JP2002238578A - Rat edg7 receptor - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a rat EDG7 receptor protein and a polynucleotide encoding rat EDG7 receptor and further provide an oligonucleotide hybridizing under stringent conditions with the polynucleotide, an antibody binding to the protein or its fragment, and a method for screening a substance having activity inhibiting binding of the protein to LPA. SOLUTION: This invention relates to the rat EDG7 receptor protein, the polynucleotide encoding the protein, the oligonucleotide hybridizing under stringent conditions with the polynucleotide, the antibody binding to the protein or its fragment and a method for screening the substance having activity inhibiting binding of the protein to LPA.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a rat EDG7.
(Endothial differential
ion gene-7, and so on. A) a receptor protein, a polynucleotide encoding the protein, an oligonucleotide that hybridizes with the polynucleotide under stringent conditions, an antibody or a fragment thereof that binds to the protein, a protein and lysophosphatidic acid (hereinafter referred to as LPA). And a pharmaceutical composition containing one selected from the group consisting of the polynucleotide, the antibody or a fragment thereof, and a substance selected by the screening method. .

[0002]

2. Description of the Related Art EDG7 receptor is a G protein-linked receptor cloned from human Jurkat T cell mRNA as an LPA receptor having various actions such as cell proliferation, cancer cell infiltration and wound healing. (Hereinafter referred to as GPCR) (Bandoh et al., J.B.
iol. Chem. , 274, 27776 (199
9)). Subsequently, the EDG7 receptor has also been cloned in mice (Ishii et al., Mol. Pha.
rmacol. , 58, 895 (2000)).

[0003] EDG2 receptor and EDG4 receptor have been known as analogous receptors of EDG7 receptor which has reactivity to LPA.
53.7% and 4 at the receptor and amino acid levels, respectively.
It has 8.8% sequence identity.

[0004] GPCRs have a molecular weight of 40,000 to 50,000, the N-terminus of which is outside the cell, and sugar chains are usually bound at two to three places, and the cell membrane is bound to 7
Once through the C-terminus is present in the cell. These transmembrane domains are represented by I-VII and describe the structural and functional characteristics of the receptor (Dohlman et al., Ann. R.
ev. Biochem. , 60, 653 (199
1)). The transmembrane region is assembled in a circle in the plasma membrane, and its central portion forms a binding pocket for accepting an agonist molecule. However, when the agonist is a macromolecule such as a protein hormone, the extracellular N-terminal Extracellular region or three extracellular loops participate in agonist recognition.

[0005] The activated receptor interacts with an intracellular G protein complex, which is further activated by intracellular signaling activities (generally by activation (Gs) or inhibition (Gi) of adenylyl cyclase). mediates the regulation of cAMP production or the activation of phospholipase Cβ (Gq) to produce inositol triphosphate).

The human and mouse EDG7 receptors are LP
It has been shown that after the reaction with A, the intracellular Ca ²⁺ ion concentration is increased through the production of inositol triphosphate by the activation of phospholipase Cβ (Bandoh).
J. et al. Biol. Chem. , 274, 27776
(1999), Ishii et al., Mol. Pharmac
ol. , 58, 895 (2000)).

[0007] LPA has a strong cell proliferation effect (van Corven et al., Cell, 59, 45 (1)
989), Jalink et al., Biochim. Biop
hys. Acta. , 1198, 185 (199
4)), and the EDG7 receptor gene is prostate in human,
Because it is highly expressed in the testis, pancreas, and testis and kidney in mice (Bandoh et al., J. Biol. Ch.
em. , 274, 27776 (1999), Ishii.
Et al., Mol. Pharmacol. , 58, 895 (2
000)), may be involved in benign prostatic hyperplasia, prostatitis, prostate cancer, testicular cancer, pancreatitis, nephritis and the like.

[0008]

GPCRs are expressed and activated in many developmental and disease processes. Identification of new GPCRs provides an opportunity to diagnose or intervene in such processes, and the receptor may trigger its activity and prolong or suppress the time it is active Can be used in screening assays for the identification of various physiological or drug molecules.

Although the sequence of the EDG7 receptor is known in humans and mice, expression in the testis and kidney of the rat has been confirmed using the human EDG7 receptor gene as a probe (Im et al., Mol. Pharmaco
l. , 57, 753 (2000)), and its sequence is not clear. It is very useful to elucidate the physiological function of rat EDG7 receptor in elucidating the physiological function in humans.

In the GPCR, the sequence identity at the amino acid level between human and rat is around 90%, but a drug molecule having a high affinity for the human receptor has a different affinity for the rat receptor. May be indicated. In such cases, elucidating the affinity of the drug molecule for the human and rat receptors is useful for clinical studies to determine the maximum tolerated and safe dose in the human body.

An object of the present invention is to provide a rat EDG7 receptor protein and a polynucleotide encoding the rat EDG7 receptor. In addition, the present invention is useful for detecting the polynucleotide, an oligonucleotide that hybridizes with the polynucleotide under stringent conditions, an antibody or a fragment thereof that binds to the protein useful for detecting the protein, the protein And a pharmaceutical composition comprising one selected from the group consisting of the polynucleotide, the antibody or a fragment thereof, and a substance obtained by the screening method. The purpose is to do.

[0012]

Means for Solving the Problems The present inventors have proposed a human ED.
In the course of research involving the G7 receptor, rat testis cDN
Novel rat EDG7 receptor cDN from A library
We isolated A and thought that it could be used for regulating cell growth, studying invasion of cancer cells, and creating new drugs, and completed the present invention.

That is, the gist of the present invention is (1)
(A) an amino acid sequence represented by SEQ ID NO: 1; (b) an amino acid sequence having a substitution, deletion, addition, or insertion of at least one residue in the amino acid sequence of (a); a) an amino acid sequence having at least 98% sequence identity with the amino acid sequence of a), a protein having one amino acid sequence selected from the group consisting of: (2) (A) SEQ ID NO: 1 (B) SEQ ID NO: in the sequence listing
(C) substitution of at least one base in the base sequence of (A) or (B),
(D) a base sequence having a deletion, addition or insertion; (D) a base sequence different from any of the base sequences (A) to (C) through degeneracy; (E) a base sequence (A) to (D) A nucleotide sequence of a nucleic acid capable of hybridizing under stringent conditions with an antisense strand of a nucleic acid having any of the following nucleotide sequences; and (F) at least the sequence identity with the amino acid sequence shown in SEQ ID NO: 1 A polynucleotide having one kind of base sequence selected from the group consisting of a base sequence encoding a sequence of 98%, (3) the polynucleotide according to the above (2), which is a cDNA or a chemically synthesized DNA; (4) an oligonucleotide capable of hybridizing with the polynucleotide of (2) or (3) under stringent conditions; (5) a polynucleotide of (2) or (3); (6) a prokaryotic or eukaryotic cell comprising the recombinant vector of (5); (7) a step of culturing the cell of (6); And (8) a recombinant protein obtainable by the method described in the above (7), (9) a recombinant protein obtainable by the method described in the above (7),
(10) A method for screening a substance having an activity of inhibiting the binding between the protein of (1) or (8) and LPA, the antibody or a fragment thereof capable of specifically binding to the protein of (i). A) contacting the protein according to (1) or (8) with LPA in the presence of a test substance; and (ii) evaluating the binding between the protein according to (1) or (8) and LPA. If the degree of binding between the protein described in (1) or (8) and LPA in the presence of the test substance is lower than that in the absence of the test substance, the decrease in the binding is indicated as an index. A screening method for determining that the test substance has an inhibitory activity, (11) the binding between the protein of (1) or (8) and LPA, which can be selected by the method of (10). Substance having an inhibiting activity,
(12) A drug comprising one selected from the group consisting of the polynucleotide according to (2) or (3), the antibody or fragment thereof according to (9), and the substance according to (11). A composition.

[0014]

DESCRIPTION OF THE PREFERRED EMBODIMENTS As a result of cloning from a rat testis cDNA library for the purpose of isolating the rat EDG7 receptor, a novel rat EDG7 receptor cDNA was obtained.
Was found. Examination of the amino acid sequence of such a protein revealed that it has relatively high sequence identity with humans and mice. The present invention relates to the protein.

That is, one embodiment of the protein of the present invention has a property of specifically binding to LPA having various actions such as cell proliferation, infiltration of cancer cells, and wound healing. Proteins determine the substrate specificity of LPA and act to promote cell proliferation, cancer cell invasion, wound healing, and the like. Therefore, since the protein of the present invention can control cell proliferation and progression of invasion of cancer cells, it is possible to treat various diseases caused by abnormal cell proliferation, diseases which can reduce symptoms by regulating cell proliferation, and the like. An excellent effect of being able to be applied to a semiconductor device can be exhibited.

The protein of the present invention includes, for example,
A protein having the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing is exemplified. The protein having the amino acid sequence of SEQ ID NO: 1 is referred to as “rat EDG7 receptor”
(Hereinafter referred to as REDG). Further, the protein of the present invention may be, for example, a protein having an amino acid sequence having at least one substitution, deletion, addition or insertion in the amino acid sequence of REDG, and SEQ ID NO: 1. Is preferably at least 97%, more preferably 98% or more, and even more preferably 99% or more.
% Or more.

In the present specification, "sequence identity" refers to a residue between two sequences in the case of an amino acid sequence, or a base between two sequences in the case of a polynucleotide sequence. Refers to sequence similarity. Such sequence identity is determined by comparing two properly aligned sequences. Here, the sequence to be compared may have an addition or a deletion (for example, a gap or the like) as compared with a reference sequence (for example, a consensus sequence or the like) for optimal alignment of the two sequences.

Specifically, the same nucleobases or amino acid residues present in both sequences are determined to determine the number of matching sites, and then the total number of bases or amino acid residues in the sequence region to be compared. The number of matching sites is divided by the total number of groups, and the obtained value is multiplied by 100 to calculate sequence identity (%). Optimizing the alignment when comparing both sequences was performed by Smith and Waterman (Wat).
erman) local homology algorithm (Lo
cal homology algorithm) (A
dd. APL. Math. , 2: 482 (198
1)), Needleman and Wunsch homology alignment algorithm (Homology alignment algorithm)
ritm) (J. Mol. Biol., 48, 443).
(1970)), a study on the method of identity between Pearson and Lipman (Se).
arch for similarity metho
d) (Proc. Natl. Acc. Sci., 85,
2444 (1988)).

Such sequence identity can be determined, for example, by using sequence analysis software, specifically, BLASTver. It is measured using 2.0 or the like. Such software is available at the website address: http: // www. ncbi. nlm.
nih. gov / BLAST / is generally available. According to BLAST, default parameters for comparing two sequences are, for example, preferably 4, more preferably 5, and preferably 1, and more preferably 2, the open gap weight.

The “amino acid sequence having a substitution, deletion, addition or insertion” may be any amino acid sequence of a GPCR that regulates the binding specificity of LPA to REDG, even if it is a naturally occurring sequence, May be a sequence obtained by introducing a substitution, deletion, addition or insertion into the sequence.

The term "at least one residue" means one or more or more residues. The number of “substitutions, deletions, additions or insertions” can be appropriately selected as long as the protein having the amino acid sequence is an amino acid sequence of a GPCR that regulates the binding specificity of LPA to REDG. The number of “substitution, deletion, addition or insertion” is preferably 1 to 20, more preferably 1 to 10
Individual.

The introduction of artificial substitutions, deletions, additions or insertions can be performed by, for example, molecular cloning:
Laboratory Manual 2nd Edition (Molecular
Cloning: A Laboratory Manu
al 2nd ed. ) (Sambrook
k) et al., (1989)).

Further, in another embodiment of the protein of the present invention, G protein regulating the binding specificity of LPA to REDG is used.
In the case of PCR, proteins that are related to alternative splicing variants of all the above proteins are also included.

The REDG of the present invention has a function of binding to LPA and has a property of specifically binding to LPA.
Therefore, the protein of the present invention is considered to be one of the factors that control cell proliferation and cancer cell invasion through LPA binding.

As described above, considering the possibility that can be assumed from the existence of the protein of the present invention, diseases caused by abnormalities of cancer or other cell proliferation, and diseases whose symptoms can be alleviated by regulating cell proliferation. It is important to examine the expression mode of the protein in the above. That is, depending on the state of the organs and cells (for example, normal cells and cancer cells), various proteins involved in cell proliferation and invasion of cancer cells through the binding of LPA to REDG are considered to occur. Proteins and genes encoding them are useful for the diagnosis and treatment of cancer and other diseases caused by abnormal cell proliferation, diseases that can reduce symptoms by regulating cell proliferation, and the development of new pharmaceuticals. is there.

Diseases caused by abnormal cell growth and diseases whose symptoms can be reduced by regulating cell growth include, for example, prostatitis, benign prostatic hyperplasia, prostate cancer, testicular cancer, pancreatitis, rheumatoid arthritis. , Various autoimmune diseases, P
Examples include vascular restenosis after TCA, pulmonary fibrosis, glomerulonephritis and the like.

[0027] The polynucleotide of the present invention comprises L
A polynucleotide encoding a GPCR that regulates the binding specificity of PA to REDG also includes a polynucleotide having a nucleotide sequence having at least one substitution, deletion, addition or insertion in the nucleotide sequence of the polynucleotide. It is.

The “base sequence having a substitution, deletion, insertion or addition” may be a naturally-occurring sequence, if it encodes a GPCR that regulates the binding specificity of LPA to REDG. It may be a sequence obtained by introducing a substitution, deletion, addition or insertion.

"At least one base" means one or more bases or more. The number of “substitutions, deletions, additions or insertions” is determined by the number of Gs that the resulting polynucleotide regulates the binding specificity of LPA to REDG.
As long as it encodes a PCR, it can be appropriately selected within a range that encodes a GPCR that regulates the binding specificity of LPA to REDG. The number of “substitution, deletion, addition or insertion” is preferably 1 to 20, more preferably 1 to 10.

To confirm whether or not the protein encodes the protein, for example, a protein is expressed from a polynucleotide as described below, and
Whether or not the GPCR regulates the binding specificity to EDG can be determined by determining according to the method described in Example 3 described later.

The introduction of artificial substitutions, deletions, additions or insertions can be performed, for example, by using molecular cloning:
Laboratory Manual 2nd Edition (Molecular
Cloning: A Laboratory Manu
ak 2nd ed.) (Sambrook
k) et al., (1989)).

[0032] The polynucleotide of the present invention may also be a polynucleotide having a base sequence different from the base sequence of the polynucleotide through degeneracy.

Furthermore, a polynucleotide having a nucleotide sequence encoding a GPCR that regulates the binding specificity of LPA to REDG can hybridize under stringent conditions to the antisense strand of the nucleotide sequence of the polynucleotide. Polynucleotides having different nucleotide sequences are also included in the present invention.

The hybridization can be carried out, for example, by the method described in Molecular Cloning: A Laboratory Manual, 2nd edition. Also, "stringent conditions" include:
Molecular cloning: A Laboratory
The conditions described in the second edition of the manual and the like are mentioned. For example, the conditions for hybridization are 42 ° C. and 24 hours in 6 × SSC containing 50% formamide. The washing conditions were 0.1 × SSC, 68 ° C., 3
0 minutes, etc.

The polynucleotide of the present invention includes GPC that regulates the binding specificity of LPA to REDG.
If it encodes R, the sequence identity with the protein, for example, the amino acid sequence shown in SEQ ID NO: 1, is preferably at least 97%, more preferably 98% or more, and still more preferably 99% or more. May have a base sequence encoding the amino acid sequence of SEQ ID NO: 2, and the sequence identity to the base sequence shown in SEQ ID NO: 2 is preferably at least 98%, more preferably 99% or more. May have the following base sequence:

[0036] The polynucleotide of the present invention is not limited to its origin, and may be, for example, a gene derived from humans, mice and the like.

According to the polynucleotide of the present invention, abnormalities in cancer or cell proliferation can be caused by detecting mRNA that is detected when the protein of the present invention is expressed, for example, for each organ or each cell. Diagnosis of a disease or a disease in which symptoms can be alleviated by regulating cell proliferation can be performed. Further, the translation of the mRNA is inhibited by using an antisense strand or the like, the progress of cell proliferation is regulated by suppressing the expression of the protein of the present invention, and diseases or cells caused by cancer or abnormal cell proliferation are controlled. It is also possible to treat diseases in which the symptoms can be alleviated by regulating proliferation.

In the case of cDNA or chemically synthesized DNA,
It can be used to produce a large amount of the protein of the present invention. Further, the protein can be used to produce an antibody against the protein, or a screening system for a substance that inhibits the binding of the protein to LPA can be constructed. It can be useful and useful. In addition, it is expected that cell growth can be regulated by introducing the gene into a living body and expressing the same, and diseases caused by abnormalities in cancer or cell proliferation, and symptoms can be reduced by regulating cell proliferation. It is expected to be applied to the treatment of various diseases.

The cDNA of the present invention is, for example, mRNA
Can be prepared by RT-PCR or the like using as a template.
Further, chemically synthesized DNA can be synthesized, for example, using a DNA synthesizer.

The present invention further provides an oligonucleotide capable of hybridizing with the polynucleotide of the present invention under stringent conditions.

The oligonucleotide of the present invention has a part of a sequence complementary to the base sequence of the polynucleotide of the present invention. The length of the oligonucleotide depends on the purpose,
Although it can be selected as appropriate, 10 to 500 bases, preferably 30 bases
Desirably, it is 300 bases. A hapten for detection after hybridization may be introduced into such an oligonucleotide. Examples of haptens include digoxigenin, FITC, biotin, and the like. In addition, it may be labeled with a radioisotope for detection. Further, from the viewpoint of enabling each of the polynucleotides encoding the protein of the present invention to hybridize to a unique nucleotide sequence of each of the polynucleotides, such oligonucleotides are all or a part of the polynucleotide of the present invention. It is desirable to design to include a base sequence or a complementary sequence thereof.

The sequence of the oligonucleotide can be appropriately selected depending on its Tm value and sequence specificity as long as it specifically hybridizes to the base sequence of the polynucleotide of the present invention.

Here, the stringent conditions include, for example, the conditions for oligonucleotides for probes or primers described in the aforementioned Molecular Cloning: Laboratory Manual, 2nd edition.

The oligonucleotide of the present invention comprises a primer for specifically amplifying the polynucleotide of the present invention or a probe for detecting the polynucleotide, for example, mRNA encoding EDG7 receptor expressed in each tissue. It can be used as a probe for in situ hybridization to be detected or as a probe for Northern blotting, and is applied to diagnosis of diseases caused by abnormalities of cancer or cell growth, and diseases whose symptoms can be reduced by regulating cell growth. It is also possible and useful.

The protein of the present invention can be prepared by a method known to those skilled in the art as a natural protein or as a recombinant protein prepared by using a gene recombination technique.

When the protein is prepared as a natural protein, for example, an immunoprecipitation method using an anti-REDG antibody, or an antibody obtained by immunizing a small animal with the prepared recombinant protein is immobilized on a column. It can be prepared by subjecting an extract of a tissue or cell expressing a protein to affinity chromatography using the column.

In addition, the protein of the present invention can be produced in a large amount if prepared using a gene recombination technique. Such proteins are also included in the present invention.

To express a polynucleotide of the present invention, for example, a polynucleotide having the nucleotide sequence shown in SEQ ID NO: 2, to produce a protein, for example,
Molecular cloning described above: It can be carried out based on many books and documents such as a laboratory manual. A translation initiation codon is added upstream of the gene to be expressed and a translation stop codon is added downstream, and a promoter sequence (eg, trp, lac, T7) that controls transcription is added.
And the like, and by adding a control gene to the appropriate vector, a recombinant vector that replicates and functions in a host cell is produced.

[0049] A recombinant vector containing the polynucleotide encoding the protein of the present invention is also included in the present invention. The vector used for the recombinant vector of the present invention can be selected according to the cell used as the host cell. When the host cell is an E. coli cell, the vector may be pUC
118, pUC119, pBR322, pCR3, pc
Plasmid vectors such as MV SPORT, λZAP
And phage vectors such as λgt11. When the host cell is a yeast cell, the vector may be p
YES2, pYEUra3, etc., for insect cells, pAcSGHisNT-A, and for animal cells, pKCR, pEFBOS, pEFCROS
cDM8, pCEV4 and the like. Such a vector may appropriately have factors such as an inducible promoter, a selection marker gene, and a terminator.

In order to facilitate isolation and purification, Hi
An s tag and a sequence that can be expressed as a GST fusion protein may be added. In this case, a suitable promoter (lac, tac, trc, tr) that functions in the host cell is used.
GST (glutathione S-transferase) fusion protein vector (pGEX4T etc.) or Tag having p, CMV, SV40 early promoter etc.
(Myc, HisA, etc.)
cDNA 3.1 / Mys-His) can be used.

Next, the recombinant vector is introduced into an appropriate host cell to obtain a transformant. Examples of the host cell include cells derived from prokaryotes or eukaryotes, for example, Escherichia coli cells, yeast cells, animal cells, insect cells, and the like. Examples of Escherichia coli include Escherichia coli K-12 strains HB101, C600, JM109, and DH5.
α strain, DH10B strain, XL-1 Blue MRF ′
Strain, TOP10F strain and the like. Examples of yeast cells include Saccharomyces cerevisiae. Animal cells include L, 3T3, FM3A, CH
O 2, COS, Ver, Hela, NIH3T3 and the like. Insect cells include sf9. Moreover, as a method for introducing a gene into a host cell, a biological method, a physical method, a chemical method, and the like are known to those skilled in the art, and these methods can be used. Examples of the biological method include a method using a virus vector, and examples of the physical method include an electroporation method and a microinjection method. Examples of the chemical method include a lipofection method, a calcium phosphate method, and a DEAE-dextran method. The target protein is produced by culturing the obtained transformant in a suitable medium. The protein obtained as described above can be isolated and purified by a general biochemical method, for example, a method using an ion exchange column, an affinity column, or the like.

[0052] The present invention also includes cells derived from prokaryotic or eukaryotic cells that carry the recombinant vector.

It is presumed that the protein of the present invention is expressed for each organ or each cell according to the state of each cell, and therefore, is considered to be able to function as a marker for determining the state of a cell, It is expected to be applied to diagnosis of diseases caused by cancer or abnormal cell growth, and diseases whose symptoms can be alleviated by regulating cell growth.

The present invention further provides an antibody against the protein or a fragment thereof. The antibody or a fragment thereof is also included in the present invention.

The antibody of the present invention may have any ability to specifically bind to the protein of the present invention, and may be a polyclonal antibody or a monoclonal antibody or a fragment thereof. At least three, preferably 10 to 200, of the amino acid sequences of the protein of the present invention.
And more preferably an antibody or a fragment thereof capable of specifically binding to a peptide having 10 to 50 consecutive amino acids. In addition, from the viewpoint that the unique amino acid sequence of each protein of the present invention can be more specifically recognized and identified, the peptide is preferably a portion characteristic of the amino acid sequence of the protein of the present invention.

Further, as the antibody of the present invention, an antibody modified by a known technique or an antibody derivative, for example, a humanized antibody, a Fab fragment, a single-chain antibody and the like can be used. The antibody of the present invention was obtained, for example, in 1992 by John Wiley & Sons, Inc.
ns, Inc), John E. Corrigan (John
E. FIG. Coligan) Edit, Current Protocols in Immunology (Current Protocol)
ol in Immunology) and the like, and can be easily prepared by immunizing a rabbit or mouse with all or a part of the protein of the present invention. Antibodies can also be produced by genetic engineering. When used for human therapy, humanized antibodies are desirable from the viewpoint of immunogenicity.

Further, the obtained antibody is purified and then treated with peptidase or the like to obtain an antibody fragment. Applications of the obtained antibodies or fragments thereof include detection of the protein of the present invention, affinity chromatography, screening of various libraries (for example, cDNA libraries), application to medicines, research reagents, and the like.

When the antibody of the present invention or a fragment thereof is used for diagnostic purposes, the antibody or the fragment of the present invention is used to facilitate detection by enzyme immunoassay, fluorescence immunoassay, luminescence immunoassay and the like. The fragment may be subjected to various modifications.

The present invention can further provide a method for detecting the protein of the present invention. In the method for detecting the protein of the present invention, the antibody of the present invention or a fragment thereof is added to a test sample, and when the protein of the present invention is present in the test sample, the detection is easily performed using the antigen-antibody reaction as an index. Can be. Such a detection method is expected to be applied to diagnosis of diseases caused by cancer or abnormal cell proliferation, and diseases in which the symptoms can be alleviated by regulating cell proliferation.

The antigen-antibody reaction can be detected by, for example, an enzyme immunoassay, a fluorescence immunoassay, a luminescence immunoassay or the like.

According to the present invention, a method for detecting a polynucleotide encoding the protein of the present invention can also be provided. By detecting the polynucleotide, particularly the mRNA after splicing, it becomes possible to diagnose the condition of an organ or a cell.

The polynucleotide of the present invention can be detected by Southern blot analysis or Northern blot analysis using the oligonucleotide of the present invention as a probe, PCR using the oligonucleotide as a primer,
It can be performed by analysis using a DNA chip or the like. In the case of Southern blot analysis or Northern blot analysis, the presence or absence of a polynucleotide can be detected using the presence or absence of hybrid formation as an indicator. In the case of PCR, the presence or absence of a polynucleotide can be detected using the presence or absence of an amplification product as an index.

Further, according to the present invention, a kit capable of detecting the protein of the present invention can be provided. According to such a kit, a disease caused by abnormality of cancer or cell proliferation,
Diseases whose symptoms can be reduced by regulating cell proliferation can be easily diagnosed. The kit may include at least the antibody of the present invention or a fragment thereof.

Further, according to the present invention, a kit capable of detecting the polynucleotide of the present invention can be provided. According to such a kit, it is possible to easily diagnose a disease caused by an abnormality of cancer or cell growth, or a disease whose symptoms can be reduced by regulating cell growth. The kit may contain at least the oligonucleotide of the present invention.

Further, the present invention relates to the protein of the present invention and L
Provided is a method for screening a substance having an activity of inhibiting the binding to PA. The screening method of the present invention comprises:
(1) a step of bringing the protein of the present invention into contact with LPA in the presence of the test substance; and (2) a step of evaluating the binding between the protein of the present invention and LPA.

Examples of test substances used in the screening include purified proteins (including antibodies), expression products of gene libraries, libraries of synthetic peptides, cell extracts, cell culture supernatants, and live low-molecular-weight compounds. Rally and the like, but are not limited thereto. The substance may be a chemically synthesized substance,
It may be a naturally occurring substance.

The phrase "evaluating the binding between the protein of the present invention and LPA" means measuring the presence or absence of the binding between the protein of the present invention and LPA and the degree of such binding.

In the screening of the present invention, the binding was evaluated by adding an RI-labeled (eg, ³ H-labeled) LPA to the protein of the present invention in the presence of a test substance, washing after incubation, and measuring the radioactivity of the bound LPA. By measuring, the presence or absence of inhibition of the binding of the protein of the present invention to LPA can be examined.

As a result of the screening, when the degree of binding between the protein of the present invention and LPA in the presence of the test substance is lower than in the absence of the test substance,
Using this as an index, it is determined that the test substance has an activity of inhibiting the binding between LPA and the protein of the present invention.

[0070] The substances obtained by these screening methods are also included in the present invention.

The screening method of the present invention is particularly effective for screening a substance that can be used in a pharmaceutical composition for suppressing cell proliferation and progression of cancer cell invasion.

According to the present invention, there is provided a pharmaceutical composition for further suppressing the progress of cell proliferation. As the pharmaceutical composition of the present invention, a composition containing one selected from the group consisting of the polynucleotide of the present invention, an antibody or a fragment thereof, and a substance having an activity of inhibiting the binding of LPA to the protein of the present invention is used. No.

As a method for administering the pharmaceutical composition containing the polynucleotide of the present invention, the gene is incorporated into a viral vector from the viewpoint of easy introduction of a sense nucleic acid or an antisense nucleic acid of the polynucleotide into cells. The oral construct, an inhalation administration, an intravenous injection, a subcutaneous injection, an intraorgan injection, or the like, or a composition containing an expression plasmid carrying a sense nucleic acid or an antisense nucleic acid of the polynucleotide of the present invention directly into a muscle. Intracellular administration (DNA vaccine method), lipofection method, microinjection method, calcium phosphate method, electroporation method and the like.

In addition, liposomes or membrane-fused liposomes containing the polynucleotide of the present invention (Sendai virus
(HIVJ) -liposomes) can be in the form of a liposome preparation such as a suspension, a freezing agent, or a centrifugal concentrated frozen agent.

The polynucleotide used in the pharmaceutical composition is
To increase the translocation into the cell or the stability in the cell,
For example, phosphothioate, phosphorodithioate,
Alkyl phosphotriesters, alkyl phosphonates,
It may have a chemically modified nucleic acid such as an alkylphosphamidate. The sense nucleic acid or antisense nucleic acid is preferably designed to include a sequence complementary to the 5 'translation initiation region of mRNA.

The content of the active ingredient in the pharmaceutical composition, that is, one content selected from the group consisting of the polynucleotide of the present invention, the antibody or a fragment thereof, and a substance having an activity of inhibiting the binding between LPA and the protein of the present invention. Can be appropriately adjusted within a range where the progress of cell proliferation can be suppressed.

The pharmaceutical composition of the present invention can control various phenomena related to the interaction between the protein of the present invention and LPA (for example, progression of cell proliferation). Therefore, the composition can be used to treat cancers and other diseases caused by abnormal cell proliferation, and diseases in which the symptoms can be alleviated by regulating cell proliferation.

The pharmacological effect of the pharmaceutical composition is determined as follows. The pharmaceutical composition is allowed to act on various established cancer cells in vitro, and its growth inhibitory effect is determined by an XTT assay. Also in vi
In vo, the pharmaceutical composition is intraperitoneally administered to nude mice into which various cancer cells have been implanted subcutaneously, and the size or mortality of the implanted tumor mass is determined by comparing with the control.

The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to these examples.

Example 1 Rat EDG7 Receptor cDNA
Isolation of clones To isolate the rat EDG7 receptor cDNA,
mR of human EDG7 receptor registered in nBank
NA base sequence (Accession No. AF12)
7138) and a human brain cDNA library (Li
The cloning of the gene for the protein was attempted using a 1081 base fragment having an open reading frame of the human EDG7 receptor, which was amplified using the following PCR primers as a template and a 1081 base fragment (manufactured by Fe Technologies) as a template. Forward primer: 5'-CGGGATCCATGAATGAGTGTCACTAT
G-3 '(SEQ ID NO: 3) Reverse primer: 5'-GCTCTAGAATCCAGAGTTTAGGAAGT
GC-3 '(SEQ ID NO: 4)

A rat testis cDNA library (1 × 1
0 ⁶ plaque-formigunits, Str
atagene) is fixed to a nylon filter (manufactured by Daiichi Kagaku) by a conventional method, and then cDN is obtained by a plaque hybridization method using the above probe.
The A clone was selected to obtain the cDNA clone.

Specifically, the probe was used as a Random
Primer DNA Labeling Kit Ve
r. 2 (manufactured by Takara Shuzo) and [α- ³² P] dCTP
(Amersham Pharmacia Biote
(Ch. company). Using this labeled probe, 5X
SSC, 1% SDS, 2X Denhard't so
lution, containing 1 mg / ml salmon sperm DNA
After hybridization with the filter in 0% formamide at 42 ° C. for 16 hours, the plate was washed twice with 0.1% SDS containing 2 × SSC at 42 ° C. for 2 minutes. Thereafter, the filter was applied to an X-ray film (Koda
After that, positive plaques were determined by performing autoradiography at -80 ° C for 16 hours to obtain one positive plaque.

A phagemid of a cDNA clone was obtained from the positive plaque by an in vivo cleavage process. In this process, the host strain was co-infected with both library phage and fl helper phage. Polypeptides or enzymes derived from both library-containing phage and helper phage will cut into the DNA and produce new DNA from defined sequences on the target DNA.
The synthesis was initiated and a smaller single-stranded circular phagemid DNA molecule containing the entire DNA sequence of the pBluescript phagemid and cDNA insert was generated. This phagemid DNA is released from the cells and is produced to produce new host cells (SOLR®, S
Tratagene). Double stranded DNA was produced in this host cell.

The nucleotide sequence of the cDNA clone was determined using a DNA sequencer LIC4200 (manufactured by Li-cor).

The obtained nucleotide sequence information was incorporated into gene information analysis software GeneWorks (manufactured by Teijin System Technology Co., Ltd.) and compared with the sequence of GenBank. As a result, it was found that the homolog was a human and mouse EDG7 receptor homolog. However, it was found that the C-terminal portion was deficient in 67 amino acids and 68 amino acids, respectively, as compared with human and mouse. Therefore, to clarify the nucleotide sequence of the C-terminal part of the cDNA clone, 3 ′
-The RACE method was performed.

Specifically, Marathon-Read
y cDNA rat tests (Clontech
And the following forward primer prepared based on the sequence of the cDNA clone
The following reverse primer (Nested adapt) attached to the on-Ready cDNA rat testis
or Primer 2) was used to perform a nested polymerase chain reaction (Nested PCR). Forward primer: 5'-CGGAATTCGACGGTGATGACTGTCT
TAG-3 '(SEQ ID NO: 5) Reverse primer: 5'-ACTCACTATAGGGCTCGAGCGGC-
3 '(SEQ ID NO: 6)

Advantage as a polymerase
2 Polymerase Mix (Clontech) was used. The composition of the reaction solution was 10x cDNA PC
R Reaction Buffer 5 μl, template DN
A 5 μl, each primer 10 μM, Advantage
e2 Polymerase Mix (50X) and d
1 μl of NTP (10 mM), balance 50 μl with sterile distilled water
l. The amplification reaction was incubated at 94 ° C. for 30 seconds, followed by 30 cycles at 94 ° C. for 5 seconds and 68 ° C. for 4 minutes.

Next, using a part of the reaction mixture of the nested PCR as a template, the following forward primer prepared based on the sequence of the cDNA clone and Marath
The following reverse primer (Adaptor Pri) attached to on-Ready cDNA rat testis
mer 2) using a secondary polymerase chain reaction (2n
d PCR) to try to amplify the target gene. Forward primer: 5'-CGGAATTCAATGTGCAGCACGTGAA
GCG-3 '(SEQ ID NO: 7) Reverse primer: 5'-CCATCCTAATACGACTCACTATAGG
GC-3 '(SEQ ID NO: 8)

Specifically, a mixture of 5 μl of the nested PCR reaction mixture and 245 μl of 10 mM Tris · HCl (pH 7.5) containing 1 mM EDTA was used as a template. The composition of the reaction solution is 10 × cD
NA PCR Reaction Buffer 5μ
1, 5 μl of the mixture, 10 μM of each primer, A
dvantage 2 Polymerase Mix
(50X) and 1 μl of dNTP (10 mM), the balance being 50 μl in sterile distilled water. The amplification reaction was performed at 94 ° C for 30 minutes.
After incubating for 5 seconds, 5 seconds at 94 ° C. and 4 seconds at 68 ° C.
Twenty cycles were performed with one minute as one cycle.

The reaction solution after completion of the reaction is treated with phenol / chloroform and precipitated with ethanol to perform PCR.
The product was obtained. After digesting the obtained PCR product with EcoRI,
The sample was subjected to 1% agarose electrophoresis. After electrophoresis, Gene
Target PC using Clean II (Funakoshi)
The R product was recovered from the gel. The recovered PCR product is Ec
After subcloning into pBluescript SK (-) digested with oRI, the nucleotide sequence was determined.

Example 2 Analysis of Nucleotide Sequence When the partial nucleotide sequence of the cDNA clone was analyzed in the same manner as in Example 1, it was found that the cDNA was a partial sequence of rat EDG7 receptor cDNA having a stop codon. From the partial nucleotide sequence and the partial sequence of Example 1, the amino sequence of the open reading frame of rat EDG7 receptor cDNA and the nucleotide sequence encoding the same were revealed.

Based on the amino acid sequence and the base sequence, a comparison with the sequence of GenBank was performed in the same manner as in (1), and as a result, the human EDG7 receptor (Accession) was obtained.
No. AF127138) and the mouse EDG7 receptor (Accession No. AF293845) were found to have 91% and 96% sequence identity at the amino acid level, respectively. Figure 1 shows the rat EDG7
Receptor and human EDG7 receptor, and FIG.
The figure which compared the amino acid of G7 receptor and mouse EDG7 receptor is shown.

The nucleotide sequence encoding the open reading frame of the rat EDG7 receptor is expressed by human EDG7
It had 84% and 93% sequence identity with the receptor and the mouse EDG7 receptor, respectively.

As a result of searching for a sequence match using BLAST based on the base sequence and amino acid sequence of the rat EDG7 receptor cDNA, a GP with unknown function was found in GenBank.
Rat snGPCR32 (A registered as CR
ccession No. AF097733).

[0094] The above sequence identity indicates that at the amino acid level, rat snGPCR32 lacks nine amino acids at the N-terminal compared to rat EDG7 receptor.
All were identical except that the fourth alanine was replaced with valine, and at the nucleotide sequence level was 98%. FIG.
2 shows a diagram comparing the amino acids of rat EDG7 receptor and rat snGPCR32. FIGS. 4 and 5 show diagrams comparing the nucleotide sequences encoding the open reading frames of rat EDG7 receptor and rat snGPCR32.

Example 3 Functional solution of rat EDG7 receptor
Analysis (Elevation of intracellular Ca ²⁺ ion concentration by LPA) Transient expression of eukaryotic cell expression vector incorporating rat EDG7 receptor gene in cultured mammalian cells,
The intracellular Ca ²⁺ ion concentration increasing effect of LPA was examined.

[0096] Specifically, cDNA Synthesi
s System (Life technologies
Rat EDG7 receptor cDN using cDNA synthesized from rat kidney poly A + mRNA
A cDNA fragment having an open reading frame was prepared by a PCR reaction using the following forward primer and reverse primer prepared based on the nucleotide sequence of A. Forward primer: 5'-CGGGATCCATGAATGAGTGTCACTA
TGAC-3 '(SEQ ID NO: 9) Reverse primer: 5'-CGGAATTCGTCCCTGGCTTAGGAGC
TGC-3 '(SEQ ID NO: 10)

Advantage as a polymerase
2 Polymerase Mix (Clontech) was used. The composition of the reaction solution was 10x cDNA PC
R Reaction Buffer 5 μl, template DN
A 5 μl, each primer 10 μM, Advantage
e2 Polymerase Mix (50X) and d
1 μl of NTP (10 mM), balance 50 μl with sterile distilled water
l. The amplification reaction was incubated at 94 ° C. for 30 seconds, followed by 30 cycles at 94 ° C. for 5 seconds and 68 ° C. for 4 minutes.

The reaction solution after completion of the reaction was treated with phenol / chloroform and precipitated with ethanol to perform PCR.
The product was obtained. The obtained PCR product was used for BamHI / Eco
After RI digestion, a PCR product was obtained in the same manner as in Example 1. Next, the PCR product was subcloned into pBluescript SK (-) digested with BamHI / EcoRI, and the nucleotide sequence was determined.

From the above vector, BamHI / EcoR
The reading frame extracted by digestion with I was digested with pcDNA3.1 (+) (Inv), an eukaryotic cell expression vector.
(Itrogen) at the BamHI / EcoRI site.

The rat EDG constructed by the above operation
7 receptor expression vector by Lipofectamine
e Plus Reagent (Life techno
RH7777 cells (Ame)
rican Type Culture Correct
(manufactured by ION Co., Ltd.). At this time, pcDNA3.1 (+) alone was introduced at the same time, and used as a negative control. 48 hours after gene introduction, 5 × 10 ⁴ cells / well were placed in a 96-well plate.
And cultured overnight. Standard buffer (130 mM NaCl, 2 mM CaCl ₂ , 5
mM KCl, 10 mM glucose, 0.45 mM
_{KH 2 PO 4, 8mM MgSO 4} , 4.2mM NaH
Fluo-3 AM (Molecular P) in CO ₃ , 20 mM HEPES, 10 μM probenecid).
robes) was incubated for 1 hour at 37 ° C. in the presence of 0.1% fetal calf serum, and the cells were washed twice with a standard buffer. Then L
The transient change in Ca ²⁺ ion concentration caused by the addition of PA was measured using the FLIPR system (Fluoromet).
(Ric Imaging Plate Reader: manufactured by Molecular Devices Co., Ltd.). As a result, RH7 transiently expressed rat EDG7 receptor.
In 777 cells, intracellular Ca by concentration-dependent LPA
^{A 2+} ion concentration increasing effect was observed (FIG. 6). On the other hand, p
RH777 transfected only with cDNA 3.1 (+)
In 7 cells, the above reaction was not observed at all. From this, the rat EDG7 receptor is a human and mouse ED.
After the reaction with LPA as in the case of the G7 receptor, it was revealed that the intracellular Ca ²⁺ ion concentration was increased.

Example 4 C using FLIPR system
Rat EDG7 receptor and LP by a ²⁺ ion concentration change
Screening drug having activity of inhibiting binding to A
By adding various compounds to the extracellular solution simultaneously with the addition of LPA at a concentration of 3-10 μM in Example 3, LP
Antagonists that inhibit the activity of A can be screened.

Example 5 Rat EDG7 Receptor and LPA
Screening of a substance having an activity of inhibiting the binding to EGF7 A cell membrane fraction prepared from RH7777 cells transiently expressing the rat EDG7 receptor of Example 3 (10 μg /
ml) and 10 nM [ ^3] dissolved in Dulbecco's phosphate buffer (Sigma) containing 0.25% bovine serum albumin.
H] LPA (1 × 10 ⁶ cpm / ml (Daiichi Pure Chemicals)) is mixed and incubated at 0 ° C. for 45 minutes.
Next, the reaction mixture was applied to a GF / C filter (Whatman
After washing with Dulbecco's phosphate buffer containing 0.05% Tween-20 three times, and then measuring the radioactivity. Thus, an antagonist that inhibits the activity of LPA can be screened by adding various compounds to the reaction solution simultaneously with LPA.

Sequence Listing Free Text SEQ ID NOs: 3 and 4 are probe sequences designed based on the nucleotide sequence of human EDG7 receptor.

SEQ ID NOs: 5 and 7 are primer sequences designed based on the nucleotide sequence of rat EDG7 receptor cDNA.

SEQ ID NOs: 6 and 8 correspond to Marathon-
This is the sequence of the primer attached to the Ready cDNA rat testis.

SEQ ID NOs: 9 and 10 are primer sequences designed based on the nucleotide sequence of rat EDG7 receptor cDNA.

[0107]

Industrial Applicability According to the present invention, there is provided a novel protein and its polynucleotide which are LPA receptors having various actions such as cell proliferation, invasion of cancer cells, and wound healing. Further, according to the present invention, the protein of the present invention and L
It is also possible to screen for substances that inhibit PA binding. Since the proteins and polynucleotides of the present invention are considered to be involved in cell proliferation, invasion of cancer cells, and wound healing, the proteins and polynucleotides of the present invention, and further, antibodies and polynucleotides that bind to the proteins of the present invention Oligonucleotides that can hybridize to and substances that inhibit the binding of the protein of the present invention to LPA include cancer and other diseases caused by abnormal cell growth,
It is expected to elucidate the molecular mechanism of diseases that can alleviate symptoms by regulating cell proliferation, and to use them for diagnosis and treatment of these diseases.

[0108]

[Sequence List] SEQUENCE LISTING <110> Nippon-Shinyaku Co., Ltd. <120> Lysophosphatidic acid receptor <130> B-343 <160>

<210> 1 <211> 354 <212> PRT <213> Rattus norvegicus <400> 1 Met Asn Glu Cys His Tyr Asp Lys Arg Met Asp Phe Phe Tyr Asn 1 5 10 15 Arg Ser Asn Thr Asp Thr Ala Asp Glu Trp Thr Gly Thr Lys Leu 20 25 30 Val Ile Val Leu Cys Val Gly Thr Phe Phe Cys Leu Phe Ile Phe 35 40 45 Phe Ser Asn Ser Leu Val Ile Ala Ala Val Ile Thr Asn Arg Lys 50 55 60 Phe His Phe Pro Phe Tyr Tyr Leu Leu Ala Asn Leu Ala Ala Ala 65 70 75 Asp Phe Phe Ala Gly Ile Ala Tyr Val Phe Leu Met Phe Asn Thr 80 85 90 Gly Pro Val Ser Lys Thr Leu Thr Val Asn Arg Trp Leu Leu Arg 95 100 105 Gln Gly Leu Leu Asp Thr Ser Leu Thr Ala Ser Leu Ala Asn Leu 110 115 120 Leu Val Ile Ala Val Glu Arg His Met Ser Ile Met Arg Met Arg 125 130 135 Ile His Ser Asn Leu Thr Lys Lys Arg Val Thr Leu Leu Ile Leu 140 145 150 Leu Val Trp Ala Ile Ala Ile Phe Met Gly Ala Val Pro Thr Leu 155 160 165 Gly Trp Asn Cys Leu Cys Asn Ile Ser Ala Cys Ser Ser Leu Ala 170 175 180 Pro Ile Tyr Ser Arg Ser Tyr Leu Ile Phe Trp Thr Val Ser Asn 185 190 195 Leu Leu Ala Phe Phe Ile Met Val Val Val Tyr Val Arg Ile Tyr 200 205 210 Met Tyr Val Lys Arg Lys Thr Asn Val Leu Ser Pro His Thr Ser 215 220 225 Gly Ser Ile Ser Arg Arg Arg Ala Pro Met Lys Leu Met Lys Thr 230 235 240 Val Met Thr Val Leu Gly Ala Phe Val Val Cys Trp Thr Pro Gly 245 250 255 Leu Val Val Leu Leu Leu Asp Gly Leu Asn Cys Lys Gln Cys Asn 260 265 270 Val Gln His Val Lys Arg Trp Phe Leu Leu Leu Ala Leu Leu Asn 275 280 285 Ser Val Met Asn Pro Ile Ile Tyr Ser Tyr Lys Asp Glu Asp Met 290 295 300 Tyr Asn Thr Met Arg Lys Met Ile Cys Cys Ala Pro His Asp Ser 305 310 315 Asn Ala Glu Arg His Pro Ser Arg Ile Pro Ser Thr Ile His Ser 320 325 330 Arg Ser Asp Thr Gly Ser Gln Tyr Leu Glu Asp Ser Ile Ser Gln 335 340 345 Gly Gln Val Cys Asn Lys Ser Ser Ser 350 354

[0110] <210> 2 <211> 1106 <212> DNA <213> Rattus norvegicus <400> 2 tcttgtccac catgaatgag tgtcactatg acaagcggat ggactttttc tacaacagga 60 gcaacacaga cacggcggac gagtggacgg ggacgaagct cgtgatcgtc ctgtgcgttg 120 ggacgttctt ctgcctcttt atattttttt ctaactccct ggtcatcgca gcagtgatca 180 cgaataggaa gttccacttt cccttctact acctgctggc taacttggcg gctgcggatt 240 tctttgccgg aatcgcttat gtgttcctga tgtttaacac cggcccggtg tcgaaaacct 300 tgactgtcaa ccgctggctt ctccgccagg ggctcctaga cacaagcctg acggcctccc 360 tggccaatct gctggtaatt gctgtggaaa gacacatgtc aatcatgagg atgagaatcc 420 acagcaactt gaccaaaaaa cgggtgacgc tgctcatcct gctggtgtgg gccatagcca 480 ttttcatggg ggcggtcccc acgctgggct ggaattgcct ctgcaacatc tcagcctgct 540 cctctctggc cccgatttac agcaggagtt acctcatctt ctggacggtg tcaaacctcc 600 tggccttctt catcatggtg gtggtgtacg tgcgcatcta catgtatgtc aagaggaaaa 660 ccaacgtcct ctctccgcac acgagtggct ccatcagccg ccggagggcc cccatgaagc 720 taatgaagac ggtgatgact gtcttaggag ccttcgtggt gtgctggacc ccgggtctcg 780 tggtgctgct gctggacggc ctgaactgca agcagtgtaa tgtgcagcac gtgaagcgct 840 ggttcctgct gctggcgctg ctcaactccg tcatgaaccc catcatctac tcgtacaagg 900 acgaggacat gtacaacacc atgcggaaga tgatctgctg tgccccgcac gacagcaacg 960 ccgagaggca cccctcccgc atcccctcta ccatccacag caggagcgac acgggcagcc 1020 agtacctgga ggacagcatc agccagggcc aggtgtgcaa taaaagcagc tcctaagcca 1080 gggacgcctc cgccctctcc ccctgg 1106

<210> 3 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Designed probe based on nucleotide sequence of Human EDG7 mRNA <400> 3 cgggatccat gaatgagtgt cactatg 27

<210> 4 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Designed probe based on nucleotide sequence of Human EDG7 mRNA <400> 4 gctctagaat ccagagttta ggaagtgc 28

<210> 5 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Designed primer based on nucleotide sequence of Rat EDG7 cDNA <400> 5 cggaattcga cggtgatgac tgtcttag 28

<210> 6 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Primer affiliated to Marathon-Ready cDNA rat testis <400> 6 actcactata gggctcgagc ggc 23

<210> 7 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Designed primer based on nucleotide sequence of Rat EDG7 cDNA <400> 7 cggaattcaa tgtgcagcac gtgaagcg 28

<210> 8 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Primer affiliated to Marathon-Ready cDNA rat testis <400> 8 ccatcctaat acgactcact atagggc 27

<210> 9 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Designed primer based on nucleotide sequence of Rat EDG7 cDNA <400> 9 cgggatccat gaatgagtgt cactatgac 29

<210> 10 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Designed primer based on nucleotide sequence of Rat EDG7 cDNA <400> 10 cggaattcgt ccctggctta ggagctgc 28

[Brief description of the drawings]

FIG. 1. Rat EDG7 receptor and human EDG7.
2 shows an alignment of the amino acid sequence of the receptor. The part where the sequence matches is displayed with a frame.

FIG. 2 shows rat EDG7 receptor and mouse EDG.
7 shows an alignment of the amino acid sequences of the seven receptors. The part where the sequence matches is displayed with a frame.

FIG. 3. Rat EDG7 receptor and rat snG.
3 shows an alignment of the amino acid sequence of PCR32. The part where the sequence matches is displayed with a frame.

FIG. 4. Rat EDG7 receptor and rat snG.
3 shows an alignment of the base sequence encoding the open reading frame of PCR32. The part where the sequence matches is displayed with a frame.

FIG. 5. Rat EDG7 receptor and rat snG.
The alignment (continued) of the base sequence encoding the open reading frame of PCR32 is shown. The part where the sequence matches is displayed with a frame.

FIG. 6 shows changes in the Fluo-3 fluorescent signal, which indicates changes in intracellular calcium concentration induced by LPA in RH7777 cells transiently expressing rat EDG7 receptor. The vertical axis indicates the fluorescence intensity (count), the horizontal axis indicates the reaction time (second), and the arrow in the figure indicates the time when LPA was added. In addition, among the five line graphs, LPs of 10, 3, 1, 0.3, and 0 μM were arranged in order from the one having the highest fluorescence intensity.
The response to the A stimulus is shown.

Continuation of the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat II (reference) A61K 48/00 A61P 1/18 4C085 A61P 1/18 9/00 4C086 9/00 13/08 4H045 13/08 19/02 19/02 29/00 101 29/00 101 35/00 35/00 37/02 37/02 C07K 14/71 C07K 14/71 16/28 16/28 C12N 1/15 C12N 1/15 1/19 1 / 19 1/21 1/21 C12P 21/02 C 5/10 G01N 33/15 Z C12P 21/02 33/50 Z G01N 33/15 33/566 33/50 C12P 21/08 33/566 (C12P 21/02 C // C12P 21/08 C12R 1:91) (C12P 21/02 C12N 15/00 ZNAA C12R 1:91) 5/00 A F term (reference) 2G045 AA40 DA12 DA13 DA36 FB02 FB03 FB08 4B024 AA01 AA11 BA63 CA04 CA12 DA02 EA03 EA04 GA13 GA18 HA15 4B064 AG20 AG26 AG27 CA10 CA19 CA20 CC24 DA01 DA13 4B065 AA90X AA91Y AB01 BA05 CA24 CA44 CA46 4C084 AA13 AA17 MA01 NA14 ZA362 ZA662 ZA812 ZA962 ZB152 ZB262 ZC4124A0A85 A85 AA01 AA02 AA03 AA04 EA16 MA01 MA04 NA14 ZA36 ZA66 ZA81 ZA96 ZB15 ZC41 4H045 AA10 AA11 AA20 AA30 BA10 CA40 DA51 DA75 DA76 EA28 EA50 FA74

Claims (12)

[Claims] (1) an amino acid sequence represented by SEQ ID NO: 1; (b) an amino acid sequence having a substitution, deletion, addition or insertion of at least one residue in the amino acid sequence of (a); (C) a protein having one amino acid sequence selected from the group consisting of an amino acid sequence having at least 98% sequence identity with the amino acid sequence of (a). (A) a base sequence shown in SEQ ID NO: 2 in the sequence listing; (B) a base sequence encoding the amino acid sequence shown in SEQ ID NO: 1; (C) the above (A) or (B) In the nucleotide sequence of
A base sequence having at least one base substitution, deletion, addition, or insertion; (D) a base sequence different from any of the base sequences (A) to (C) through degeneracy; (A) a nucleotide sequence of a nucleic acid capable of hybridizing under stringent conditions with an antisense strand of a nucleic acid having any one of the nucleotide sequences of (A) to (D); and (F) an amino acid sequence represented by SEQ ID NO: 1. A polynucleotide having one kind of base sequence selected from the group consisting of a base sequence encoding a sequence having a sequence identity of at least 98%. 3. The polynucleotide according to claim 2, which is cDNA or chemically synthesized DNA. 4. An oligonucleotide capable of hybridizing with the polynucleotide according to claim 2 or 3 under stringent conditions. 5. A recombinant vector comprising the polynucleotide according to claim 2 or 3. 6. A prokaryotic or eukaryotic cell comprising the recombinant vector of claim 5. 7. A method for producing a recombinant protein, comprising a step of culturing the cell according to claim 6, and a step of separating and purifying the produced protein. 8. A recombinant protein obtainable by the method according to claim 7. 9. An antibody or a fragment thereof capable of specifically binding to the protein according to claim 1 or 8. 10. The protein according to claim 1 or 8 and L.
A method for screening a substance having an activity of inhibiting binding to PA, comprising: (i) a step of contacting the protein according to claim 1 or 8 with LPA in the presence of a test substance; and (ii) a step of: Assessing the binding of the protein of claim 1 or 8 to LPA, the degree of binding of the protein of claim 1 or 8 to LPA in the presence of the test substance compared to in the absence of the test substance. A screening method for determining that the test substance has an inhibitory activity using the decrease in binding as an index when the test substance decreases. 11. A substance having an activity of inhibiting the binding of the protein according to claim 1 or 8 to LPA, which can be selected by the method according to claim 10. 12. A pharmaceutical composition comprising the polynucleotide according to claim 2 or 3, the antibody or a fragment thereof according to claim 9, and one selected from the group consisting of the substance according to claim 11.