1992 Fiscal Year Final Research Report Summary
Studies on Leukotriene Biosynthesis and Signal Transduction.
| Project/Area Number |
02454143
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| Research Category |
Grant-in-Aid for General Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Research Field |
General medical chemistry
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| Research Institution | The University of Tokyo |
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Principal Investigator |
SHIMIZU Takao The University of Tokyo, Faculty of Medicine, Professor, 医学部(医) 教授 (80127092)
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| Co-Investigator(Kenkyū-buntansha) |
IZUMI Takashi The University of Tokyo, Faculty of Medicine, Assistant, 医学部(医), 助手 (70232361)
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| Project Period (FY) |
1990 – 1992
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| Keywords | PAF / Receptor / Leukotrienes / Phospholipases / Leukotriene A4 hydrolase / Antagonists |
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| Research Abstract |
PAF and leukotrienes are lipid mediators involved in inflammatory/allergic response as well as various cardiovascular and immunological disorders. We have carried out experiments on biosyntheses and signal transduction of these lipid mediators.
1. Isolation of PAF and leukotriene receptors.
By expression cloning using Xenopus oocyte system, we isolated a cDNA of PAF receptor as the first successful example of receptor cloning of lipid mediators. A human homologue was also obtained. They are composed of 342 amino acid residues with putative seven transmembrane spanning domains. These receptors were expressed in COS cells or CHO cells, and cellular signalings are extensively studied. It was shown that PAF receptor couples to phospholipases, adenylate cyclase, and MAP kinase via different types of G- proteins. Leukotriene receptors (B4, and D4) were solubilized from guinea pig leukocyte and lung membrane, respectively in an active form. They activate phospholipase A_2 and C via Gi proteins.
… More
Molecular cloning of these receptors by two strategies (protein purification and expression cloning) is currently ongoing.
2. Leukotriene A_4 hydrolase. a bifunctional enzyme.
Leukotriene B_4, a potent chemotactic factor, is produced by an enzyme termed leukotriene A4 hydrolase. By cDNA cloning and elucidation of the primary structure of the enzyme have shown that the enzyme is highly homologous to various types of metalloproteinases. In fact, it was shown that the enzyme contains a zinc ion, which is essential for both intrinsic aminopeptidase activity and leukotriene A_4 hydrolase. To determine the active site of the enzyme, site-directed mutagenesis experiments were performed. The mutation of His-296, or Glu-315 yielded loss of zinc ion with concomitant disappearance of both enzyme activities. However, by changing Glu-297 to other amino acids. two enzyme activities were clearly separated. The mechanism of incorporation of water molecule as well as the physiological significance of bifunctional role of enzyme remain unclear. Less
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[Publications] Ohishi, N., Minami, M., Kobayashi, J., Seyama, Y., Hata, J., Yotsumoto, H., Takaku, F., and Shimizu, T.: "Immunological quantitation and immunohistochemical localization of leukotriene A4 hydrolase." J. Biol. Chem.265. 7520-7525 (1990)
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[Publications] Honda, Z., Nakamura, M., Miki, I., Minami, M., Watanabe, T., Okado, H., Toh, H., Ito, K., Miyamoto, T. and Shimizu, T.: "Cloning by functional expression of guinea-pig lung platelet-activating factor receptor." Nature. 349. 342-346 (1991)
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[Publications] Watanabe, T., Yatomi, Y., Sunaga, A., Miki, I., Seyama, Y. Ogura, M., Saito, H., Kurokawa, K., and Shimizu, T.: "Characterization of partially-purified prostaglandin E and thromboxane receptors expressed on a megakaryo- blastic leukemia cell line." Blood. 78. 1-9 (1991)
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[Publications] Bito, H., Nakamura, M., Honda, Z., Izumi, T., Iwatsubo, T. Seyama, Y., Ogura, A., Kudo, H., and Shimizu, T.: "Platelet activating factor receptor in rat brain; PAF mobilizes intracellular Ca in hippocampal neuron" Neuron. 9. 285-294 (1992)
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