Regulation of Epithelial Cell Polarity by Ral-Exocyst Complexes

项目总结/摘要 胞吐作用的时空调节对于建立和维持细胞间连接和细胞间粘附是必不可少的。 表面极性，对上皮组织至关重要的特性。未能准确地提供和维持正确水平的 在适当位点的膜蛋白是人类代谢疾病和癌症的共同特征。 尽管对蛋白质分选、运输和周转机制的理解取得了进展，但主要问题是， 坚持这些过程。我们研究的长期目标是了解环境线索 向细胞骨架和运输机构发出信号，以组织上皮细胞中的运输途径。的 我们的研究重点是外囊，一种多功能支架，和调节外囊的Ral GTP酶 活动根据我们以前的工作结果，我们已经开发了一个工作假设。我们提出 RalA和RalB通过细胞-细胞粘附协调调节，并且它们控制不同的阶段， 外泌囊依赖的膜运输在胞吐和胞吞途径，通过控制 支架和其他蛋白质之间的关联。我们发现了两种这样的蛋白质。Munc18c，a 囊泡融合的调节因子，以RalA依赖的方式结合外囊，这种相互作用是 基底外侧胞吐。USP 9 X是一种去泛素化酶，以RalB依赖性方式与外囊结合， 在细胞间连接装配期间稳定质膜中的蛋白质。因此，我们建议， 通过两种相关的GTP酶（RalA和RalB）协调调节共同的效应复合物（外囊） 提供了环境线索（细胞-细胞粘附）和上皮细胞生物发生之间的关键分子联系。 细胞特定结构。这项建议的总体目标是剖析Ral GTP酶和胞外膜复合物控制细胞间连接和极化膜的形成 结构域。本研究的具体目的是：1）确定E-cadherin-1的作用机制， 介导的细胞粘附调节Ral GT3活性; 2）确定RalA如何与外囊结合， 复合物，以促进基底外侧胞吐作用;和3）定义RalB如何参与胞吐调节 顶端连接复合体的内吞作用。总的来说，我们在这里提出的研究旨在 揭示了将细胞间粘附与信号通路激活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膜运输和选择性蛋白稳定 极性鉴于我们将关注的分子在后生动物中是保守的，我们相信， 研究它们如何合作建立和维持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中质膜蛋白复合物组装的机制， 有机体此外，由于Ral GTP酶和外囊组分参与许多癌症， 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 治疗人类疾病，其中这些因素被破坏。

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.