1995 Fiscal Year Final Research Report Summary
Molecular Cloning and Expression of a cDNA of the Bovine Prostaglandin F_<2alpha> Receptor
| Project/Area Number |
06670177
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| Research Category |
Grant-in-Aid for General Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Research Field |
Pathological medical chemistry
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| Research Institution | University of Tsukuba (1995)
Osaka Bioscience Institute (1994) |
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Principal Investigator |
SAKAMOTO Kazuichi Univ.of Tsukuba, Inst of Biological Sciences, Associate Professor, 生物科学系, 助教授 (90235169)
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| Co-Investigator(Kenkyū-buntansha) |
ITO Seiji Kansai Medical Univ., Dept.Medical Chemistry, Professor, 医化学教室, 教授 (80201325)
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| Project Period (FY) |
1994 – 1995
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| Keywords | Prostaglandine F_<2alpha> / gene expression / regression |
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| Research Abstract |
In order to elucidate the structure and function of prostaglandine F_<2alpha> receptor, we have used a polymerase chain reaction (PCR) approach to isolate a cDNA encoding the PGF_<2alpha> receptor from a bovine corpus luteal cDNA library. The clone SN463 which was isolated by PCR method by using several pairs of degenerated primers carried the homologous sequence which covered transmembrane motif IV to VI of thromboxane (TX) A_2 receptor. This PCR product was used as a probe DNA for following cross hybridization and a clone BC2211 carrying a 2.2-kbp insert DNA was isolated form bovine corpus luteal cDNA library. This clone encodes a protein of 362 amino acid residues (Mr 40,983) with seven potential transmembrane domains. Injection of the mRNA synthesized in vitro from the cloned cDNA into a Xenopus oocyte elicited electrophysiological response to PGF_<2alpha>, representing BC2211 encodes functional full-length cDNA.Ligand-binding displacement in membranes of mammalian COS-7 cells transfected with the cDNA indicated the rank order of affinity of the receptor to PGs : PGF_<2alpha>>PGD_2>PGE_2>STA_2, a TXA_2 agonist. PGF_<2alpha> activated InsP formation in COS-7 cells transfected with receptor cDNA.Northern blot analysis and in situ hybridization indicated that the PGF_<2alpha> receptor mRNA is highly expressed and accumulated in corpus luteum and mRNA level of PGF_<2alpha> receptor was regulated in corpus luteum during the sexual cycle and pregnancy. These data should help further understanding of molecular mechanism of the receptor function.
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