Interaction of multiple phospholipase C and GTP-binding proteins in platelet signal transduction

通常已知激动剂-受体相互作用后磷酸肌醇周转的刺激是通过质膜中推定的GTP结合蛋白激活磷酸肌醇特异性磷脂酶C（PI-PLC）启动的。血小板经常被用作研究跨膜信号传导的潜在有用的模型。本研究的目的是阐明人血小板中PI-PLC的活化机制，其中PI-PLC同工酶和GTP结合蛋白被纯化并以适当的组合进行重构。人血小板胞浆和膜组分的PI-PLC的有效分辨率显示了五个不同的活性峰。用不同抗体对PI-PLC同工酶进行蛋白质印迹分析，结果表明，PLC-β、PLC-γ和PLC-δ同工酶被鉴定出来，另外两个未鉴定的活性峰从胞质组分中分离出来，PLC-β主要存在于细胞膜中 ...更多信息 .此外，从膜级分中纯化未鉴定的mPLC-II。从人血小板膜中纯化并鉴定了两种异源三聚体GTP结合蛋白，Gi 2（主要组分）和Gi 3（次要组分），它们是百日咳毒素的底物。用抗GL α抗体进行Western印迹分析，检测百日咳毒素不敏感的GTP结合蛋白（Gq）。从人血小板胞浆和膜中纯化出各种类型的低分子量GTP结合蛋白（c21 KG、c25 KG、m22 KGI、II）。它们被鉴定为smg 21 A（Krev-1）、rap 1B和ralA。获得了新的c25 KG基因的cDNA，命名为ram。已知GTP结合蛋白参与凝血酶和血栓烷A2介导的人血小板中肌醇磷酸的产生。最近有报道，血栓素A2受体结合百日咳毒素不敏感的GTP结合蛋白（Gq）和PLC-β 1被Gq α特异性激活。我们以前观察到，人血小板膜相关PLC被Gi和Go激活。PLC-β 2同工酶是GTP结合蛋白的激活β γ亚基。凝血酶受体与两种不同的GTP结合蛋白（Gi和Gq）偶联。这些结果表明，血栓素A2刺激通过Gq激活PLC-β 1，而凝血酶通过Gi 2的β γ亚基介导激活PLC（β 2）。我们研究了PLC-γ与胞浆组分中的肌动蛋白-凝溶胶蛋白复合物相关，并表明凝溶胶蛋白可能在人血小板PLC活性的调节中起作用。少

It has generally been known that the stimulation of phosphoinositide turnover after the agonist-receptor interaction is initiated by the activation of phosphoinositide-specific phospholipase C (PI-PLC) through putative GTP-binding protein in plasma membranes. Blood platelets have frequently been used as a potentially useful model for studying transmembrane signaling. The purpose of this study was to clarify the mechanism of activation of thePI- PLC in human platelets, where PI-PLC isozymes and GTP-binding proteins were purified and reconstituted with adequate combination. The effective resolution of PI-PLCs of human platelet cytosolic and membrane fractions revealed five distinct activity peaks. The results of Western blotting analysis with various antibodies against PI-PLC isozymes showed that PLC-beta, PLC-gamma and PLC-delta isozymes were identified and two other unidentified activity peaks were separated from the cytosolic fraction and PLC-beta was mainly conatined in the membranes … More . Furthermore, unidentified mPLC-II was purified from the membrane fractions. Two heterotrimeric GTP-binding proteins, Gi2 (main component) and Gi3 (miner component) were purified and identified as substrates of pertussis toxin from human platelet membranes. The purtussis toxin-insensitive GTP-binding protein (Gq) was detected by Western blotting analysis with anti-GLalpha antibody. Various types of low molecular weight GTP- binding proteins were purified from cytosol and membrane of human platelets (c21KG, c25KG, m22KGI,II). These are identified to be smg21A (Krev-1), rap1B and ralA. The cDNA of novel c25KG was obtained and namedas ram. GTP-binding proteins have been known to be involved in thrombin and thromboxane A2-mediated production of inositol phosphates in human platelets. It has been recently reported that thromboxane A2 receptor bound pertussis toxin-insensitive GTP-binding protein (Gq) and PLC-beta 1 was specifically activated by the Gq alpha . We previously observed that human platelet membrane-associated PLC was activated by Gi and Go. The PLC-beta2 isozyme was activated beta gamma subunit of GTP-binding protein. Thrombin receptor coupled with two different GTP-binding proteins (Gi and Gq). These results suggested that thromboxane A2-stimulation activate PLC-beta1 via Gq and thrombin induced the PLC (beta2) activation mediated by beta gamma subunit of Gi2. We investigated that PLC-gamma was associated with actin-gelsolin complex in cytosolic fraction and suggested that gelsolin may play a role in regulation of PLC activity in human platelets. Less