Regulation of Clathrin Adaptor Function

描述(由申请人提供)：膜边界定义了不同的 真核细胞与其同源异构体之间的结构和功能 环境，以及细胞本身。物质和信息的流动 在这些隔室之间是由囊泡运输和蛋白质调节的 涂层，其中笼状蛋白涂层膜是一种范例，总是 与这些进程相关联。最近，磷脂酰肌醇也被 认识到在调节膜贩运方面发挥重要作用。 重要的问题是肌苷在局部存在的机制。 在行动地点积累或产生的，以及它们如何与 效应器蛋白。在上一次的颁奖期间，我们发现胞质蛋白结合了 具体涉及一种新的II类PI 3-激酶，PI3K-C2a，该结合 增强蛋白质的酶活性。这就引出了假设 PI3K-C2pha的网状蛋白募集和激活诱导局部 肌苷的生成。我们还发现3-磷脂酰肌醇结合 具体到AP-2Cx亚基上的一个离散位点，并且该完整性 对于AP-2正确地招募到涂覆的坑和 受体介导的内吞作用。一个主要目标将是确定 PI3K-C2a参与的膜转运途径及其如何 与笼状蛋白相互作用来完成这些功能。这将通过以下方式实现 检测PI3K-C2a在细胞内的超微结构定位 它优先与CP的特定阶段相关的可能性 形成，并解剖在完整细胞膜运输途径中的作用 PI3K-C2a的脂蛋白激酶和网状蛋白结合活性的研究。的研究 将携带介导分子筛蛋白-PI3K-C2pha相互作用的决定因素 为这些研究提供显性阴性试剂，并提供 用于结晶和结构分析的材料。最后，肌醇二肽 将检查AP-2α结合域的结合属性，并 启动了结构研究。在谈到衣物的管理方式时 在完整细胞中的功能和囊泡运输，这个项目的重点是一个 细胞生物学中与正常生物过程相关的中心问题 所有的真核细胞和许多疾病中的紊乱。

DESCRIPTION (provided by applicant): Membrane boundaries define distinct structural and functional compartments between the eukaryotic cell and its environment, and within the cell itself. Movement of material and information between these compartments is mediated by vesicular transport and protein coats, of which the clathrin coated membrane is a paradigm, are invariably associated with these processes. Recently, phosphoinositides have also been recognized to play important roles in the regulation of membrane trafficking. Important questions have been the mechanism by which inositides are locally accumulated or generated at sites of action, and how they interact with effector proteins. During the last award period, we found that clathrin binds specifically to a novel Class II PI 3-kinase, PI3K-C2a, and that this binding enhances the enzymatic activity of the protein. This leads to the hypothesis that clathrin recruitment and activation of PI3K-C2alpha induces local generation of inositides. We also showed that 3-phosphoinositides bind specifically to a discrete site on the AP-2cx subunit, and that the integrity of this site is required both for proper recruitment of AP-2 to coated pits and for receptor-mediated endocytosis. A major goal will be to determine the membrane trafficking pathways in which PI3K-C2a participates and how it interacts with clathrin to accomplish these functions. This will be attained by determining the ultrastructural localization of PI3K-C2a in cells, examining the possibility that it is preferentially associated with specific stages of CP formation, and dissecting the role in intact cell membrane trafficking pathways of the lipid kinase and clathrin binding activities of PI3K-C2a. Study of determinants mediating the clathrin-PI3K-C2alpha interaction will be carried out to provide dominant-negative reagents for these studies, and to provide material for crystallization and structural analysis. Finally, the inositide binding properties of the AP-2alpha binding domain will be examined and structural studies initiated. In addressing the mode of regulation of coat function and vesicular transport in intact cells, this project focus upon a central issue in cell biology with relevance for normal biological processes in all eukaryotic cells and derangement in many diseases.