purpose of reducing non-specific binding.
Surfactants such as PS, Tween-80, digitonin, deoxycholic acid and various proteins such as bovine serum albumin and gelatin can also be added to the buffer. In addition, PMSF, leupeptin, E
Protease inhibitors such as -64 and pepstatin can also be added. 10 μl to 10 ml of the polypeptide preparation is allowed to coexist with a radioactive test substance labeled with a radioisotope ( ³ H, ¹²⁵ I, ¹⁴ C, ³⁵ S, ³² P, etc.). A reaction tube containing a large excess of unlabeled test substance is also prepared to determine the amount of non-specific binding (NSB). The reaction is carried out at about 0 to 50 ° C, preferably about 4 to 37 ° C, for about 20 minutes to 24 hours, preferably for about 30 minutes to 3 hours. After the reaction, the mixture is filtered with a glass fiber filter or the like, washed with an appropriate amount of the same buffer, and the radioactivity remaining on the glass fiber filter is measured with a liquid scintillation counter or a γ-counter. From the total binding amount (B) to the non-specific binding amount (NS
A test substance having a count (B-NSB) less than 0 cpm minus B) can be selected as a substance that binds to the polypeptide of the present invention. Among them, the binding activity to the cell containing the polypeptide of the present invention or the cell membrane fraction of the cell is strong, and the binding activity to the cell not containing the polypeptide of the present invention or the cell membrane fraction of the cell is weak. Substances can be selected as ligands for the polypeptide of the invention.

In the above method, as a cell containing the polypeptide or the partial peptide of the present invention, a host cell not expressing the polypeptide of the present invention is transformed into a vector DNA by encoding a DNA encoding the polypeptide or the partial peptide.
Using the polypeptide or the partial peptide-expressing cells obtained by introducing the recombinant DNA incorporated into the host cell as a cell not containing the polypeptide of the present invention, by introducing only a vector into the host cell. By using control cells that do not express the polypeptide, the ligand can be more accurately determined. (II) Method for measuring the amount of GTPγS bound to Gα protein As shown in (c) above, a test substance is brought into contact with the membrane fraction of cells containing the polypeptide of the present invention, and labeled G
By measuring the amount of TPγS bound to the Gα protein (membrane fraction), the ligand of the polypeptide can be searched or determined [Molecular Pharmacology, 47 , 84].
8 (1995), WO98 / 46995].

As the test substance, any substance can be used. For example, a known peptide, a known GPCR ligand,
Biological samples and biological samples such as known proteins, recombinant proteins produced using recombinant techniques, cell extracts and purified products derived from the extracted solutions, cell culture supernatants and purified products derived from the supernatants, serum and the like Use of a purified product derived from a microorganism, a cell extract of a microorganism, a purified product derived from the extract, a microorganism culture supernatant or a purified product derived from the supernatant, a known compound, a compound synthesized using combinatorial chemistry, or the like. Can be. GTP labeled
As γS, for example, GTPγS labeled with ³⁵ S can be used.

As the cells containing the polypeptide of the present invention or the membrane fraction of the cells, those described in the above (I) can be used.

Hereinafter, specific examples will be described.

The polypeptide preparation of the present invention was prepared using the above (I)
Prepared by the method described in The 10Myueru～10 ml of the polypeptide preparation, test compound, a radioactive isotope ⁽³⁵ S
) And a fixed amount of radioactivity of GTPγS and GDP. If necessary, prepare a reaction tube to which a large excess of unlabeled GTPγS has been added in order to know the amount of non-specific binding (NSB). Count (B-NS) obtained by subtracting the non-specific binding amount (NSB) from the total binding amount (B)
B) is the specific binding amount. The reaction is carried out at about 0 to 50 ° C, preferably about 4 to 37 ° C, for about 20 minutes to 24 hours, preferably for about 30 minutes to 3 hours. After the reaction, the mixture is filtered with a glass fiber filter or the like, washed with an appropriate amount of the same buffer, and the radioactivity remaining on the glass fiber filter is measured with a liquid scintillation counter. When the cell membrane fraction of the cell containing the polypeptide of the present invention is used, the activity of enhancing the binding of GTPγS to the membrane fraction is strong, and the cell membrane fraction of the cell not expressing the polypeptide of the present invention is used. When is used, a substance having a weak activity of enhancing the binding to the membrane fraction can be selected as a ligand of the polypeptide of the present invention.

In the above method, the cells containing the polypeptide of the present invention may be obtained by introducing, into a host cell that does not express the polypeptide of the present invention, a recombinant DNA obtained by incorporating a DNA encoding the polypeptide into a vector. Ligand determination can be performed by using the obtained polypeptide-expressing cell and using a control cell that does not express the polypeptide prepared by introducing only the vector into the host cell as a cell not containing the polypeptide of the present invention. It can be done more accurately. (III) Method for measuring GTPase activity As shown in (d) above, a test substance is brought into contact with the membrane fraction of cells containing the polypeptide of the present invention, and GTPas
By measuring the e-activity, the ligand of the polypeptide of the present invention can be searched or determined [J. Biol.
Chem., 271 , 1857 (1996), J. Biol. Chem., 271 , 185
7 (1996), WO98 / 46995].

As the test substance, the substances described in the above (II) can be used.

As the cells containing the polypeptide of the present invention or the membrane fraction of the cells, those described in the above (I) can be used.

A specific example will be described below.

A sample of the polypeptide of the present invention is prepared by the method described in the above (I). A test compound, a radioisotope (10 μl to 10 ml) are added to the polypeptide preparation.
³² P, etc.) in the coexistence of labeled fixed amount of radioactivity GTP (e.g. [gamma ³² P] GTP). The reaction is performed at about 0 ° C to 5
The reaction is carried out at 0 ° C., preferably about 4-37 ° C., for about 20 minutes to 24 hours, preferably for about 30 minutes to 3 hours. After the reaction, the supernatant of the reaction solution is recovered, and the radioactivity of the released [γ ³² P] Pi is measured with a liquid scintillation counter. After filtering the reaction solution with a glass fiber filter or the like and washing with an appropriate amount of the same buffer, the radioactivity in the filtrate may be measured with a liquid scintillation counter. When using the cell membrane fraction of cells containing the polypeptide of the present invention, GTPas
When the cell membrane fraction of cells that have strong e-enhancing activity and do not express the polypeptide of the present invention is used, G
A substance having a weak activity of enhancing TPase activity can be selected as a ligand of the polypeptide of the present invention.

In the above method, the cells containing the polypeptide of the present invention may be obtained by introducing a recombinant DNA obtained by incorporating a DNA encoding the polypeptide into a vector into a host cell that does not express the polypeptide of the present invention. Ligand determination can be performed by using the obtained polypeptide-expressing cells and using, as cells not containing the polypeptide of the present invention, control cells that do not express the polypeptide produced by introducing only the vector into the host cell. It can be done more accurately. (IV) Method for Detecting Cell Response As shown in (e) above, a test substance is brought into contact with a cell expressing the polypeptide of the present invention, and the activation of the polypeptide is detected using the cell response as an index. By doing so, the ligand of the polypeptide can be searched or determined. Cell responses include, for example, arachidonic acid release, acetylcholine release, intracellular Ca ²⁺ release, intracellular c
AMP production, intracellular cAMP reduction, intracellular cGMP production, inositol phosphate production, cell membrane potential fluctuation, intracellular protein phosphorylation, c-fos activation, pH decrease, cell growth activity, melanin pigment aggregation or diffusion, Or the expression level of a reporter gene can be raised (J.
Biol. Chem., 271 , 1857 (1996), Science, 268 , 98.
(1995), Journal of Pharmacology and Experimental
Therapeutics, 275 , 1274 (1995), J. Biol. Chem., 27
2 , 1822 (1997), Journalof Receptor and Signal Tran
sduction Research, 17 , 57 (1997), Endocrinology 13
8 , 1400 (1997), Endocrinology 138 , 1471 (1997), N
at.Biotechnol., 16 , 1334 (1998), Biochem. Biophys.
Res. Commun., 251 , 471 (1998), Brit. J. Pharmaco
l., 125 , 1387 (1998), Trends Biotechnol., 15 , 487
(1997), Anal.Biochem., 252 , 115 (1997), Nature, 3
58 , 325 (1992), Nature, 393 , 272 (1998), Cell, 92 ,
573 (1998), J. Biol. Chem., 272 , 27497 (1997), Tre
ndsPharmacol.Sci., 18 , 430 (1997), Trends Pharmac
ol. Sci., 20 , 370 (1999), WO98 / 46995].

When the response of a cell is monitored using a reporter system, for example, a cell expressing the polypeptide of the present invention may be appropriately placed downstream of a promoter sequence of a gene whose expression is induced by activation of the polypeptide. By introducing a DNA linked to any reporter gene, the activation of the polypeptide can be measured by the expression of the reporter gene. As the promoter, for example, IC
AM-1 gene promoter, c-fos promoter, Krox-24 promoter [Biochem. J., 3
20 , 145 (1996)]. Further, the promoter may be an artificial promoter consisting of a binding sequence of an appropriate transcription factor and a basic promoter. Examples of transcription factor binding sequences include CRE (CREB binding element),
TRE (TPA responsive element), SRE (serum re
sponsive element) can be used. Reporter genes include chloramphenicol acetyltransferase gene, β-galactosidase gene,
A lactamase gene, a luciferase gene, a green fluorescent protein gene and the like can be used.

As the cells containing the polypeptide of the present invention used in the above method, those described in the above (I) can be used.

As the cells expressing the polypeptide of the present invention, as described in the above (5), a plasmid obtained by introducing a recombinant DNA containing a DNA encoding the polypeptide into an appropriate host cell is used. Cells expressing the polypeptide in large amounts, such as transformed cells, can also be used.
Examples of the host cell include microorganisms such as Escherichia coli, Bacillus subtilis, and yeast, as well as insect cells, frog oocytes, frog melanocytes, animal cells, plant cells, and the like. In order for the polypeptide to retain its higher-order structure and retain its binding property and functionality with a ligand, it is expressed in yeast, insect cells, frog oocytes, frog melanocytes, animal cells, plant cells, etc. It is preferred that Further, a mutant strain of yeast, a yeast expressing the modified Gα protein, or the like can also be used as a host [Tren
ds in Biotechnology, 15 , 487 (1997), Mol. Cell. Bi
ol., 15 , 6188 (1995), Mol. Cell. Biol., 16 , 4700.
(1996)].

As the test substance, the substances described in the above (II) can be used.

A specific example will be described below.

The cells expressing the polypeptide of the present invention or the transformed cells expressing the polypeptide of the present invention are cultured in a multiwell plate or the like. If necessary, the medium is replaced with a fresh medium or an appropriate buffer that is not toxic to cells, and the test compound is added and incubated for a certain period of time. Thereafter, the cellular response (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-
fos activation, pH reduction, cell proliferation activity, aggregation or diffusion of melanin pigment, activity to promote or suppress reporter gene expression, etc.) are measured. For example, using a cell extract or supernatant, a product produced by the response of the cell is quantified according to each method. When the production of a substance (for example, arachidonic acid or the like) as an indicator of cell stimulating activity is difficult due to a degrading enzyme contained in a cell, an assay may be performed by adding an inhibitor against the degrading enzyme. In addition, for activities such as cAMP production suppression, the cells c
It can be detected as a production inhibitory effect on cells that have increased AMP production.

When cells containing the polypeptide of the present invention were used, cell responses were strongly detected, and when cells not expressing the polypeptide of the present invention were used, almost no cell responses were detected. Substances that are not used can be selected as ligands for the polypeptides of the invention.

In the above method, the cells containing the polypeptide of the present invention can be obtained by introducing a recombinant DNA having the DNA encoding the polypeptide into a vector into a host cell that does not express the polypeptide of the present invention. Ligand determination can be performed by using the obtained polypeptide-expressing cells and using, as cells not containing the polypeptide of the present invention, control cells that do not express the polypeptide produced by introducing only the vector into the host cell. Can be performed more accurately. (7) Kit for Determining Ligand for G Protein-Coupled Receptor Polypeptide of the Present Invention The kit for determining a ligand that binds to the polypeptide of the present invention or a salt thereof comprises the polypeptide of the present invention or a partial peptide thereof or a partial peptide thereof. It contains a salt, a cell containing the polypeptide or the partial peptide of the present invention, or a membrane fraction of a cell containing the polypeptide or the partial peptide of the present invention. Examples of the ligand determination kit of the present invention include the following. (A) Reagent for ligand determination Measurement buffer and washing buffer Hanks' Balanced Salt Solution (manufactured by Gibco Co.)
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. Preparation of polypeptide or partial peptide of the present invention CHO cells expressing the polypeptide or partial peptide of the present invention were subcultured on a 12-well plate at 5 × 10 ⁵ cells / well at 37 ° C., 5% CO ₂ 95 % Cultivated for 2 days. Labeled test compound A compound labeled with commercially available [ ³ H], [ ¹²⁵ I], [ ¹⁴ C], [ ³⁵ S], or the like, or a compound labeled by an appropriate method. An aqueous solution of the compound at 4 ° C or -20
Store at ℃ and dilute to 1 μmol / L with a measuring 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 compound as the labeled compound was prepared at a concentration 100 to 1000 times higher. (B) Measurement method CHO cells expressing the polypeptide of the present invention cultured on a 12-well tissue culture plate were mixed with 1 ml of a measurement buffer solution.
After washing twice, 490 μl of measurement buffer is added to each well. 5 μl of the labeled test compound is added and reacted at room temperature for 1 hour. To determine the amount of non-specific binding, add 5 μl of unlabeled test compound. Remove the reaction solution and wash 3 times with 1 ml of washing buffer. The labeled test compound bound to the cells was 0.2N NaOH
1% SDS, 4 ml of liquid scintillator A
(Made by Wako Pure Chemical Industries). Liquid scintillation counter (Beckman)
The radioactivity is measured using.

The ligand of the polypeptide of the present invention includes, for example, substances present in the brain, hypothalamus, pituitary gland, pancreas and the like. The ligands of the present invention include the following known ligands. Not included. Known ligands include angiotensin, bombesin, cannabinoid, cholecystokinin, glutamine, serotonin, melatonin, neuropeptide Y, opioid, purine, vasopressin, oxytocin, PACAP, secretin, glucagon, calcitonin, adrenomedullin, somatostatin, GHRH, CRF, ACTH,
Melanin stimulation hormone, GRP, PT
H, VIP, dopamine, motilin, amylin, bradykinin, CGRP, leukotriene, pancreastatin, prostaglandin, thromboxane, adenosine, adrenaline, α and β-chemokines (eg,
IL-8, GROα, GROβ, GROγ, NAP-
2, ENA-78, PF4, IP10, GCP-2, M
CP-1, HC14, MCP-3, I-309, MIP
1α, MIP-1β, RANTES, etc.), endothelin, enterogastrin, histamine, neurotensin, TRH, pancreatic polypeptide, galanin, urotensins I and II, neuropeptide F
F, orexin and melanin concentrating hormone. (8) Method for Quantifying Ligands for G Protein-Coupled Receptor Polypeptides of the Present Invention Since the polypeptides of the present invention or their partial peptides or salts thereof have binding properties to ligands, they can be used in vivo. The ligand concentration can be quantified with high sensitivity. The quantification method of the present invention can be used, for example, by combining it with a competition method. That is,
The ligand concentration in the subject can be measured by contacting the subject with the polypeptide of the present invention or a partial peptide thereof or a salt thereof. Specifically, for example, it is carried out according to a known method ("Radio Immunoassay" edited by Hiroshi Irie (Kodansha, published in 1974), "Radio Immunoassay" (edited by Hiroshi Irie) (published in Kodansha, 1974) or a method according thereto. be able to. (9) Screening method for agonist, antagonist or functional modifier of G protein-coupled receptor polypeptide of the present invention Expressing the polypeptide of the present invention or its partial peptide or a salt thereof, or the polypeptide or partial peptide of the present invention The cell or the membrane fraction of the cell is useful as a reagent for searching for an agonist, antagonist or functional modifier for the polypeptide of the present invention.

Methods for screening for agonists, antagonists or functional modifiers of the polypeptide of the present invention include, for example, (A) a method in which the polypeptide of the present invention or its partial peptide or a salt thereof is contacted with a ligand. Comparing the polypeptide of the present invention or its partial peptide or a salt thereof with a ligand and a test substance, and selecting an agonist, antagonist or function modifying substance of the polypeptide of the present invention from the test substance (B) a method in which a cell expressing the polypeptide of the present invention or a partial peptide thereof or a cell membrane fraction thereof is contacted with a ligand, and the polypeptide of the present invention or a partial peptide thereof. Expressing cells or the cell membrane fraction, ligand and test substance And (c) selecting an agonist, antagonist or function modifier of the polypeptide of the present invention from the test substance. (C) (a) to (a) of (6) above. a method characterized by using the same method as the method for searching for the ligand of the polypeptide of the present invention described in d).

The method for searching for a ligand of the polypeptide of the present invention described in (a) to (d) of (6) above is characterized by using the constitutively active mutant polypeptide described in (6). Screening methods for agonists, antagonists or functional modifiers of the polypeptide of the present invention.

More specifically, (a) the case where the labeled ligand is brought into contact with the polypeptide of the present invention or the partial peptide or a salt thereof, and the case where the labeled ligand and the test substance are brought into contact with the polypeptide of the present invention or the same. When contacted with a partial peptide or a salt thereof, the amount of a labeled ligand bound to the polypeptide or a partial peptide or a salt thereof is measured and compared, and an agonist of the polypeptide of the present invention is obtained from the labeled ligand. A method for screening for an agonist, antagonist or functional modifier of the polypeptide of the present invention, which comprises selecting an antagonist or functional modifier; (b) a labeled ligand containing the polypeptide or partial peptide of the present invention; Contacted with cells or membrane fraction of the cells When the labeled ligand and the test substance are brought into contact with the cell or the membrane fraction of the cell containing the polypeptide or partial peptide of the present invention, the amount of the labeled ligand bound to the cell or the membrane fraction Measuring and comparing the agonist, antagonist or function modifier of the polypeptide of the present invention from the labeled ligand, screening method of agonist, antagonist or function modifier of the polypeptide of the present invention, (C) When the ligand is brought into contact with the cell containing the polypeptide of the present invention or the membrane fraction of the cell, and when the ligand and the test substance are brought into contact with the cell containing the polypeptide of the present invention or the membrane fraction of the cell. Labeled GTPγS when contacted
Is measured and compared to the Gα protein (membrane fraction),
A method of screening for an agonist, antagonist or function-modifying substance of the polypeptide of the present invention, which comprises selecting an agonist, antagonist or function-modifying substance of the polypeptide of the present invention from a test substance; GTPase activity was measured in the case of contacting with a cell containing the or a membrane fraction of the cell, and in the case of contacting a ligand and a test substance with a cell containing the receptor protein of the present invention or a membrane fraction of the cell. (E) a method of screening for an agonist, antagonist or function-modifying substance of the polypeptide of the present invention, which comprises selecting an agonist, antagonist or function-modifying substance of the polypeptide of the present invention from the test substance. Ligand is added to a cell containing the polypeptide of the present invention or the cell. A cell containing the polypeptide or partial peptide of the present invention or a cell mediated by the polypeptide of the present invention in a case where the ligand and the test substance are brought into contact with the membrane fraction of the cell. Stimulation activity (eg, arachidonic acid release, acetylcholine release, intracellular Ca ²⁺ release, intracellular cAMP production, intracellular cGMP production, inositol phosphate production, cell membrane potential fluctuation, intracellular protein phosphorylation, c-fos activation ,
pH decrease, cell proliferation activity, aggregation or diffusion of melanin pigment, or activity to promote or suppress the expression level of a reporter gene, etc.). A method for screening for an agonist, antagonist or function-modifying substance of the receptor protein of the present invention, which comprises selecting an antagonist or a function-modifying substance; (f) a cell containing the polypeptide of the present invention as a test substance or the cell; GT in contact with the membrane fraction of
Measuring the amount of binding of PγS to Gα protein (membrane fraction) and selecting an agonist or functional modifier of the polypeptide of the present invention from the test substance; A method for screening a substance, (g) measuring the GTPase activity when a test substance is brought into contact with a cell containing the polypeptide of the present invention or a membrane fraction of the cell, and determining whether an agonist of the polypeptide of the present invention or A method for screening for an agonist or a function-modifying substance of the polypeptide of the present invention, which comprises selecting a function-modifying substance; and (h) a method for contacting a test substance with a cell containing the polypeptide of the present invention. Cell stimulating activity via the polypeptides or partial peptides of the invention (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-
fos activation, pH reduction, melanin pigment aggregation or diffusion, or activity to promote or suppress reporter gene expression, etc.) is measured, and an agonist or functional modifier of the polypeptide of the present invention is determined from the test substance. A method of screening for an agonist or a function modifier of the polypeptide of the present invention, characterized by selecting
(I) In the above (a) to (h), an agonist, antagonist or function-modifying substance of the polypeptide of the present invention, characterized in that a polypeptide mutated to a constitutively active form is used as the polypeptide of the present invention. Screening methods.

The substances selected in the above (a) and (b) can be obtained by using the above-mentioned methods (c) to (i)
It is possible to distinguish between antagonists or functional modifiers. On the other hand, among the substances selected by the above methods (a) to (i), substances which are not agonists or antagonists of the polypeptide of the present invention but which can modify the function of the polypeptide of the present invention (function modifying substances) ) Is also included. For example, among the substances selected by the methods (a) to (i),
It is considered that a substance that inhibits or enhances coupling between the polypeptide of the present invention and a G protein is also included. Also,
It is considered that the substances selected by the methods (c) to (i) include substances that inhibit or enhance signals downstream of the polypeptide of the present invention. These functional modifiers are also useful as drug candidates.

A detailed description of the method for screening for an agonist, antagonist or functional modifier of the polypeptide of the present invention by the above method will be given below. (I) Method for Measuring the Amount of Ligand Binding As shown in (a) and (b) above, the polypeptide or partial peptide of the present invention or a salt thereof, or the polypeptide or partial peptide of the present invention is expressed. A test substance and a labeled ligand are allowed to act on cells, and the amount of the ligand bound to the polypeptide or partial peptide of the present invention or a salt thereof is measured. Screening can be performed.

As the labeled ligand, a labeled ligand, a labeled ligand analog compound and the like can be used. For example, [ ³ H], [ ¹²⁵ I], [ ¹⁴ C], [
³⁵ S] or the like.

As the test substance, any substance can be used. For example, a known peptide, a known GPCR ligand,
Biological samples and biological samples such as known proteins, recombinant proteins produced using recombinant techniques, cell extracts and purified products derived from the extracted solutions, cell culture supernatants and purified products derived from the supernatants, serum and the like Use of a purified product derived from a microorganism, a cell extract of a microorganism, a purified product derived from the extract, a microorganism culture supernatant or a purified product derived from the supernatant, a known compound, a compound synthesized using combinatorial chemistry, or the like. Can be.

The polypeptide or partial peptide of the present invention used in the present method may be any as long as it contains the polypeptide or partial peptide, and may be a cell containing the polypeptide or partial peptide. The polypeptide or the partial peptide purified from the above may be used, or a cell containing the polypeptide or the partial peptide itself or a cell membrane fraction thereof may be used. When cells containing the polypeptide or partial peptide are used, the cells may be immobilized with glutaraldehyde, formalin, or the like.

The polypeptide or partial peptide of the present invention may be a naturally occurring polypeptide or partial peptide, or a recombinant polypeptide or recombinant partial peptide prepared by using a gene recombination technique. Was constitutively active in the polypeptide encoded by the mutated DNA obtained by introducing a mutation into the DNA having the base sequence represented by SEQ ID NO: 2 by the method described in (3). Variant polypeptides are particularly useful.

As described in (6) (I), the GPCR mutated to the constitutively active form is known to have an increased affinity with an agonist in the mutant GPCR. Constitutively active mutant GPCRs are useful in the search for agonists. Searching for an antagonist usually requires the use of a ligand, but in the case of a GPCR whose ligand is unknown (called an orphan GPCR), the ligand cannot be used. However, if a natural or mutant constitutively active GPCR is used, it is possible to search for an antagonist without a ligand. For example, an antagonist can be selected by searching for a signal that flows when a constitutively active GPCR polypeptide is overexpressed in a cell or a substance that suppresses activation of a G protein. At this time, a GPCR function modifier may be selected together with the antagonist.
In addition, by searching for a signal that flows when a constitutively active GPCR polypeptide is overexpressed in a cell or a substance that enhances activation of a G protein, an agonist or a function modifying substance can also be selected.

A number of diseases caused by mutations in GPCRs and changes to constitutively active forms are known [Japanese clinical practice, 56 , 1658 (1998), Japanese clinical practice, 56 , 1843 (1998),
Japanese clinical, 56 , 1856 (1998), Japanese clinical, 56 , 1931 (199)
8), Trends in Endocrinology and Metabolism, 9 , 27
(1998), Endocrinology, 137 , 3936 (1996)]. Antagonists capable of suppressing the activity of these constitutively active mutant GPCRs (called inverse agonists) are useful for treating diseases caused by constitutively active mutant GPCRs. Antagonists are classified into neutral antagonists and inverse agonists. Inverse agonists can suppress the activity of constitutively active GPCRs, whereas neutral antagonists cannot suppress constitutive activity. The constitutively active mutant GPCR is useful for searching for an inverse agonist.

The cell membrane fraction used in the above method can be prepared by the method described in the above (6) (I).

As the cells expressing the polypeptide or partial peptide of the present invention, as described in the above (5), a recombinant DNA containing the DNA encoding the polypeptide is introduced into an appropriate host cell. Cells expressing the polypeptide in large amounts, such as the resulting transformed cells, can also be used. Examples of the host cell include microorganisms such as Escherichia coli, Bacillus subtilis, and yeast, as well as insect cells, frog oocytes, frog melanocytes, animal cells, and plant cells. In order that the polypeptide of the present invention expressed by the transformed cell retains a higher-order structure and retains binding with a ligand, yeast, insect cells, frog oocytes, frog melanocytes, animal cells, It is preferably expressed in plant cells and the like. A mutant strain of yeast or a yeast expressing the modified Gα protein can also be used as a host [Tr
ends in Biotechnology, 15 , 487 (1997), Mol. Cell.
Biol., 15 , 6188 (1995), Mol. Cell. Biol., 16 , 4700.
(1996)].

The amount of the GPCR in the cell containing the polypeptide or the partial peptide of the present invention or the cell membrane fraction thereof is as follows:
It is preferably 10 ³ to 10 ⁸ molecules per cell, preferably 10 ⁵ to 1 molecule.
0 ⁷ a molecule of is preferable. 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. .

The following is a specific example.

A sample of the polypeptide or partial peptide of the present invention is prepared by the method described in (I) of (6) above. A test substance and a radioisotope ( ³ H, ¹²⁵ I, ¹⁴
C, ³⁵ S, ³² P, etc.) in the presence of a certain amount of radioactive ligand. A reaction tube containing a large excess of unlabeled ligand is also prepared to determine the amount of non-specific binding (NSB). The reaction is carried out at about 0-50 ° C, preferably about 4-37 ° C.
For about 20 minutes to 24 hours, preferably for about 30 minutes to 3 hours. After the reaction, the mixture is filtered through a glass fiber filter or the like and washed with an appropriate amount of the same buffer.
Measure with a counter. The count (B-NSB) obtained by subtracting the non-specific binding amount (NSB) from the total binding amount (B) is the specific binding amount. A specific binding amount of the ligand in the absence of the test substance, and comparing the specific binding amount of the ligand in the presence of the test substance, a substance that decreases the specific binding amount of the ligand, an agonist of the receptor protein of the present invention, It can be selected as an antagonist or a function modifier.

In the above method, as a cell containing the polypeptide or the partial peptide of the present invention, a host cell not expressing the polypeptide of the present invention is transformed into a vector DNA by encoding a DNA encoding the polypeptide or the partial peptide.
By using cells expressing a large amount of the polypeptide or the partial peptide obtained by introducing the recombinant DNA incorporated into the receptor, the agonist, antagonist or functional modifier of the receptor protein of the present invention can be selected with higher sensitivity. Can be. In addition, cells that do not express the polypeptide obtained by introducing only the vector into the same host cell, and other cells that are obtained by introducing an expression plasmid of another G protein-coupled receptor polypeptide into the same host cell. By performing similar experiments using cells expressing the G protein-coupled receptor polypeptide, it is possible to examine the specificity of the obtained agonist, antagonist or functional modifier for the G protein-coupled receptor polypeptide of the present invention. it can. (II) Method for measuring the amount of GTPγS bound to Gα protein As shown in (c) above, a test substance and a ligand are brought into contact with the membrane fraction of a cell containing the polypeptide of the present invention,
By measuring the amount of labeled GTPγS bound to the Gα protein (membrane fraction), it is possible to screen for agonists, antagonists or functional modifiers of the GPCR [Molecular Pharmacology, 47 , 848-854 (19)
95), WO98 / 46995].

Further, as shown in (f) above, the test substance was brought into contact with the membrane fraction of the cells containing the polypeptide of the present invention, and the amount of labeled GTPγS bound to the Gα protein (membrane fraction). By measuring the molecular weight, an agonist or a functional modifier of the polypeptide can be screened [Molecular Pharmacology, 47 , 848-854 (1992).
5), WO98 / 46995].

As a test substance, any substance can be used. For example, a known peptide, a known GPCR ligand,
Biological samples and biological samples such as known proteins, recombinant proteins produced using recombinant techniques, cell extracts and purified products derived from the extracted solutions, cell culture supernatants and purified products derived from the supernatants, serum and the like Use of a purified product derived from a microorganism, a cell extract of a microorganism, a purified product derived from the extract, a microorganism culture supernatant or a purified product derived from the supernatant, a known compound, a compound synthesized using combinatorial chemistry, or the like. Can be. GTP labeled
As γS, for example, GTPγS labeled with ³⁵ S can be used.

As the cells containing the polypeptide of the present invention or the membrane fraction of the cells, those described in the above (I) can be used.

Hereinafter, specific examples will be described.

The polypeptide preparation of the present invention was prepared using the above (6)
It is prepared by the method described in (I).

In screening for agonists, 10
A test substance, a radioactive isotope (such as ³⁵ S) -labeled GT with a certain amount of radioactivity
PγS and GDP coexist. If necessary, prepare a reaction tube to which a large excess of unlabeled GTPγS has been added in order to know the amount of non-specific binding (NSB). The count (B-NSB) obtained by subtracting the non-specific binding amount (NSB) from the total binding amount (B) is the specific binding amount. The reaction is about 0-5
The reaction is carried out at 0 ° C., preferably about 4-37 ° C., for about 20 minutes to 24 hours, preferably for about 30 minutes to 3 hours. After the reaction, the mixture is filtered with a glass fiber filter or the like, washed with an appropriate amount of the same buffer, and the radioactivity remaining on the glass fiber filter is measured with a liquid scintillation counter. A substance having an activity of enhancing the binding of GTPγS to the membrane fraction can be selected as an agonist or a function modifier of the polypeptide of the present invention.

When screening for antagonists or functional modifiers, a ligand, a test substance, a radioactive isotope (such as ³⁵ S) -labeled GTPγS, And G
A similar experiment is performed in the presence of DP. Comparing the amount of GTPγS binding in the absence of the test substance with the amount of ligand binding in the presence of the test substance, and selecting a substance that reduces the amount of GTPγS binding as an antagonist or a functional modifier of the polypeptide of the present invention. Can be. On the other hand, a substance that increases the amount of GTPγS bound can be selected as a function-modifying substance or an agonist of the polypeptide of the present invention.

As a cell containing the polypeptide of the present invention, a polypeptide obtained by introducing a recombinant DNA obtained by incorporating a DNA encoding the polypeptide into a vector into a host cell that does not express the polypeptide of the present invention. By using cells expressing a large amount of the above, an agonist, antagonist or functional modifier of the receptor protein of the present invention can be selected with higher sensitivity. In addition, cells that do not express the polypeptide obtained by introducing only the vector into the same host cell, and other cells that are produced by introducing an expression plasmid for another G protein-coupled receptor polypeptide into the same host cell. By performing the same experiment using cells expressing the G protein-coupled receptor polypeptide, it is possible to examine the specificity of the obtained agonist, antagonist or functional modifier for the G protein-coupled receptor polypeptide of the present invention. it can. (III) Method for measuring GTPase activity As shown in (d) above, a ligand and a test substance are brought into contact with a membrane fraction of a cell containing the polypeptide of the present invention,
By measuring the GTPase activity, an agonist, antagonist or functional modifier of the polypeptide can be screened [J. Biol. Che., 27
1 , 1857-1860 (1996), WO98 / 46995].

Further, as shown in the above (e), the test substance is brought into contact with the membrane fraction of the cell containing the polypeptide of the present invention, and the GTPase activity is measured to determine the agonist or function of the polypeptide. Modifiers can be screened [J. Biol. Che., 271 , 1857-18.
60 (1996), WO 98/46995].

As a test substance, any substance can be used. For example, a known peptide, a known GPCR ligand,
Biological samples and biological samples such as known proteins, recombinant proteins produced using recombinant techniques, cell extracts and purified products derived from the extracted solutions, cell culture supernatants and purified products derived from the supernatants, serum and the like Use of a purified product derived from a microorganism, a cell extract of a microorganism, a purified product derived from the extract, a microorganism culture supernatant or a purified product derived from the supernatant, a known compound, a compound synthesized using combinatorial chemistry, or the like. Can be.

As the cells containing the polypeptide of the present invention or the membrane fraction of the cells, those described in the above (I) can be used.

Hereinafter, specific examples will be described.

The polypeptide preparation of the present invention was prepared according to the above (6)
It is prepared by the method described in (I).

When screening for agonists, 10
the Myueru～10 ml of the polypeptide preparation, test compound coexist GTP radioactive isotopes (such as ³² P) constant amount of radioactivity labeled with (e.g. [γ ³² P] GTP). The reaction is carried out at about 0 to 50 ° C, preferably about 4 to 37 ° C, for about 20 minutes to 24 hours, preferably for about 30 minutes to 3 hours. After the reaction, the supernatant of the reaction solution was recovered, and the released [γ ³² P] Pi
Is measured with a liquid scintillation counter. After filtering the reaction solution with a glass fiber filter or the like and washing with an appropriate amount of the same buffer, the radioactivity in the filtrate may be measured with a liquid scintillation counter. GTPase
A substance having an activity of enhancing the activity can be selected as an agonist or a function modifier of the polypeptide of the present invention.

When screening for antagonists or functional modifiers, 10 μl to 10 ml of the polypeptide preparation is added to a ligand, a test compound, a radioactive isotope (such as ³² P), and a certain amount of radioactive GTP ( For example, [γ ³²
P] GTP) is used in the same experiment. By comparing the GTPase activity in the absence of the test substance with the GTPase activity in the presence of the test substance, a substance that decreases the GTPase activity can be selected as an antagonist or a function modifier of the polypeptide of the present invention. On the other hand, a substance that increases GTPase activity can be selected as a function modifier or an agonist of the polypeptide of the present invention.

As the cell containing the polypeptide of the present invention, a recombinant DNA obtained by introducing a DNA encoding the polypeptide into a vector is introduced into a host cell that does not express the polypeptide of the present invention. By using cells expressing a large amount of the peptide, the agonist, antagonist or functional modifier of the receptor protein of the present invention can be selected with higher sensitivity. Also,
A cell that does not express the polypeptide obtained by introducing only a vector into the host cell, or another G protein that is obtained by introducing an expression plasmid for another G protein-coupled receptor polypeptide into the host cell By performing a similar experiment using cells expressing the conjugated receptor polypeptide, the specificity of the obtained agonist, antagonist, or function modifying substance for the G protein-coupled receptor polypeptide of the present invention can be examined. (IV) Method of Detecting Cell Response As shown in (e) above, a cell expressing the polypeptide of the present invention is brought into contact with a ligand and a test substance, and the activation of the polypeptide is used as an indicator of the cell response. As a result, an agonist, antagonist or functional modifier of the polypeptide can be screened.

Further, as shown in the above (h), a test substance is brought into contact with cells expressing the polypeptide of the present invention,
By detecting the activation of the polypeptide using the response of the cell as an index, an agonist or a functional modifier of the polypeptide can be screened.

The response of the cell includes, for example, arachidonic acid release, acetylcholine release, intracellular Ca ²⁺ release, intracellular cAMP generation, intracellular cAMP reduction, intracellular cGMP
Production, inositol phosphate production, cell membrane potential fluctuations, phosphorylation of intracellular proteins, c-fos activation, pH reduction,
Cell proliferation activity, melanin pigment aggregation or diffusion, etc. are measured. The response of cells can also be monitored using a reporter system. For example, by introducing into a cell that expresses a functional polypeptide of the present invention, a DNA linked with an appropriate reporter gene downstream of the promoter sequence of a gene whose expression is induced by activation of the polypeptide, Activation of the polypeptide can be measured by expression of the reporter gene. Examples of the promoter include a promoter of ICAM-1 gene, c-
A fos promoter, a Krox-24 promoter and the like can be used. Further, the promoter may be an artificial promoter consisting of a binding sequence of an appropriate transcription factor and a basic promoter. As the binding sequence of the transcription factor, for example, CRE, TRE, SRE and the like can be used. As the reporter gene, a chloramphenicol acetyltransferase gene, a β-galactosidase gene, a β-lactamase gene, a luciferase gene, a green fluorescent protein gene and the like can be used.

As the cells expressing the polypeptide of the present invention, as described in the above (5), a plasmid obtained by introducing a recombinant DNA containing a DNA encoding the polypeptide into an appropriate host cell is used. Cells expressing the polypeptide in large amounts, such as transformed cells, can also be used. Examples of the host cell include microorganisms such as Escherichia coli, Bacillus subtilis, and yeast, as well as insect cells, frog oocytes, frog melanocytes, animal cells, and plant cells. In order that the polypeptide of the present invention expressed by the transformed cell retains a higher-order structure and retains binding and functionality with a ligand, yeast, insect cells, frog oocytes, and frog melanocytes It is preferably expressed in animal cells, plant cells and the like. A yeast mutant or a yeast expressing the modified Gα protein can also be used as a host.

As a test substance, any substance can be used. For example, a known peptide, a known GPCR ligand,
Biological samples and biological samples such as known proteins, recombinant proteins produced using recombinant techniques, cell extracts and purified products derived from the extracted solutions, cell culture supernatants and purified products derived from the supernatants, serum and the like Use of a purified product derived from a microorganism, a cell extract of a microorganism, a purified product derived from the extract, a microorganism culture supernatant or a purified product derived from the supernatant, a known compound, a compound synthesized using combinatorial chemistry, or the like. Can be.

Specific examples are shown below. Agonist screening method Cells expressing the polypeptide of the present invention are cultured in a multiwell plate or the like. If necessary, replace with a fresh medium or an appropriate buffer that is not toxic to cells, add the test substance, and incubate for a certain period of time. afterwards,
Cell response (eg, arachidonic acid release, acetylcholine release, intracellular Ca ²⁺ release, intracellular cAMP production, intracellular cGMP production, inositol phosphate production, cell membrane potential fluctuation, intracellular protein phosphorylation, c-fos activity , P
H reduction, melanin pigment aggregation or diffusion, or the activity of promoting or suppressing the expression level of a reporter gene, etc.). For example, using a cell extract or supernatant, a product produced by the response of the cell is quantified according to each method. When the production of a substance (for example, arachidonic acid or the like) as an indicator of cell stimulating activity is difficult due to a degrading enzyme contained in a cell, an assay may be performed by adding an inhibitor against the degrading enzyme. In addition, activities such as cAMP production suppression can be detected as a production suppression effect on cells whose cAMP production has been increased by forskolin or the like. A substance having an activity of enhancing a cellular response can be selected as an agonist or a function modifier of the polypeptide of the present invention. Screening for antagonists or functional modifiers Cells expressing the polypeptide of the present invention are cultured in a multiwell plate or the like. If necessary, replace the medium with a fresh medium or an appropriate buffer that is not toxic to cells, add the ligand and the test substance, and incubate for a certain period of time. Thereafter, the response of the cells is measured by the same method as described above. By comparing the cellular response in the absence of the test substance with the cellular response in the presence of the test substance, a substance that reduces the cellular response can be selected as an antagonist or a functional modifier of the polypeptide of the present invention. On the other hand, substances that increase the cellular response can be selected as functional modifiers or agonists of the polypeptide of the present invention.

A cell containing the polypeptide of the present invention
Host cells that do not express the polypeptide of the present invention.
DNA encoding the repeptide was incorporated into the vector
The size of the polypeptide obtained by introducing the recombinant DNA
The receptor protein of the present invention is obtained by using
Agonists, antagonists or functional modifiers
Can be selected with high sensitivity. In addition, the host cell
The polypeptide obtained by introducing only the vector
Cells that do not express the peptide or host cells that contain other G proteins
Introduce an expression plasmid for a coupled receptor polypeptide
G protein-coupled receptor polypeptide obtained by the method
Similar experiments were performed using peptide-expressing cells.
Acquired agonists, antagonists or functional
G protein-coupled receptor polypeptide of the present invention as a decorating substance
The specificity can be determined. (10) The G protein-coupled receptor polypeptide of the present invention
Agonists, antagonists or functional modifiers
Leaning kit Agonists, antagonists and the like of the polypeptide of the present invention.
Or a kit for screening for a functional modifier
Or a partial peptide thereof or
Or a polypeptide of the present invention or a partial peptide thereof.
A cell containing a tide-containing cell or a membrane fraction of the cell.
And so on. The screening kit of the present invention
For example, the following reagents and measurement methods can be used.
You. (A) Screening reagent Measurement buffer and washing buffer Hanks' Balanced Salt Solution (Gibco)
05% bovine serum albumin (Sigma) was added.
of. Sterilized by filtration through a 0.45 μm filter, 4 ° C
Or may be prepared at the time of use. Polypeptide preparation of the present invention CHO cells expressing the polypeptide of the present invention
5 × 10 on hole plate ^FiveSubculture at 37 ° C, 5%
CO_Two, Cultured for 2 days at 95% air. Labeled ligand^ThreeH], [¹²⁵I], [¹⁴C], [³⁵S]
Labeled ligand. Aqueous solution at 4 ° C or
Store at −20 ° C., and use 1 μmol /
Dilute to L. Ligand standard solution 0.1% bovine serum albumin (Sigma)
And dissolved at 1 mmol / L in PBS containing -20 ° C.
To save. (B) Measuring method The poly of the present invention cultured on a 12-well tissue culture plate
Peptide-expressing CHO cells were washed twice with 1 ml of measurement buffer.
After washing, 490 μl of measurement buffer was added to each well.
You. 10^-3-10^-Ten Add 5 μl of mol / L test substance solution
After adding 5 μl of labeled ligand, react for 1 hour at room temperature
Let it. To know the amount of non-specific binding
10^-3 Add 5 μl of mol / L ligand. Remove the reaction solution and wash 3 times with 1 ml of washing buffer
You. 0.2 mol / L NaO was added to the labeled ligand bound to the cells.
Dissolve in H-1% SDS, 4 ml 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. PBM = (B-NSB) ÷ B0 × 100 PMB: Percent Maximum Binding B: Value when a sample is added NSB: Non-specific Binding (non-specific binding amount) B0: Maximum binding amount The screening method of the above (9) Or book screenin
Substance or its salt obtained using the kit for
An agonist, antagonist, or
It is a function modifier. Such substances include peptides, tan
Park, non-peptide compounds, synthetic compounds, fermentation products
And these substances may be novel substances
However, the known substance may be a known substance.
Light agonists, antagonists or function modifiers
Is not included. Agonis for polypeptides of the invention
Is a ligand for the polypeptide of the present invention.
Since it has the same effect as the physiological activity,
It is useful as a safe and low toxic pharmaceutical composition
You. Conversely, antagonis to the polypeptides of the invention
Is a ligand for the polypeptide of the present invention.
Since the physiological activity can be suppressed, the ligand activity
It is useful as a safe and low toxic pharmaceutical composition
You. In addition, the functional modifier of the polypeptide of the present invention
Physiological activity of the ligand for the polypeptide of the invention
Can be enhanced or suppressed.
Safe and low toxic pharmaceutical composition to enhance or suppress
And useful.

When the compound obtained by using the screening method of the above (9) or the present screening kit or a salt thereof is used as the above pharmaceutical composition, it can be formulated into a pharmaceutical preparation according to a conventional method. For example, tablets, capsules, elixirs, microcapsules, and the like, optionally coated with sugar, aseptic, or aseptic solution or suspension in water or other pharmaceutically acceptable liquids It can be used parenterally in the form of injections.
For example, the compound or a salt thereof is mixed with physiologically acceptable carriers, flavors, excipients, vehicles, preservatives, stabilizers, binders and the like in a unit dosage form required for generally accepted pharmaceutical practice. Can be manufactured. The amount of the active ingredient in these preparations is such that an appropriate dose in the specified range can be obtained. Examples of additives that can be mixed with tablets, capsules, and the like include binders such as gelatin, corn starch, tragacanth, and acacia, excipients such as crystalline cellulose, corn starch, gelatin, and alginic acid. Useful bulking agents, lubricants such as magnesium stearate, sweeteners such as sucrose, lactose or saccharin, flavoring agents such as peppermint, reddish oil or cherry and the like are used. When the preparation unit form is a capsule, a liquid carrier such as oil and fat can be further contained in the above-mentioned type of material. Sterile compositions for injection include the active substance in a vehicle such as water for injection, sesame oil,
It can be formulated in accordance with normal pharmaceutical practice such as dissolving or suspending naturally occurring vegetable oils such as coconut oil.

As the aqueous solution for injection, for example, physiological saline, isotonic solution containing glucose and other adjuvants (eg, D-sorbitol, D-mannitol, sodium chloride, etc.) and the like are used. It may be used in combination with a solubilizing agent, for example, an alcohol (for example, ethanol), a polyalcohol (for example, propylene glycol, polyethylene glycol), or a nonionic surfactant (for example, polysorbate 80 (TM) or HCO-50). Good. As the oily liquid, for example, sesame oil, soybean oil and the like are used, and may be used in combination with solubilizers such as benzyl benzoate and benzyl alcohol. 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.), storage Agents (eg, benzyl alcohol, phenol, etc.), antioxidants and the like. The prepared injection is usually filled in a suitable ampoule. Since the thus obtained preparation is safe and has low toxicity, it can be administered to, for example, humans and mammals (eg, rats, rabbits, sheep, pigs, cows, cats, dogs, monkeys, etc.). . The dose of the compound or a salt thereof varies depending on the administration subject, the target organ, the condition, the administration method, and the like. However, in the case of oral administration, generally, in an adult (60 kg), about 0.1 per day is used.
-100 mg, preferably about 1.0-50 mg, more preferably about 1.0-20 mg. In the case of parenteral administration, the single dose varies depending on the administration subject, target organ, symptoms, administration method and the like. About 0.01 to 30 mg, preferably about 0.1
Conveniently, about 20 mg, more preferably about 0.1 to 10 mg is administered by intravenous injection. In the case of other animals, the dose can be administered in terms of 60 kg. (11) Use of DNA or Oligonucleotide of the Present Invention for Treatment or Diagnosis of Disease Using DNA or Oligonucleotide of the Present Invention ~
(V). (I) Acquisition of a chromosomal gene encoding the polypeptide of the present invention and an expression control region of the gene Using the DNA or oligonucleotide of the present invention as a probe, a known method [Cancer Research Division, Institute of Medical Science, The University of Tokyo; Cell Engineering Experiment Protocol, Shujunsha (1993
Year)] can be used to obtain a chromosomal gene encoding the polypeptide of the present invention and an expression control region of the gene.

The human cDNA encoding the polypeptide of the present invention can also be compared by comparing the sequence of the human cDNA encoding the polypeptide of the present invention with the sequence of the human chromosomal gene registered in a public database. Chromosomal genes could be identified and their structure revealed. If a chromosomal gene sequence that matches the cDNA sequence is registered, the exon and intron structures of the chromosomal gene encoding the polypeptide of the present invention are compared by comparing the sequence of the chromosomal gene with the sequence of the DNA of the present invention, and The expression control region (eg, promoter region, etc.) of the chromosomal gene can be determined.

Examples of the promoter region include all promoter regions involved in transcription of a gene encoding the polypeptide of the present invention in mammalian cells.
Specifically, for example, in the human pituitary gland, human hippocampus, human pancreas, human lung, human thymus, human colorectal cancer cells, human pancreatic cancer cells or human prostate cancer cells, the gene encoding the polypeptide of the present invention, Examples include a promoter region involved in transcription.

As shown in the following (14), the promoter region is useful for screening for a substance that controls the transcription of a gene encoding the polypeptide of the present invention. Further, using the sequence information of the promoter region, a decoy DNA [Nippon Rinsho-Japanese Journal o] for suppressing transcription of a gene encoding the polypeptide of the present invention.
f Clinical Medicine, 56 , 563 (1998), Circulation R
esearch, 82 , 1023 (1998), Experimental Nephrology,
5 , 429 (1997), Nippon Rinsho-Japanese Journal o
f Clinical Medicine, 54 , 2583 (1996)]. (II) Measurement of Transcript Amount of the Gene Encoding the G Protein-Coupled Receptor Polypeptide of the Present Invention Northern Hybridization (Molecular Cloning 2nd Edition), PCR [PCR Protocols, Acade
mic Press (1990)), Real Time PCR (JunkoStevens,
The amount of the transcript of the gene encoding the polypeptide of the present invention can be measured by a method such as Experimental Medicine (Extra Number), 15 , 46-51 (1997)]. In particular, quantitative PCR (Proc. Natl. Acad. Sci. USA, 87 , 2725 (1990)) and R
The eal Time PCR method is a preferable method because of its excellent quantitative property. By quantifying the transcript, it is possible to diagnose a disease based on abnormal expression of the polypeptide of the present invention. Therefore, the DNA or oligonucleotide of the present invention is useful as a genetic diagnostic agent for quantifying a transcript of a gene encoding the polypeptide of the present invention. (III) Detection of Mutations and Polymorphisms in the Gene Encoding the G Protein-Coupled Receptor Polypeptide of the Present Invention It is known that mutations and polymorphisms exist in the GPCR gene or the expression control region of the GPCR gene. For example,
It is known that the mutation of the GPCR gene inactivates or activates the function of the GPCR and causes various diseases [Japanese clinical, 56 , 1658 (1998), Japanese clinical, 56 , 1836.
(1998), Japanese clinical practice, 56 , 1843 (1998), Japanese clinical practice, 56 , 1
848 (1998), Japanese clinical study, 56 , 1856 (1998), Japanese clinical study, 5
6 , 1871 (1998), Japanese clinical practice, 56 , 1876 (1998), Japanese clinical practice, 56 , 1931 (1998), Trends in Endocrinology and Me
tabolism, 9 , 27 (1998)]. In addition, GPCR gene or GPCR gene expression control region mutation
Expression level increases or decreases, and various diseases may occur. Therefore, by examining the mutation of the gene encoding the polypeptide of the present invention or the expression control region of the gene, inactivation or activation of the function of the receptor protein of the present invention, or expression of the polypeptide of the present invention, Diagnosis of a disease based on an increase or decrease can be made.

In addition, depending on the polymorphism of the GPCR gene or the expression control region of the GPCR gene,
It is known that the expression level of the disease changes, and the disease incidence rate and progression rate differ (Cancer. Res., 59 , 3561 (1999), A
nnu. Rev. Immunol., 17 , 657 (1999), Japanese clinical practice, 56 ,
1871 (1998)]. For example, the beta ₃ adrenergic receptor 64
It is known that a person having the mutant receptor protein in which the second tryptophan is substituted with arginine has a high incidence of obesity and diabetes. Therefore, by examining the mutation of the gene encoding the G protein-coupled receptor polypeptide of the present invention or the expression control region of the gene, the disease incidence rate based on changes in the properties and expression level of the polypeptide of the present invention can be improved. A prediction of the speed of progress can be made.

Many of the drugs currently used are G
Although it is targeted for PCR, the GPR gene and the polymorphism of the expression control region of the GPCR gene may cause
It is known that the properties of CR and the expression level of GPCR change, and the sensitivity to drugs changes [Journal of Pharmac.
olgy and Experimental Therapeutics, 275 , 1274 (199
5), J. Bilo. Chem., 272 , 1822 (1997), Pharmacogene
tics, 5 , 318 (1995), J. Bilo. Chem., 274 , 12670 (1
Natl. Acad. Sci. USA, 95 , 9608 (199).
8), Science, 286 , 487 (1999)]. Therefore, by examining the mutation of the gene encoding the polypeptide of the present invention or the expression control region of the gene, it is possible to predict drug sensitivity based on changes in the properties and expression level of the polypeptide of the present invention.

Detection of mutations or polymorphisms in the gene encoding the polypeptide of the present invention or the expression control region of the gene can be performed using the nucleotide sequence information of the gene or the control region. Specifically, gene mutations and polymorphisms can be detected by Southern blotting, direct sequencing, PCR, DNA chip method, etc. [Clinical test, 42 , 1507-1517 (1998), clinical test , 42 , 15
65-1570 (1998)].

By using a DNA encoding the polypeptide of the present invention or a partial peptide thereof and a DNA having a nucleotide sequence in the expression control region of a gene encoding the polypeptide of the present invention as a probe or a primer, Alternatively, it is possible to detect a gene encoding the polypeptide of the present invention in a mammal (eg, rat, rabbit, sheep, pig, cow, cat, dog, monkey, etc.) and a mutation or polymorphism in the expression control region of the gene. it can. Therefore, a DNA encoding the polypeptide of the present invention or a partial peptide thereof, and a DNA having a nucleotide sequence in the expression control region of the gene encoding the polypeptide of the present invention may be a gene for detecting the above mutation or polymorphism. Useful as a diagnostic agent. (IV) A therapeutic agent for a disease in which the expression level or function of the G protein-coupled receptor polypeptide of the present invention is enhanced. Antisense RNA / DNA technology [Bioscience and Industry, 50 , 322 (1992), Chemistry, 46 , 681]. (199
1), Biotechnology, 9 , 358 (1992), Trends in Biotec
hnology, 10 , 87 (1992), Trends in Biotechnology,
10 , 152 (1992), Cell Engineering, 16 , 1463 (1997)), Triple Helix Technology (Trends in Biotechnology, 10 ,
132 (1992)), ribozyme technology (Current Opinion in
ChemicalBiology, 3 , 274 (1999), FEMS Microbiology
Reviews, 23 , 257 (1999), Frontiers in Bioscience,
4 , D497 (1999), Chemistry & Biology, 6 , R33 (199
9), Nucleic Acids Research, 26 , 5237 (1998), Trend
s In Biotechnology, 16 , 438 (1998)) or decoy DNA method [Nippon Rinsho-Japanese Journal of Cli
nical Medicine, 56 , 563 (1998), Circulation Resear
ch, 82 , 1023 (1998), Experimental Nephrology, 5 , 4
29 (1997), Nippon Rinsho-Japanese Journal of Cli
nical Medicine, 54 , 2583 (1996)].

For example, the DN of the present invention described in the above (2)
A, using an oligonucleotide or a derivative thereof, suppresses transcription of a DNA encoding the polypeptide of the present invention;
Alternatively, translation of a transcript encoding the polypeptide of the present invention can be suppressed. That is, by administering the DNA, oligonucleotide or derivative thereof of the present invention, production of the polypeptide of the present invention can be suppressed.

For example, when there is a patient whose receptor-mediated physiological action is enhanced due to increased expression of the polypeptide of the present invention or increased expression of a ligand of the polypeptide,
The physiological action can be suppressed by administering the DNA, oligonucleotide or derivative thereof of the present invention to a patient. That is, the DNA, oligonucleotide, or derivative thereof of the present invention is useful as a therapeutic agent for a disease in which the expression level or function of the polypeptide of the present invention is enhanced.

When the DNA, oligonucleotide or derivative thereof of the present invention is used as the above therapeutic agent, the DNA, oligonucleotide or derivative thereof of the present invention may be used alone or in a retrovirus vector, adenovirus vector, adenovirus associated virus vector. After inserting into an appropriate vector such as (10)
Can be formulated, formulated and administered according to the usual methods described in (1). (V) Gene preventive / therapeutic agent for diseases in which the expression level or function of the G protein-coupled receptor polypeptide of the present invention is reduced. The DNA encoding the polypeptide of the present invention is expressed in the amount or function of the polypeptide of the present invention. Can be used as a medicament such as a gene preventive / therapeutic agent for a disease in which is decreased. For example, when there is a patient who cannot expect the physiological action of the ligand due to a decrease in the expression or mutation of the polypeptide of the present invention, (i) DNA encoding the polypeptide of the present invention
Is administered to the patient and expressed, or (ii) after inserting and expressing the DNA encoding the polypeptide of the present invention in cells of interest and transplanting the cells into the patient, etc. Can increase the amount of the polypeptide of the present invention in the body of the subject and sufficiently exert the action of the ligand. Therefore, the DNA encoding the polypeptide of the present invention is useful as a safe and low-toxic gene preventive / therapeutic agent for diseases in which the expression level or function of the polypeptide of the present invention is reduced.

DNA encoding the polypeptide of the present invention
When used as the above prophylactic / therapeutic agent, the D of the present invention
NA can be formulated alone, or inserted into an appropriate vector such as a retrovirus vector, an adenovirus vector, or an adenovirus associated virus vector, and then formulated, formulated, and administered according to the conventional method described in (IV) above.

(12) Production of Antibody Recognizing the Polypeptide of the Present Invention (I) Preparation of Polyclonal Antibody The polypeptide or partial peptide obtained by the method described in (5) above is used as an antigen and administered to animals. Thus, a polyclonal antibody can be prepared.

Animals to which the antigen is administered include rabbits,
Goats, rats, mice, hamsters, etc., 3 to 20 weeks old can be mentioned. The dose of the antigen is preferably 50 to 100 μg per animal. When a peptide is used as an antigen, the peptide is used as keyhole limpet hemocyanin
(ole limpet haemocyanin) or bovine thyroglobulin, which is covalently bonded to a carrier protein. A peptide serving as an antigen can also be synthesized by a peptide synthesizer.

The administration of the antigen is performed 1-2 times after the first administration.
Perform 3 to 10 times every week. Blood is collected from the fundus venous plexus 3 to 7 days after each administration, and the reaction of the serum with the antigen used for immunization is determined by enzyme immunoassay [enzyme immunoassay (ELIS)].
A method): 1976, published by Medical Shoin, Antibodies, A Labora
tory Manual, Cold Spring Harbor Laboratory (198
8)] and so on.

[0205] A serum is obtained from the above animal whose serum shows a sufficient antibody titer against the antigen used for immunization, and the serum is separated and purified to obtain a polyclonal antibody.

Methods for separation and purification include centrifugation,
Salting out with 40-50% saturated ammonium sulfate, caprylic acid precipitation [Antibodies, A Laboratory manual, Cold Spr.
ingHarbor Laboratory, (1988)] or DEAE-Sepharose column, anion exchange column, protein A
Alternatively, there is a method in which chromatography using a G-column or a gel filtration column or the like is performed alone or in combination. (II) Preparation of Monoclonal Antibody (a) Preparation of Antibody-Producing Cells The above animal whose serum showed a sufficient antibody titer against the partial peptide of the polypeptide of the present invention used for immunization was used as a source of antibody-producing cells. Can be served as

The spleen is excised 3 to 7 days after the final administration of the antigenic substance to the above animal showing the antibody titer. The spleen is shredded in a MEM medium (manufactured by Nissui Pharmaceutical Co., Ltd.), loosened with forceps, centrifuged at 1,200 rpm for 5 minutes, and the supernatant is discarded. The spleen cells in the obtained precipitate fraction are treated with a Tris-ammonium chloride buffer (pH 7.65) for 1 to 2 minutes to remove erythrocytes, and then washed three times with a MEM medium. Use as cells. (b) Preparation of myeloma cells As myeloma cells, cell lines obtained from mice or rats are used.

For example, 8-azaguanine-resistant mice (BA
LB / c-derived) myeloma cell line P3-X63Ag8-U1 (hereinafter P3-U
1) [Curr. Topics. Microbiol. Immunol., 81 ,
1 (1978), Europ. J. Immunol., 6 , 511 (1976)), SP2
/ 0-Ag14 (SP-2 ) [Nature, 276, 269 (1978)], P3-X63-Ag
8653 (653) [J. Immunol., 123 , 1548 (1979)], P3-X63
-Ag8 (X63) [Nature, 256 , 495 (1975)] or the like can be used.

These cell lines were cultured in an 8-azaguanine medium [glutamine (1.5 mmol) in RPMI-1640 medium.
/ L), 2-mercaptoethanol (5 × 10 ⁻⁵ mmol /
L), gentamicin (10 μg / ml) and fetal calf serum (FCS) (manufactured by CSL, 10%) (8% azaguanine (15 μg / ml) were further added to a medium (hereinafter referred to as a normal medium). Medium), and cultured in a normal medium 3 to 4 days before cell fusion, and use 2 × 10 ⁷ or more of the cells for fusion. (c) Preparation of hybridoma The antibody-producing cells obtained in (a) and the myeloma cells obtained in (b) were cultured in MEM medium or PBS (disodium phosphate 1.
83 g, monopotassium phosphate 0.21 g, salt 7.65
g, 1 liter of distilled water, pH 7.2)
The cells are mixed so that the number of cells becomes antibody-producing cells: myeloma cells = 5 to 10: 1, centrifuged at 1,200 rpm for 5 minutes, and the supernatant is discarded.

[0210] The cells of the obtained precipitate fraction were well loosened,
To the cell group, while stirring, at 37 ° C., 10 ⁸ antibody-producing cells per polyethylene of glycol -1000 (P
EG-1000), 0.2 to 1 ml of a mixed solution of 2 g of MEM, 2 ml of MEM and 0.7 ml of dimethylsulfoxide (DMSO) is added, and 1-2 ml of MEM medium is added several times every 1 to 2 minutes. After the addition, a MEM medium is added to adjust the total volume to 50 ml.

After centrifuging the preparation at 900 rpm for 5 minutes, the supernatant is discarded. The cells of the obtained precipitate fraction are loosened loosely, and then slowly sucked and blown out with a mesipette into a HAT medium [hypoxanthine (1 in normal medium).
0 ^-4 mmol / L), thymidine (1.5 × 10 ^-5 mmol / L)
Medium supplemented with aminopterin (4 × 10 ⁻⁷ M)
Suspend in 100 ml.

The suspension was added to a 96-well culture plate in an amount of 100
aliquots per well, in a 5% CO ₂ incubator,
Incubate at 37 ° C for 7-14 days. After culturing, a part of the culture supernatant is removed and used for antibodies (Antibodies, A Laboratory
manual, Cold Spring Harbor Laboratory, Chapter 14
(1988)).
A hybridoma that specifically reacts with a partial peptide of the polypeptide of the present invention is selected.

Specific examples of the enzyme immunoassay include the following methods. In the case of immunization, a partial peptide of the polypeptide of the present invention used as an antigen is coated on an appropriate plate, and the hybridoma culture supernatant or After reacting the purified antibody obtained in (d) below as a first antibody, and further reacting an anti-rat or anti-mouse immunoglobulin antibody labeled with biotin, an enzyme, a chemiluminescent substance or a radioactive compound as a second antibody A reaction according to the labeling substance is performed, and a substance that specifically reacts with the polypeptide of the present invention is selected as a hybridoma that produces a monoclonal antibody against the polypeptide of the present invention.

Using the hybridoma, cloning was repeated twice by the limiting dilution method (the first time, an HT medium (a medium obtained by removing aminopterin from the HAT medium), and the second time, a normal medium was used). Those having a high antibody titer are selected as hybridoma strains producing anti-polypeptide antibody of the polypeptide of the present invention. (d) Preparation of Monoclonal Antibody To a mouse or a nude mouse of 8 to 10 weeks old, which was treated with pristane (0.5 ml of 2,6,10,14-tetramethylpentadecane (Pristane) was intraperitoneally administered and bred for 2 weeks). , 5-20 hybridoma cells producing the polypeptide monoclonal antibody of the present invention obtained in (c)
× 10 ⁶ cells / mouse are injected intraperitoneally. In 10 to 21 days, the hybridoma becomes ascites cancer.

[0215] Ascites was collected from the mouse with ascites cancer,
Centrifuge at 3,000 rpm for 5 minutes to remove solids. From the obtained supernatant, a monoclonal antibody can be purified and obtained by a method similar to the method used for polyclonal.

The subclass of the antibody is determined using a mouse monoclonal antibody typing kit or a rat monoclonal antibody typing kit. The protein content is
It is calculated from the Lowry method or the absorbance at 280 nm. (13) Use of the Antibody of the Present Invention (a) Immunological Detection and Quantification of the Polypeptide of the Present Invention Using the Antibody of the Present Invention As a method for immunological detection of the polypeptide of the present invention, an ELISA using a microtiter plate is used. law, fluorescent antibody method, Western blot, immunohistochemical staining [Supplement experimental medicine, The protocol Series, immunostaining · in sit
u hybridization, YODOSHA (1997), Journal o
f Immunological Methods, 150 , 5, (1992)].

As an immunological quantification method, a sandwich E using two kinds of monoclonal antibodies having different epitopes among antibodies reacting with the polypeptide of the present invention in the liquid phase was used.
LISA, radioimmunoassay using the polypeptide of the present invention labeled with a radioisotope such as ¹²⁶ I, and an antibody recognizing the polypeptide of the present invention, and the like.

The above detection or quantification method can be used for diagnosis of colon cancer, pancreatic cancer, lung cancer, prostate cancer and the like.
In addition, cells or cell lines that express the polypeptide of the present invention can be identified using the above detection or quantification methods. Cells or cell lines that express the polypeptide of the invention are
It is useful for searching for a ligand, agonist or antagonist of the polypeptide and for analyzing the function of the polypeptide. (B) Medicine containing the antibody of the present invention The antibody of the present invention is a medicine, for example, colon cancer, pancreatic cancer, lung cancer,
It can be used as a therapeutic drug for diseases such as prostate cancer.

The pharmaceutical containing the antibody of the present invention can be administered alone as a therapeutic agent, but is usually mixed with one or more pharmacologically acceptable carriers. It is desirable to provide it as a pharmaceutical preparation manufactured by any method well known in the field of pharmaceutical science.

It is desirable to use the most effective route for treatment.
Parenteral administration, such as in the respiratory tract, rectally, subcutaneously, intramuscularly and intravenously, can be mentioned. Administration forms include sprays, capsules, tablets, granules, syrups, emulsions, suppositories, injections, ointments, tapes and the like.

Formulations suitable for oral administration include emulsions, syrups, capsules, tablets, powders, granules and the like. Liquid preparations such as emulsions and syrups include water, sugars such as sucrose, sorbitol, fructose, glycols such as polyethylene glycol, propylene glycol, oils such as sesame oil, olive oil, soybean oil, p-hydroxybenzoate. It can be produced using preservatives such as acid esters and flavors such as strawberry flavor and peppermint as additives. Capsules, tablets, powders, granules, etc. are excipients such as lactose, glucose, sucrose, mannitol, disintegrants such as starch, sodium alginate, lubricants such as magnesium stearate, talc, polyvinyl alcohol, hydroxy It can be produced using a binder such as propylcellulose and gelatin, a surfactant such as fatty acid ester, and a plasticizer such as glycerin as additives.

Preparations suitable for parenteral administration include injections, suppositories, sprays and the like. For example, injections
It is prepared using a carrier comprising a salt solution, a glucose solution, or a mixture of both. Suppositories are prepared using carriers such as cocoa butter, hydrogenated fats or carboxylic acids. Also,
Sprays are prepared using the compound itself or a carrier which does not irritate the oral and respiratory tract mucosa of the recipient and which disperses the compound as fine particles to facilitate absorption. Specific examples of the carrier include lactose and glycerin. Formulations such as aerosols and dry powders are possible depending on the properties of the compound and the carrier used. Also, in these parenteral preparations, the components exemplified as additives in the oral preparation can be added.

The dosage or number of administrations varies depending on the intended therapeutic effect, administration method, treatment period, age, body weight, etc., but is usually from 10 μg / kg to 8 mg / kg per adult per day.
It is. (14) A method for screening a substance that controls transcription or translation of a gene encoding a G protein-coupled receptor polypeptide of the present invention.

A compound that promotes or suppresses the process of transcription of the gene encoding the polypeptide of the present invention or the process of translating a transcript into a protein can control the expression of the polypeptide, thereby controlling the expression of the polypeptide. It is possible to control the cell functions exerted. Therefore, a compound that promotes or suppresses the transcription process of a gene encoding the polypeptide of the present invention or the translation process from a transcript to a protein can enhance or suppress the physiological activity of the ligand for the polypeptide of the present invention. Therefore, it is useful as a safe and low toxic pharmaceutical composition that enhances or suppresses the activity of the ligand.

The compound can be obtained by the following methods (a) to (e). (A) [i] a cell expressing the polypeptide of the present invention;
[Ii] In the presence of the test substance, cells expressing the polypeptide of the present invention are cultured for 2 hours to 1 hour by the culture method described in (5) above.
After culturing for a week, the amount of the polypeptide in the cells is determined according to the above (1).
A method of measuring and comparing using the antibody of the present invention described in 2), and selecting and obtaining a compound having an activity of increasing or decreasing the amount of the polypeptide.

As a measuring method using the antibody of the present invention, for example, an ELISA using a microtiter plate is used.
Method, fluorescent antibody method, Western blot method, immunohistochemical staining and the like. (B) [i] cells expressing the polypeptide of the present invention;
[Ii] In the presence of the test substance, cells expressing the polypeptide of the present invention are cultured for 2 hours to 1 hour by the culture method described in (5) above.
After weekly culture, DN encoding the polypeptide in the cells
The amount of the transcript of A is measured and compared using a method such as the Northern hybridization method or the PCR method described in (11) above, and a compound having an activity of increasing or decreasing the amount of the transcript is selected and obtained. Method. (C) A plasmid in which a reporter gene-linked DNA has been incorporated downstream of the promoter obtained in (11) above is prepared by a known method, and introduced into animal cells according to the method described in (5) above. Obtain a transformant.
[I] In the presence of the transformant and [ii] a test substance, the transformant is cultured for 2 hours to 1 hour by the culture method described in the above (5).
After culturing for a week, the expression level of the reporter gene in the cells can be determined by a known method (New Cancer Engineering Protocol, Shujunsha (1993), Biotechniques, Ed.
20 , 914 (1996), J. Antibiotics, 49 , 453 (1996),
A method for selecting and obtaining a compound having an activity of increasing or decreasing the expression level by measuring and comparing the results using Trendsin Biochemical Sciences, 20 , 448 (1995), Cell Engineering, 16 , 581 (1997)].

Examples of the reporter gene include a chloramphenicol acetyltransferase gene, a β-galactosidase gene, a β-lactamase gene, a luciferase gene, a green fluorescent protein (GFP) gene and the like. (15) Preparation of an animal deficient or substituted for the DNA of the present invention An animal deficient or substituted for the DNA of the present invention has all or a part of the gene containing the DNA of the present invention deleted or substituted, An animal in which the expression level of the polypeptide has changed. The animal, or an organ, tissue or cell of the animal is used for evaluation of a medicament obtained by the above method, for example, a therapeutic drug for a disease such as colon cancer, pancreatic cancer, lung cancer, prostate cancer or pituitary abnormality. It is useful as a drug evaluation model animal, organ, tissue or cell.

As the animal, a vector containing the DNA of the present invention can be used to produce an animal of interest, for example, cow, sheep, goat,
Pigs, horses, chickens, embryonic stem cells of non-human mammals such as mice (embryonic stem cells), a DNA encoding the polypeptide of the present invention on the chromosome in a known homologous recombination technique (for example, Nature, 326 , 295 (1987), Cell,
51 , 3, 503 (1987), etc.] to produce a mutant embryonic stem cell clone inactivated or substituted with an arbitrary sequence [Nature, 350 , 243 (1991)].

Using the embryonic stem cell clone thus prepared, chimera consisting of embryonic stem cell clone and normal cells by a technique such as injection chimera method or collective chimera method of injecting fertilized eggs of animals into blastcysts. Individuals can be created. By crossing the chimeric individual with a normal individual, it is possible to obtain an individual having an arbitrary mutation in the DNA encoding the polypeptide of the present invention on the chromosome of whole body cells. A homozygous individual (knockout animal) having both mutations can be obtained.

[0230] In this manner, in an animal individual, a mutation can be introduced into the chromosome at any position of the DNA encoding the polypeptide of the present invention. For example, by introducing a mutation such as deletion, substitution or addition of a base into the translation region of the DNA encoding the polypeptide of the present invention on the chromosome, the activity of the product can be changed.

By introducing similar mutations into the expression control region, the degree, timing, tissue specificity, etc. of expression can be altered. Furthermore, by combining with the Cre-loxP system, the expression timing, expression site, expression amount, and the like can be more positively controlled.

[0232] Examples of such a case include the use of a promoter expressed in a specific region of the brain and deletion of the target gene only in that region [Cell, 87 , 1317 (1996)] and C
Using adenovirus expressing re, at the desired time,
An organ-specific deletion of a target gene [Science, 27
8 , 5335 (1997)].

Thus, the expression of the DNA encoding the polypeptide of the present invention on the chromosome can be controlled at any time or in any tissue, or the deletion, substitution or addition of any base can be controlled by its translation region or expression. It is possible to create an animal individual having the control region.

Such an animal can induce various diseases caused by the polypeptide of the present invention, for example, symptoms such as cancer and pituitary abnormality, at any time, at any degree, or at any site.

Therefore, the knockout animal of the present invention becomes an extremely useful animal model in the treatment and prevention of various diseases caused by the polypeptide of the present invention, for example, cancer and pituitary abnormality. In particular, it is very useful as a therapeutic drug, a preventive drug, and a model for evaluating functional foods, health foods and the like.

[0236]

EXAMPLES The methods described in the following Examples were carried out according to the method described in Molecular Cloning, Second Edition, unless otherwise specified. Example 1 A novel G protein-coupled receptor polypeptide (P
Cloning of cDNA encoding GMO334 polypeptide) (1) Preparation of expression vector pAMo-d Expression vector p for preparing a cDNA library
AMo-d was produced by the method shown below. (I) Construction of plasmid pAMo-nd '(see FIG. 11) pAMo [J. Biol. Chem., 268 , 22782 (1993), aka p
AMoPRC3Sc (Japanese Unexamined Patent Publication No. 05-336963)]
After digestion with HindIII and Asp 718, 8.7 kb
It has acquired the Hin dIII- Asp 718 fragment. Also,
After cutting the pAMo with restriction enzyme Not I, by partial cleavage with Bam HI, 2.5 kb of Bam HI- No
The tI fragment was obtained.

[0237] To produce a linker connecting Hin dIII cleavage site and Bam HI cleavage site, the DNA. DNA of SEQ ID NO: 4 SEQ ID NO: 3 according to the four synthetic DNA (SEQ ID NO: 3-6 Base number 5 of the described DNA
(SEQ ID NO: 6 has a complementary base sequence to the base sequence represented by base numbers 5 to 26 of the DNA described in SEQ ID NO: 5). Was synthesized. Among them, the DNA having the nucleotide sequence represented by SEQ ID NO: 4 or SEQ ID NO: 5 was phosphorylated using T4 polynucleotide kinase. Next, the synthetic DNA of SEQ ID NO: 3 and the phosphorylated DNA of SEQ ID NO: 4 were mixed and annealed to obtain double-stranded DNA. Also, by mixing and annealing the synthetic DNA of SEQ ID NO: 6 and the phosphorylated DNA of SEQ ID NO: 5, double-stranded D
NA.

In order to prepare a linker connecting the Not I cleavage site and the Asp 718 cleavage site, four kinds of synthetic DNAs (DNAs of SEQ ID NOs: 7 to 10; DNA of SEQ ID NO: 8 corresponds to SEQ ID NO: 8) 7. having a complementary base sequence to the base sequence represented by base numbers 5 to 44 of the DNA according to 7,
The DNA of SEQ ID NO: 10 is the DN of SEQ ID NO: 9.
A having a complementary base sequence to the base sequence represented by base numbers 6 to 34 of A). Among them, the DNA having the nucleotide sequence represented by SEQ ID NO: 8 or SEQ ID NO: 9 was phosphorylated using T4 polynucleotide kinase. Next, the synthetic DNA of SEQ ID NO: 7 and the phosphorylated DNA of SEQ ID NO: 8 were mixed and annealed to obtain double-stranded DNA. Also, by mixing and annealing the synthetic DNA of SEQ ID NO: 10 and the phosphorylated DNA of SEQ ID NO: 9,
Double-stranded DNA was used.

[0239] Hin of 8.7kb from pAMo dII
I- Asp 718 fragment, 2.5 kb B from pAMo
The ligation of the am HI- Not I fragment and the four double-stranded DNAs prepared above resulted in plasmid pAM.
o-nd 'was created. (Ii) Construction of plasmid pAMo-nd (see FIG. 11) pAMo-nd ′ constructed in (i) above was replaced with restriction enzyme Sf
cleaved with i I and Xho I, the 6.0 kb Sfi I- Xh
The oI fragment was obtained. After cutting pAMo-nd 'with restriction enzymes Xho I and Nru I, 4.0 kb Xho I
-The Nru I fragment was obtained. After pAMo-nd ′ was cleaved with restriction enzymes Nru I and Sfi I, a 1.4 kb N
The ru I- Sfi I fragment was obtained. By ligating the above three fragments, a plasmid pAMo-nd was constructed. (Iii) Construction of plasmid pAMo-d (see FIG. 12) pAMo was replaced with restriction enzymes Xho I, Asp 718 and Bam H.
After digestion with I, a 5.9 kb Xho I- Asp 718 fragment was obtained. Also, the pAMo-n created in the above (ii)
d is cleaved with restriction enzymes ScaI and XhoI and then partially cleaved with SfiI to obtain a 5.4 kb XhoI- Sf
The iI fragment was obtained.

In order to prepare a linker connecting the Sfi I cleavage site and the Asp 718 cleavage site, DNA having the nucleotide sequences represented by SEQ ID NOS: 11 and 12 was synthesized (the DNA described in SEQ ID NO: 12 was No. 11). Among them, SEQ ID NO: 1
The DNA described in No. 2 was phosphorylated using T4 polynucleotide kinase. Next, the synthetic DNA of SEQ ID NO: 11
And phosphorylated DNA of SEQ ID NO: 12 were mixed and annealed to obtain double-stranded DNA.

[0241] of 5.9kb from pAMo Xho I- A
sp 718 fragment, X of 5.4kb from pAMo-nd
By combining the ho I- Sfi I fragment, the double-stranded DNA described above, and the double-stranded DNA prepared from the synthetic DNA of SEQ ID NO: 10 and the phosphorylated DNA of SEQ ID NO: 9 in (i) above, plasmid pAMo was obtained. -D was created. (2) Preparation of cDNA library derived from human pituitary gland Poly (A) ⁺ RNA derived from human pituitary (Clontech)
Oligo (dT) -SfiI primer (DNA having the base sequence of SEQ ID NO: 13)
3.2 μg was added, and distilled water was added to make 6.8 μl.
After heating at 70 ° C. for 10 minutes, the mixture was rapidly cooled in ice. In the solution,
5x reverse transcriptase reaction buffer (LifeTechnol)
4), 2 μl of 100 mmol / L DTT, and a dNTP mixed solution [10 mmol / L dATP, 1 μm).
0 mmol / L dGTP, 10 mmol / L dTTP, 5 mmol
/ L 5-methyl dCTP] as a tracer and [α- ³² P] dATP (110 TBq / m
mol; AmershamPharmacia Bio
Tech) (1 μl) was added. After incubating at 37 ° C for 2 minutes, reverse transcriptase Superscript II RNa
seH ^- Reverse transscriptas
e (manufactured by Life Technologies)
1 (1,000 units) was added and reacted at 44 ° C. for 1 hour to synthesize cDNA.

[0242] 0.8 µl of 0.5 mol / L was added to the reaction solution.
EDTA (pH 8.0) was added to stop the reaction, and the mixture was treated with phenol / chloroform and chloroform, and then a cDNA / mRNA hybrid was recovered by ethanol precipitation. The precipitate is dissolved in 17 μl of distilled water, and a 5 × reaction buffer (Life Technologies)
5 μl, 100 μM dGTP 2.5 μl, and 15 units / μl of terminal deoxynuc
leotidyl transferase (Life
Technologies (produced by Technologies) was added in an amount of 0.5 μl. The reaction was carried out at 37 ° C. for 30 minutes, and oligo dG was added to the 3 ′ end of the cDNA. Add 5 μl of 0.5 mol to the reaction solution.
/ L EDTA (pH 8.0) was added to stop the reaction, and treated with phenol / chloroform and chloroform.
Ethanol precipitation was performed. 20.7 μl of the obtained precipitate
Of the reaction buffer A [200 mmol / L
Tris · HCl (pH 8.75), 100 mmol / L
KCl, 100 mmol / L (NH ₄ ) ₂ SO ₄ , 20 mmol / L
MgSO ₄ , 1% Triton X-100, 1mg
/ Ml BSA] and 1.5 μl of reaction buffer B [20
0 mmol / L Tris · HCl (pH 9.2), 600 m
mol / L KCl, 20 mmol / L MgCl ₂ ]
1, oligo (dC) -Sfi I primer (SEQ ID NO: 1)
0.3 μg of the DNA having the nucleotide sequence described in
0.75 μl of 10 mmol / L dNTP mixture, 10 mmol
/ L β-NAD to add 1.5 μl to a total volume of 27.4.
The volume was 5 μl. After keeping at 55 ° C for 5 minutes, 5 units / μ
1.5 μl of ExTaq DNA polymerase (Takara Shuzo Co., Ltd.) and 100 units / μl of Amplig
case (Epicentre) 0.75 μl, 5
Unit / μl of Hybridase (Epicentre
Was added. Thermal cycler DN
Using A engine (manufactured by MJ Research), slowly lower the temperature from 55 ° C. to 35 ° C. at a rate of 0.3 ° C. per minute, and then keep the temperature at 35 ° C. for 15 minutes to obtain the 3 ′ end of the cDNA. The primer was annealed to the oligo dG added to. Then 72 ° C
For 15 minutes to perform an extension reaction of the second strand DNA. This cycle of annealing and extension reaction was repeated three more times to synthesize double-stranded cDNA.

The reaction solution was added to 0.5 mol / L EDTA (p
H8.0) 0.5 μl, 10% SDS 0.5 μl,
0.5 μl of 20 μg / μl Proteinase K
The mixture was incubated at 45 ° C. for 15 minutes to inactivate the enzyme and stop the reaction. After phenol / chloroform treatment and chloroform treatment, ethanol precipitation was performed, and the obtained precipitate was dissolved in 44 μl of distilled water. 5 μl of a 10 × reaction buffer (manufactured by New England Biolabs) and Sfi I (10 units / μl; New E)
ngland Biolabs) (1 μl),
The reaction was carried out at 37 ° C. for 8 hours to cut the Sfi I site derived from the oligo (dT) -Sfi I primer and the oligo (dC) -Sfi I primer. The double-stranded cDNA after the treatment with the restriction enzyme was subjected to electrophoresis using an autoclaved 0.7% agarose gel, and 1 to 2 kb (Lo).
w fraction) cDNA and 2 kb or more (Hig
hfractoin) cDNA. Recovery of the cDNA from the gel was performed using the QIAquick Gel E
xtraction Kit (manufactured by QIAGEN) was used. CDNA of each fraction was 100 μl of 10 μm
It was dissolved in mol / L Tris-HCl (pH 8.3).

[0244] For cDNA total amount of Low fraction, expression vectors were cut with Sfi I pAMo-d
Was added, and ethanol precipitation was performed. After washing with 70% ethanol, the precipitate was dissolved in 10 μl of sterile water.
To this, 10 μl of Ligation High (manufactured by Toyobo Co., Ltd.) was added, and a ligation reaction was performed at 16 ° C. for 12 hours. After that, it is heated at 70 ° C for 30 minutes.
e was deactivated. After phenol / chloroform treatment and chloroform treatment, the supernatant was passed through a Millipore filter (UFC
3LYK00; manufactured by Millipore) and centrifuged (8000 rpm, 4 ° C, 10 ° C).
Min) to capture the DNA on the filter. After removing the filtrate, 10 mmol / L Tr was added to the filter cup.
is-HCl (pH 8.0) and 1 mmol / L EDT
The filter was washed by adding 300 μl of a buffer solution consisting of A (sodium ethylenediaminetetraacetate) (hereinafter abbreviated as TE buffer solution) and centrifuging (8000 rpm, 4 ° C., 10 minutes). This washing operation was repeated twice. T
50 μl of E buffer was added to the filter, and the DNA captured by the filter was eluted by pipetting. Invert the filter cup into a 15 ml tube, and centrifuge (2000 rpm, 4 ° C., 15 seconds) to remove D.
The NA solution was collected.

2 μl of the solution was introduced into Escherichia coli MC1061A (Molecular Cloning, 2nd edition) by electroporation to prepare a cDNA library. (3) Random Sequence Plasmid DNA was obtained from each of the E. coli clones obtained in the above (2) according to a conventional method, and the 5′-terminal and 3′-terminal nucleotide sequences of cDNA contained in each plasmid were determined.
The nucleotide sequence can be determined using a commercially available kit (Dye Terminator Cyc
le SequencingFS Ready Reaction Kit, dRhodamine Ter
minator Cycle Sequencing FS Ready Reaction Kit or BigDye Terminator Cycle Sequencing FS Ready Reac
The kit was performed using a DNA Kit (ABI PRISM 377, manufactured by PE Biosystems) and a tion kit (manufactured by PE Biosystems). As a primer, T3 primer (manufactured by Stratagene) and T7 primer (manufactured by Stratagene) were used.

As described above, 5 ′ of about 300 cDNAs
The nucleotide sequences at the termini and the 3 'termini were determined. The homologous genes and proteins were analyzed using a program such as the FrameSearch method for the amino acid sequence deduced from the nucleotide sequence obtained from the obtained nucleotide sequence. As a result, it was considered that the cDNA contained in the plasmid named pAMo-PGMO334 encodes a protein having homology to human galanin receptors (GALR1, GALR2, GALR3), human somatostatin receptor, and the like. (4) Determination of the entire nucleotide sequence of the cDNA contained in pAMo-PGMO334 Based on the cDNA sequence information derived from pAMo-PGMO334 determined in (3) above, a DNA having a nucleotide sequence represented by SEQ ID NO: 15 ( Having a base sequence represented by SEQ ID NO: 16) and a base sequence represented by SEQ ID NO: 16
NA (base number 402 in the base sequence described in SEQ ID NO: 2)
~ 421) is designed and synthesized, and by using the DNA as a primer, p
The entire nucleotide sequence of the cDNA contained in AMo-PGMO334 was determined. To determine the nucleotide sequence, use a commercially available kit (Dy
e Terminator Cycle Sequencing FS Ready Reaction Ki
t, dRhodamine Terminator Cycle Sequencing FS Ready
Reaction Kit or BigDye Terminator Cycle Sequenc
ing FS Ready Reaction Kit, PE Biosystems) and D
An NA sequencer (ABI PRISM 377, manufactured by PE Biosystems) was used.

The pAMo-PG determined as described above
The entire nucleotide sequence of the cDNA contained in MO334 (1458)
bp) is shown in SEQ ID NO: 2. The cDNA encoded a polypeptide consisting of 361 amino acids. This polypeptide is called PGMO334 polypeptide, and the amino acid sequence is shown in SEQ ID NO: 1. From the hydropathy profile, the polypeptide was considered to have seven membrane-bound regions (FIG. 1). The membrane-bound region of a GPCR can be predicted based on homology with a known GPCR [EMBO J., 12 , 1693 (1993)]. FIGS. 2 to 10 show the membrane-bound region predicted by the method. In addition, a SOSUI system (ver. 1.
0/10, Mar. , 1996) [Shigeki Mitaku et.al. A
Theoretical Method to Distinguish between Membran
e and Soluble Proteins by Physicochemical Approac
h. (submitted), Takatsugu Hirokawa.et.al.SOSUI: C
lassification and Secondary Structure Prediction S
ystem for Membrane Proteins, Bioinformatics (forme
rly CABIOS) 14 , 378 (1998)], it was verified that the above prediction was valid. The software is available from Mitsui Information Development.

The polypeptide had homology at the amino acid level to known G protein-coupled receptors.
LR3), human somatostatin receptor (SSTR1, SSTR1)
STR2, SSTR3, SSTR4, SSTR5), human orphanin receptor, human opioid receptor (μ,
δ, κ), human melanin concentrating hormone receptor (GPR24), human urotensin II receptor (GPR14), human orphan G protein-coupled receptor GPR7, human orphan G protein-coupled receptor GPR
8 showed high homology. The polypeptide and the known G
FIG. 13 shows a dendrogram created using the amino acid sequence of the protein-coupled receptor. Dendrogram CLUS
TAL X Multiple Sequence Alignment Program ( ftp: // f
tp-igbmc.u-strasbg.fr/pub/ClustalX/ ). The polypeptide has, at the amino acid level, human galanin receptors (GALR1, GALR2, GALR3) and 2
0.5-26.0%, human somatostatin receptor (SS
TR1, SSTR2, SSTR3, SSTR4, SST
R5) and 19.4-23.0%, human orphanin receptor and 20.5%, human opioid receptor (μ, δ, κ)
19.4-20.8%, human melanin concentrating hormone receptor (GPR24) and 21.3%,
Human urotensin II receptor (GPR14) and 20.5
%, Human orphan G protein-coupled receptor GPR7 and 2
3.3%, human orphan G protein-coupled receptor GPR
8 and 19.7% homology.

The above results revealed that the polypeptide was a novel G protein-coupled receptor polypeptide. Human galanin receptor (GALR1, GALR
2, GALR3), human somatostatin receptor (SST
R1, SSTR2, SSTR3, SSTR4, SSTR
5), human orphanin receptor, human opioid receptor (μ, δ, κ), human melanin concentrating hormone receptor (GPR24), human urotensin II
Since all natural ligands of the receptor (GPR14) are peptides, it was estimated that the ligand of the G protein-coupled receptor of the present invention was also a peptide. Further, the G protein-coupled receptor is different from the known G protein-coupled receptor by 1
Since it shows 9.4 to 26.0% homology, it was estimated that the ligand of the G protein-coupled receptor was different from the ligand of the known G protein-coupled receptor.

CD contained in pAMo-PGMO334
A plasmid pBS-PGMO334 in which NA was subcloned into pBluescript II SK (+) was constructed (see FIG. 14). After cutting the pAMo-PGMO334 with restriction enzymes Sph I, the digested ends were converted to blunt ends with Klenow fragment, were obtained Hin dIII- Sph I (blunt) fragment of 1.25kb and further digested with restriction enzyme Hin dIII . Also, pAMo-PGMO33
4 was cleaved with restriction enzymes Hin dIII and Sna BI to obtain an 8.8 kb Hin dIII- Sna BI fragment. By combining the above two fragments, pAMo-PG
MO334m was created. pAMo-PGMO334m
H cleavage after 1.25kb with Hin dIII and Not I
in dIII- Not acquired the I fragment. Also, pBlues
cript II SK (+) was acquired the Hin dIII- Not I fragment cleaved after 2.9kb with restriction enzymes Hin dIII and Not I. By combining the above two fragments, pBS-P
GMO334 was created. Escherichia coli DH5α / pBS-PGMO334, which is Escherichia coli containing pBS-PGMO334
Was deposited as FERM BP-6966 with the National Institute of Bioscience and Human Technology on December 8, 1999. Example 2 Investigation of the expression level of the PGMO334 gene transcript in various human cells
Protocols, Academic Press (1990)].

Also, the transcript of β-actin, which is considered to be expressed to the same extent in all cells, was simultaneously determined, and differences in the amount of mRNA between cells and the difference between mRNAs by reverse transcriptase between samples were measured. It was confirmed that there was no significant difference in the conversion efficiency to strand cDNA. The quantification of β-actin transcript is performed by a conventional method [Proc. Natl. Acad. Sci. USA, 87 , 2725
(1990); J. Biol. Chem., 269 , 14730 (1994);
6-181759] according to the quantitative PCR method. (1) Synthesis of single-stranded cDNA derived from various cells and cell lines As human cell lines, T cell lines (Molt-3, Molt-4, HUT
78), B cell lines (Namalwa KJM-1, Daudi, Raji), granulocyte / monocyte cell lines (HL-60, U-937, THP-1), vascular endothelial cell lines (IVEC, HUVEC), melanoma Cell line (WM266-
4, WM115), neuroblastoma cell line SK-N-MC, lung cancer cell line (PC-9, HLC-1, QG90), prostate cancer cell line PC-3, gastric cancer cell line KATOIII, pancreatic cancer cell line ( Capan-1, Capan-2) and colon cancer cell lines (Colo205, SW1116, LS180). QG90
And SW1116 were obtained from Aichi Cancer Center. HLC-1 was obtained from Osaka University Cancer Institute. KATO III and PC-9 were obtained from the Institute for Immunobiology. HUVEC (human umbel
ical vascular endothelial cell) was obtained from Crabo. IVEC [J. Cell. Physiol., 157 , 41 (1993)] is described in N.W. T. L. Obtained from FRANCE. Molt-4, Da
udi was obtained from JCRB. Other cells are from the American Type Culture Collection (American Type Culture Collection).
Type Culture Collection).

The total RNA of each cell was determined by a conventional method [Biochemistry,
18 , 5294 (1977)]. Synthesis of single-stranded cDNA from total RNA is performed using a kit (SUPERSCRIPT ^™ Prea
mplification System; manufactured by BRL).
For cell lines, 5 μg of total RNA to single-stranded cDNA
Was synthesized, diluted 50-fold with water, and used as a template for PCR. As a primer, an oligo (dT) primer was used.

In addition, mRNA derived from various human organs (Clon
tech) was synthesized in the same manner.
Single-stranded cDNA was synthesized from 1 μg of mRNA, and
A 40-fold dilution was used as a template for PCR. As a primer, an oligo (dT) primer was used. mR
As the NA, mRNA derived from the following 35 organs was used. 1 adrenal gland, 2 brains, 3 caudate nuclei, 4 hippocampus, 5 substantia nigra, 6
Thalamus, 7 kidneys, 8 pancreas, 9 pituitary gland, 10 small intestine, 11 bone marrow, 12 amygdala, 13 cerebellum, 14 corpus callosum, 15 fetal brain, 1
6 fetal kidney, 17 fetal liver, 18 fetal lung, 19 heart, 20
Liver, 21 lungs, 22 lymph nodes, 23 mammary glands, 24 placentas, 2
5 prostate, 26 salivary glands, 27 skeletal muscle, 28 spinal cord, 29 spleen, 30 stomach, 31 testis, 32 thymus, 33 thyroid, 34 trachea, 35 uterus. (2) Preparation of Standard and Internal Control for Quantitative PCR pBS-PGMO334 was replaced with cDNA by restriction enzyme Hi.
converts was cleaved with n dIII and Not I linearized DNA, was used as a standard for quantification. After confirming that the plasmid was completely cleaved, it was used after being diluted stepwise with water containing yeast transfer RNA at 1 μg / ml.

Further, pUC119-ACT and pUC
Restriction enzyme of each cDNA portion of 119-ACTd
Converts was cleaved with Hin dIII and Asp 718 in a linear DNA, it was used as the standard and internal control for transcript quantification of β-actin, respectively [J.
Biol. Chem., 269 , 14730 (1994), JP-A-06-18175
9]. After confirming that each plasmid was completely cleaved, it was used by diluting it stepwise with water containing yeast transfer RNA at 1 μg / ml. (3) Quantification of PGMO334 gene transcript using PCR method Single-chain c derived from various tissues and cell lines prepared in (1)
PCR was performed using DNA as a template. In PCR, DNA having the nucleotide sequence represented by SEQ ID NO: 16 or 17 was used as a primer set, and the reaction was performed using Recombinant Taq DNA Polymerase manufactured by Takara and the attached 10 × Taq.
Using Buffer and 2.5 mmol / L dNTP Mixture,
Performed according to instructions. The reaction was performed in 20 μl,
Dimethyl sulfoxide was added to a final concentration of 5%.

Further, a calibration curve was prepared by similarly performing PCR using the standard prepared in the above (2) as a template.

The single-stranded cDNA (5 μl) synthesized in (1)
l), as a forward primer, SEQ ID NO: 1
10 pmol of the DNA having the nucleotide sequence represented by 6,
10 pmol, 2.5 mmol of DNA having the base sequence represented by SEQ ID NO: 17 as a reverse primer
/ L dNTP mixture 1.6 μl, dimethyl sulfoxide 1 μl, 5 units / μl Recombinant Taq DNA Po
Lysase (Takara Shuzo Co., Ltd.) was added in an amount of 0.1 μl, 10 × reaction buffer (Takara Shuzo Co., Ltd.) in an amount of 2 μl, and sterile water was added to adjust the total volume to 20 μl. Thermal cycler DNA e
ngine (manufactured by MJ Research) and 94
After denaturing the DNA by heat treatment at 3 ° C. for 3 minutes, a reaction consisting of 94 ° C. for 1 minute, 60 ° C. for 1 minute, and 72 ° C. for 1 minute was performed for 25 to 33 cycles. Part of the reaction solution (8 μ
After l) was subjected to agarose gel electrophoresis, the gel was
Green I nucleic acid stain (Molecular Probes)
Stained. FluorImager SI (Molecular Dynamics)
And the amount of the amplified DNA fragment was measured. In order to more accurately quantify the transcript, the same PCR was performed by changing the number of PCR cycles. The amount of the standard was changed according to the number of PCR cycles.

The quantification of the transcript of β-actin has been reported previously [J. Biol. Chem., 269 , 14730 (1994), JP-A-6-18
1759].

The transcript of the PGMO334 gene was most frequently expressed in the pituitary gland in normal human tissues, followed by the lung and mammary gland. Expression was also found in the caudate nucleus, hippocampus, substantia nigra, thalamus, pancreas, amygdala, spinal cord, and uterus (FIG. 15). It is well known that the secretion of anterior pituitary hormone is controlled by various ligands released from the hypothalamus acting on the corresponding G protein-coupled receptor present on the anterior pituitary cells. . Therefore, P
GMO334 polypeptide (a novel G protein-coupled receptor polypeptide) may also regulate the release of hormones and neuropeptides from pituitary cells in response to stimulation of ligands released from the hypothalamus etc. .

In the human cultured cell line, a colon cancer cell line (LS-1
80, Colo205), lung cancer cell line (QG90), prostate cancer cell line (PC-3), and pancreatic cancer cell lines (Capan-1, Capan-2). No expression in other cell lines
(FIG. 16). Example 3 Expression analysis of PGMO334 gene in pancreatic β cell line Somatostatin receptor and galanin receptor having homology to PGMO334 polypeptide are expressed in pancreatic β cells, and are involved in regulation of insulin secretion from pancreatic β cells. (Annu.Rep.Med.Chem., 33 ,
41 (1998), Life Sci., 60 , 1523 (1997), Life Sci.,
57 , 1249 (1995), N. Engl. J. Med., 334 , 246 (199
6), Diabetes, 48 , 77 (1999), American Journal of P
hysiology, 271 , C1781 (1996), Frontiers in Neuroen
docrinology, 20 , 157 (1999), Annals of the New Yor
k Academy of Sciences, 263 , 129 (1998), Annals of
theNew York Academy of Sciences, 527 , 29 (198
8)]. That is, somatostatin and galanin suppress insulin secretion from pancreatic β cells stimulated by glucose. On the other hand, it has been reported that some galanin partial peptides, on the other hand, promote insulin secretion (Annals o
f the New York Academy of Sciences, 527 , 29 (198
8)].

Therefore, it was considered that the PGMO334 polypeptide was also involved in the control of insulin secretion from pancreatic β-cells.
The expression of transcripts of 34 genes was examined. Since human pancreatic β cells are not readily available, the expression of transcripts of the PGMO334 gene in the mouse pancreatic β cell line MIN6 [Endocrinology, 127 , 126 (1990)] was examined.

The expression was examined by a conventional method [PCR Protocols, Aca
demic Press (1990)]. PCR was performed using DNAs having the nucleotide sequences represented by SEQ ID NOS: 18 and 19 as a primer set. The design of the primer was performed as follows. PGMO334cDN in the public database
Since a partial sequence derived from a mouse having homology to the nucleotide sequence of A was registered, this sequence was used for mouse PGMO334.
It was considered a partial sequence of the gene. Next, this partial sequence was compared with the sequence of the human PGMO334 gene, and the above primer was prepared using the well-conserved partial sequence.

The total RNA of MIN6 was obtained by a conventional method [Biochemistry, 1
8 , 5294 (1977)]. The synthesis of single-stranded cDNA from total RNA was performed in the same manner as the synthesis of single-stranded cDNA from the cell line of Example 2. In addition, mouse chromosome DNA was used as a template as a negative control. The standard prepared in Example 2 was used as a standard.

The PCR reaction was performed using Ex Taq (Takara Shuzo), the attached 10 × Buffer and 2.5 mmol / L dNTP Mixt.
ure was used according to the instructions. The reaction was performed in 20 μl, and dimethyl sulfoxide (dimethyl sulfo) was used.
xide) was added to a final concentration of 5%. The synthesized single-stranded cDNA (5 μl) was added as a forward primer with a D having the base sequence represented by SEQ ID NO: 18.
NA was 10 pmol, and a DNA having a base sequence represented by SEQ ID NO: 19 was used as a reverse primer.
1.6 μm of the pmol, 2.5 mmol / L dNTP mixture
1, 1 μl of dimethyl sulfoxide, 0.1 μl of ExTaq (manufactured by Takara Shuzo) at 5 units / μl, 2 μl of 10 × reaction buffer (manufactured by Takara Shuzo), and add sterile water to make a total volume of 20 μl. Prepared. Thermal cycler DNA
Using an engine (manufactured by MJ Research), the DNA was denatured by a heat treatment at 94 ° C. for 5 minutes, and then 35 cycles of a reaction consisting of 94 ° C. for 30 seconds, 60 ° C. for 1 minute, and 72 ° C. for 2 minutes were performed. After a part (10 μl) of the reaction solution was subjected to agarose gel electrophoresis, 10 μl of the solution after the reaction was electrophoresed on a 1% agarose gel. After staining the gel with ethidium bromide, pictures were taken. By scanning the photograph using an NIH image system or the like, the intensity of staining of the amplified fragment was measured, and the amount of amplified DNA was quantified.

As a result of the analysis, it was found that the PGMO334 gene
It was found to be expressed in MIN6, a cell line. No band was amplified when the mouse genome was used as a template.

From the above results, it was suggested that the PGMO334 gene may be expressed in pancreatic β cells and may be involved in the control of insulin secretion in pancreatic β cells. Example 4 Construction of Human Cell Line Expressing PGMO334 Polypeptide Control plasmid (pAMo) and PGMO3
34 polypeptide expression plasmid (pAMo-PGM
O334) was adjusted to 1 μg / μl with TE
After dissolving in water, electroporation (Cytotechno
logy, 3 , 133 (1990)], Namalwa KJM-1 cells (Cyto
technology, 1 , 151 (1988)] to obtain transformed cells.

After introducing 4 μg of plasmid per 1.6 × 10 ⁶ cells, 8 ml of RPMI1640.ITPS
G medium [7.5% NaHCO ₃ in 1/40 volume, 200 mmol / L L-glutamine solution (GIBCO) 3%, penicillin / streptomycin solution (GIBCO, 5000 units / ml penicillin, 5000 μg / ml streptomycin) 0.5%, N-2-
Hydroxyethylpiperazine-N'-2-ethanesulfonic acid (N-2-hydroxyethylpiperazine-N'-
2-hydroxypropane-3-sulfonic acid; HEPES) (10 mmo
l / L), insulin (3 μg / ml), transferrin (5 μg / ml), sodium pyruvate (5 mmol / L),
The cells were suspended in RPMI1640 medium (manufactured by Nissui Pharmaceutical Co., Ltd.) supplemented with sodium selenite (125 nmol / L) and galactose (1 mg / ml), and were suspended in a CO ₂ incubator at 37 ° C. for 24 hours.
Cultured for hours. Then, G418 (manufactured by Gibco) was added at 0.5 mg / ml.
And cultured for 14 days to obtain a stable transformant. The transformant was subcultured in RPMI1640.ITPSG medium containing 0.5 mg / ml G418.

The stable transformant or its membrane fraction is
It can also be used to search for ligands, agonists, or antagonists of GMO334 polypeptide (a novel G protein receptor). The specific search method is as described in the detailed description.

[0268]

According to the present invention, the G protein-coupled receptor polypeptide of the present invention or a partial peptide of the polypeptide, or a ligand for the polypeptide using the polypeptide or a DNA encoding the partial peptide Screening methods and screening kits for agonists, antagonists or functional modifiers, pharmaceutical compounds effective for ameliorating pathological conditions caused by the screening methods or the polypeptides selected using the screening kits, And a model animal in which all or part of the gene containing the DNA of the present invention has been deleted or replaced, and an antibody against the polypeptide of the present invention that is effective for diagnosing a disease state caused by the polypeptide of the present invention. .

[0269]

[Sequence List Free Text]

 SEQ ID NO: 3-Description of Artificial Sequence: Synthetic DNA SEQ ID NO: 4-Description of Artificial Sequence: Synthetic DNA SEQ ID NO: 5-Description of Artificial Sequence: Synthetic DNA SEQ ID NO: 6-Description of Artificial Sequence: Synthetic DNA SEQ ID NO: 7-Artificial Sequence Description: Synthetic DNA SEQ ID NO: 8-Description of Artificial Sequence: Synthetic DNA SEQ ID NO: 9-Description of Artificial Sequence: Synthetic DNA SEQ ID NO: 10-Description of Artificial Sequence: Synthetic DNA SEQ ID NO: 11-Description of Artificial Sequence: Synthetic DNA Sequence No. 12—Description of Artificial Sequence: Synthetic DNA SEQ ID No. 13—Description of Artificial Sequence: Synthetic DNA SEQ ID No. 14—Description of Artificial Sequence: Synthetic DNA SEQ ID No. 15—Description of Artificial Sequence: Synthetic DNA SEQ ID No. 16—Description of Artificial Sequence Description: Synthetic DNA SEQ ID NO: 17-Description of Artificial Sequence: Synthetic DNA SEQ ID NO: 18-Description of Artificial Sequence: Synthetic DNA SEQ ID NO: 19-Description of Artificial Sequence: Synthetic DNA

[Sequence List] SEQUENCE LISTING <110> KYOWA HAKKO KOGYO CO., LTD <120> Novel Polypeptides <130> H11-1901A4 <140> <141> <160> 19 <170> PatentIn Ver.2.0 <210> 1 <211 > 361 <212> PRT <213> Homo sapiens <400> 1 Met Ser Pro Glu Cys Ala Arg Ala Ala Gly Asp Ala Pro Leu Arg Ser 1 5 10 15 Leu Glu Gln Ala Asn Arg Thr Arg Phe Pro Phe Phe Ser Asp Val Lys 20 25 30 Gly Asp His Arg Leu Val Leu Ala Ala Val Glu Thr Thr Val Leu Val 35 40 45 Leu Ile Phe Ala Val Ser Leu Leu Gly Asn Val Cys Ala Leu Val Leu 50 55 60 Val Ala Arg Arg Arg Arg Arg Gly Ala Thr Ala Cys Leu Val Leu Asn 65 70 75 80 Leu Phe Cys Ala Asp Leu Leu Phe Ile Ser Ala Ile Pro Leu Val Leu 85 90 95 Ala Val Arg Trp Thr Glu Ala Trp Leu Leu Gly Pro Val Ala Cys His 100 105 110 Leu Leu Phe Tyr Val Met Thr Leu Ser Gly Ser Val Thr Ile Leu Thr 115 120 125 Leu Ala Ala Val Ser Leu Glu Arg Met Val Cys Ile Val His Leu Gln 130 135 140 Arg Gly Val Arg Gly Pro Gly Arg Arg Ala Arg Ala Val Leu Leu Ala 145 150 155 160 Leu Ile Trp Gly Tyr Ser Ala Val Ala Ala L eu Pro Leu Cys Val Phe 165 170 175 Phe Arg Val Val Pro Gln Arg Leu Pro Gly Ala Asp Gln Glu Ile Ser 180 185 190 Ile Cys Thr Leu Ile Trp Pro Thr Ile Pro Gly Glu Ile Ser Trp Asp 195 200 205 Val Ser Phe Val Thr Leu Asn Phe Leu Val Pro Gly Leu Val Ile Val 210 215 220 Ile Ser Tyr Ser Lys Ile Leu Gln Ile Thr Lys Ala Ser Arg Lys Arg 225 230 235 240 Leu Thr Val Val Ser Leu Ala Tyr Ser Glu Ser His Gln Ile Arg Val Ser 245 250 255 Gln Gln Asp Phe Arg Leu Phe Arg Thr Leu Phe Leu Leu Met Val Ser 260 265 270 Phe Phe Ile Met Trp Ser Pro Ile Ile Ile Thr Ile Leu Leu Ile Leu 275 280 285 285 Ile Gln Asn Phe Lys Gln Asp Leu Val Ile Trp Pro Ser Leu Phe Phe 290 295 300 Trp Val Val Ala Phe Thr Phe Ala Asn Ser Ala Leu Asn Pro Ile Leu 305 310 315 320 Tyr Asn Met Thr Leu Cys Arg Asn Glu Trp Lys Lys Ile Phe Cys Cys 325 330 335 Phe Trp Phe Pro Glu Lys Gly Ala Ile Leu Thr Asp Thr Ser Val Lys 340 345 350 Arg Asn Asp Leu Ser Ile Ile Ser Gly 355 360 <210> 2 <211> 1458 <212> DNA <213> Homo sapiens <400 > 2 gagtcgcctc ccagatgagc actctctcag accgctgcgg gccgccaggc gccggga atg 60 Met 1 tcc cct gaa tgc gcg cgg gca gcg ggc gac gcg ccc ttg cgc agc ctg 108 Ser Pro Glu Cys Ala Arg Ala Ala Gly Asp Ala Pro Leu Arg Serg cgc ttt ccc ttc ttc tcc gac gtc aag ggc 156 Glu Gln Ala Asn Arg Thr Arg Phe Pro Phe Phe Ser Asp Val Lys Gly 20 25 30 gac cac cgg ctg gtg ctg gcc gcg gtg gag aca acc gtg ctg Astg ctg Arct Leu Val Leu Ala Ala Val Glu Thr Thr Val Leu Val Leu 35 40 45 atc ttt gca gtg tcg ctg ctg ggc aac gtg tgc gcc ctg gtg ctg gtg 252 Ile Phe Ala Val Ser Leu Leu Gly Asn Val Cys Ala Leu Val Leu Val 50 55 60 65 gcg cgc cga cga cgc cgc ggc gcg act gcc tgc ctg gta ctc aac ctc 300 Ala Arg Arg Arg Arg Arg Gly Ala Thr Ala Cys Leu Val Leu Asn Leu 70 75 80 ttc tgc gcg gac ctg atcc gc ctg atcc gc cct ctg gtg ctg gcc 348 Phe Cys Ala Asp Leu Leu Phe Ile Ser Ala Ile Pro Leu Val Leu Ala 85 90 95 gtg cgc tgg act gag gcc tgg ctg ctg ggc ccc gtt gcc tgc cac ctg 396 Val Arg Trp Thr Glu Ala eu Leu Gly Pro Val Ala Cys His Leu 100 105 110 ctc ttc tac gtg atg acc ctg agc ggc agc gtc acc atc ctc acg ctg 444 Leu Phe Tyr Val Met Thr Leu Ser Gly Ser Val Thr Ile Leu Thr Leu 115 120 125 gcc gcg gtc agc ctg gag cgc atg gtg tgc atc gtg cac ctg cag cgc 492 Ala Ala Val Ser Leu Glu Arg Met Val Cys Ile Val His Leu Gln Arg 130 135 140 145 ggc gtg cgg ggt cct ggg cgg cgg gcg cgg gca ggt ctc 540 Gly Val Arg Gly Pro Gly Arg Arg Ala Arg Ala Val Leu Leu Ala Leu 150 155 160 atc tgg ggc tat tcg gcg gtc gcc gct ctg cct ctc tgc gtc ttc ttc 588 Ile Trp Gly Tyr Ser Ala Val Ala Ala Cys Val Phe Phe 165 170 175 cga gtc gtc ccg caa cgg ctc ccc ggc gcc gac cag gaa att tcg att 636 Arg Val Val Pro Gln Arg Leu Pro Gly Ala Asp Gln Glu Ile Ser Ile 180 185 190 tgc aca ctg att tcc att cct gga gag atc tcg tgg gat gtc 684 Cys Thr Leu Ile Trp Pro Thr Ile Pro Gly Glu Ile Ser Trp Asp Val 195 200 205 tct ttt gtt act ttg aac ttc ttg gtg cca gga ctg gtc att gtg atc 732 Ser Phe Val Thr L eu Asn Phe Leu Val Pro Gly Leu Val Ile Val Ile 210 215 220 225 agt tac tcc aaa att tta cag atc aca aag gca tca agg aag agg ctc 780 Ser Tyr Ser Lys Ile Leu Gln Ile Thr Lys Ala Ser Arg Lys Arg Leu 230 235 240 acg gta agc ctg gcc tac tcg gag agc cac cag atc cgc gtg tcc cag 828 Thr Val Ser Leu Ala Tyr Ser Glu Ser His Gln Ile Arg Val Ser Gln 245 250 255 cag gac ttc cgg ctc ttc cgc acc ctc ttc atg gtc tcc ttc 876 Gln Asp Phe Arg Leu Phe Arg Thr Leu Phe Leu Leu Met Val Ser Phe 260 265 270 ttc atc atg tgg agc ccc atc atc atc acc atc ctc ctc atc ctg atc 924 Phe Ile Met Trp Ser Pro Ile Thr Ile Leu Leu Ile Leu Ile 275 280 285 cag aac ttc aag caa gac ctg gtc atc tgg ccg tcc ctc ttc ttc tgg 972 Gln Asn Phe Lys Gln Asp Leu Val Ile Trp Pro Ser Leu Phe Phe Trp 290 295 g 305 305 g ttc aca ttt gct aat tca gcc cta aac ccc atc ctc tac 1020 Val Val Ala Phe Thr Phe Ala Asn Ser Ala Leu Asn Pro Ile Leu Tyr 310 315 320 aac atg aca ctg tgc agg aat gag tgg aag aaa att ttt tgc tgc ttc Asn Met Thr Leu Cys Arg Asn Glu Trp Lys Lys Ile Phe Cys Cys Phe 325 330 335 tgg ttc cca gaa aag gga gcc att tta aca gac aca tct gtc aaa aga 1116 Trp Phe Pro Glu Lys Gly Ala Ile Leu Thr Asp Thr Ser Val Lys Arg 340 345 350 aat gac ttg tcg att att tct ggc taatttttct ttatagccga gtttctcaca 1170 Asn Asp Leu Ser Ile Ile Ser Gly 355 360 cctggcgagc tgtggcatgc ttttaaacag agttcatttc cagtaccctc catcagtgca 1230 ccctgcttta agaaaatgaa cctatgcaaa tagacatcca cagcgtcggt aaattaaggg 1290 gtgatcacca agtttcataa tattttccct ttataaaagg atttgttggc caggtgcagt 1350 ggttcatgcc tgtaatccca gcagtttggg aggctgaggt gggtggatca cctgaggtca 1410 ggagttcgag accaacctga ccaacatggt gagacccccg tctctact 1458 <210> 3 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: synthetic DNA <400gtgac gct acgcac gcac gct acg <210> 4 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: synthetic DNA <400> 4 cgtgcgtacg gtcgacactg gccgtcgttt taca 3 4 <210> 5 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: synthetic DNA <400> 5 cacgtgtacg taggcctgtg aggccg 26 <210> 6 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: synthetic DNA <400> 6 gatccggcct cacaggccta cgtaca 26 <210> 7 <211> 44 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: synthetic DNA <400> 7 ggccatggcc tcactggcct acgtagcggc cgcggtaccc ccta 44 <210> 8 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: synthetic DNA <400> 8 cactataggg ggtaccgcgg ccgctacgta ggccagtgag gccat 45 <210> 9 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: synthetic DNA <400> 9 tagtgagtcg tattaggtca tagctgtttc ctgt 34 <210> 10 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: synthetic DNA <400> 10 gtacacagga aacagctatg acctaatacg act 33 <210> 11 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: synthetic DNA <400> 11 gatggcctac atggcctacg tagcggccgc ggtaccccct a 41 <210> 12 <211> 49 <212> DNA <213 > Artificial Sequence <220> <223> Description of Artificial Sequence: synthetic DNA <400> 12 cactataggg ggtaccgcgg ccgctacgta ggccatgtag gccatcgtg 49 <210> 13 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: synthetic DNA <400> 13 gcggctgaag acggccttgt tggccttttt tttttttttt 41 <210> 14 <211> 46 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: synthetic DNA <400> 14 gagagagaga gagagaggcc tgtgaggcca ctagtccccc cccccc 46 <210> 15 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: synthetic DNA <400> 15 tgcctggtac tcaacctctt ctgc 24 <210> 16 <211 > 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: synthetic DNA <400> 16 ctacgtgatg accctgag cg 20 <210> 17 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: synthetic DNA <400> 17 ttccactcat tcctgcacag tgtc 24 <210> 18 <211> 24 <212 > DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: synthetic DNA <400> 18 gaccaggaaa ttccgatttg caca 24 <210> 19 <211> 24 <212> DNA <213> Artificial Sequence <220> <223 > Description of Artificial Sequence: synthetic DNA <400> 19 tgtaaaaatg gctcccttct ctgg 24

[Brief description of the drawings]

FIG. 1 shows a hydropathy plot of a PGMO334 polypeptide. This plot was created using analysis software (MacMolly). The predicted seven transmembrane regions (TM1 to TM7) are shown in the figure.

FIG. 2. PGMO334 polypeptide and known GPCR
FIG. 3 is a view showing an alignment of the amino acid sequences of FIG. Known G
As the PCR, human galanin receptor (GALR1, G
ALR2, GALR3), human somatostatin receptor (SSTR1, SSTR2, SSTR3, SSTR4,
SSTR5), human orphanin receptor (orphan
in receptor), human opioid receptor (o
bioreceptor μ, δ, κ), human melanin concentrating hormone receptor (GPR2
4) Human urotensin II receptor (GPR14), human orphan G protein-coupled receptor GPR7, and human orphan G protein-coupled receptor GPR8 were used. The alignment is CLUSTAL X Multiple Sequence Alignment
Program ( ftp://ftp-igbmc.u-strasbg.fr/pub/ClustalX
/ ). The seven possible transmembrane regions (TM1-TM7) in the PGMO334 polypeptide are underlined. * Amino acids conserved in all GPCRs
Indicated by

FIG. 3 is a view showing the same thing as the above, and is a continuation of FIG. 2;

FIG. 4 is a view showing the same thing as the above, and is a continuation of FIG. 3;

FIG. 5 is a diagram showing the same thing as the above, and is a continuation of FIG. 4;

FIG. 6 is a view showing the same thing as the above, and is a continuation of FIG. 5;

FIG. 7 is a view showing the same thing as the above, and is a continuation of FIG. 6;

8 is a view showing the same thing as the above, and is a continuation of FIG. 7;

9 is a view showing the same thing as the above, and is a continuation of FIG.

10 is a view showing the same thing as the above, and is a continuation of FIG. 9;

FIG. 11 is a diagram showing the steps of constructing pAMO-nd ′ and pAMo-nd.

FIG. 12 is a view showing a step of forming pAMo-d.

FIG. 13: PGMO334 polypeptide and known GPC
3 shows a dendrogram prepared using the amino acid sequence of R. Known GPCRs include the human galanin receptor (GA
LR1, GALR2, GALR3), human somatostatin receptor (SSTR1, SSTR2, SSTR3, SS
TR4, SSTR5), human orphanin receptor (or
phanin receptor, human opioid receptor (opioid receptor μ, δ,
κ), human melanin concentrating hormone receptor (GPR24), human urotensin II receptor (G
PR14), human orphan G protein-coupled receptor GP
R7 and human orphan G protein-coupled receptor GPR8 were used. The dendrogram is CLUSTAL X Multiple Seque
nce Alignment Program ( ftp: //ftp-igbmc.u-strasbg.f
r / pub / ClustalX / ).

FIG. 14 is a view showing a construction process of pBS-PGMO334.

FIG. 15 is an electrophoresis diagram showing the results of examining the expression levels of PGMO334 transcripts in 35 types of human organs using the PCR method. The number of PCR cycles is 25. The arrow indicates the position of the target amplified fragment (650 bp). The samples in each lane of the electrophoretogram are as follows. nc: Negative control, 1: Adrenal Grand
(Adrenal gland), 2: Brain, 3: Brain, caudate nucle
us (brain, caudate nucleus), 4: Brain, hippocampus (brain, hippocampus), 5: Brain, substantia nigra (brain, substantia nigra), 6:
Brain, thalamus (brain, thalamus), 7: Kidney (kidney),
8: Pancreas (pancreas), 9: Pituitary gland (pituitary gland), 10: Small intestine (small intestine), 11: Bone ma
rrow (bone marrow), 12: Brain, amygdala (brain, amygdala),
13: Brain, cerebellum (brain, cerebellum), 14: Brain, co
rpus callosum (brain, corpus callosum), 15: Fetal brain (fetal brain), 16: Fetal kidney (fetal kidney), 17: Fetal
liver 18: Fetal lung, 1
9: Heart, 20: Liver, 21: Lung
(Lung), 22: Lymph node (lymph node), 23: Mammar
y gland (breast gland), 24: Placenta (placenta), 25: Pro
state (prostate), 26: Salivary gland (salivary gland),
27: Skeletal muscle (skeletal muscle), 28: Spinal cord
(Spinal cord), 29: Spleen (spleen), 30: Stomach
(Stomach), 31: Testis (testis), 32: Thymus (thymus), 33: Thyroid (thyroid), 34: Trachea (trachea), 35: Uterus (uterus), 36: Standard 10 fg
(Standard sample 10 fg), 37: Standard 50 fg, 38: St
andard 125 fg, M .: size marker

FIG. 16 is an electrophoresis diagram showing the results of examining the expression levels of PGMO334 transcripts in various human cell lines using the PCR method. The number of PCR cycles is 25. The arrow indicates the position of the target amplified fragment (650 bp). The sample of each lane in the figure is as follows. 1: Molt-3, 2: Molt-4, 3: Hut78, 4: Namalwa K
JM-1, 5: Daudi, 6: Raji, 7: HL-60, 8: U-93
7, 9: THP-1, 10: IVEC, 11: HUVEC, 12: WM26
6-4, 13: WM115, 14: SK-N-MC, 15: PC-9,
16: HLC-1, 17: QG-90, 18: PC-3, 19: KAT
O-III, 20: Capan-1, 21: Capan-2, 22: Colo
205, 23: SW1116, 24: LS180, 25: Standard 1 f
g (standard sample 1 fg), 26: Standard 10 fg, 27: St
andard 50 fg, 28: Standard 125 fg, M .: size marker

[Explanation of symbols]

bp: base pairs kb: kilobase pairs G418 / Km: G418 derived from transposon 5 (Tn5), kanamycin resistance gene Ap: pBR322-derived ampicillin resistance gene Tc: pBR322-derived tetracycline resistance gene Sp.BG: Rabbit β globin gene splicing signal A.BG: Rabbit β globin gene poly A addition signal A.SE: Simian virus 40 (SV4
0) Early gene polyaddition signal Pmo: Moloney murine leukemia virus long terminal repeat (LTR) promoter oriP: Epstein-Barr virus (Epstein)
PGMO334: DNA encoding the PGMO334 polypeptide obtained in the present invention.

Continued on the front page (51) Int.Cl. ⁷ Identification code FI Theme coat II (Reference) A61K 39/395 A61K 45/00 4B065 A61P 5/08 4C084 45/00 35/00 4C085 A61P 5/08 C07K 14/705 4H045 35/00 16/28 C07K 14/705 C12N 1/15 16/28 1/19 C12N 1/15 1/21 1/19 C12P 21/02 C 1/21 21/08 5/10 C12Q 1/68 A C12P 21/02 Z 21/08 G01N 33/15 Z C12Q 1/68 33/50 Z 33/566 G01N 33/15 C12N 15/00 ZNAA 33/50 A61K 37/02 33/566 C12N 5/00 BC (72 ) Inventor Satoshi Saeki 3-6-6 Asahicho, Machida-shi, Tokyo Inside Kyowa Hakko Kogyo Co., Ltd. (72) Inventor Kazumi Miura 3-6-6 Asahicho, Machida-shi, Tokyo Kyowa Hakko Hakko Kogyo Co., Ltd. (72) Inventor Susumu Sekine 3-6-6 Asahicho, Machida-shi, Tokyo F-term in Kyowa Hakko Hakko Kogyo Co., Ltd. 2B030 AA02 AD08 CA06 CA17 CA19 CD02 CD07 CD09 2G045 AA40 BB10 BB16 BB18 BB20 BB60 DA12 DA13 DA14 DA36 FB01 FB02 FB03 FB08 4B024 AA01 AA11 AA12 BA43 BA63 CA04 DA03 DA06 EA04 GA05 Q14 QR02 Q14 QR2 QS34 QX02 QX07 4B064 AG20 AG27 CA02 CA10 CA19 CA20 CC24 DA01 DA05 DA13 4B065 AA26X AA92X AA93X AA93Y AB01 AB05 BA03 CA24 CA25 CA44 CA46 4C084 AA02 AA06 AA07 AA17 BA01 BA08 BA22 CA13 CA17 ZC18 ZC25 CA25 CA14 Z15 BB11 CC02 CC03 DD62 DD63 EE01 GG01 GG08 4H045 AA10 AA11 AA20 AA30 BA10 CA45 DA50 DA76 EA28 EA50 EA51 FA72 FA74

Claims (31)

[Claims] 1. A G protein-coupled receptor polypeptide having the amino acid sequence of SEQ ID NO: 1. 2. The polypeptide according to claim 1, which has an amino acid sequence in which one or more amino acids have been deleted, substituted or added in the amino acid sequence of the polypeptide set forth in SEQ ID NO: 1. A polypeptide having substantially the same activity. 3. A partial peptide which is a partial peptide of the G protein-coupled receptor polypeptide according to claim 1 or 2, and has a binding ability to a ligand, agonist, antagonist or functional modifier of the polypeptide. 4. A DNA encoding the G protein-coupled receptor polypeptide according to claim 1 or 2. 5. A DNA encoding a partial peptide of the G protein-coupled receptor polypeptide according to claim 3. 6. In the DNA of SEQ ID NO: 2,
DNA having a base sequence represented by base numbers 58 to 1140. 7. D according to any one of claims 4 to 6,
A DNA that hybridizes with a DNA selected from NA under stringent conditions and encodes a polypeptide having substantially the same activity as the G protein-coupled receptor polypeptide according to claim 1. 8. D according to any one of claims 4 to 7,
A recombinant DNA obtained by incorporating a DNA selected from NA into a vector. 9. A transformed cell, transformed plant or transformed non-human animal having the recombinant DNA according to claim 8. 10. Using the transformed cell, transformed plant or transformed non-human animal according to claim 9, (1) culturing the transformed cell in a medium, and (2) culturing the transformed cell. Cultivating the transformed plant in the plant or (3) breeding the transformed non-human animal to produce the polypeptide of claim 1 or 2 or the partial peptide of claim 3 in the animal The method for producing the polypeptide according to claim 1 or 2, wherein the polypeptide or the partial peptide is collected from the culture, the plant, or the animal. . An antibody which recognizes the polypeptide according to claim 1 or 2, or the partial peptide according to claim 3. 12. A method for immunologically quantifying the polypeptide according to claim 1 or 2, or the partial peptide according to claim 3, using the antibody according to claim 11. 13. A method for diagnosing cancer or pituitary dysfunction using the quantification method according to claim 12. 14. A method for diagnosing cancer or pituitary dysfunction by measuring the amount of mRNA encoding the polypeptide according to claim 1 or 2. 15. A method for diagnosing cancer or pituitary dysfunction by detecting deletion, substitution or insertion of the gene encoding the polypeptide according to claim 1 or 2. 16. The polypeptide according to claim 1 or 2, wherein the polypeptide according to claim 1 or 2 or the partial peptide according to claim 3 is contacted with a test sample. The method for screening a ligand, agonist, antagonist or function modifying substance for a polypeptide according to claim 1 or 2, wherein an agonist, antagonist or function modifying substance is selected. 17. The method according to claim 1, wherein (i) the polypeptide according to claim 1 or 2 or the partial peptide according to claim 3 is brought into contact with a ligand; and (ii) the polypeptide according to claim 1 or 2. A peptide or a partial peptide according to claim 3 is contacted with a ligand and a test sample, and the agonist, antagonist or function modifying substance of the polypeptide according to claim 1 or 2 is compared with the test sample. 3. A method for screening for an agonist, antagonist or function-modifying substance of the polypeptide according to claim 1, wherein the method is selected. 18. A ligand, agonist or antagonist of the polypeptide according to claim 1 or 2, which comprises the polypeptide according to claim 1 or 2 or the partial peptide according to claim 3. Or a kit for screening for a functional modifier. 19. A ligand, agonist, antagonist or function of the polypeptide according to claim 1 or 2, obtained by using the screening method according to claim 16 or 17 or the screening kit according to claim 18. Modifiers or pharmacologically acceptable salts thereof. 20. The antibody according to claim 11, wherein the antibody is used.
A therapeutic agent for cancer or pituitary dysfunction, comprising a substance selected from the ligands, agonists, antagonists and function-modifying substances described in 1 or a pharmacologically acceptable salt thereof. 21. A case where a test sample is brought into contact with (i) a cell expressing the polypeptide of claim 1 or 2 and (ii) a cell expressing the polypeptide of claim 1 or 2 with a test sample. And claim 1 or 2 from the test sample.
3. A method for screening a compound that changes the expression level of a DNA encoding the polypeptide according to claim 1 or 2, wherein the method selects a compound that changes the expression of the gene encoding the polypeptide according to 1. 22. A method according to claim 12, wherein the fluctuation of the expression level is measured by the method according to claim 12 or the method according to claim 1 or 2 for quantifying the amount of mRNA encoding the polypeptide. 23. The screening method according to 22. 23. A test sample is contacted with a transformant containing a reporter gene-linked DNA downstream of a region controlling transcription of the gene encoding the polypeptide according to claim 1 or 2, and 3. The method according to claim 1, wherein a compound that changes the expression level of the DNA encoding the polypeptide according to claim 1 or 2 is selected from the sample. A method for screening a compound to be varied. 24. A compound obtained by a method selected from the screening methods according to claim 21 or a pharmacologically acceptable salt thereof. (25) A therapeutic agent for cancer or pituitary disorder comprising the compound according to (24) or a pharmacologically acceptable salt thereof. 26. An oligonucleotide having the same sequence as 5 to 60 consecutive bases in a base sequence of a DNA selected from the DNA of claim 4 or 6 and the DNA of SEQ ID NO: 2, and complementary to the oligonucleotide. Selected from oligonucleotides having specific sequences and derivative oligonucleotides of these oligonucleotides. 27. The DN according to claim 4, wherein the DN is
A transcription or mRN of a DNA encoding the polypeptide according to claim 1 or 2, using a DNA selected from A.
A method for suppressing the translation of A. 28. A gene in which the expression level of the polypeptide according to claim 1 or 2 has been changed by deleting or replacing all or a part of the gene containing the DNA encoding the polypeptide according to claim 1 or 2. Deleted or substituted non-human animals. 29. A method of contacting the animal according to claim 28, or an organ, tissue or cell of the animal with a test sample, and selecting a drug for treating cancer or pituitary disorder from the test sample. , A method for screening or evaluating a therapeutic agent for cancer or pituitary abnormality. 30. A compound obtained by the screening method according to claim 29 or a pharmacologically acceptable salt thereof. 31. A therapeutic agent for cancer or pituitary abnormality, comprising the compound according to claim 30 or a pharmacologically acceptable salt thereof.