Interaction of multiple phospholipase C and GTP-binding proteins in platelet signal transduction
Interaction of multiple phospholipase C and GTP-binding proteins in platelet signal transduction
批准号:
02454544
负责人:
NOZAWA Yoshinori
金额:
$4.35万
依托单位:
依托单位国家:
日本
项目类别:
Grant-in-Aid for General Scientific Research (B)
财政年份:
1990
资助国家:
日本
项目状态:
已结题
起止时间:
1990 至 1992
中文摘要
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
英文摘要
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
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Y.Banno: "Effects of gelsoin on human platelet cytosolic phosphoinositide-phospholipase C isozymes" J.Biol.Chem.267. 6488-6494 (1992)
Y.Banno:“凝溶胶对人血小板胞质磷酸肌醇-磷脂酶 C 同工酶的影响”J.Biol.Chem.267。
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S.Nakashima: "Thrombin induces a biphasic 1,2-diacylglycerol production in human platelets." Biochem. J.275. 355-361 (1991)
S.Nakashima:“凝血酶诱导人血小板产生双相 1,2-二酰基甘油。”
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Nakashima,S.: "Genisteine,a protein tyrosin kinase inhibitor,inhibits thromboxane A2ーmediated humna platlet responses" Mol.Pharmacol. 39. 475-480 (1991)
Nakashima, S.:“Genisteine,一种蛋白酪氨酸激酶抑制剂,抑制血栓素 A2 介导的人类血小板反应”Mol.Pharmacol 39. 475-480 (1991)
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Banno,Y.: "Isolation of a polyphosphoinositideーphospholipase C(Type)" Platelets.
Banno, Y.:“多磷酸肌醇-磷脂酶 C(类型)的分离”血小板。
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Nozawa,Y.: "Phospholipidーmediated signaling in receptor activation of human platelets" Biochim.Biophys.Acta. 1082. 219-238 (1991)
Nozawa, Y.:“人血小板受体激活中的磷脂介导的信号传导”Biochim.Biophys.Acta.1082.219-238(1991)
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共 42 条
REGULATORY MECHANISM BY PHOSPHOLIPASE D IN OXIDANT-STRESS INDUCED SURVIVAL SIGNALING
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批准号:16390098
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项目类别:Grant-in-Aid for Scientific Research (B)
-
资助金额:$9.09万
-
财政年份:2004
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负责人:NOZAWA Yoshinori
-
依托单位:
CROSS-TALK OF MEMBRANE LIPID SIGNALING IN CELL DEATH AND SURVIVAL
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批准号:14370064
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项目类别:Grant-in-Aid for Scientific Research (B)
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资助金额:$8.13万
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财政年份:2002
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负责人:NOZAWA Yoshinori
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依托单位:
MECHNISM OF APOPTOSIS INDUCED BY MEMBRANE LIPID SYGNALING
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批准号:12470042
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项目类别:Grant-in-Aid for Scientific Research (B)
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资助金额:$7.94万
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财政年份:2000
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负责人:NOZAWA Yoshinori
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依托单位:
Functional analysis of the new signal transduction enzyme PLD by the molecular genetic technique
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批准号:10212204
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项目类别:Grant-in-Aid for Scientific Research on Priority Areas (B)
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资助金额:$21.57万
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财政年份:1998
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负责人:NOZAWA Yoshinori
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依托单位:
Molecular mechanisms for regulation and physiological role of phospholipase D
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批准号:09480162
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项目类别:Grant-in-Aid for Scientific Research (B)
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资助金额:$9.6万
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财政年份:1997
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负责人:NOZAWA Yoshinori
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依托单位:
Molecular mechanisms for membrane lipid signaling in apoptosis
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批准号:07457036
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项目类别:Grant-in-Aid for Scientific Research (B)
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资助金额:$4.67万
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财政年份:1995
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负责人:NOZAWA Yoshinori
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依托单位:
Studies on Functions of bioactive phospholipids
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批准号:06304050
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项目类别:Grant-in-Aid for Scientific Research (A)
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资助金额:$17.66万
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财政年份:1994
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负责人:NOZAWA Yoshinori
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依托单位:
Action mechanisms and roles of small GTP-binding proteins
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批准号:05271103
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项目类别:Grant-in-Aid for Scientific Research on Priority Areas
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资助金额:$111.87万
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财政年份:1993
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负责人:NOZAWA Yoshinori
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依托单位:
Studies on the conformation and functions of ras-related low Mr GTP-binding proteins.
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批准号:03304052
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项目类别:Grant-in-Aid for Co-operative Research (A)
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资助金额:$4.86万
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财政年份:1991
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负责人:NOZAWA Yoshinori
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依托单位:
Adaptation mechanism of membrane lipids and its genetic control
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批准号:61480465
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项目类别:Grant-in-Aid for General Scientific Research (B)
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资助金额:$3.97万
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财政年份:1986
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负责人:NOZAWA Yoshinori
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依托单位:
Development of Medically ApplicableLiposomes as Enzyme or Drug Carriers
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批准号:58870126
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项目类别:Grant-in-Aid for Developmental Scientific Research
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资助金额:$2.5万
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财政年份:1983
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负责人:NOZAWA Yoshinori
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依托单位:
海外基金