Regulation of Epithelial Cell Polarity by Ral-Exocyst Complexes

项目摘要/摘要 胞吐作用的时空调节对于建立和维持细胞间连接和 表面极性，对上皮组织至关重要的特性。未能准确交付并保持正确的 膜蛋白在适当的位置是人类代谢性疾病和癌症的共同特征。 尽管在了解蛋白质分类、贩运和周转的机制方面取得了进展，但主要问题 坚持这些过程。我们研究的长期目标是理解环境线索是如何 向细胞骨架和运输机械发出信号，以组织上皮细胞的运输途径。这个 我们的研究重点集中在多功能支架--外囊和调节外囊的ral GTP酶上。 活动。根据我们先前工作的结果，我们提出了一个工作假说。我们建议 Rala和RalB受细胞间黏附的协调调节，它们控制着不同阶段的 通过控制胞外和胞内途径的胞外依赖膜转运 支架和其他蛋白质之间的结合。我们已经鉴定出两种这样的蛋白质。Munc18c，a 囊泡融合的调节剂，以依赖于Rala的方式结合外囊，这种相互作用是 基底侧方胞吐。USP9X是一种脱泛素酶，它以RalB依赖的方式与胞囊结合，并 在细胞间连接组装过程中稳定质膜中的蛋白质。因此，我们建议 两个相关的GTP酶(Rala和RalB)对共同效应复合体(外囊)的协调调节 在环境线索(细胞-细胞黏附)和上皮细胞的生物发生之间提供了关键的分子联系 细胞特有的结构。这项建议的总体目标是剖析RAR的分子机制 GTP酶和胞外复合体控制细胞间连接和极化膜的形成 上皮细胞中的结构域。这项研究的具体目的是：1)确定E-钙粘附素- 介导的细胞黏附调节Ral GTPase活性；2)确定Rala如何参与外囊 促进基侧胞吐的复合体；以及3)确定RalB如何与胞囊结合来调节 顶端连接复合体各成分的内吞作用。总体而言，我们在这里提出的研究旨在 揭示将细胞间黏附与激活信号通路联系起来的机制的重要细节 在上皮细胞建立过程中促进极化的膜转运和选择性的蛋白质稳定 两极。鉴于我们将关注的分子在后生动物中是保守的，我们相信 研究它们如何协作建立和维护上皮结构将为以下方面提供重要的见解 调节许多其他细胞类型和细胞中质膜蛋白复合体组装的机制 有机体。此外，由于Ral GTP酶和胞囊成分与许多癌症有关， 代谢紊乱和糖尿病，这项工作的意义在于它将指导我们对新的 这些因素被破坏的人类疾病的治疗方法。

Project Summary/Abstract Spatio-temporal regulation of exocytosis is essential for establishing and maintaining intercellular junctions and surface polarity, properties vital to epithelial tissues. Failure to accurately deliver and maintain correct levels of membrane proteins at appropriate sites is a common feature of human metabolic diseases and cancers. Despite progress in understanding mechanisms of protein sorting, trafficking and turnover, major questions persist about these processes. The long-term goal of our research is to understand how environmental cues signal to the cytoskeleton and transport machinery to organize trafficking pathways in epithelial cells. The focus of our research is on the exocyst, a multi-functional scaffold, and Ral GTPases, which regulate exocyst activities. Based upon results of our previous work, we have developed a working hypothesis. We propose that RalA and RalB are coordinately regulated by cell-cell adhesion, and that they control distinct stages of exocyst-dependent membrane trafficking in both the exocytic and endocytic pathways by controlling the association between the scaffold and additional proteins. We have identified two such proteins. Munc18c, a regulator of vesicle fusion, binds the exocyst in a RalA-dependent manner and this interaction is required for basolateral exocytosis. USP9X, a deubiquitinase, engages the exocyst in a RalB-dependent manner and stabilizes proteins in the plasma membrane during intercellular junction assembly. Therefore, we propose that coordinated regulation of a common effector complex (exocyst) by two related GTPases (RalA and RalB) provides a key molecular link between an environmental cue (cell-cell adhesion) and biogenesis of epithelial cell specific structures. The overall objective of this proposal is to dissect molecular mechanisms by which Ral GTPases and exocyst complexes control the formation of intercellular junctions and polarized membrane domains in epithelial cells. The specific aims of this study are: 1) to identify mechanisms by which E-cadherin- mediated cell adhesion regulates Ral GTPase activities; 2) to determine how RalA engages exocyst complexes to facilitate basolateral exocytosis; and 3) to define how RalB engages the exocyst to regulate endocytosis of components of apical junctional complexes. Collectively, the studies we propose here seek to uncover important details of a mechanism that links cell-cell adhesion to activation of a signaling pathway that promotes polarized membrane trafficking and selective protein stabilization during establishment of epithelial polarity. Given that the molecules on which we will focus are conserved across metazoans, we believe that studying how they collaborate to establish and maintain epithelial structures will provide important insights into mechanisms that regulate assembly of plasma membrane protein complexes in many other cell types and organisms. Moreover, because Ral GTPases and exocyst components are involved in many cancers, metabolic disorders and diabetes, the significance of this work is that it will guide our thinking about new therapies for human diseases in which these factors are disrupted.