Mechanisms of CFTR Internalization

描述（由申请人提供）：布拉德伯里博士的长期目标 实验室的目的是了解离子通道的内吞机制，使用 囊性纤维化跨膜传导调节因子的内吞作用 作为一个实验系统。CFTR在氯离子调节中起重要作用 cAMP介导的第二信使级联的分泌，以及CFTR的突变 引起人类遗传性疾病囊性纤维化（CF）。我们的工作模式 预测cAMP不仅增加CFTR的开放概率（Po）， 还调节质膜中CFTR分子的数量（N）。 CFTR的内吞作用是急性调节细胞增殖的主要机制。 CFTR在质膜和亚细胞器之间的分布 例如内体和循环囊泡。因此，内吞作用可能部分 确定对cAMP介导的激动剂的分泌反应的大小。 然而，CFTR内吞的具体步骤的机制尚不清楚， 完全理解拟议的研究将集中在步骤和分子 CFTR的内吞作用所必需的相互作用。项目的重点 将研究网格蛋白接头分子的特性 其允许CFTR与质膜网格蛋白包被有效相互作用 小窝是内吞器官的关键元件。这些国家的总体战略 研究福尔斯两个相关的方向。我们将确定哪些网站上 CFTR直接与质膜衔接蛋白结合，并确定 衔接蛋白的亚基负责这种相互作用。 接头蛋白结合位点的鉴定将为 了解CFTR内吞作用的特异性机制。我们将 分析CFTR磷酸化在这种相互作用中的作用，以确定 CFTR的磷酸化状态调节其对衔接蛋白的亲和力， 从而提高内吞作用效率。这些研究将伴随着动力学 表达缺乏CFTR突变体的细胞中CFTR内吞作用的分析 质膜接头结合基序。类似地， CFTR磷酸化突变体将在存在和不存在以下物质的情况下进行评估： cAMP升高剂。对CFTR内化动力学的研究， 反过来，帮助确定其他蛋白调节CFTR内吞作用。结果 我们的研究应该使我们能够设计实验方法来防止 CFTR突变体的内吞作用，这些突变体到达质膜但具有低的开放水平。 激活后的概率。

DESCRIPTION (provided by applicant): The long-term goal of Dr. Bradbury's laboratory is to understand the mechanism of endocytosis of ion channels, using endocytosis of the cystic fibrosis transmembrane conductance regulator (CFTR) as an experimental system. CFTR is important in the regulation of chloride secretion by the cAMP-mediated second messenger cascade, and mutations in CFTR give rise to the human genetic disease cystic fibrosis (CF). Our working model predicts that cAMP not only increases the open probability (Po) of CFTR, but also regulates the number (N) of CFTR molecules in the plasma membrane. Endocytosis of CFTR is a major mechanism for acutely regulating the distribution of CFTR between the plasma membrane and subcellular organelles such as endosomes and recycling vesicles. Therefore endocytosis may partly determine the magnitude of the secretory response to cAMP mediated agonists. However, the mechanisms of the specific steps of CFTR endocytosis are not yet fully understood. Proposed research will focus on the steps and molecular interactions necessary for the endocytosis of CFTR. The emphasis of the project will be on the search for and characterization of clathrin adaptor molecules that allow effective interaction of CFTR with a plasma membrane clathrin coated pit, a key element of the endocytic apparatus. The general strategy of these studies falls into two related directions. We will determine which sites on CFTR directly bind to plasma membrane adaptor proteins, and determine which subunits of the adaptor proteins are responsible for this interaction. Identification of binding sites for adaptor proteins should provide a basis for understanding the mechanisms of specificity in CFTR endocytosis. We will analyze the role of CFTR phosphorylation in this interaction, to determine if phosphorylation status of CFTR modulates its affinity for adaptor proteins and hence efficiency of endocytosis. These studies will be accompanied by a kinetic analysis of CFTR endocytosis in cells expressing mutants of CFTR which lack plasma membrane adaptor binding motifs. Similarly the endocytic kinetics of CFTR phosphorylation mutants will be evaluated in the presence and absence of cAMP raising agents. Studies of the dynamics of CFTR internalization may, in turn, help identify other proteins regulating CFTR endocytosis. Results from our studies should allow us to design experimental approaches to prevent the endocytosis of CFTR mutants that reach the plasma membrane but have a low open probability upon activation.