Membrane Targeting of G Proteins

描述（由申请方提供）：细胞内信号传导途径取决于其组成蛋白的适当和独特的亚细胞位置。外周膜蛋白可逆靶向不同细胞膜的机制知之甚少。这项研究经费将集中在几个关键问题的机制，可逆质膜定位的异源三聚体（α β γ）G蛋白。G蛋白作为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蛋白必须定位在质膜的细胞质表面，至少在最初是这样。G蛋白α亚基（G α）通过脂肪酸肉豆蔻酸酯和/或棕榈酸酯的共价连接而被修饰，并且β γ二聚体的γ亚基通过阿呢基或香叶基香叶基脂质部分而被修饰。这些附着的脂质可能作为疏水锚来促进与细胞膜的结合;然而，对于异源三聚体G蛋白的另外的膜靶向信号还没有很好地描述。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，这些都是尚未回答的关键问题。一旦异源三聚体G蛋白在质膜上被激活，G α和β γ就会解离，G α可以快速脱棕榈酰化。对于一种Galalpha α，受体活化也促进其易位离开质膜并进入细胞的细胞质。这种G蛋白运输的潜在机制和细胞途径也知之甚少。因此，本提案的主要目的是1）确定Get在Gbata γ的质膜靶向中的作用; 2）确定合成后Ga和Gbeta γ运输至质膜中所使用的细胞途径; 3）确定N-末端多元簇在Ga的膜靶向中的作用;和4）确定激活诱导的Galphas再分布的机制。这些目标集中在G蛋白的细胞生物学的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，并将采用许多技术，包括免疫荧光显微镜，活细胞的荧光显微镜，亚细胞分级分离，和许多生化测定，以确定结构-功能关系的机制，可逆的膜靶向G蛋白。

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.