Membrane Targeting of G Proteins

G 蛋白的膜靶向

基本信息

批准号：
6751595
负责人：
PHILIP B WEDEGAERTNER
金额：
$ 25.83万
依托单位：
THOMAS JEFFERSON UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
1998
资助国家：
美国
起止时间：
1998-08-01 至 2007-07-31
项目状态：
已结题

项目摘要

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.

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。对不同细胞膜的可逆性靶向外周膜蛋白的机制知之甚少。这项研究赠款将重点介绍有关异源三聚体（alpha beta伽马）G蛋白的可逆质膜定位机制的几个关键问题。 G蛋白充当分子开关，从细胞表面受体传递到适当的效应蛋白。为了传输信号，必须至少在最初将G蛋白定位在质膜的细胞质面上。 G蛋白α亚基（GALPHA）通过脂肪酸和/或棕榈酸盐的共价附着来改变，以及βgamma二聚体的伽马亚基通过芳香基或黄烷基果仁糖基部分修饰。这些附着的脂质可能充当疏水锚固，以促进与细胞膜结合。然而，尚未很好地描述异源三聚体G蛋白的其他膜靶向信号。此外，在细胞内部执行异构体的何时何地，最初形成了异构体，而G蛋白用于到达质膜的细胞途径是什么，是尚未得到回答的关键问题。一旦在质膜，galpha和beta伽马分离的杂点G蛋白被激活，而Galpha可以经历快速的去氨木酰化。对于一个Galpha alpha，受体激活还可以促进其从质膜和细胞细胞质中的易位。该G蛋白运输的基本机制和细胞途径也很少了解。因此，该提案的主要目标是1）定义GET在GBATA伽马靶向质膜中的作用； 2）定义Galpha和Gbeta Gamma在合成后运输到质膜中使用的细胞途径； 3）定义N末端多比亚簇在Galpha膜靶向中的作用； 4）定义激活引起的GALLASE重新分布的机制。这些目标集中在G蛋白的细胞生物学的明显但高度相关的问题上。这项研究将利用培养的哺乳动物细胞作为模型系统，并将采用多种技术，包括免疫荧光显微镜，活细胞的荧光显微镜，亚细胞分馏和大量的生化测定来定义结构 - 官方触发关系，以可逆的G型G蛋白的机制来定义结构 - 功能关系。