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Physiological significance of G protein gamma subunit heterogeneity

G蛋白γ亚基异质性的生理意义

基本信息

批准号：
08458201
负责人：
ASANO Tomiko
金额：
$ 4.67万
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 (B)
财政年份：
1996
资助国家：
日本
起止时间：
1996 至 1998
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-08458201/
关键词：
G protein gamma subunit beta subunit Protein kinase C Phosphorylation F-actin Motility Localization リゾホスファチジン酸組織分布細胞内分布 F-アクチン

项目摘要

Heterotrimeric G proteins play a major role in signal transduction from cell-surface receptors to intracellular effectors. The p and y subunits are tightly bound under physiological conditions, and so far, 11 isoforms of gamma have been found. To elucidate the physiological significance of gamma subunit heterogeneity, we searched the differences among gamma isoforms and obtained following results. 1. In fibroblasts, the gamma12 subunit associated with F-actin, while the gamma5 localized at focal adhesions, suggesting functional differences for the two gamma isoforms. 2. The phosphorylation site of gamma12 by protein kinase C was determined to be Ser-2.3. The gamma12 was phosphorylated in the cells by the stimulation with various reagents, such as phorbol ester, serum, lysophosphatidic acid and growth factors. 4. To examine the roles of gamma subunits, various gamma isoforms were overexpressed in NIH 3T3 cells together with the beta1 subunit. Expression of gamma12 induced cell rounding and increase of cell migration, but expression of other gamma subunits did not induce significant changes. 5. Deletion of the N-terminal region or replacement by alanine of Ser-2 of gamma12 eliminated these effects of gamma12, while a mutant in which Ser-2 was replaced with glutamic acid showed effects equivalent to wild-type gamma12. These results indicated that phosphorylation of gamma12 at Ser-2 enhances the motility of cells. 6. Immunohistochemical studies showed that gamma3 was localized in neuronal cells, while gamma12 was localized some glial cells in the brain. 7. We identified the beta4 protein in the preparation of betagamma5 complex purified from bovine lung. The beta4 was shown to interact selectively with gamma5 and gamma12.

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转导中发挥着重要作用。 p和y亚基在生理条件下紧密结合，迄今为止，已发现11种γ亚型。为了阐明γ亚基异质性的生理意义，我们搜索了γ亚型之间的差异并获得了以下结果。 1. 在成纤维细胞中，gamma12 亚基与 F-肌动蛋白相关，而 gamma5 位于粘着斑处，表明两种 gamma 亚型存在功能差异。 2.确定蛋白激酶C对γ12的磷酸化位点为Ser-2.3。通过佛波酯、血清、溶血磷脂酸和生长因子等各种试剂的刺激，细胞中的γ12被磷酸化。 4. 为了检查 γ 亚基的作用，各种 γ 亚型与 β1 亚基一起在 NIH 3T3 细胞中过表达。 γ12的表达诱导细胞变圆和细胞迁移增加，但其他γ亚基的表达不诱导显着变化。 5. 缺失伽马12的N端区域或用丙氨酸替换伽马12的Ser-2消除了伽马12的这些影响，而其中Ser-2被谷氨酸替换的突变体显示出与野生型伽马12相同的效果。这些结果表明，γ12 Ser-2 位点的磷酸化增强了细胞的运动性。 6. 免疫组织化学研究表明，gamma3 定位于神经元细胞，而 gamma12 定位于大脑中的一些神经胶质细胞。 7. 我们鉴定了从牛肺纯化的βamma5复合物制备过程中的β4蛋白。 beta4 被证明可以选择性地与 gamma5 和 gamma12 相互作用。