Mechanisms of CFTR Internalization

描述(申请人提供)：布拉德伯里博士的长期目标 实验室是为了了解离子通道的内吞机制，使用 囊性纤维化跨膜电导调节因子(CFTR)的内吞作用 作为一个实验系统。Cftr在氯离子的调节中很重要。 CAMP介导的第二信使级联分泌和cftr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不仅增加了CFTR的开放概率(Po)，而且 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周期的主要机制。 CFTR在质膜和亚细胞细胞器之间的分布 例如内小体和回收小泡。因此，内吞作用可能部分地 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。建议的研究将集中在步骤和分子上 CFTR的内吞作用所必需的相互作用。该项目的重点 将致力于寻找和表征网状蛋白接头分子 使CFTR与包被的质膜笼状蛋白有效地相互作用 PIT是内吞细胞器的关键元件。这些公司的总体战略是 研究分为两个相互关联的方向。我们将确定哪些站点位于 Cftr直接与质膜接头蛋白结合，并确定哪些 接头蛋白的亚基负责这种相互作用。 接头蛋白结合位点的鉴定应为 了解CFTR内吞作用中的特异性机制。我们会 分析CFTR磷酸化在这种相互作用中的作用，以确定是否 CFTR的磷酸化状态调节其与接头蛋白的亲和力 因此，内吞作用的效率。这些研究将伴随着一个动力学 表达缺失cftr突变体细胞的CFtR内吞作用分析 质膜接头结合基序。类似地，细胞的内吞动力学 CFTR磷酸化突变体将在存在和不存在的情况下进行评估 军营募捐人员。对CFTR内化动力学的研究可能会在 反过来，帮助识别调节CFTR内吞作用的其他蛋白质。结果来自 我们的研究应该允许我们设计实验方法来预防 到达质膜但低开放的cftr突变体的内吞作用 激活时的概率。

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.