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WO1999067383A1 - Nouvelle proteine receptrice couplee a la proteine g, adn correspondante et leur utilisation - Google Patents

Nouvelle proteine receptrice couplee a la proteine g, adn correspondante et leur utilisation Download PDF

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Publication number
WO1999067383A1
WO1999067383A1 PCT/JP1999/003306 JP9903306W WO9967383A1 WO 1999067383 A1 WO1999067383 A1 WO 1999067383A1 JP 9903306 W JP9903306 W JP 9903306W WO 9967383 A1 WO9967383 A1 WO 9967383A1
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Prior art keywords
protein
coupled receptor
dna
seq
receptor protein
Prior art date
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PCT/JP1999/003306
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English (en)
Japanese (ja)
Inventor
Yuko Nozaki
Takayuki Naito
Original Assignee
Japan Tobacco Inc.
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Publication date
Application filed by Japan Tobacco Inc. filed Critical Japan Tobacco Inc.
Priority to AU41694/99A priority Critical patent/AU4169499A/en
Publication of WO1999067383A1 publication Critical patent/WO1999067383A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/72—Receptors; Cell surface antigens; Cell surface determinants for hormones
    • C07K14/723—G protein coupled receptor, e.g. TSHR-thyrotropin-receptor, LH/hCG receptor, FSH receptor
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/72—Assays involving receptors, cell surface antigens or cell surface determinants for hormones
    • G01N2333/726—G protein coupled receptor, e.g. TSHR-thyrotropin-receptor, LH/hCG receptor, FSH
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value

Definitions

  • the present invention relates to a novel G protein-coupled receptor protein and its DNA. Further, a method for screening the agonist of the G protein-coupled receptor protein and the gonist, the agonist or the agonist obtained by the screening, an antibody against the G protein-coupled receptor protein and use thereof, The present invention also relates to a method of using the DNA fragment of the G protein-coupled receptor protein as a probe or primer. Background art
  • G protein-coupled receptor proteins Because they transduce intracellular signals through activation of G-coupled proteins. ing.
  • G protein-coupled receptor proteins are present on the surface of various functional cells in living cells and organs, and are important targets for hormones, neurotransmitters, and biologically active substances that regulate the functions of those living cells and organs. Has a role.
  • phospholipases When a stimulus is applied to the cell membrane, phospholipases are activated, the constituent lipids of the cell membrane and the like are degraded, and physiological activities such as arachidonic acid derivatives, platelet activating factor, lysophosphatidic acid, sphingosine-1-monophosphate, and anandamide Lipid mediators such as fats are produced.
  • Lysophosphatidic acid one of the lipid mediators, is glycerol It has a fatty acid at the 1- or 2-position of the skeleton and a phosphate group at the 3-position.
  • Lysophosphatidic acid includes 1-acyl lysophosphatidic acid, 1-alkyl lysophosphatidic acid, and 1-alkenyl lysophosphatidic acid having a fatty acid at position 1 of the glycerol skeleton. Lysophosphatidic acid and the like. And there is diversity depending on the type of fatty acid.
  • Lysophosphatidic acid along with sphingosine 1-phosphate, is also collectively referred to as lysophospholipids.
  • Lysophospholipids bind to G-protein-coupled receptors present on the surfaces of functional cells in living cells and organs to perform intracellular signal transduction, resulting in cell differentiation / proliferation, blood pressure regulation, inflammatory immune response, It is thought to regulate a variety of biological functions, including the regulation of nerve function.
  • G protein-coupled receptors such as Edg-1, Edg-2, Edg-3, Edg-4, and AGR16 have been known as receptors using lysophospholipids as ligands (Cell Science Vol. 17, No. 5 (1998)). Although these receptors use lysophosphatidic acid or sphingosine-1 1-phosphate as a ligand, some receptors are described in Nature 365, 557-560 (1993) and Nature 381 800-803 (1996). It has been reported that both lysophosphatidic acid and sphingosine-1-phosphate can be used as ligands. However, the relationship between these receptors and lysophospholipids, The functions of the body are not yet fully understood. Disclosure of the invention
  • An object of the present invention is to provide a G protein-coupled receptor using lysophosphatidic acid as a ligand, and a novel G protein-coupled receptor belonging to a group different from these known G protein-coupled receptors.
  • the present invention provides a method for screening an agonist binding to the G protein-coupled receptor, a method for screening an agonist of the G protein-coupled receptor, and an agonist and an agonist obtained by the screening.
  • the present invention provides an antibody useful for detecting the G protein-coupled receptor or regulating the activity of the G protein-coupled receptor.
  • Another object of the present invention is to provide a DNA fragment useful for cloning a G protein-coupled receptor using lysophosphatidic acid as a ligand.
  • the present inventors focused on the DNA sequence in the known transmembrane region of the lysophospholipid receptor, designed a primer, and isolated a fragment of cDNA encoding a human-derived G protein-coupled receptor protein. The nucleotide sequence was determined. Further primers were prepared based on the nucleotide sequence of the obtained fragment, and cDNA encoding human-derived G protein-coupled receptor protein was isolated.
  • the cDNA encoding the G protein-coupled receptor protein has a seven-time transmembrane region characteristic of the G protein-coupled receptor protein, and has a known lysophosphatidic acid receptor and sphingosine-1-phosphorus.
  • the homology between the DNA sequence of the acid receptor and the amino acid sequence indicated that the DNA encodes a human G protein-coupled receptor protein.
  • the expression tissue is different from the known G protein-coupled receptor that uses lysophosphatidic acid as a ligand.
  • an evolutionary phylogenetic tree based on the amino acid sequence was created, it was found that the gene belongs to a different group from the known G protein-coupled receptor that uses lysophosphatidic acid as a ligand. From this, it is considered that the protein-coupled receptor protein has a different function in vivo from the known one.
  • 1-oleoyl lysophosphatidic acid which is a kind of lysophosphatidic acid, was found to have an agonist activity.
  • the present inventors have used a receptor protein in which DNA encoding the human G protein-coupled receptor protein has been expressed by an appropriate means, and used any one of the following (1) to (3). By using the method described above, it was found that agonists and angoniists of the human G protein-coupled receptor protein can be easily screened from in vivo or natural and unnatural compounds.
  • intracellular signals such as second messenger such as intracellular calcium ion release and intracellular cAMP production
  • encoding the reporter plasmid was inserted Lucifera Ichize downstream of z if 2 6 8 promoter, EF-1 alpha downstream of promoter Isseki scratch ⁇ Hi Bok G protein-coupled receptor protein
  • the ⁇ -inserted receptor expression vector was introduced into PC12h cells together with the test compound, and the luciferase activity was measured.
  • the 1-oleoyl lysophosphatidic acid was ligated. Found to act as a
  • IL-6 and ET-1 are also known as growth factors for prostate support cells (J. Urol. 151 (5): 1396-1399 (1994), Eur J Pharmacol 349 (1): 123-8 ( 1998), Prostate 34 (4): 241-250).
  • the human G protein-coupled receptor protein is expressed in secretory cells and controls cell growth and secretion.
  • the prostate it can be expected to be a novel G protein-coupled receptor that not only controls secretory functions but also is involved in diseases such as prostate hypertrophy, prostate cancer, and prostatitis.
  • G protein-coupled receptor protein consisting of the amino acid sequence represented by SEQ ID NO: 1 or lysophosphatidine consisting of an amino acid sequence in which one or several amino acids have been deleted, replaced or added in SEQ ID NO: 1 G protein-coupled receptor protein with acid as the ligand.
  • a protein containing a G protein-coupled receptor protein comprising the amino acid sequence represented by SEQ ID NO: 1 or an amino acid sequence wherein one or several amino acids are deleted, substituted or added in SEQ ID NO: 1
  • a protein containing a G protein-coupled receptor protein that has lysophosphatidic acid as a ligand
  • a partial peptide of the G protein-coupled receptor protein according to (1) (4) a DNA containing a DNA encoding a G protein-coupled receptor protein consisting of the amino acid sequence represented by SEQ ID NO: 1; Or a DNA containing a G protein-coupled receptor encoding a lysophosphatidic acid consisting of an amino acid sequence in which several amino acids have been deleted, substituted or added, as a ligand.
  • a DNA comprising the G protein-coupled receptor protein DNA according to (4).
  • G protein-coupled receptor protein DNA represented by SEQ ID NO: 2.
  • a vector comprising the DNA according to (4).
  • An agonist selected by the screening method according to (11) above is a compound selected from lysophosphatidic acid.
  • a probe comprising a DNA fragment having a sequence of at least 15 consecutive nucleotides in the nucleotide sequence encoding the G protein-coupled receptor protein described in (1) or a nucleotide sequence complementary thereto.
  • the primer length is 15 to 40 bases.
  • the distance on the base sequence of the G protein-coupled receptor protein DNA corresponding to the selected primer is 100 to 3000 bases.
  • a G protein conjugate characterized in that a substance to be detected is present in a detection system containing the antibody according to (23), and the degree of reaction between the substance to be detected and the antibody is measured.
  • a method for detecting a type receptor protein is provided.
  • amino acid sequence in which one or several amino acids are deleted, substituted or added '' 1 to 20 amino acids, preferably 1 to 10 amino acids, more preferably 1 to 10 amino acids Amino acid sequences in which at least 5 and at most 5 amino acids have been deleted, substituted or added are preferred.
  • lysophosphatidic acid refers to a compound that has a fatty acid at the first or second position of the glycerol skeleton and has a structure in which a phosphate group is bonded at the third position, and a phosphate at the third position is cyclically placed at the second position And cyclic phosphatidic acid, which is also bound.
  • Compounds having a fatty acid at the 1-position of the glycerol skeleton include 1-silyl lysophosphatidic acid, 1-alkyl lyso-phosphatidic acid, 1-alkenyl lyso-phosphatidic acid, etc. And sillyzophosphatidic acid.
  • Fatty acids may be straight-chain or branched, and may be saturated or unsaturated. Fatty acids have from 12 to 24 carbon atoms. Preferably, it is lysophosphatidic acid having an unsaturated fatty acid having 12 to 24 carbon atoms, and more preferably, 1-acyl lysophosphatidic acid having a linear unsaturated fatty acid having 12 to 24 carbon atoms. Specifically, it is 1-oleoyl lysophosphatidic acid.
  • These peptide fragments may be selected from a region other than the transmembrane region, that is, a region protruding on the cell surface or a region protruding on the inner surface of the cell. It may be selected from a region where a region that is present or a region that protrudes into the cell inner surface is continuous. It is preferably a region protruding from the cell surface or a peptide fragment selected from the region.
  • the transmembrane region is approximately 32 to 55 (region I), approximately 68 to 88 (region II), approximately 107 from Met.
  • region III To the region of about 125th position (region III), the region of about 144th position to about 165th position (region IV), the region of about 192th position to about 221st position (region V region), a force region estimated to be in the region of about 241st to about 261st (region VI) and a region of about 278th to 295th position (region VII) About 1 to 20, preferably about 1 to 10, and more preferably about 1 to 5.
  • the region protruding from the cell surface is a hydrophilic region, which is a region from the 1st Met to about 31st, about 89 to about 106, and about 166 to about 106 It is estimated to be in the region of the 1st, 1st, about 26 2nd to about 277th positions, but it is about 1 to 20, preferably about 1 to 10, and more preferably about 1 to 5 Things are also included.
  • Partial DNA means DNA encoding the above-mentioned partial peptide, or a fragment of DNA encoding the amino acid sequence of SEQ ID NO: 1, which can be used as a probe or primer. Including fragments.
  • “Signal” means arachidonic acid release, a second messenger expressed through a G protein-coupled receptor, intracellular calcium ion release, Not only means intracellular signals such as intracellular cAMP production, but also includes indicators that can easily detect the expression of these intracellular signals. Examples of this indicator include repo overnight genes such as luciferase, aequorin, CAT, GUS, and ve-galactosidase.
  • the protein and the partial peptide in the present invention include not only a case where the terminal carboxyl group is a free carboxyl group but also a salt, ester or amide thereof.
  • the protein or partial peptide has the N-terminus (amino terminus) at the left end and the C-terminus (carboxyl terminus) at the right end according to the convention of peptide notation. Therefore, the proteins and partial peptides of the present invention, including the protein containing the amino acid sequence represented by SEQ ID NO: 1, usually have a carboxyl group (—COOH) or a carboxylate (_ C ⁇ _ ), But the C-terminus may be an amide or ester. The same applies to carboxyl groups in side chains other than the C-terminal.
  • the salt of the protein or partial peptide of the present invention is particularly preferably a physiologically acceptable acid addition salt.
  • Such salts include, for example, salts with inorganic acids (eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid) or organic acids (eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, Salts with co-octanoic acid, tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid) are used.
  • Esters include ethyl ester, and amides include CO NH 2 .
  • the protein, partial peptide or a salt thereof of the present invention can be produced from human or mammalian cells or tissues by a known method for purifying a protein or peptide, or a DNA encoding the protein of the present invention described below. It can also be produced by culturing a transformant containing the transformant. Alternatively, it can be produced according to a peptide synthesis method known per se.
  • human or mammalian tissues or cells are homogenized, extracted with an acid, etc., and the extract is subjected to salting out, dialysis, gel filtration, reverse phase chromatography. It can be purified and isolated by combining chromatography such as ion exchange chromatography.
  • the present invention provides a novel G protein-coupled receptor protein and a partial peptide thereof, as well as DNA encoding the G protein-coupled receptor protein and a partial DNA thereof.
  • DNA encoding the G protein-coupled receptor protein of the present invention was obtained as follows.
  • PCR was performed on human genomic DNA using a primer designed based on the DNA sequence in the known transmembrane region of the lysophospholipid receptor to obtain two gene fragments.
  • SEQ ID NO: 2 shows the nucleotide sequence of the G protein-coupled receptor protein of the present invention.
  • the amino acid sequence deduced from the nucleotide sequence of the receptor is shown in SEQ ID NO: 1. Furthermore, when an evolutionary phylogenetic tree based on the amino acid sequences of these genes was created, it was found that the gene belongs to a different group from the known G protein-coupled receptors that use lysophosphatidic acid as a ligand.
  • screening was performed using a system that detects the production of the second messenger as luciferase activity.
  • a reporter plasmid into which luciferase was inserted downstream of the zif268 promoter and a human G protein-coupled receptor protein of the present invention downstream of the EF-1 ⁇ promoter were encoded.
  • a receptor expression vector into which the DNA to be inserted was inserted was prepared.
  • the repo overnight plasmid and the receptor expression plasmid were both introduced into the PC12h cells, and the test compound was added to the resulting medium containing the transformed cells, and the luciferase activity was measured.
  • 1-oleoyl lysophosphatidic acid classified as 1-acyl lysophosphatidic acid acts as a ligand.
  • the expression distribution of the G protein-coupled receptor protein of the present invention was examined, it was confirmed that the expression distribution was high in the prostate gland and the trachea.
  • the G protein-coupled receptor protein of the present invention was expressed in main secretory cells but not in supporting cells, and (b) 1-oleoyl lysozyme was expressed.
  • phosphatidic acid was added to the medium of prostate secretory cells, it was found that the concentrations of IL-6 and endoselin-11 (ET-1) increased.
  • IL-16 and ET-1 are also known as growth factors for prostate supporting cells (J. Urol. 151 (5): 1396-1399 (1994), Eur J Pharmacol 349 (1): 123- 8 (1998), Prostate 34 (4): 241-250).
  • the G protein-coupled receptor protein of the present invention is expressed in secretory cells and controls cell growth or secretion.
  • the G protein-coupled receptor protein and the DNA encoding it are useful for controlling normal function in prostate glands and investigating the cause of disease, and for developing drugs that are effective in controlling normal function and treating and preventing disease. It is very useful for Furthermore, it is useful for investigating the causes of various diseases in the expressed tissues such as the trachea, and for developing pharmaceuticals effective for treating and preventing these diseases.
  • DNA encoding the G protein-coupled receptor protein of the present invention can be used, for example, to introduce disease into cells isolated from a patient who does not express the receptor of the present invention by using retrovirus or adenovirus to cause disease. It can also be used for gene therapy to be treated.
  • the lysophosphatidic acid is preferably lysophosphatidic acid having an unsaturated fatty acid having 12 to 24 carbon atoms, and more preferably having a linear unsaturated fatty acid having 12 to 24 carbon atoms.
  • the number of amino acids to be deleted, substituted or added is 1 or more and 20 or less, preferably 1 or more and 10 or less, more preferably 1 or more and 5 or less.
  • the present invention includes any DNA encoding the amino acid sequence shown in SEQ ID NO: 1. Also, as described above, it is a well-known fact that the physiological activity of a protein having biological activity may be maintained even when the amino acid sequence of the protein is slightly changed. Therefore, even if a G protein-coupled receptor protein in which one or more amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 1, G protein coupling using lysophosphatidic acid as a ligand DNA encoding the type receptor protein is included in the scope of the present invention.
  • the number of amino acids to be deleted, substituted or added is 1 or more and 20 or less, preferably 1 or more and 10 or less, more preferably 1 or more and 5 or less.
  • the DNA of the present invention includes a DNA that hybridizes to the nucleotide sequence of SEQ ID NO: 2 under controlled conditions and encodes a G protein-coupled receptor protein having lysophosphatidic acid as a ligand.
  • the controlled conditions are described, for example, in Sambrook et al., Molecula rCloning: AL aboratory Manual, 2nd du cation, Vol. 1, 101-104, (1986). Means. More specifically, for example, washing conditions at 1 ⁇ SSC, 0.5% SDS, and a temperature of 65 ° C. are included.
  • Mutants by deletion, substitution or addition of amino acids can be produced, for example, by site-directed mutagenesis (for example, Nucl 1. Aid Search, vol. 20, 6487-6500, 19.92).
  • Site-directed mutagenesis is, for example, a synthetic oligonucleotide primer complementary to the single-stranded phage DNA to be mutated, which is the desired mutation. Can be performed.
  • the method of deleting, substituting or adding an amino acid sequence includes a method of treating a gene with a mutagen or a method of cleaving a gene with a restriction enzyme and selecting a selected gene. There are also methods of removing, adding or substituting child fragments, and then ligating.
  • a variant may include conservatively substituted sequences, which means that a particular amino acid residue may be replaced by a residue having similar physicochemical characteristics.
  • conservative substitutions include substitutions between aliphatic chain containing amino acid residues, such as substitutions of I 1 e, Val, Leu or A1 a, or Lys and Arg. Substitutions between polar basic amino acid residues, such as mutual substitutions, are included.
  • the G protein-coupled receptor protein of the present invention or a partial peptide thereof is chemically synthesized, or an expression vector depending on the host is used to prepare a DNA encoding the G protein-coupled receptor protein of the present invention or a partial peptide thereof. It can be obtained by inserting, transforming, culturing, and isolating the host.
  • the G protein-coupled receptor or partial peptide of the present invention obtained as described above can be used as an antigen for the binding test of the G protein-coupled receptor protein of the present invention or the antigen for the production of antibodies, or as an antigen for producing an antibody. Can be used.
  • the G protein-coupled receptor protein or its partial peptide of the present invention may be performed by a conventional method of a gene recombination technique.
  • the G protein of the present invention may be added to an expression vector selected according to a host cell to be used, and a G protein of the present invention downstream of a suitable transcription or translation regulatory nucleotide sequence derived from a mammal, a microorganism, a virus, an insect gene, or the like. Insert a DNA sequence encoding a coupled receptor protein or a fragment thereof.
  • regulatory sequences include transcription promoters, operators, or enhancers, mRNA liposome binding sites, and appropriate transcriptional and translational initiation and termination controls. Sequences.
  • Prokaryotic cells, yeasts or higher eukaryotic cells can be used as host cells.
  • Suitable cloning and expression vectors for use in bacterial, fungal, yeast, and mammalian cell hosts are described, for example, in Pouwels et al., Cloning Vectors: A Laboratory Manual, Elsevier, New York, (1985).
  • Prokaryotes include gram negative or gram positive bacteria, for example, E. coli or Bacillus subtilis.
  • Expression vectors used in prokaryotic host cells generally contain one or more phenotype selectable marker genes.
  • a phenotypic selectable marker gene is, for example, a gene that confers antibiotic resistance or auxotrophy.
  • suitable plasmid vectors for prokaryotic host cells include commercially available plasmids such as pBR322 (ATCC 37017), or those derived therefrom.
  • PBR 322 contains genes for ampicillin and tetracycline resistance, making it easy to identify transformed cells.
  • An appropriate promoter and the DN ⁇ sequence of the] 3-galactosid ⁇ 2,6-sialyltransferase gene are inserted into this pBR322 vector.
  • Other commercially available vectors include, for example, pKK223-3 (Pharmacia Fine Chemicals, Epsala, Sweden) and pGEMl (Promega Biotec, Madison, Wis., USA).
  • Promoter sequences commonly used in expression vectors for prokaryotic host cells include tac promoter, i3_lactamase (penicillinase), lactose promoter (Chang et al., Nature 275: 615). And Goeddel et al., Nature 281: 544, 1979).
  • a particularly useful prokaryotic host cell expression system employs the phage ⁇ PL promoter and c I 857 ts thermostable repressor sequence.
  • Plasmid vectors available from the American Type Culture Collection that incorporate derivatives of the ⁇ PL promoter include: Includes plasmid PHUB2 (located in E. coli strain JMB9 (ATCC 37092)) and pPLC28 (located in E. coli RPI (ATCC 53082)).
  • yeast vector contains 2 // sequence of replication origin from yeast plasmid, autonomously replicating sequence (ARS), promoter region, sequence for polyadenylation, sequence for transcription termination, and selectable marker. Often contains genes. Other leader sequences suitable for promoting secretion of recombinant polypeptides from yeast hosts are also known. Methods for transforming yeast are described, for example, in Hinnen et al., Proc. Natl. Acad. Sci. USA 75: 1929, 1978.
  • a mammalian cell line When expressing the G protein-coupled receptor protein of the present invention or its partial peptide using a mammalian or insect host cell culture system, a mammalian cell line may be used. Transcription and translation control sequences for mammalian host cell expression vectors can be obtained, for example, from the viral genome. Commonly used promoter and enhancer sequences are derived from polyoma virus, adenovirus 2 and the like. For expression of structural gene sequences in mammalian host cells using the SV40 viral genome, e.g., DNA sequences derived from the SV40 origin, early and late promoters, enhancers, splice sites, and polyadenylation sites. Other genetic elements may be provided. Expression vectors for use in mammalian host cells can be constructed, for example, according to the method of Okayama and Berg (Mol. Cell. Biol. 3: 280, 1983).
  • DNA encoding G protein-coupled receptor protein of the present invention can be used as a probe or primer.
  • a probe may be designed based on either the sequence of SEQ ID NO: 2 or SEQ ID NO: 19.
  • the length is desirably at least 15 consecutive bases or more.
  • the probe can be labeled by a conventional method, for example, with a detectable enzyme such as a radioisotope or digoxigenin.
  • a detectable enzyme such as a radioisotope or digoxigenin.
  • radioactive P when using a cDNA fragment, it is convenient to label it with a random priming label, and when using a synthetic oligoprobe, it is convenient to label it at the 5 'end with a kinase.
  • the probe thus labeled is hybridized with a cDNA library or a genomic library and cloned. Hybridization can be carried out according to commonly used methods and conditions. Generally, cleaning at moderate strength, eg, 1X SSC, 0.5% SDS, 65 ° C.
  • the cDNA library to be used may be derived from animals including mammals, but is preferably derived from human tissues and cells.
  • two primers may be selected from SEQ ID NO: 2 or SEQ ID NO: 19 so as to satisfy the following conditions. I just need.
  • the primer length is 15 to 40 bases, preferably 20 to 30 bases.
  • the ratio of guanine to cytosine in the primer is 40% to 60%, preferably 45% to 55%, more preferably about 50%.
  • adenine, thymine, guanine, and cytosine in the primer sequence should not be partially biased.
  • a region in which guanine and cytosine are repeatedly distributed is considered to have low specificity, so that it may be used as a primer. Not appropriate.
  • the distance on the base sequence corresponding to the selected primer is 100 to 300 bases, preferably 150 to 100 bases, and more preferably 150 to 500 bases. That. and
  • the DNA may be synthesized using a commercially available DNA synthesizer, for example, PerkinElmer.
  • the present invention also provides a method for screening an agonist and an agonist for G protein-coupled receptor protein, and an agonist and an angel gonist selected by the screening.
  • the screening of agonists is performed by allowing a test sample to act in a system that expresses the G protein-coupled receptor protein of the present invention and detects a signal that mediates the receptor, and detects a signal downstream of the receptor. Can be performed by
  • agonists are screened by using a system that expresses a G protein-coupled receptor protein and a target gene induced downstream of a signal that mediates the G protein-coupled receptor. be able to.
  • This target gene expression system requires cells that express the G protein-coupled receptor of the present invention on the cell membrane.
  • a cell may be, for example, a natural cell line that expresses the G protein-coupled receptor of the present invention on the cell membrane, or a host transformed with DNA encoding the G protein-coupled receptor protein of the present invention. Cells or the like may be used.
  • the screening operation can be performed, for example, by detecting a target gene or a protein that is a target gene product.
  • the presence or absence of target protein expression can be easily determined without directly measuring the target gene or target protein.
  • a system for detecting another index that can be detected at the same time may be used.
  • a reporter gene such as luciferase, aequorin, CAT, GUS, or beta-galactosidase may be used to perform repo overnight gene access.
  • the repo overnight gene is a plasmid bound after the promoter region of the target gene, and this plasmid is used to transform cells expressing the G protein-coupled receptor of the present invention, and to detect the indicator. To build.
  • the screening of an anthony gonist is based on the expression of a target gene product when a ligand is allowed to act on the receptor in a system that expresses a G protein-coupled receptor protein and the target gene, and the reaction between the ligand and a test sample.
  • the expression can be compared with the expression of the target gene product.
  • cells expressing the G protein-coupled receptor of the present invention on the cell membrane are required as in the screening of agonists.
  • a natural cell line that expresses the G protein-coupled receptor of the present invention on a cell membrane, a cell produced by a genetic recombination method, and the like may be used.
  • Screening is performed by detecting a target gene or a protein that is a product of the target gene, but when the target protein is produced, it is possible to perform the screening in the same manner as agonist screening without directly measuring the target protein.
  • a system for measuring another index that can easily detect the presence or absence of expression of the target protein may be used.
  • An agonist selected by agonist screening can be used as the ligand. Specifically, it is 1-oleoyl lysophosphatidic acid.
  • the agonist and angyu gonist selected by the above screening are used for treatment and treatment of prostate diseases such as prostatic hypertrophy, prostate cancer, and prostatitis. It is useful as a drug effective for prevention and prevention.
  • the agonist or angelic gonist is formulated into a solid preparation (for example, a tablet) or a liquid preparation (for example, an injection) according to a method known per se, and is orally or parenterally (for example, an injection). The pharmacologically necessary and sufficient amount is administered to the patient. Little toxicity.
  • the present invention further provides an antibody against the G protein-coupled receptor protein of the present invention or a partial peptide of the protein.
  • An antibody for example, a polyclonal antibody or a monoclonal antibody
  • an antiserum against the G protein-coupled receptor protein or a partial peptide thereof of the present invention uses the G protein-coupled receptor protein of the present invention or a partial peptide thereof as an antigen.
  • a monoclonal antibody can be produced according to the following method. (a) Preparation of monoclonal antibody-producing cells
  • the G protein-coupled receptor protein or its partial peptide of the present invention is administered to a warm-blooded animal together with a carrier and a diluent.
  • a portion other than the transmembrane region of the receptor protein may be selected as the antigen.
  • the intramembrane transmembrane region is the region of about 32 to about 55 from Met (region I), the region of about 68 to about 88 position (region II), about 107 Approximately 125th region (region III), approximately 146th region to approximately 165th region (region IV), approximately 192th region to approximately 221st region (region V region), the region of approximately 2241st to approximately 2661th (region VI) and the region of approximately 278th to approximately 295th position (region VII). Other regions or fragments of these regions can be used as antigens.
  • the region protruding from the cell surface that is, the region from the first Met to about the 31st position, about 89th position Region from about 166 to about 191; about 262 to about 277; about 1 to 20, preferably about 1 to 10, more preferably about 1 to 5 May be.
  • FIG. 2 shows the putative structure of the receptor of the present invention.
  • the underlined part in the figure is the estimated transmembrane region.
  • the G protein-coupled receptor protein or its partial peptide of the present invention can be obtained by a protein expression method by chemical synthesis or gene recombination as described above.
  • Complete Freund's adjuvant / incomplete Freund's adjuvant may be administered in order to enhance the antibody-producing ability upon antigen administration to the animal.
  • the administration is usually performed once every 2 to 6 weeks, for a total of about 2 to 10 doses.
  • the warm-blooded animal used includes, for example, monkeys, rabbits, dogs, guinea pigs, mice, rats, sheep, goats, and chickens, and mice and rats are preferably used.
  • a monoclonal antibody-producing hybridoma When preparing monoclonal antibody-producing cells, select a warm-blooded animal immunized with the antigen, for example, an individual with an antibody titer from a mouse, collect the spleen or lymph nodes 2 to 5 days after the final immunization, and include them in By hybridizing the antibody-producing cells to myeloma cells, a monoclonal antibody-producing hybridoma can be prepared. The measurement of the antibody titer in the antiserum is performed, for example, by reacting the labeled lysophospholipid receptor described below with the antiserum, and then measuring the activity of the labeling agent bound to the antibody.
  • the fusion operation can be carried out according to known methods, for example, the method of Köller and Milstein (Nature, 256, 495 (1975)).
  • the fusion promoter include polyethylene glycol (PEG) and Sendai virus, and PEG is preferably used.
  • PEG polyethylene glycol
  • myeloma cells include NS-1, P3 Ul, SP 2/0, AP-1, and the like, with P3U1 being preferred.
  • the number of antibody producing cells (spleen cells) used and the number of myeloma cells The preferred ratio is about 1: 1 to 20: 1, and PEG (preferably PEG 1000 to PEG6000) is added to a degree of about 10 to 80%, and 20 to 40: preferably 30 to 37 to 1 to 30. Cell incubation can be performed efficiently by incubating for 10 minutes.
  • a variety of methods can be used to screen for anti-G protein-coupled receptor antibody-producing hybridomas.
  • the hybridoma culture supernatant is placed on a microplate on which a G protein-coupled receptor antigen is directly or adsorbed together with a carrier.
  • an anti-immunoglobulin antibody or protein A labeled with a radioactive substance or an enzyme is added, and a method for detecting an anti-G protein-coupled receptor monoclonal antibody bound to a microplate, an anti-immunoglobulin antibody or protein Add the hybridoma culture supernatant to the microplate to which A has been adsorbed, add a G protein-coupled receptor labeled with radioactive substances, enzymes, etc., and detect the anti-G protein-coupled receptor monoclonal antibody bound to the microplate And the like.
  • Selection of the anti-G protein-coupled receptor monoclonal antibody can be performed according to a known method or a method analogous thereto. It is usually performed in a medium for animal cells supplemented with HAT (hypoxanthine, aminobuterin, thymidine).
  • HAT hyperxanthine, aminobuterin, thymidine
  • any medium can be used as long as it can grow a hybridoma.
  • RPMI 1640 medium containing 1-20%, preferably 10-20% fetal calf serum, G1T medium containing 1-10% fetal calf serum (Wako Pure Chemical Industries, Ltd.) or hybridoma
  • a serum-free culture medium SFM-101, Nissui Pharmaceutical Co., Ltd.
  • SFM-101 Nissui Pharmaceutical Co., Ltd.
  • the cultivation temperature is usually 20 to 40 t, preferably about 37.
  • the culture time is usually 5 days to 3 weeks, preferably 1 week to 2 weeks. Culture is usually performed under 5% carbon dioxide gas.
  • the antibody titer of the hybridoma culture supernatant was identified in the same manner as the measurement of the anti-G protein-coupled receptor antibody titer in the antiserum described above. it can.
  • Separation and purification of the anti-G protein-coupled receptor monoclonal antibody can be carried out in the same manner as normal polyclonal antibody separation and purification.Immunoglobulin separation and purification [e.g., salting out, alcohol precipitation, isoelectric precipitation, electrophoresis Adsorption / desorption using an ion exchanger (e.g., DEAE), ultracentrifugation, gel filtration, antigen-binding solid phase, or active antibody such as protein A or protein G. This is performed according to a specific purification method for obtaining an antibody.
  • an ion exchanger e.g., DEAE
  • the G protein-coupled receptor antibody of the present invention produced according to the above method can specifically recognize the G protein-coupled receptor, quantification of the G protein-coupled receptor in a test solution, particularly It can be used for quantification by sandwich immunoassay.
  • the antibody to be used the antibody molecule itself may be used, or F (ab ') 2, Fab' or Fab fraction of the antibody molecule may be used.
  • the assay method using the antibody of the present invention is not particularly limited, and may be an antibody, an antigen, or an antibody-antigen complex corresponding to the amount of antigen (eg, the amount of G protein-coupled receptor) in the test solution.
  • Any measurement method may be used as long as the amount is detected by chemical or physical means and the amount is calculated from a standard curve prepared using a standard solution containing a known amount of antigen.
  • the nephrometry, competition method, immunometric method, and San Germanti method are suitably used, but the sandwich method described below is particularly preferable in terms of sensitivity and specificity.
  • Examples of the labeling agent used in the measurement method using a labeling substance include a radioisotope, an enzyme, a fluorescent substance, and a luminescent substance.
  • a radioisotope for example, 125 I, 3 H, "C and the like are preferable.
  • the enzyme a stable and large specific activity is preferable.
  • 3-galactosidase 3-darcosidase, a Lucariphosphatase, peroxidase, malate dehydrogenase, etc .
  • fluorescent substances such as fluorescamine and fluorescein isothiocyanate
  • luminescent substances such as luminol, luminol derivatives, luciferin, lucigenin, etc.
  • a biotin-avidin system can be used for binding the antibody or antigen to the labeling agent.
  • the insolubilization of the antigen or antibody physical adsorption may be used, or a method using a chemical bond usually used for insolubilizing and immobilizing proteins or enzymes may be used.
  • the carrier include insoluble polysaccharides such as agarose, dextran, and cellulose, synthetic resins such as polystyrene, polyacrylamide, and silicon, and glass.
  • sandwich method a test solution is reacted with an insolubilized anti-G protein-coupled receptor antibody (primary reaction), and further reacted with a labeled anti-G protein-coupled receptor antibody (secondary reaction).
  • the amount of the G protein-coupled receptor in the test solution can be determined.
  • the antibody used for the solid phase antibody or the labeling antibody is not necessarily one kind, and a mixture of two or more kinds of antibodies is used for the purpose of improving the measurement sensitivity and the like. May be used.
  • the anti-G protein-coupled receptor antibody used in the primary reaction and the secondary reaction has a site where the G protein-coupled receptor binds. Different antibodies are desirably used.
  • the antibodies used in the primary reaction and the secondary reaction include, for example, an antibody used in the secondary reaction, if the antibody used in the secondary reaction recognizes the C-terminal of the G protein-coupled receptor, Preferably, an antibody that recognizes other than the C-terminus, for example, the N-terminus, is used.
  • the G protein-coupled receptor antibody of the present invention can be quantified with high sensitivity.
  • Antibodies can also be used to control prostate function and to treat and prevent disease.
  • FIG. 1 is a view showing the expression status of mRNA analyzed by dot printing conducted to examine the expression distribution of the receptor of the present invention.
  • FIG. 2 is a diagram inferring the amino acid sequence and base sequence of the receptor of the present invention and the seven transmembrane regions.
  • Fig. 3 shows an evolutionary phylogenetic tree based on the amino acid sequence of a gene group highly homologous to GLM01, using the evolutionary phylogenetic tree creation program, SINCA (Fujitsu).
  • FIG. 4 is a diagram showing that the G protein-coupled receptor of the present invention increases luciferase activity in a concentration-dependent manner by 1-oleoyl lysophosphatidic acid.
  • FIG. 5 is a diagram showing the expression status of mRNA in human prostate tissue by in situ hybridization using an antisense probe.
  • FIG. 6 is a diagram showing the expression status of mRNA in human prostate tissue by in situ hybridization using a sense probe.
  • FIG. 7 is a diagram showing the results of examining changes in cell numbers by the action of 1-oleoyl lysophosphatidic acid on human prostate epithelial cells.
  • FIG. 8 is a diagram showing the results of examining the change in the concentration of IL-16 in the medium by allowing 1-oleoyl lysophosphatidic acid to act on human prostate epithelial cells.
  • Figure 9 shows that 1-oleoyl lysophosphatidic acid was added to human prostate epithelial cells.
  • FIG. 4 is a graph showing the results of examining the change in the concentration of ET_1 in a culture medium when activated.
  • Human genomic DNA (0.1 g / l: CL0NTECH Co., Ltd.) 101 is used as type III, and then added to sterile water 23 and 10 times concentrated solution of PCR buffer (lOOmMTris-HCl (pH8.3), 500 mM KCK 15 mM MgC12: Takara Shuzo Deoxynucleotides (dATP, dCTP, dGTP, dTTP, 2.5 mM each: Takara Shuzo) 51 1, Taq DNA polymerase (5u /: Takara Shuzo) 1 and edg.DF2 (SEQ ID NO: 3) ) Sense degenerate mix primer and edg.DRl (SEQ ID NO: 4) antisense degenerate 3 JL (500 M) of each mixed primer was added to prepare 50 reaction solutions.
  • PCR buffer lOOmMTris-HCl (pH8.3)
  • 500 mM KCK 15 mM MgC12 Takara Shuzo De
  • PCR was performed under the same conditions using the edg.DF3 (SEQ ID NO: 5) sense primer and the edg.DR3 (SEQ ID NO: 6) antisense primer.
  • the two PCR products thus obtained were separated by electrophoresis using a 2% agarose gel, and two bands of approximately 180 bp and 150 bp were excised from each, and the QIAEX II Agarose Gel Extraction Kit (manufactured by QIAGEN) ) was used to purify the PCR product.
  • PCR product cloned into pGEMT-Vector was reacted with Dye Deoxy terminator Cycle Sequencing Kit FS (ABI), and the nucleotide sequence was determined using a fluorescent autosequencer (ABI373A: ABI).
  • nucleotide sequence two novel nucleotide sequences were obtained in addition to the known nucleotide sequence of the G protein-coupled receptor used in designing the degenerate mix primer.
  • the obtained nucleotide sequence had homology to a known G protein-coupled receptor (SEQ ID NOs: 7 and 8; the nucleotide sequence of the primer portion was not included).
  • CDNA was synthesized as follows using human poly (A) RNA synthesis type II derived from human heart and testis (manufactured by CL0NTECH) and Superscript Preamplification System for First Strand cDNA Synthesis (manufactured by GIBCO BRL).
  • RNA 1 g of poly (A) RNA (manufactured by CL0NTECH) is added to a 10-fold concentrated solution of synthesis buffer (200 mM Tris-HCl (pH 8.4), 500 mM KC1) ⁇ 0u 25 mM MgC12 10 K 0.1 M DTT 10 l, Deoxy Nucleotides (dATP, dCTP, dGTP, dTTP, lOmM, respectively) 51, oligo dT12-18 primer (0. ⁇ g / 1) 51, reverse transcriptase Superscript II RT (200u / i1) 5 The reaction was for 50 minutes at 42. Then, E. Coli RNaseH 1) Add 5 1 to decompose RNA. The reaction solution was adjusted to 100/1 at the final stage.
  • synthesis buffer 200 mM Tris-HCl (pH 8.4), 500 mM KC1) ⁇ 0u 25 mM MgC12 10 K 0.1 M DTT 10 l,
  • the thus prepared human cDNA 41 was used as type II, and this was added to a PCR buffer (100 mM Tris-HCl (pH 8.3), 500 mM KCK 15 mM MgC12: Takara Shuzo) in sterile water 10.27 10 times concentrated solution.
  • a PCR buffer 100 mM Tris-HCl (pH 8.3), 500 mM KCK 15 mM MgC12: Takara Shuzo
  • nucleotide sequence fragment overlaps with SEQ ID NO: 7 upstream and SEQ ID NO: 8 downstream, it contains the nucleotide sequences (SEQ ID NOs: 7 and 8) of the two PCR products analyzed above and between them. It was confirmed that it was a connection. Therefore, it became clear that these three base sequences were derived from the same transcript. In addition, based on homology analysis with known G protein-coupled receptors, etc., it is speculated that SEQ ID NO: 11 is responsible for the transmembrane domains III to VII of the novel G protein-coupled receptor. Was done.
  • Hybridization was performed on a Human RNA Master Blot (manufactured by CLONTECH) in which poly (A) RNA derived from 50 tissues of humans was dotted on a nylon membrane, and the expression distribution of this gene was examined.
  • Plasmid DNA 1 (2.5 ng / 1) was added as type II, and 2 l of PCR buffer (100 mM Tris-HC1 (pH 8.3), 500 mM KC1, 15 mM MgC12: Takara Shuzo Co., Ltd.) Deoxynucleotides (dATP, dCTP, dGTP, dTTP, 2.5 mM each: Takara Shuzo) 2 zl, Taq DNA polymerase (5u / u ⁇ : Takara Shuzo) 0.25 1 and F05F1 (SEQ ID NO: 9) Sense primer and antisense primer of F05R1 (SEQ ID NO: 12) (10 zM) to prepare 201 reaction solutions.
  • PCR buffer 100 mM Tris-HC1 (pH 8.3), 500 mM KC1, 15 mM MgC12: Takara Shuzo Co., Ltd.
  • Deoxynucleotides dATP, dCTP, dGTP,
  • the solution was subjected to PCR under the following conditions: [2 minutes at 94 ⁇ (30 seconds ⁇ 55: 30 seconds ⁇ 2 minutes at 72) X 35 times ⁇ 10 minutes at 72].
  • GeneAmp PCR System 9600 (ABI) was used as a PCR apparatus.
  • the obtained PCR product was separated by electrophoresis using 4% NuSieve 3: 1 Gel (manufactured by FMC), a 118 bp band was cut out, and the PCR product was separated using the QIAEX II Agarose Gel Extraction Kit (manufactured by QIAGEN). Was purified.
  • Probe labeling was performed using [-32P] dCTP as follows using only the antisense primer.
  • PCR product 11 (20 ng / l) was used as type II, and this was added to sterile water 301, a 10-fold concentrated PCR buffer (1 OOmM Tris-HCl (pH8.3), 500 mM KC1, 15 mM MgC 12: 5 1, dATP, dGTP, dTTP (0.2 mM each: Takara Shuzo) 1 ⁇ 1, each [a-32P] dCTP (3.3 mM: Amasham) 51 1, Taq DNA polymerase (5 u / H) : Takara Shuzo Co., Ltd.) and 50 ⁇ l of a reaction solution to which 5 zl (lOAiM) of an antisense primer of F05R1 (SEQ ID NO: 12) was added.
  • a 10-fold concentrated PCR buffer (1 OOmM Tris-HCl (pH8.3), 500 mM KC1, 15 mM MgC 12: 5 1, dATP, dGTP, dT
  • This probe was subjected to gel filtration purification using ProbeQuant G-50 Micro Columns (Pharmacia).
  • Blot Fil Yuichi was prehybridized in a 15 ml ExpressHyb buffer (CL0NTECH) containing 1.5 mg salmon sperm DNA (CL0NTECH) heat denatured at 94 for 5 minutes for 55 hrs. . Thereafter, 200 ⁇ l containing the above probe and 150 ⁇ g salmon sperm DNA (CL0NTECH) was heat-denatured in 1945 minutes to 6 ml of ExpressHyb buffer (CL0NTECH) and hybridized at 55 overnight. . After this, the filter was washed with a washing solution containing 0.1XSSC (15mM sodium chloride, 1.5mM sodium citrate) and 0.1% SDS at 65. Was cleaned. Hybridization signals were visualized using Bio-imaging Analysis System 2000 (Fujifilm).
  • LA-TaqPCR buffer 1 (Takara Shuzo) 51 in sterile water 29.10 times concentrated solution, deoxynucleotide (dATP, dCTP, dGTP, dTTP, 2.5xM: Takara Shuzo) 8 x U LA-Taq DNA polymerase (5u / il: Takara Shuzo) 0.5 1 and 5'-RACE F05R2 (SEQ ID NO: 13) antisense primer 1 ⁇ l (10 ⁇ l) of each adapter API (CLONTECH) was added to prepare a 50 / il reaction system.
  • the resulting two PCR products were separated by electrophoresis using 0.8% agarose gel, bands of about 800 bp and 500 bp were cut out, and purified using QIAEX II Agarose Gel Extraction Kit (QIAGEN).
  • the PCR product was transferred to 2 1 (p.1611 ⁇ 2 / 1), sterile water 2 z1, pGEM T-Vector
  • SEQ ID NO: 17 The nucleotide sequence obtained by 5'-RACE (SEQ ID NO: 17) and the nucleotide sequence obtained by 3'-RACE (SEQ ID NO: 18) match the previously obtained nucleotide sequence (SEQ ID NO: 11). There was an area to do. By further assembling these, a single connected base sequence (SEQ ID NO: 19) was obtained.
  • SEQ ID NO: 19 Has a large open reading frame, which has homology with known G protein-coupled receptors and has been shown to encode a novel G protein-coupled receptor.
  • Marathon-Ready Human Prostate 51 type 1 was added to sterile water 29.5 ti, 10 times concentrated solution of Ex-Taq PCR buffer 1 (Takara Shuzo) 5 ⁇ 1, deoxynucleotide (dATP, dCTP, dGTP, dTTP) 2.5 mM each: Takara Shuzo Co., Ltd.
  • the PCR product was subjected to buffer exchange with MicroSpin S-200 HR Columns (Pharmacia) and then treated with restriction enzymes (Hpal, Xbal). This was separated by electrophoresis using 1% agarose gel, one band of about 1200 bp was cut out, and purified using QIAEX II Agarose Gel Extraction Kit (manufactured by QIAGEN).
  • the restriction-enzyme-treated PCR product approximately 2 ng / zl
  • pGEM3zf 50 ng / l
  • 11 were incubated with LigationKit Ver.2 solution I (Takara Shuzo) 5 // 1 at 16 for 3 hours and ligated. .
  • the plasmid thus obtained was transformed into Escherichia coli DH5a competent cells (manufactured by Toyobo) to obtain clones. Plasmid DNA was purified from this E. coli clone using the QIAGEN Plasmid Mini Kit (QIAGEN).
  • Plasmid MA cloned into pGEM3zf was reacted with Dye Deoxy terminator Cycle Sequencing Kit FS (manufactured by ABI), and the nucleotide sequence was determined using a fluorescent autosequencer (ABI373A: manufactured by ABI) (SEQ ID NO: 22). It was confirmed to be identical to the 19 partial sequences.
  • the highest homology was human edg-2 and human edg-4, with 47.5% and 46.1% homology at the amino acid level, and 57.2% and 56.9% homology at the nucleic acid level. There was one.
  • the activity of the ligand was measured by the reporter gene method by activating the zif268 promoter.
  • a repo overnight rasmid (pGL2-zif) having luciferase inserted downstream of the zif 268 promoter was prepared by the following procedure.
  • primer F 1 5'- PCR was performed using 3 ') (SEQ ID NO: 23) and primer Rl (5, one AGAGAGAAG CTTGAAGCTACTGAGGGCACACT-3') (SEQ ID NO: 24), and the promoter region of the zif 268 gene [to the transcription start site— 526 to 227 (Proc. Nat 1. Acad. Sci., 86, 377-381, 1989)].
  • the amplified DNA fragment was digested with a restriction enzyme SacII and then blunt-ended, and then further digested with a restriction enzyme KpnI to obtain fragments of -526 to 97.
  • a receptor expression vector in which the receptor gene represented by SEQ ID NO: 2 was inserted downstream of the EF-1 ⁇ promoter was prepared.
  • PC12h cells (Develomental Brain Research, 6 (3), 243-250 (1983)) were seeded in a collagen-coated culture flask (IWAKI) so that they became semi-confluent the next day, and cultured at 37. did.
  • the medium used was (containing 10% horse serum, 5% fetal calf serum, benicillin 50 units Zml, streptomycin 50 / X gZm 1)! -MEM.
  • Receptor expression vector (pEF-humanGLMOl) 50 ng / ⁇ , reporter plasmid (pGL2-zif) 20 ng / ⁇ , G16 expression vector (pME-G16) 2.5 ng / ⁇ , empty
  • the prepared cell solution (100 1 / ⁇ ) was added to the DNA-SuperFect solution, and the cells were seeded on a collagen-coated 96-well plate (IWAKI).
  • an expression vector (pEF-106r) containing no receptor gene was subjected to cotransfection with repo overnight plasmid under the conditions described above.
  • Luciferase activity was measured using a luciferase reporter gene assay kit (Behringer Mannheim) as a substrate and a LUMINOUS CT-9000D (DIA-IATRON) as a measuring instrument.
  • luciferase activity was shown to increase depending on the concentration of LPA (C18: l) as compared to the control (Fig. 4).
  • the circles in the figure represent the average of three measurements, and the bars represent the standard errors.
  • the human prostate gland removed by surgery was cut into blocks of about 5 mm square, and fixed by immersion in 4 fixative (4% paraformaldehyde / 0.1 M phosphate buffer [pH 7.4]). Fix the fixed tissue block in 4% with 10% sucrose / 0.1 M phosphate buffer [pH 7.4], 15% sucrose / 0.1 M phosphate buffer [ ⁇ 7.4], 20% sucrose / 0.1 ⁇
  • the cells were immersed in phosphate buffer [ ⁇ 7.4] and 30% sucrose / 0.10 phosphate buffer [ ⁇ 7.4] sequentially for 30 minutes to perform sucrose replacement.
  • the sucrose-substituted human prostate tissue block was replaced with 0.CT coimmediate oimd
  • the frozen human prostate tissue block was cut into 6 m thick sections using CRY0CUT 1800 (manufactured by Leica) and mounted on a slide glass coated with 3-aminopropyltriethoxysilane. After air drying for 1 hour, it was dried at 45 for 2 hours.
  • a part of the gene of the present invention was amplified by PCR so that a T7 promoter was added to its end.
  • F05-F1 primer (5'-GAGGCACATGTCMTCATGAGG-3 ') (SEQ ID NO: 9) 10 picomoles
  • T7-AS primer (5'-
  • TAATACGACTCACTATAGGGTTCTCCTGAGAGAAGC-3 ′ (SEQ ID NO: 25) 10 pmol, 0.4 ig of plasmid DNA cloned with the gene of the present invention, four types of deoxynucleotide triphosphates (dATP, dCTP, dGTP, TTP) each at 6.25 nmol, and rTaq
  • dATP deoxynucleotide triphosphates
  • dCTP deoxynucleotide triphosphates
  • dGTP dGTP
  • TTP deoxynucleotide triphosphates
  • rTaq A reaction solution containing 2.5 units of DNA polymerase (Takara Shuzo) was prepared.
  • reaction solution was heated for 2 minutes at 95 using GeneAmp 9600 (manufactured by PE Applied Biosystems), and then subjected to 25 cycles of 95 minutes for 30 seconds, 55 seconds for 30 seconds, and 72 minutes for 72 minutes, and further heated for 72 minutes for 10 minutes. .
  • the reaction solution was subjected to agarose gel electrophoresis, and a 570 bp amplified DNA fragment was separated and purified using QIAEX II (manufactured by QIAGEN).
  • DIG RNA labeling kit (Rochedai Digoxigenin-labeled RNA probe was prepared using the same method as described above.
  • Reaction solution 201 containing 200 ng of purified DNA fragment, 20 units of RNase inhibitor, 20 nm of ATP, 20 nm of CTP, 20 nm of GTP, 13 nm of UTP, 7 nm of digoxigenin-labeled UTP, and 20 units of T7 RNA polymerase Prepared. The reaction solution was kept warm for 372 hours to react.
  • a part of the gene of the present invention was amplified by PCR so that an SP6 promoter was added to the end.
  • the reaction mixture was heated using GeneAmp 9600 (manufactured by PE Applied Biosystems) for 95 t: 2 min., followeded by 95 30 s '55 30 s' 72 ⁇ : 2 min 25 cycles, followed by heating for another 72 10 min did.
  • GeneAmp 9600 manufactured by PE Applied Biosystems
  • the reaction solution was subjected to agarose gel electrophoresis, and a 433 bp amplified DNA fragment was separated and purified using QIAEX II (manufactured by QIAGEN).
  • a reaction solution 201 containing 160 ng of the purified DNA fragment, 20 units of RNase inhibitor, 20 nm of ATP, 20 nm of CTP, 20 nm of GTP, 13 nm of UTP, 7 nm of digoxigenin-labeled UTP, and 20 units of SP6 RNA polymerase was prepared. . The reaction solution was kept warm for 372 hours to react.
  • Frozen sections of human prostate tissue were immersed in phosphate buffered saline three times for 5 minutes each and washed.
  • the substrate was immersed in 0.2 N hydrochloric acid for 20 minutes.
  • the Proteinase K solution was placed on a section of human prostate tissue and incubated for 37 15 minutes.
  • the cells were washed and then immersed twice in phosphate buffered saline containing 2 mg / ml glycine for 15 minutes.
  • the cells were sequentially immersed in 50% ethanol, 70% ethanol, and 95% ethanol, and then air-dried at room temperature for 10 minutes.
  • 50% deionized formamide / 4xSSC containing 2 g / ml probe was mounted on a section of human prostate tissue.
  • Ix TBK buffer solution 8 g / l NaCl, 0.2 g / 1 KCl, 3 g / 1 Tris base [pH 7.4]) for 10 minutes.
  • the blocking solution was placed on a section of human prostate tissue and left at room temperature for 30 minutes.
  • Alkaline phosphatase-labeled anti-digoxigenin antibody (Roche Diagnostics) was diluted 500-fold with a blocking solution, mounted on a section of human prostate tissue, and reacted overnight at 4: 5.
  • the plate was immersed in 1 ⁇ TBK buffer at room temperature three times for 10 minutes each and washed.
  • reaction was stopped by immersion in phosphate buffered saline.
  • LPA LPA is applied to normal human prostate epithelial cells (PrE (Clonetics)) to increase cell proliferation, basic fibroblast growth factor (bFGF) secretion, interleukin-6 (IL-6) secretion, interleukin-6 soluble receptor (IL-6sR) secretion, endothelin-1 (ET-1) secretion, PSA (prostate specific antigen) secretion, TGF (trans forming growth factor) -vehicle 1 secretion, and vascular endothelial growth factor (VEGF ) Changes in secretion were measured, with significant effects on cell proliferation, interleukin 6 (IL-6) secretion, and endothelin-11 (ET-1) secretion.
  • bFGF basic fibroblast growth factor
  • IL-6 interleukin-6
  • IL-6sR interleukin-6 soluble receptor
  • EGF vascular endothelial growth factor
  • Changes in secretion were measured, with significant effects on cell proliferation, interleuk
  • PrEC The PrEC to 1 day, PrEGM medium; 5Xl0 to (Clonetics Corp. BPE, hydrocortisone, human EGF, Epinefurin, transferrin, insulin, retinoic acid, preparative Riyo one Gasironin, PrEBM medium Clonetics Corporation plus GA- 100) 4 /
  • the cells were suspended at a concentration of 2 ml and 2 ml was inoculated into each well of a 6-well multiplate.
  • PrEGM medium or LPA l-oleoyl-2-hydroxy-sn-glycero-3-phosphate; Avanti Polar Lipids ⁇
  • 10 M3 ⁇ 4 2 ml of calyzed PrEGM medium was added.
  • the medium was removed and the cells were washed with 1 ml of phosphate buffered saline. To this, 0.5 ml of a trypsin EDTA solution was added to release the cells. The removed medium was used to measure bFGF secretion, IL-6 secretion, endothelin-1 secretion, and EGF secretion.
  • the released cells were suspended in ISOTON II (Beckman Cole Yuichisha), and the number of cells was counted using a Koruyuichi Counter ZM (former Cole Yuichisha, current Beckman Cole Yuichisha).
  • Fig. 7 shows the average value and standard error of the three measurements. The cell number was reduced when LPA was added.
  • the amount of IL-6 in the culture medium was measured by sandwich ELISA using Quanticain Human IL-6 Immunoassay Kit (manufactured by R & D Systems). The concentration was expressed as pg / ml of IL-6 concentration in the medium.
  • Fig. 8 shows the average value and standard error of the three measurements.
  • the IL-6 concentration increased when LPA was added.
  • the amount of ET-1 in the medium was measured by the sandwich ELI SA method using the human ET-1 ELI SA system (manufactured by Amersham Pharmacia Biotech), and the ET-1 concentration in the medium was measured as f mo 1 e / m1.
  • Figure 9 shows the average value and standard error of the three measurements.
  • the ET-1 concentration increased when LPA was added.
  • the present invention is extremely useful for the development of a medicament that can be used for diagnosis, treatment and prevention of prostate diseases. That is, by using the G protein-coupled receptor of the present invention, it became possible to screen an antagonist of the G protein-coupled receptor. Further, by using the G protein-coupled receptor of the present invention and its ligand lysophosphatidic acid, it became possible to easily screen for an agonist of the G protein-coupled receptor.

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Abstract

L'invention concerne une nouvelle protéine réceptrice couplée à la protéine G qui peut servir au développement de médicaments ainsi que l'ADN codante pour cette protéine. L'invention concerne également la nouvelle protéine réceptrice couplée à la protéine G dont le ligand est l'acide lysophosphatidique ainsi que l'ADN codant pour ce récepteur. De même, l'invention se rapporte à un procédé de recherche d'un antagoniste et d'un agoniste de cette protéine réceptrice, une sonde et une amorce basées sur cette ADN ainsi qu'un anticorps dirigé contre la nouvelle protéine réceptrice.
PCT/JP1999/003306 1998-06-22 1999-06-21 Nouvelle proteine receptrice couplee a la proteine g, adn correspondante et leur utilisation WO1999067383A1 (fr)

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1151006A4 (fr) * 1998-12-18 2002-07-24 Smithkline Beecham Corp Recepteur couple de proteine g humaine
EP1138693A4 (fr) * 1998-12-11 2002-09-04 Takeda Chemical Industries Ltd Nouvelle proteine recepteur couplee a une proteine g et son adn
WO2003013605A1 (fr) * 2001-08-07 2003-02-20 Japan Tobacco, Inc. Remedes pour maladies prostatiques
WO2003104480A1 (fr) * 2002-06-08 2003-12-18 Aventis Pharma Deutschland Gmbh Procede pour identifier des agonistes ou des antagonistes du recepteur de type gpr45/gpr63
US7300764B2 (en) 2002-06-08 2007-11-27 Sanofi-Aventis Deutschland Gmbh Method for identifying agonists and antagonists of the GPR45-like/GPR63 receptor

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JPH1118788A (ja) * 1997-05-13 1999-01-26 Smithkline Beecham Corp Ｇタンパク質共役受容体 ｈｏｆｎｈ３０

Patent Citations (1)

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JPH1118788A (ja) * 1997-05-13 1999-01-26 Smithkline Beecham Corp Ｇタンパク質共役受容体 ｈｏｆｎｈ３０

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AN S, ET AL.: "MOLECULAR CLONING OF THE HUMAN EDG2 PROTEIN AND ITS IDENTIFICATION AS A FUNCTIONAL CELLULAR RECEPTOR FOR LYSOPHOSPHATIDIC ACID", BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, ACADEMIC PRESS INC. ORLANDO, FL, US, vol. 231, 1 January 1997 (1997-01-01), US, pages 619 - 622, XP002926347, ISSN: 0006-291X, DOI: 10.1006/bbrc.1997.6150 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1138693A4 (fr) * 1998-12-11 2002-09-04 Takeda Chemical Industries Ltd Nouvelle proteine recepteur couplee a une proteine g et son adn
EP1151006A4 (fr) * 1998-12-18 2002-07-24 Smithkline Beecham Corp Recepteur couple de proteine g humaine
WO2003013605A1 (fr) * 2001-08-07 2003-02-20 Japan Tobacco, Inc. Remedes pour maladies prostatiques
WO2003104480A1 (fr) * 2002-06-08 2003-12-18 Aventis Pharma Deutschland Gmbh Procede pour identifier des agonistes ou des antagonistes du recepteur de type gpr45/gpr63
US7300764B2 (en) 2002-06-08 2007-11-27 Sanofi-Aventis Deutschland Gmbh Method for identifying agonists and antagonists of the GPR45-like/GPR63 receptor

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