G Protein Beta-Gamma and Beta-RGS Dimers--Structure and

G 蛋白 Beta-Gamma 和 Beta-RGS 二聚体——结构和

• 批准号: 6983887
• 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/6983887
• 关键词: G protein; PC12 cells; biological signal transduction; cell membrane; confocal scanning microscopy; dimer; guanine nucleotide binding protein; immunocytochemistry; phospholipase C; protein localization; protein structure function; receptor coupling; 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 (Gbeta5-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. Immunoaffinity purification of Gbeta5 from detergent-extracted membranes of mouse brain identified regulators of G protein signaling RGS6 and RGS7 as tightly bound partners. We characterized the expression of Gbeta5 in brain and in several neuroendocrine cell lines to learn more about the function and regulation of Gbeta5 in native systems. One Gbeta5-positive cell line was rat pheochromocytoma PC12 cells. We recently demonstrated nuclear expression of Gbeta-5 in PC12 cells and brain (Zhang, J. H., Barr, V. A., Mo, Y., Rojkova, A. M., Liu, S., and Simonds, W. F. (2001) J. Biol. Chem. 276, 10284-10289). To gain further insight into the mechanism of Gbeta-5 nuclear localization, we generated a Gbeta-5 mutant deficient in its ability to interact with RGS7 while retaining its ability to bind Ggamma, and we compared its properties to the wild-type Gbeta-5. In HEK-293 cells co-transfection of RGS7 but not Ggamma-2 supported expression in the nuclear fraction of transfected wild-type Gbeta-5. In contrast the Ggamma-preferring Gbeta-5 mutant was not expressed in the HEK-293 cell nuclear fraction with either co-transfectant. The Ggamma-selective Gbeta-5 mutant was also excluded from the cell nucleus of transfected PC12 cells analyzed by laser confocal microscopy. These results define a requirement for RGS protein binding for Gbeta-5 nuclear expression. Current work focuses on the function of the Gbeta-5 homolog in fly, using Drosophila melanogaster as a model system.

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（Gbeta5-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。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。我们对大脑和几种神经内分泌细胞系中 Gbeta5 的表达进行了表征，以了解有关 Gbeta5 在天然系统中的功能和调节的更多信息。一种 Gbeta5 阳性细胞系是大鼠嗜铬细胞瘤 PC12 细胞。我们最近证明了 PC12 细胞和大脑中 Gbeta-5 的核表达（Zhang, J. H.、Barr, V. A.、Mo, Y.、Rojkova, A. M.、Liu, S. 和 Simonds, W. F. (2001) J. Biol. Chem. 276、10284-10289）。为了进一步了解 Gbeta-5 核定位的机制，我们生成了一种 Gbeta-5 突变体，该突变体缺乏与 RGS7 相互作用的能力，同时保留其结合 Ggamma 的能力，并将其特性与野生型 Gbeta-5 进行了比较。在 HEK-293 细胞中，共转染 RGS7 而不是 Ggamma-2，支持转染的野生型 Gbeta-5 的核级分中的表达。相反，Ggamma 偏好的 Gbeta-5 突变体在具有任一共转染子的 HEK-293 细胞核级分中均不表达。通过激光共聚焦显微镜分析，Ggamma 选择性 Gbeta-5 突变体也被排除在转染 PC12 细胞的细胞核之外。这些结果定义了 Gbeta-5 核表达的 RGS 蛋白结合的要求。目前的工作重点是使用果蝇作为模型系统，研究 Gbeta-5 同源物在果蝇中的功能。