Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
2000-05-11
2001-06-26
Whisemont, Ethan (Department: 1655)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C435S069100, C435S091100, C435S252300, C435S320100
Reexamination Certificate
active
06252056
ABSTRACT:
TECHNICAL FIELD
The present invention relates to a lysophosphatidic acid (LPA) receptor and use thereof. More specifically, it relates to (1) a human LPA receptor, (2) a method for screening a human LPA-like substance, a human LPA antagonist and an antagonist or agonist for a phospholipid other than LPA capable of responding to the human LPA receptor, comprising using a human LPA receptor protein, (3) an LPA inhibitor or an inhibitor for a phospholipid other than human LPA, comprising the human LPA receptor, (4) a method for producing the human LPA receptor, (5) a monoclonal or polyclonal antibody against the human LPA receptor, (6) a cDNA encoding the human LPA receptor, (7) a replication or expression vector carrying the above cDNA, and (8) a host cell transformed with the above replication or expression vector.
BACKGROUND ART
It is known that LPA receptors bind LPA to transmit a signal into cells and induce various physiological phenomena, such as cell proliferation, dedifferentiation of vascular cells, inhibition of cell proliferation, and the like. Thus, if a human LPA receptor can be provided, not only its application as a medicament or diagnostic drug for vasospasm and the like can be expected using the human LPA receptor per se but also its broad contribution to the development of medicaments, such as screening and evaluation of medicaments, such as a new LPA receptor antagonist and the like, can be expected. Although it is known that plural kinds of LPA receptors are present, all kinds of human LPA receptors have not been isolated yet, and the actions and structures have not been specified.
Cells recognize various physiologically active substances, such as proliferation factors, hormones, neurotransmitters, and the like, via a receptor and response thereto in various manner. LPA receptors which are present on the cell membrane bind to LPA and transmit a signal into the cell via a G protein conjugated with the receptor. Gi, Gq and the like are known as G proteins which can be conjugated with LPA receptors, and it is considered that these receptors take part in responses, such as cell proliferation promoting action or, conversely, proliferation inhibiting action, and the like. Additionally, it has been found that an MAP-kinase system is connected with the downstream of the G protein and the LPA receptors transmit various signals.
Receptors using LPA as the ligand have been isolated from mouse and Xenopus. For example, Tigyi et al. have obtained an LPA receptor, called PSP24 LPA receptor, for the first time from Xenopus oocyte (
Proc. Natl. Acad. Sci. U.S.A.,
93: 14367-14372 (1996)). Also, another kind of LPA receptor has been cloned from a mouse, which is called vzg-1 (
J. Cell. Biol.,
135:1071-1083 (1996)).
As human origin LPA receptors, Edg-2 which is a homologue of vzg-1 has been reported (
Biochem. Bioph. Res. Commun.,
231:619-622 (1997)); however, there are no reports on the PSP24 in human.
SUMMARY OF THE INVENTION
With the aim of cloning a human counterpart of the PSP24 LPA receptor which is expressed in Xenopus oocyte, the present inventors have conducted intensive studies and, as a result, succeeded in cloning a cDNA encoding a PSP24 human LPA receptor derived from a human brain tissue and in determining the complete amino acid sequence of the LPA receptor.
When the known nucleotide sequences registered in the nucleotide sequence database were searched by BLASTN, and the amino acid sequences of known polypeptides registered in the amino acid sequence database by BLASTP, there was no sequence which coincided with the nucleotide sequence encoding the PSP24 human LPA receptor. Also, it was expected based on a hydrophobic plotting analysis that the polypeptide of the LPA receptor has seven transmembrane domains. Accordingly, it was confirmed that this polypeptide is a novel membrane protein.
The present invention relates to:
(1) a protein comprising the amino acid sequence shown in SEQ ID NO:1, a homologue thereof, a fragment thereof, or a fragment of the homologue thereof;
(2) a human lysophosphatidic acid (human LPA) receptor comprising the amino acid sequence shown in SEQ ID NO:1 or a homologue thereof;
(3) a method for screening a human LPA-like substance, a human LPA antagonist, and an antagonist or agonist for a phospholipid other than LPA capable of responding to the human LPA receptor, comprising using the protein according to the above (1) or (2);
(4) a human LPA inhibitor or an inhibitor for a phospholipid other than human LPA, comprising the protein according to the above (1) or (2) as an active ingredient;
(5) a method for producing a human LPA receptor, comprising culturing a cell transformed with an expression vector carrying a DNA fragment encoding the protein according to the above (1) or (2), and recovering the protein from the cultured mixture;
(6) a monoclonal or polyclonal antibody against the protein according to the above (1) or (2) or a peptide comprising a partial sequence of the protein;
(7) a cDNA encoding the polypeptide according to the above (1) or (2);
(8) a fragment which selectively hybridizes to the cDNA according to the above (7) comprising the nucleotide sequence shown in SEQ ID NO:2, or a sequence thereof;
(9) a fragment which selectively hybridizes to the cDNA according to the above (7) comprising the nucleotide sequence shown in SEQ ID NO:3, or a sequence thereof;
(10) a replication or expression vector carrying the cDNA according to any one of the above (6) to (9); and
(11) a host cell transformed with the replication or expression vector according to the above (10).
REFERENCES:
Proc. Natl. Acad. Sci. USA, vol. 93 (1996), Z. Guo et al., “Molecular cloning of a high-affinity receptor for the growth factor-like lipid mediator lysophosphatidic acid from Xenopus oocytes”, pp. 14367-14372.
R.W. Ould, S.B. Primrose, “Principles of Gene Manipulation”, Feb. 25, 1991, K.K. Baifukan, p. 115.
Neuroscience Res., Suppl. 22 (Oct., 1998), Y. Kawasawa et al., “Cloning and Characterization of Mouse LPA Receptor (PSP24)”, p. S89.
Faseb J., vol. 12 (Apr., 1998), Y. Kawasawa et al., “Cloning and Characterization of A Mouse Homologue of Xenopus PSP24 LPA Receptor”, p. A1460.
Biochemical and Biophysical Research Communications, vol. 231, (1997), Article No. RC976150, S. An et al., “Molecular Cloning of the Human Edg2 Protein and Its Identification as a Functional Cellular Receptor for Lysophosphatidic Acid”, pp. 619-622.
Fukushima Daikichi
Haga Hisanori
Nakade Shinji
Lu Frank W.
Ono Pharmaceutical Co. Ltd.
Sughrue Mion Zinn Macpeak & Seas, PLLC
Whisemont Ethan
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