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，其通过将细胞外刺激的受体偶联至细胞内效应物（酶、离子通道）而作为跨膜信号转导物起作用。G蛋白组成了一个多样化的家族，其特征在于特异性受体和效应物相互作用，而这又由三个组成亚基的结构决定。α亚基结合鸟嘌呤核苷酸，并在效应子调节中具有明确的作用。β亚基和γ亚基作为β-γ复合物紧密结合，包含单个功能实体，其与α亚基一样，是G蛋白与受体相互作用所绝对需要的。β-γ复合物的效应调节作用在几个系统中变得越来越明显。本研究强调β-γ复合物在G蛋白介导的信号转导中的作用。一种结构上不同的神经表达的G β亚基β-5由Mel Simon及其同事从大脑中克隆出来，后来在视网膜中发现了一种选择性剪接的“长”形式（G β 5-L）。最近发现G β-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，G β-5/γ-2抑制AC II型，并选择性地与G α-q亚型相互作用，这是迄今为止研究的G β-γ复合物中的新特性。从洗涤剂提取的小鼠脑膜中对Gbeta 5进行免疫亲和纯化，将G蛋白信号转导调节剂RGS 6和RGS 7鉴定为紧密结合的伴侣。我们表征了Gbeta 5在脑和几种神经内分泌细胞系中的表达，以了解更多关于Gbeta 5在天然系统中的功能和调节。一种Gbeta 5阳性细胞系是大鼠嗜铬细胞瘤PC 12细胞。我们最近证明了在PC 12细胞和脑中G β-5的核表达（Zhang，J.H.，巴尔，弗吉尼亚州，莫，Y.，Rojkova，A. M.，Liu，S.，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，我们产生了一种Gbeta-5突变体，它缺乏与RGS 7相互作用的能力，同时保留了它结合Ggamma的能力，我们将其特性与野生型Gbeta-5进行了比较。在HEK-293细胞中，RGS 7而非Ggamma-2的共转染支持转染的野生型G β-5的核级分中的表达。相比之下，Ggamma偏好的Gbeta-5突变体在具有任一共转染子的HEK-293细胞核部分中不表达。通过激光共聚焦显微镜分析，G γ-选择性G β-5突变体也从转染的PC 12细胞的细胞核中排除。这些结果定义了Gbeta-5核表达对RGS蛋白结合的要求。目前的工作集中在功能的Gbeta-5同系物在苍蝇，使用果蝇作为模型系统。