| Project/Area Number |
12670049
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
General physiology
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| Research Institution | Department of Physiology, Kinki University School of medicine |
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Principal Investigator |
FUKAO Hideharu Department of Physiology, Kinki University School of Medicine, Assistant, 医学部, 助手 (70218874)
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| Co-Investigator(Kenkyū-buntansha) |
OKADA Kiyotaka Department of Physiology, Kinki University School of Medicine, Assistant, 医学部, 助手 (20185432)
UESHIMA Shigeru Department of Physiology, Kinki University School of Medicine, Assistant Professor, 医学部, 助教授 (30193791)
MATSUO Osamu Department of Physiology, Kinki University School of Medicine, Professor, 医学部, 教授 (40030879)
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| Project Period (FY) |
2000 – 2001
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| Project Status |
Completed (Fiscal Year 2001)
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| Budget Amount *help |
¥3,300,000 (Direct Cost: ¥3,300,000)
Fiscal Year 2001: ¥1,500,000 (Direct Cost: ¥1,500,000)
Fiscal Year 2000: ¥1,800,000 (Direct Cost: ¥1,800,000)
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| Keywords | Vascular endothelial cells / Fibrinolytic system / tissue-type plasminogen activator (t-PA) / t-PA receptor (t-PAR) / Antithrornbotic property / Intracellular signal transduction / Tyrosine phosphorylation / Mitogen-activated protein kinase (MAPK) / t-PA受容体 / 細胞内伝達系 / チロシンリン酸化蛋白 |
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| Research Abstract |
We previously demonstrated tissue-type plasminogen activator (t-PAR) that is expressed by vascular endothelial cells. In the present studies, the specific binding of t-PA to t-PAR on cultured human umbilical vein endothelial cells (HUVECs) was further demonstrated by plasmon resonance biosensor system (lAsys), and the molecular cloning as well as the analysis of signal transduction function of t-PAR were performed. In addition, the binding epitope(s) of t-PA to t-PAR was also investigated by using t-PA varients. The immobilized t-PA was interacted with suspended HUVECs in which only excess t-PA could interfer the binding, indicating that the cell surfacet-PAR recognized only t-PA. Since the lysine analogue reduced the t-PA binding to HUVECs and Kringle2 (K2)-exposed t-PA mutant most efficiently bound to the cells, the lysine binding sites Of K2 is most likely the critical epitope for t-PAR. It was also shown that the major part of t-PA preferably bound to type 1 plasminogen activator inhibitor (PAI-1) on the extracellular matrix of monolayer HUVECs with high affinity. Several candidate genes encoding t-PAR were obtained, of which the translated molecules are intracellular proteins. The final identification of t-PAR molecule is now on going. The t-PAR induced tyrosine phosphorylation by 15 min that is involved in protein C pathway. However, interestingly, this signal also induced the down-regulation of mitogen-activated protein kinase (MAPK, p42/44) by 30 min after"the binding of t-PA. The signal was dependent on the active site of t-PA, since a pannel of monoclonal antibodies which recognized protease domain of t-PA abolished the t-PA-induced tyrosine phosphorylation. These findings indicate that the t-PAR functions as a cell surface receptor for t-PA modulating the bound t-PA activity and biological actions of endothelial cells.
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