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

G 蛋白 β-γ 和 β-RGS 二聚体——结构和功能

• 批准号: 6289793
• 负责人: WILLIAM F SIMONDS
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6289793
• 关键词: Dictyostelium; G protein; biological signal transduction; dimer; guanine nucleotide binding protein; phospholipase C; protein structure function; receptor coupling; recombinant proteins; site directed mutagenesis; tissue /cell culture; transfection

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

鸟嘌呤核苷酸结合调节蛋白（G 蛋白）是异源三聚体，通过将细胞外刺激受体与细胞内效应器（酶、离子通道）偶联而发挥跨膜信号转导器的作用。 G 蛋白构成了一个多样化的家族，其特征在于特定的受体和效应子相互作用，而这些相互作用又由三个组成亚基的结构决定。 α亚基结合鸟嘌呤核苷酸并在效应器调节中具有明确的作用。 β 和 γ 亚基作为 β-γ 复合物紧密相连，包含单个功能实体，与 α 亚基一样，它是 G 蛋白与受体相互作用所必需的。 β-γ 复合物的效应调节作用在多个系统中变得越来越明显。目前的研究强调了 β-γ 复合物在 G 蛋白介导的信号转导中的作用。 Mel Simon 和他的同事从大脑中克隆了一种结构上不同的神经表达 G beta 亚基 beta-5，后来在视网膜中发现了一种选择性剪接的长形式（beta-5 L）。最近发现 G beta-5 表现出功能专门化，因为它能够激活 PLC，但不能激活 MAPK 或 JNK 级联。此外，G beta-5/gamma-2 抑制 II 型 AC 并选择性地与 G alpha-q 同工型相互作用，这是迄今为止研究的 G beta-gamma 复合物中的新颖特性。通过体外激酶测定进一步探讨了 G beta-5 的效应子选择性，并通过蛋白质免疫印迹 (WB)、原位杂交 (ISH) 和核糖核酸酶保护测定 (RPA) 分析了其在小鼠组织和培养细胞系中的表达模式。在HEK-293细胞中，共转染G beta-1/gamma-2而非G beta-5/gamma-2以渥曼青霉素敏感的方式激活表位标记的Akt/PKB。 G beta-5 未能激活 MAPK 级联和 Akt/PKB，这表明无法与常见中间体（如 PI3K gamma）相互作用可能是 G beta-5 选择性的原因。通过 Northern 印迹、RPA 和 WB 对培养细胞系进行的分析表明，G beta-5 在 alpha-T3、PC-12、SH-SY5Y 和 GT1-7 神经元细胞系中表达，但在非神经元 C6 胶质瘤、COS-7 中不表达或 HEK-293 细胞。使用 G beta-5 特异性核糖核酸探针对小鼠脑切片进行 ISH 分析，揭示了整个大脑的特异性信号，包括前脑皮质层、中脑上丘和下丘以及小脑浦肯野细胞。这些结果表明 G beta-5 的特殊功能在整个中枢神经系统中具有广泛的重要性。从去污剂提取的小鼠脑膜中免疫亲和纯化 Gbeta5，鉴定出 G 蛋白信号调节因子 RGS6 和 RGS7 作为紧密结合的伴侣。 - G蛋白、信号转导、G蛋白信号传导调节剂