Regulation of Clathrin Adaptor Function

描述（由申请人提供）：膜边界定义了不同的 真核细胞和其细胞之间的结构和功能区室 环境和细胞本身。材料和信息的流动 这些区室之间的相互作用是由囊泡运输和蛋白质介导的 涂层，其中网格蛋白涂层膜是一个范例，总是 与这些过程有关。最近，磷酸肌醇也被 被认为在膜运输的调节中发挥重要作用。 重要的问题是肌醇类药物局部作用的机制。 积累或产生的作用，以及它们如何相互作用， 效应蛋白在上一个奖项期间，我们发现网格蛋白结合 特异性地针对新的II类PI 3-激酶PI 3 K-C2 a，并且这种结合 增强蛋白质的酶活性。这就引出了一个假设 网格蛋白的募集和PI 3 K-C2 α的激活诱导局部 肌醇的生成。我们还表明3-磷酸肌醇结合 特异性地结合到AP-2cx亚基上的离散位点， 这一点是必要的，既为适当的招聘AP-2涂层坑， 用于受体介导的内吞作用。一个主要目标将是确定 PI 3 K-C2 a参与的膜运输途径以及它如何 与网格蛋白相互作用以实现这些功能。这将通过以下方式实现： 确定细胞中PI 3 K-C2 a的超微结构定位， 它可能优先与CP的特定阶段相关 形成，并剖析在完整的细胞膜运输途径的作用 的脂质激酶和网格蛋白结合活性的PI 3 K-C2 a。研究 将携带介导网格蛋白-PI 3 K-C2 α相互作用的决定簇 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 将检查AP-2 α结合结构域的结合特性， 结构研究启动。在处理大衣的监管模式， 功能和囊泡运输在完整的细胞，这个项目的重点是 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.