G PROTEIN BETA-GAMMA AND BETA-RGS DIMERS--STRUCTURE AND FUNCTION

鸟嘌呤核苷酸结合调节蛋白(G蛋白)是一种异源三聚体，通过将细胞外刺激受体与细胞内效应物(酶、离子通道)偶联，起到跨膜信号转导的作用。G蛋白是一个不同的家族，由特定的受体和效应器相互作用决定，而受体和效应器的相互作用又由三个组成亚基的结构决定。α亚基与鸟嘌呤核苷酸结合，并在效应器调节中发挥重要作用。β亚基和伽马亚基作为一个β-伽马复合体紧密联系在一起，组成一个单一的功能实体，就像阿尔法亚基一样，是G蛋白与受体相互作用必不可少的。在几个系统中，β-伽马复合体的效应调节作用正变得越来越明显。本研究强调了β-γ复合体在G蛋白介导的信号转导中的作用。梅尔·西蒙和他的同事从大脑中克隆了一个结构不同的神经元表达的Gβ亚基，β-5，后来在视网膜中发现了一个可选择性剪接的长形式(β-5 L)。最近发现G beta-5表现出功能特化，因为它能够激活PLC，但不能激活MAPK或JNK级联。此外，Gβ-5/伽马-2抑制AC II型，并选择性地与Gα-Q亚型相互作用，这是迄今为止研究的Gβ-伽马复合体中的新特性。并通过蛋白免疫印迹(WB)、原位杂交(ISH)和核糖核酸酶保护试验(RPA)分析Gβ-5在小鼠组织和培养细胞中的表达模式。在HEK-293细胞中，共转染Gβ-1/伽马-2，而不是Gβ-5/伽马-2激活的表位标记Akt/PKB以Wortmannin敏感的方式。Gβ-5不能同时激活MAPK级联和Akt/PKB表明，不能与常见的中间体如PI3K伽马相互作用可能是Gβ-5选择性的原因。Northern blotting、RPA和WB分析显示，Gβ-5在α-T3、PC-12、SH-SY5Y和GT1-7神经细胞系中表达，而在非神经性C6胶质瘤、COS-7和HEK-293细胞中不表达。用Gβ-5特异性核糖核酸探针对小鼠脑切片进行ISH分析，发现特定的信号遍及整个大脑，包括前脑的皮质层、中脑的上、下丘和小脑浦肯野细胞。这些结果表明，Gβ-5的特殊功能在整个中枢神经系统中具有广泛的重要性。从洗涤剂提取的小鼠脑膜中免疫亲和纯化Gbeta5，证实G蛋白信号转导调节因子RGS6和RGS7是紧密结合的伙伴。-G蛋白、信号转导、G蛋白信号调节因子

The guanine-nucleotide binding regulatory proteins (G-proteins) are heterotrimers which function as transmembrane signal transducers by coupling receptors for extracellular stimuli to intracellular effectors (enzymes, ion channels). G-proteins constitute a diverse family distinguished by specific receptor and effector interactions which in turn are determined by the structure of the three constituent subunits. The alpha subunit binds guanine nucleotides and has a well established role in effector modulation. The beta and gamma subunits are tightly associated as a beta-gamma complex, comprising a single functional entity which, like the alpha subunit, is absolutely required for G protein interaction with receptor. An effector modulatory role for the beta-gamma complex is becoming increasingly apparent in several systems. The present research emphasizes the role of the beta-gamma complex in G-protein-mediated signal transduction. A structurally divergent neurally expressed G beta subunit, beta-5, was cloned from brain by Mel Simon and coworkers, and later found in an alternatively spliced long form in retina (beta-5 L). G beta-5 was recently found to exhibit functional specialization, as it was able to activate PLC but not the MAPK or JNK cascades. Furthermore G beta-5/gamma-2 inhibited AC type II and interacted selectively with the G alpha-q isoform, properties novel among G beta-gamma complexes studied to date. The effector selectivity of G beta-5 was further explored by in vitro kinase assays, and its pattern of expression in mouse tissues and cultured cell lines analyzed by protein immunoblotting (WB), in situ hybridization (ISH) and ribonuclease protection assays (RPA). In HEK- 293 cells, cotransfected G beta-1/gamma-2 but not G beta-5/gamma-2 activated epitope-tagged Akt/ PKB in a wortmannin-sensitive fashion. The failure of G beta-5 to activate both the MAPK cascade and Akt/PKB suggests that the inability to interact with a common intermediate such as PI3K gamma may account for G beta-5 selectivity. Analysis of cultured cell lines by Northern blotting, RPA and WB demonstrated G beta-5 expression in alpha-T3, PC-12, SH-SY5Y and GT1-7 neuronal cell lines, but not in non-neuronal C6 glioma, COS-7 or HEK-293 cells. Mouse brain sections were analyzed by ISH with G beta-5-specific riboprobe and revealed specific signal throughout the brain including cortical layers of the forebrain, the superior and inferior colliculi in the midbrain, and cerebellar Purkinje cells. These results suggest the specialized function of G beta-5 is of widespread importance throughout the central nervous system. Immunoaffinity purification of Gbeta5 from detergent-extracted membranes of mouse brain identified regulators of G protein signaling RGS6 and RGS7 as tightly bound partners. - G proteins, signal transduction, regulators of G protein signaling