Function of RAP1 Signaling in Epithelial Morphogenesis

RAP1 信号传导在上皮形态发生中的功能

• 批准号: 6610516
• 负责人: Linda Van Aelst
• 金额: $ 33.71万
• 依托单位: COLD SPRING HARBOR LABORATORY
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-05-01 至 2008-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6610516
• 关键词: Drosophilidae; biological signal transduction; cell adhesion; cell migration; cell morphology; confocal scanning microscopy; enzyme activity; epithelium; fluorescence microscopy; green fluorescent proteins; guanosinetriphosphatases; histogenesis; laboratory mouse; molecular biology; monoclonal antibody; polymerase chain reaction; protein protein interaction; protein structure function

DESCRIPTION (provided by applicant): The major goal of this research project is to elucidate the function of the small GTPase Rap1 and the molecular mechanism(s) by which this GTPase exerts its effects on epithelial morphogenesis. Recent studies in Drosophila and in mammalian systems have implicated Rap1proteins in the control of cell spreading, adhesion and morphogenesis, but little is known about the molecular mechanisms that mediate Rap1 function. The mammalian junctional protein AF-6 was identified as a Rap1-interacting protein, but functional analyses of mammalian Rap1 and AF-6 proteins, and of their interaction, have been hampered by the fact that these proteins are largely refractory to loss- and/or gain-of-function analysis. Hence, we decided to first gain insights into their functions using a genetically more tractable Drosophila system. Our preliminary results from Drosophila implicate Canoe (the Drosophila homologue of AF-6) as an effector of Drap1 (the Drosophila homologue of Rapl) in dorsal closure (DC), a morphogenetic process that relies on elongation and migration of epithelial sheets in the embryo. Furthermore, we have recently identified a putative novel regulator of Drap1 in DC, designated DPDZ-GEF, whose vertebrate homologue is associated with epithelial adherens junctions, just as Rap1 and AF-6/Canoe are. Notably, the Drosophila homologues of Rap1 and AF-6 (Drap1 and Canoe) share a similar physical interaction, and the mammalian proteins can functionally substitute for their Drosophila counterparts. This proposal aims to define the function of Drap1, Canoe, and DPDZ-GEF in regulating cell shape changes, cell-cell adhesion, and coordinated movement of an epithelial sheet during DC. Our specific aims are: 1) To analyze the function of Drap1 and Canoe in DC and cell-cell adhesion, and 2) To characterize the function of DPDZ-GEF. Specific aim 1 is designed to provide a thorough assessment of the mechanisms that underlie the Drap1/Canoe interaction and the role of Canoe as a junctional protein in the process of DC. In aim 2, the biochemical and biological functions of the DPDZ-GEF and its epistatic relationship to Drap1 will be assessed. These studies offer a comprehensive set of approaches to define the roles of Drap1, Canoe, and DPDZ-GEF in regulating cell-cell adhesion and coordinated epithelial cell movements, and will likely lead to a better mechanistic understanding of these processes and facilitate the design of experiments addressing the role of the mammalian homologues in similar processes. Ultimately, our studies are relevant to a number of medically important issues, including wound healing and the homeostasis of epithelial cells and sheets.

描述（由申请人提供）：该研究项目的主要目标是阐明小GTPase RAP1的功能以及该GTPase对上皮形态发生的影响的分子机制。果蝇和哺乳动物系统中的最新研究已将RAP1蛋白涉及控制细胞扩散，粘附和形态发生，但对介导RAP1功能的分子机制知之甚少。哺乳动物连接蛋白AF-6被鉴定为RAP1相互作用蛋白，但是哺乳动物RAP1和AF-6蛋白的功能分析及其相互作用受到了以下事实的阻碍：这些蛋白在很大程度上是对损失和/或功能障碍分析的折叠。因此，我们决定首先使用遗传上更可行的果蝇系统获得对其功能的见解。果蝇的初步结果暗示了独木舟（AF-6的果蝇同源物）是背闭合（DC）的Drap1（Rapl的果蝇同源物）的效应子，这是一种依赖于胚胎中上皮片的伸长和迁移的形态发生过程。此外，我们最近在DC中确定了一个假定的DRAP1的新型调节剂，该调节剂指定为DPDZ-GEF，其脊椎动物同源物与上皮粘附连接相关，就像RAP1和AF-6/CANOE一样。值得注意的是，Rap1和Af-6（Drap1和独木舟）的果蝇同源物具有相似的物理相互作用，并且哺乳动物蛋白可以在功能上代替其果蝇对应物。 该建议旨在定义DRAP1，独木舟和DPDZ-GEF在调节细胞形状变化，细胞细胞粘附以及DC期间上皮纸的协调运动中的功能。我们的具体目的是：1）分析DC1和Cell-Cell粘附中DARAP1和独木舟的功能，以及2）表征DPDZ-GEF的功能。特定目标1旨在对DRAP1/独木舟相互作用的机制进行彻底评估，以及独木舟作为连接蛋白在DC过程中的作用。在AIM 2中，将评估DPDZ-GEF的生化和生物学功能及其与DARAP1的上皮关系。这些研究提供了一组综合的方法来定义DARAP1，独木舟和DPDZ-GEF在调节细胞细胞粘附和协调的上皮细胞运动中的作用，并可能会更好地理解这些过程，并促进实验在类似过程中的实验设计。最终，我们的研究与许多具有医学上重要的问题有关，包括伤口愈合以及上皮细胞和床单的稳态。