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

众所周知，在激动剂-受体相互作用后，磷酸肌醇转换的刺激是通过质膜中假定的gtp结合蛋白激活磷酸肌醇特异性磷脂酶C （PI-PLC）而启动的。血小板经常被用作研究跨膜信号传导的潜在有用模型。本研究的目的是阐明PI-PLC在人血小板中的活化机制，纯化PI-PLC同工酶和gtp结合蛋白并充分结合重组。人血小板胞浆和膜组分的pi - plc有效分辨率显示出5个明显的活性峰。用不同抗体对PI-PLC同工酶进行Western blotting分析，鉴定出plc - β、plc - γ和PLC-delta同工酶，并从细胞质部分分离出另外两个未鉴定的活性峰，plc - β主要存在于细胞膜中。此外，从膜组分中纯化了未识别的hplc - ii。从人血小板膜中分离纯化了两种异三聚体gtp结合蛋白Gi2（主要成分）和Gi3（次要成分），并鉴定其为百日咳毒素底物。采用抗glα抗体进行免疫印迹法检测百日咳毒素不敏感gtp结合蛋白（Gq）。从人血小板胞浆和膜中纯化出多种低分子量GTP结合蛋白（c21KG, c25KG, m22KGI，II）。这些被鉴定为smg21A (Krev-1)， rap1B和ralA。获得了c25KG的cDNA，命名为ram。已知gtp结合蛋白参与凝血酶和凝血素a2介导的人血小板肌醇磷酸的生成。最近有报道称，血栓素A2受体结合百日咳毒素不敏感gtp结合蛋白（Gq）和plc - β 1被Gq α特异性激活。我们之前观察到人血小板膜相关的PLC被Gi和Go激活。plc - β 2同工酶激活gtp结合蛋白的β - γ亚基。凝血酶受体偶联两种不同的gtp结合蛋白（Gi和Gq）。这些结果表明，血栓素a2刺激可通过Gq激活PLC-beta1，凝血酶可诱导由Gi2的β - γ亚基介导的PLC （beta2）激活。我们研究了PLC- γ与细胞质部分的肌动蛋白-凝胶复合物相关，并表明凝胶可能在人血小板中调节PLC活性中起作用。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