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Expression of t-PA receptor on the endothelial cell and analysis of its physiological function.

内皮细胞t-PA受体的表达及其生理功能分析。

基本信息

批准号：
15590198
负责人：
MATSUO Osamu
金额：
$ 2.3万
依托单位：
Kinki University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2003
资助国家：
日本
起止时间：
2003 至 2004
项目状态：
已结题

项目摘要

We demonstrated previously that tissue-type plasminogen activator (t-PA) bound to its specific receptor (t-PAR) on human umbilical vein endothlial cells (HUVEC) and that t-PAR interacted only with t-PA to form t-PA/ t-PAR complex. To investigate the function of t-PAR, we synthesized recombinant t-PAR (rt-PAR). Furthermore, the binding ability of t-PA to t-PAR on vascular endothelial cells was studied by using IAsys.The cDNA coding region for t-PAR was cloned into glutathione S-transferase (GST) expression vector. The target protein was expressed as the fusion protein with GST. The mature rt-PAR was obtained from cleaving fusion protein by protease. The ligand blot analysis revealed that t-PA specifically bound to rt-PAR.On the interaction analysis using IAsys, the binding signal of HUVEC to t-PA elevated in the cell concentration manner. It is considered that the t-PAR may concentrate t-PA on the surface of HUVEC and enhance the fibrinolytic properties around them.When HUVEC were treated with EACA, the binding signal of HUVEC to t-PA decreased in an EACA dependent manner. It was suggested that the binding of t-PA to endothelial cell was mediated via lysine binding site in t-PA molecule. On the other hand, when the endothelial cell was treated with receptor associated protein, the binding parameter did not change. This result indicated that low density lipoprotein receptor related protein was not involved in the binding of t-PA to HUVEC. Furthermore, polyclonal antibody to annexin II and that to α ^-enolase did not interfere the interaction between t-PA and HUVEC. It concluded that t-PAR is the novel protein which is different from annexin II and α ^-enolase.

我们以前曾证明组织型纤溶酶原激活剂（t-PA）与人脐静脉内皮细胞（HUVEC）上的t-PAR特异性受体结合，而t-PAR只与t-PA相互作用形成t-PA/ t-PAR复合物。为了研究t-PAR的功能，我们合成了重组t-PAR（rt-PAR）。将t-PAR的cDNA编码区克隆到谷胱甘肽S转移酶（GST）表达载体中，用免疫荧光分析仪（IAsys）研究t-PA与血管内皮细胞上t-PAR的结合能力。目的蛋白以GST融合蛋白的形式表达。用蛋白酶裂解融合蛋白，获得了成熟的rt-PAR。配体印迹分析显示，t-PA与rt-PAR特异性结合，IAsys分析显示，HUVEC与t-PA的结合信号以细胞浓度方式增强。认为t-PAR可使t-PA在HUVEC表面富集，增强其周围的纤溶活性，EACA处理HUVEC后，HUVEC与t-PA的结合信号呈EACA依赖性下降。提示t-PA与内皮细胞的结合是通过其分子中的赖氨酸结合位点介导的。另一方面，当内皮细胞用受体相关蛋白处理时，结合参数没有改变。提示低密度脂蛋白受体相关蛋白不参与t-PA与HUVEC的结合。annexin II多克隆抗体和α ^-烯醇化酶多克隆抗体均不干扰t-PA与HUVEC的相互作用。结论：t-PAR是不同于annexin II和α ^-烯醇化酶的新蛋白。