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分子的信号传导过程中起着重要作用。它们由α，β和γ亚基组成，每个亚基都有多种亚型。在这项研究中，我们研究了βγ亚基的功能，特别是γ亚基的不同亚型导致的功能差异。研究结果如下：1)在成纤维细胞中，G蛋白βγ亚基对应激纤维产生破坏性影响。而βγ基因可诱导上皮样细胞形成应力纤维和局灶性黏附集结。βγ诱导的应力纤维和灶性黏附的形成被Rho的显性负突变体共表达抑制，这表明βγ亚基调控Hala细胞中Rho依赖性肌动蛋白的聚合。2)百日咳毒素可阻断溶血磷脂酸(LPA)诱导的NIH3T3成纤维细胞在纤维连接蛋白上的细胞扩散。这种对细胞扩散的阻止被Consti…的表达消除了Rho家族小G蛋白的突变体Rac和CDc42更具肿瘤活性，但不是由Rho引起的。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。αi2和βγ亚单位的结构活性突变体的转染促进了百日咳毒素处理细胞的扩散。β1和γ12是成纤维细胞中的一种主要γ形式，在促进细胞扩散方面比β1γ2或β1+γ12S2A更有效，后者是一种突变体，其中蛋白激酶C的磷酸化位点Ser2被丙氨酸取代。这些发现表明，αI和βγ都通过LPA诱导的细胞扩散来刺激Rac和CDC42通路。3)大脑中大多数G蛋白亚基的浓度在发育过程中升高。而γ-5在胚胎脑中的表达水平较高，在出生后整个发育过程中表达水平较低。胚胎脑免疫组织化学染色显示，γ-5在脑室带增殖区特异表达，提示其在未分化细胞中具有特定功能。较少

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