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）是异源三聚体，通过将细胞外刺激受体偶联到细胞内效应器（酶、离子通道），起到跨膜信号转导的作用。g蛋白构成了一个多样化的家族，以特定的受体和效应相互作用为特征，而受体和效应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调节中具有良好的作用。β亚基和γ亚基紧密结合为β - γ复合物，组成一个单一的功能实体，与α亚基一样，是G蛋白与受体相互作用所必需的。在一些系统中，β - γ复合物的效应调节作用越来越明显。目前的研究强调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出了一种结构上分化的神经表达的G β亚基-5，后来在视网膜中发现了一种选择性拼接的“长”形式（Gbeta5-L）。最近发现G β -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 β -5/ γ -2抑制AC II型，并选择性地与G α -q异构体相互作用，这是迄今为止研究的G β - γ复合物中的新特性。从小鼠脑清洁剂提取膜中纯化Gbeta5，发现G蛋白信号传导调控因子RGS6和RGS7是紧密结合的伙伴。我们对Gbeta5在脑和几种神经内分泌细胞系中的表达进行了表征，以进一步了解Gbeta5在天然系统中的功能和调控。1株gbeta5阳性细胞系为大鼠嗜铬细胞瘤PC12细胞。我们最近证实了gβ -5在PC12细胞和大脑中的核表达(Zhang， J. H., Barr， V. A., Mo， Y., Rojkova， A. M., Liu， S., and Simonds， 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，我们构建了一个Gbeta-5突变体，该突变体缺乏与RGS7的相互作用能力，但保留了与gamma的结合能力，并将其与野生型Gbeta-5的特性进行了比较。在HEK-293细胞中，共转染RGS7而不转染γ -2支持转染野生型Gbeta-5核部分的表达。相比之下，具有γ偏好的gβ -5突变体在HEK-293细胞核片段中均未表达。通过激光共聚焦显微镜分析，转染PC12细胞的细胞核中也排除了γ -选择性gβ -5突变体。这些结果确定了gβ -5核表达需要RGS蛋白结合。目前的工作主要集中在gβ -5同源物在果蝇中的功能，以果蝇为模型系统。

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.