ROLE OF GI3 PROTEINS IN PROTEIN TRAFFICKING

这项提议的长期目标是提供一个基本的 对细胞内蛋白质转运的细胞生物学的认识 极化的上皮细胞，特别关注细胞外基质的作用 异三聚体G蛋白，Gi3。我们已经证明了阿尔法亚单位 Gi3，G Alpha I3，可以调节，尽管定义尚不明确 生化途径，膜蛋白在细胞水平上的运输 高尔基体在基底外侧分泌途径中。我们建议这一行动 需要一系列复杂结构的蛋白质相互作用 GαI3和辅助蛋白中的基序。这样做的最初目的是 提案是根据G阿尔法I3的结构基序定义 最近解决了其他Gα蛋白的晶体结构，即 对于1)适当地靶向高尔基膜；以及2)结合很重要 到GAIP，最近发现的RGS的GαI3结合蛋白 一家人。我们建议产生GαI3的突变体(特别是在 插入3环)和GAIP并研究其细胞内靶向性 利用免疫荧光、共聚焦和电子显微镜 研究它们使用谷胱甘肽相互结合的能力-S- 转移酶融合蛋白柱。我们已经表明，GAIP更喜欢 Gαi3的GTP绑定形式；我们不会确定 GAIP与Gαi3-GTP的相互作用导致GTP增强 水解液。Gαi3和GAIP的影响将在1中确定) 非极性细胞、上皮细胞和半完整细胞的分泌分析 使用内源性和报告的蛋白质作为分泌监控器的细胞； 以及2)在蛋白质转运分析中，利用荧光恢复 薄荷糖苷酶II/绿色荧光蛋白AS的光漂白 高尔基体内蛋白循环的标志物TGN38-GFP 跨高尔基网络和基底膜循环与生长 激素-绿色荧光蛋白监测分泌途径。此外，这个角色 GαI3和GAIP在内吞作用中的数量将使用 细胞内小泡融合的荧光分析。最后，信号 GαI3在高尔基体水平的转导途径将是 通过监测MAP激酶级联反应来探索 GαI3作用的功能及其纯化和克隆 效应器蛋白。分泌途径是至关重要的。 对细胞的生长、发育和功能，以及对上皮细胞的影响 极性的发展。了解致病的分子机制 它的调节将为调节细胞 健康和疾病方面的反应。

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.