Studies on various γ subunits of heterotrimeric GTP-binding proteins

异三聚体G蛋白在从细胞表面受体到细胞内效应器的信号传递中起着重要作用。它们由α、β和γ亚基组成，每个亚基都有多个同工型。在这项研究中，我们研究了βγ亚基的功能，特别关注了γ亚基同工异构体的功能差异。所得结果如下：1)在成纤维细胞中，G蛋白βγ亚基对应激纤维具有破坏性影响。然而，转染βγ诱导上皮样HeLa细胞的应激纤维形成和局灶黏附组装。Rho显性负突变体的共表达抑制了βγ诱导的应力纤维和局灶粘连的形成，表明βγ亚基调节了HaLa细胞中Rhodependent actin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扩散。Rho家族小G蛋白的活性更强的突变体Rac和Cdc42的表达消除了这种阻止细胞扩散的作用，但Rho没有。此外，内源性形式的激活被百日咳毒素抑制，表明gi诱导的细胞扩散是通过Rac和Cdc42途径介导的。转染αi2和βγ亚基的组成型活性突变体增强百日咳毒素处理细胞的扩散。β1与γ - 12（成纤维细胞中的一种主要γ形式）相比，β1 - γ - 2或β1 + γ - 12s2a（一种蛋白激酶C的磷酸化位点Ser2被丙氨酸取代的突变体）更有效地增加细胞扩散。这些发现表明αi和βγ都刺激Rac和Cdc42通路，通过lpa诱导细胞扩散。3)大脑中大多数G蛋白亚基的浓度在发育过程中增加。然而，γ - 5在胚胎脑中表达量高，在整个出生后发育过程中表达量低。胚胎脑免疫组化染色显示γ - 5在脑室区增殖区特异性表达，提示其在未分化细胞中具有特异性功能。少

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