ROLE OF GI3 PROTEINS IN PROTEIN TRAFFICKING

这项建议的长期目标是提供一个基本的 了解细胞内蛋白质运输的细胞生物学， 极化上皮细胞，特别关注的作用， 异源三聚体G蛋白，Gi 3。 我们已经证明了α亚基 Gi 3，G α i3，可以调节，通过尚未明确定义 生化途径，膜蛋白运输水平的 基底外侧分泌通路中的高尔基体。 我们建议这一行动 需要一系列蛋白质相互作用， G α i3和辅助蛋白中的基序。 最初的目标是 建议是基于以下定义G α i3的结构基序： 最近解决了其他G α蛋白的晶体结构， 重要的是1）正确靶向高尔基体膜;和2）结合 GAIP是最近鉴定的RGS的G α 13结合蛋白， 家人 我们建议产生G α 13的突变体（特别是在 插入物3环）和GAIP，并研究它们的细胞内靶向 利用免疫荧光、共聚焦和电子显微镜， 来研究它们利用谷胱甘肽-S相互结合的能力 转移酶融合蛋白柱。 我们已经表明，GAIP更喜欢 G α i3的GTP结合形式;我们将不确定 GAIP与G α 13-GTP的相互作用导致增强的GTP 水解 G α i3和GAIP的影响将在1）中确定 非极性和上皮细胞以及半完整细胞中的分泌测定 使用内源性和报告蛋白作为分泌监测物的细胞; 和2）在蛋白质运输测定中， 用于门糖苷酶II/绿色荧光蛋白（GFP）的光漂白， 高尔基体内蛋白质循环的标记，TGN 38-GFP作为高尔基体内蛋白质循环的标记， trans-Golgi网络和基底外侧膜循环，以及生长 用荧光素酶-GFP监测分泌途径。 此外，作用 G α 13和GAIP在胞吞作用中的含量将使用 胞内囊泡融合的荧光测定。最后，信号 在高尔基体水平的G α i3的转导途径将是 通过监测MAP激酶级联反应， G α i3作用的功能，并通过纯化和克隆G α i3 效应蛋白 分泌途径是至关重要的 细胞生长，发育和功能，以及上皮细胞 极性的发展。 了解分子机制， 它的调节将为调节细胞内 健康和疾病的反应。

The long-term objective of this proposal is to provide a fundamental understanding of the cell biology of intracellular protein trafficking in polarized epithelial cells, focusing particularly on the role of the heterotrimeric G protein, Gi3. We have shown that the alpha subunit of Gi3, G alpha i3, can regulate, through as yet poorly defined biochemical pathways, membrane protein trafficking at the level of the Golgi in the basolateral secretory pathway. We propose that this action requires a series of protein interactions mediated by complex structural motifs in G alpha i3 and auxiliary proteins. The initial aims of this proposal are to define the structural motifs of G alpha i3 based on the recently solved crystal structure of other G alpha proteins, that are important for 1) proper targeting to Golgi membranes; and 2) binding to GAIP, a recently identified G alpha i3 binding protein of the RGS family. We propose to generate mutants of G alpha i3 (especially in the Insert 3 Loop) and GAIP and to study their intracellular targeting utilizing immunofluorescence, confocal, and electron microscopy and to study their ability to bind to each other using glutathione-S- transferase fusion protein column. We have shown that GAIP prefers the GTP-bound form of G alpha i3; we will not determine whether the interaction of GAIP with G alpha i3-GTP results in enhanced GTP hydrolysis. The effects of G alpha i3 and GAIP will determine in 1) secretion assays in non-polar and epithelial cells, and in semi-intact cells using endogenous and reported proteins as monitors of secretion; and in 2) protein trafficking assays utilizing fluorescence recovery after photobleaching for mennosidase II/green fluorescent protein (GFP) as a marking for intra-Golgi protein cycling, TGN38-GFP as a marker for trans-Golgi network and basolateral membrane cycling, and growth hormone-GFP to monitor the secretory pathway. In addition, the role of G alpha i3 and GAIP in endocytosis will be quantitated utilizing a fluorescence assay of intracellular vesicle fusion. Finally, the signal transduction pathway for G alpha i3 at the level of the Golgi will be explored by monitoring the response of the MAP kinase cascade as a function of G alpha i3 action and by purifying and cloning G alpha i3 effector proteins. The secretory pathway is of fundamental importance to cell growth, development, and function, and in epithelia to the development of polarity. Understanding the molecular mechanisms for its regulation will provide new opportunities for modulating cellular responses in health and disease.