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

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表达的Gβ亚基，β-5，后来在视网膜中发现了一种可选择性剪接的“长”形式(Gbeta5-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。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。我们对Gbeta5在脑和几个神经内分泌细胞系中的表达进行了表征，以了解Gbeta5在自然系统中的功能和调节。1个Gbeta5阳性细胞系为大鼠嗜铬细胞瘤PC12细胞。我们最近证实了Gbeta-5在PC12细胞和脑中的核表达(Zhang，J.H.，Barr，V.A.，Mo，Y.，Rojkova，A.M.，Liu，S.，and Simond，W.F.(2001)J.Biol)。化学。276、10284-10289)。为了进一步了解Gbeta-5核定位的机制，我们产生了一个Gbeta-5突变体，它与RGS7相互作用的能力存在缺陷，同时保留了与GGamma结合的能力，我们将其性质与野生型Gbeta-5进行了比较。在HEK-293细胞中，共转染RGS7，但不支持GGamma-2在野生型Gbeta-5的核部分表达。相反，偏爱GGamma的Gbeta-5突变体在HEK-293细胞的细胞核中没有表达，与任何一个共转染体都没有表达。激光共聚焦显微镜分析显示，转基因PC12细胞的细胞核中也排除了GGamma选择性的Gbeta-5突变体。这些结果定义了Gbeta-5核表达对RGS蛋白结合的要求。目前的工作主要是以果蝇为模型系统，研究Gbeta-5同源基因在果蝇中的功能。

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.