Mechanisms of CFTR Internalization

CFTR 内部化机制

• 批准号: 6647747
• 负责人: Neil A Bradbury
• 金额: $ 23.45万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-09-01 至 2005-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6647747
• 关键词: biosensor device; chimeric proteins; chloride channels; clathrin; cyclic AMP; cystic fibrosis; endocytosis; ion transport; optics; phosphorylation; protein binding; protein protein interaction; protein structure function; tissue /cell culture

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.

描述（由申请人提供）： Bradbury 博士的长期目标 实验室的目的是了解离子通道的内吞作用机制，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 对于氯化物的调节很重要 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 CFTR 的主要机制 CFTR 在质膜和亚细胞器之间的分布 例如内体和回收囊泡。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强度。 但CFTR内吞作用具体步骤的机制尚不清楚 完全明白了。拟议的研究将重点关注步骤和分子 CFTR 内吞作用所必需的相互作用。项目重点 将致力于网格蛋白接头分子的搜索和表征 允许 CFTR 与质膜网格蛋白涂层有效相互作用 凹坑，内吞装置的关键元件。这些的总体策略 研究分为两个相关的方向。我们将确定哪些网站 CFTR 直接与质膜接头蛋白结合，并确定哪些 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应为以下方面提供基础： 了解 CFTR 内吞作用的特异性机制。我们将 分析 CFTR 磷酸化在此相互作用中的作用，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。这些研究将伴随着动力学 表达缺少 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，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 cAMP 升高剂。 CFTR 内化动力学的研究可能 反过来，帮助识别调节 CFTR 内吞作用的其他蛋白质。结果来自 我们的研究应该使我们能够设计实验方法来防止 CFTR 突变体的内吞作用到达质膜但开放度较低 激活时的概率。