Membrane Targeting of G Proteins

描述(由申请人提供)：细胞内信号通路取决于其组成蛋白的适当和独特的亚细胞位置。负责可逆靶向不同细胞膜的外周膜蛋白的机制还知之甚少。这项研究拨款将集中在关于异源三聚体(α-β-伽马)G蛋白可逆质膜定位机制的几个关键问题上。G蛋白充当分子开关，将信息从细胞表面受体传递到适当的效应蛋白。为了传递信号，G蛋白必须至少在一开始定位到质膜的细胞质表面。G蛋白α亚基(Galpha)通过脂肪酸肉豆蔻酸酯和/或棕榈酸酯的共价结合而修饰，而β-Gamma二聚体的Gamma亚基则由Arnyl或Geranylgeranyl脂部分修饰。这些附着的脂类可能作为疏水锚促进与细胞膜的结合；然而，针对异源三聚体G蛋白的额外的膜靶向信号尚未得到很好的描述。此外，异源三聚体最初在细胞内何时何地形成，以及G蛋白到达质膜的细胞途径是什么，这些都是仍然没有答案的关键问题。一旦异三聚体G蛋白在质膜上被激活，Galpha和βGamma就会解离，Galpha可以进行快速的脱氨基转移。对于一个Galpha阿尔法，受体的激活也促进了它的移位，使其脱离质膜进入细胞质。这种G蛋白运输的潜在机制和细胞途径也鲜为人知。因此，这项建议的主要目的是：1)确定GET在质膜靶向GABA-γ中的作用；2)定义Galpha和Gbeta-γ在合成后运输到质膜中所使用的细胞途径；3)定义N-末端多元簇在膜靶向Galpha中的作用；以及4)定义激活诱导GalpHa重新分布的机制。这些目标集中在G蛋白的细胞生物学中截然不同但高度相关的问题。这项研究将利用培养的哺乳动物细胞作为模型系统，并将使用许多技术，包括免疫荧光显微镜、活细胞的荧光显微镜、亚细胞分离和大量的生化分析，以确定G蛋白可逆膜靶向的机制的结构-功能关系。

DESCRIPTION (provided by applicant): Intracellular signaling pathways depend upon appropriate and unique subcellular locations of their constituent proteins. Mechanisms responsible for reversibly targeting peripheral membrane proteins to different cellular membranes are poorly understood. This research grant will focus on several key questions regarding the mechanisms of reversible plasma membrane localization of heterotrimeric (alpha beta gamma) G proteins. G proteins act as molecular switches to relay information from cell surface receptors to appropriate effector proteins. To transmit a signal, G proteins must be localized, at least initially, to the cytoplasmic face of the plasma membrane. G protein alpha subunits (Galpha) are modified by the covalent attachment of the fatty acids myristate and/or palmitate, and gamma subunits of the beta gamma dimers are modified by arnesyl or geranylgeranyl lipid moieties. These attached lipids likely function as hydrophobic anchors to promote binding to cellular membranes; however, additional membrane targeting signals for heterotrimeric G proteins have not been well described. Moreover, when and where inside the cell does the heterotrimer initially form, and what is the cellular pathway used by G proteins to arrive at the plasma membrane, are critical questions that remain unanswered. Once the heterotrimeric G protein is activated at the plasma membrane, Galpha and beta gamma dissociate, and Galpha can undergo rapid depalmitoylation. For one Galpha alphas, receptor activation also promotes its translocation off the plasma membrane and into the cytoplasm of the cell. The underlying mechanisms and cellular pathways of this G protein trafficking are also poorly understood. Thus, the major objectives of this proposal are 1) Define the role of Get in plasma membrane targeting of Gbata gamma; 2) Define the cellular pathway used by Galpha and Gbeta gamma in trafficking to the plasma membrane after synthesis; 3) Define the role of an N-terminal polybasic cluster in membrane targeting of Galpha; and 4) Define mechanisms of activation-induced redistribution of Galphas. These objectives focus on distinct yet highly related questions of the cell biology of G proteins. This research will utilize cultured mammalian cells as model systems and will employ a number of techniques, including immunofluorescence microscopy, fluorescence microscopy of live cells, subcellular fractionation, and numerous biochemical assays to define structure -function relationships in terms of mechanisms of reversible membrane targeting of G proteins.