REGULATION OF CLATHRIN ADAPTOR FUNCTION

描述(改编自申请人的摘要)：膜边界定义 真核细胞之间不同的结构和功能隔间 和它的环境，以及细胞本身。材料的移动和 这些隔室之间的信息是由囊泡运输介导的， 而蛋白质外衣总是与这些过程有关。多重 涂层的形式是已知的，其中笼状蛋白涂层的膜是 范例。除了氯氰菊酯外，这些涂层还含有异四聚体 称为组装、接头或相关蛋白或AP的复合体。这个 质膜适配器AP-2的α亚基具有调节高 亲和多磷肌醇(PPI)结合部位。在组装好的外套里， 该位点与PI-3，4，5-P3的产物PI-3，4，5-P3有最大的亲和力 它被激活的受体招募到笼状蛋白涂层的凹坑中， 与受体交易的调控有牵连。结合部位 已经被映射到一个75个残基区域；它显示了功能性的PPI结合 与完整的蛋白质相似。来检验这样一种假设 AP-2与PPI，特别是PI-3，4，5-P3一起调节AP-2的性质 以及它所参与的过程，主要的抑制形式 突变的AP-2α将被设计、产生和表征，以及 随后在完整的细胞中表达。在这种情况下，以及 在对PI-3激酶活性进行药理学和遗传操作后， 将对涂层膜动力学的变化进行评估。最后， AP-2αPIP3结合结构域的分离结构 亚基将用圆二色谱和核磁进行研究 共振光谱学、X射线结晶学和分子模拟。在……里面 探讨衣膜功能和囊泡运输的调节方式 在完整的细胞中，这个项目聚焦于细胞生物学中的一个中心问题 与所有真核细胞的正常生物学过程相关， 疾病中的错乱。

DESCRIPTION (Adapted from applicant's abstract): 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 are invariably associated with these processes. Multiple forms of coats are known, of which the clathrin-coated membrane is the paradigm. In addition to clathrin, these coats contain heterotetrameric complexes termed assembly, adaptor or associated proteins, or APs. The alpha subunit of the plasma membrane adaptor AP-2 has a regulatory high affinity polyphosphoinositide (PPI)-binding site. In the assembled coat, this site has greatest affinity for PI-3,4,5-P3, the product of PI-3 kinase that is recruited to clathrin coated pits by activated receptors and which has been implicated in regulation of receptor trafficking. The binding site has been mapped to a 75-residue region; it displays functional PPI binding similar to the intact protein. To test the hypothesis that interaction of AP-2 with PPIs, particularly PI-3,4,5-P3, regulates the properties of AP-2 and the processes in which it is involved, dominant inhibitory forms of mutant AP-2 alpha will be designed, generated and characterized, and subsequently expressed in intact cells. Under these conditions, as well as following pharmacologic and genetic manipulation of PI-3 kinase activity, alterations in coated membrane dynamics will be evaluated. Finally, the structure of the isolated prototypical PIP3 binding domain of the AP-2 alpha subunit will be studied using circular dichroism and nuclear magnetic resonance spectroscopy, x-ray crystallography and molecular modeling. In addressing the mode of regulation of coat function and vesicular transport in intact cells, this project focuses upon a central issue in cell biology with relevance for normal biological processes in all eukaryotic cells and derangements in disease.