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Studies on various γ subunits of heterotrimeric GTP-binding proteins

异源三聚体 GTP 结合蛋白各种 γ 亚基的研究

基本信息

批准号：
11680649
负责人：
ASANO Tomiko
金额：
$ 2.3万
依托单位：
Institute for Developmental Research, Aichi Human Service Center
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1999
资助国家：
日本
起止时间：
1999 至 2000
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-11680649/
关键词：
G protein γ subunit Stress fibers Cell spreading Protein kinase C Lysophosphatidic acid Neuroepithelial cells Rho family βγサブユニットリゾフォスファケジン酸 Rac Cdc42 百日咳毒素細胞走化性リン酸化

项目摘要

Heterotrimeric G proteins play a major role in signal tradsuction from cell-surfacereceptors to intracellular effectors. They are composed of α, β and γ subunits and multiple isoforms are identified for each subunits. In this study, we investigated functions of βγ subunits, particularly focused on the functional difference due to isoforms of the γ subunit. The obtained results are as follows.1) In fibroblasts, G protein βγ subunits exert a disruptive influence on stress fibers. However, transfection of βγ induced stress fiber formation and focal adhesion assembly in epithelial-like HeLa cells. βγ-induced stress fiber and focal adhesion formation was inhibited by co-expression of a dominant negative mutant of Rho, indicating that βγ subunits regulate Rhodependent actin polymerization in HaLa cells. 2) Pertussis toxin blocks lysophosphatidic acid(LPA)-induced cell spreading of NIH 3T3 fibroblasts on fibronectin. This prevention of cell spreading was eliminated by the expression of consti … More tutively active mutants of Rho family small G proteins, Rac and Cdc42, but not by Rho. In addition, activation of the endogenous forms was suppressed by pertussis toxin, indicating that Gi-induced cell spreading is mediated through the Rac and Cdc42 pathways. Transfection of a constitutively active mutant of αi2 and βγ subunits enhanced spreading of pertussis toxin-treated cells. β1 with γ12, a major γ form in fibroblasts, was more effective for increasing cell spreading than β1γ2 or β1 plus γ12S2A, a mutant in which Ser2, a phosphorylation site for protein kinase C, is replaced with alanine. These findings indicate that both αi and βγ stimulate Rac and Cdc42 pathways with LPA-induced cell spreading. 3) Concentrations of most G protein subunits in the brain increase during development. However, expression of γ5 was high in embryonic brain and low throughout postnatal development. Immunohistochemical staining of embryonic brains showed γ5 to be specifically expressed in the prolifereative region of the ventricular zone, suggesting its specific function in undifferentiated cells. Less

异源三聚体G蛋白在细胞表面受体到细胞内效应物的信号转导中起重要作用。它们由α、β和γ亚基组成，每个亚基鉴定出多种亚型。在本研究中，我们研究了βγ亚基的功能，特别是由于γ亚基的异构体的功能差异。结果表明：1）在成纤维细胞中，G蛋白βγ亚基对应力纤维具有破坏性影响。然而，转染βγ诱导上皮样HeLa细胞中应力纤维形成和粘着斑组装。共表达Rho的显性失活突变体可抑制βγ诱导的应力纤维和粘着斑的形成，表明βγ亚基调节HaLa细胞中Rho依赖性肌动蛋白聚合。2)百日咳毒素阻断溶血磷脂酸（LPA）诱导的NIH 3T3成纤维细胞在纤连蛋白上的细胞铺展。这种对细胞扩散的阻止作用被表达的constitutional ...更多信息 Rho家族小G蛋白Rac和Cdc42的突变体，但不是由Rho。此外，百日咳毒素抑制了内源性形式的激活，表明Gi诱导的细胞扩散是通过Rac和Cdc42途径介导的。α 12和βγ亚基组成型活性突变体的转染增强了百日咳毒素处理的细胞的扩散。β1 + γ12（成纤维细胞中的主要γ形式）比β1 + γ2或β1 + γ12S2A（蛋白激酶C的磷酸化位点Ser2被丙氨酸取代的突变体）更有效地增加细胞铺展。这些发现表明，αi和βγ都刺激Rac和Cdc42通路，伴随LPA诱导的细胞铺展。3)大多数G蛋白亚基在大脑中的浓度在发育过程中增加。γ5在胚胎脑中表达量较高，而在整个出生后发育过程中表达量较低。免疫组化染色显示γ5特异性表达于脑室带的增殖反应区，提示γ5在未分化细胞中具有特异性功能。少