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

众所周知，激动剂-受体相互作用后对磷脂酰肌醇周转的刺激是通过质膜上假定的GTP结合蛋白激活磷脂酰肌醇特异性磷脂酶C(PI-PLC)。血小板常被用作研究跨膜信号的一个潜在有用的模型。本研究的目的是通过纯化PI-PLC同工酶和GTP结合蛋白，并充分结合重组，阐明PI-PLC在人血小板中激活的机制。对人血小板胞浆和膜组分的PI-PLC的有效拆分显示出五个明显的活性峰。用不同的PI-PLC同工酶抗体进行Western blotting分析，结果表明，PLC-β、PLC-γ和PLC-Delta同工酶被鉴定出来，并从胞浆组分中分离出另外两个未知的活性峰，PLC-β主要存在于细胞膜…中更多。此外，还从膜组分中纯化了未知的mPLC-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(主成分)和Gi3(Miner成分)，并鉴定为百日咳毒素的底物。免疫印迹法检测百日咳毒素不敏感的GTP结合蛋白(GQ)。从人血小板胞浆和细胞膜(c21KG、c25KG、m22KGI、II)中纯化了各种类型的低相对分子质量GTP结合蛋白。经鉴定为smg21A(Krev-1)、Rap1B和Rala。获得了新的C25KG基因，命名为Ram。已知GTP结合蛋白参与凝血酶和血栓素A2介导的人血小板肌醇磷酸的产生。最近有报道血栓素A2受体结合百日咳毒素不敏感GTP结合蛋白(GQ)和PLC-β1被GQα特异性激活。我们先前观察到人血小板膜相关PLC被GI和GO激活。PLC-β2同工酶是GTP结合蛋白的活化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。提示血栓素A2刺激通过GQ激活PLC-β1，凝血酶诱导GI2β-γ亚基介导的PLC(β2)激活。我们研究了胞浆组份中PLC-γ与肌动蛋白-明胶蛋白复合体的关系，提示明胶蛋白可能在调节人血小板PLC活性中发挥作用。较少

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