MOLECULAR MECHANISMS OF ENDOCYTIC CFTR RETRIEVAL

编码囊性纤维化跨膜传导调节因子的基因 囊性纤维化（CF）患者的CFTR有缺陷。突变 CFTR蛋白的一级序列导致了 CF细胞中的表型，即顶膜Cl cAMP介导的第二信使级联反应。表情， 互补、突变和重组研究， 证明CFTR是一种激酶激活的Cl通道。我们有 证明除了有缺陷的cAMP依赖性调节外， 质膜Cl渗透性，CF细胞具有受损的cAMP依赖性 内吞/外吞过程的调节。两种cAMP依赖性 当CF细胞转染一种 正常CF基因（wtCFTR）。为了整合这些观察结果，我们 已经提出顶端膜Cl渗透性可以被调节 间接通过插入和检索CFTR Cl通道， 胞内膜囊泡池。本项目的总体目标是 为了了解维持一个细胞的分子机制， 细胞膜中DFTR分子的适当数量， 分泌和休息时。具体而言，我们将调查监管机构 CFTR通过内吞从血浆中回收的机制 通过网格蛋白依赖性和网格蛋白非依赖性机制来调节细胞膜。 为了研究这些机制，我们将重点关注两个具体目标。我们 将检验网格蛋白包被囊泡的水平 相关的CFTR与内吞事件同时调节，因此 与细胞的Cl分泌状态相平行。我将决定。 CFTR水平在CCV之前，期间，和后毛喉素刺激。在 此外，我们将确定CCV相关的磷酸化状态， CFTR在细胞的每个分泌期，即之前，期间和之后， 刺激后。我们还将确定CFTR是否可以从 质膜通过网格蛋白独立机制，即，通过洞穴。 其次，我们将研究CFTR是内吞的假设， 通过在网格蛋白包被的凹坑中聚类而被回收到CCV中。因此，我们将 测定CFTR与质膜适配器相互作用的能力 复合物，并确定结合效率是否取决于 CFTR的磷酸化状态，即，是否在涂层凹坑中聚集， 这取决于细胞的分泌状态。的结果予以 研究将提供重要的见解，细胞的过程， CFTR周转和正常细胞运输的基础机制。 这些结果将为今后的周转研究提供依据 和转染到CF的受影响上皮中的wtCFTR的运输 患者，这将是必要的，以提供一个完整的理由，遗传 治疗干预了解分子机制 这是维持适当数量的CFTR分子的基础， 质膜还可以提供另外的治疗手段 对CF患者进行干预。

The gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) is defective in patients with cystic fibrosis (CF). Mutations in the primary sequence of the CFTR protein lead to a characteristic phenotype in CF cells, namely defective regulation of apical membrane Cl channels by the cAMP mediated 2nd messenger cascade. Expression, complementation, mutation, and reconstitution studies have collectively demonstrated that CFTR is a kinase activated Cl channel. We have demonstrated that in addition to defective cAMP-dependent regulation of plasma membrane Cl permeability, CF cells have an impaired cAMP-dependent regulation of endocytic/exocytic processes. Both of the cAMP-dependent regulatory defects are restored when CF cells are transfected with a normal CF gene (wtCFTR). In an effort to integrate these observations, we have proposed that apical membrane Cl permeability can be regulated indirectly by the insertion and retrieval of CFTR Cl channels from an intracellular membrane vesicle pool. The general goal of this project is to understand the molecular mechanisms underlying the maintenance of an appropriate number of DFTR molecules in the plasma membrane during secretion and at rest. Specifically, we will investigate the regulatory mechanisms by which CFTR is endocytically retrieved from the plasma membrane by both clathrin-dependent and clathrin-independent mechanisms. To investigate these mechanisms, we will focus on two specific aims. We will test the hypothesis that the levels of clathrin coated vesicle associated CFTR are regulated concomitantly with endocytic events and thus parallel the Cl secretory status of the cell. Thus we will determine the levels of CFTR in CCV before, during, and after forskolin stimulation. In addition, we will determine the phosphorylation status of CCV associated CFTR at each of the secretory phases of the cell, i.e, before, during, and after stimulation. We will also determine if CFTR can be removed from the plasma membrane by clathrin independent mechanisms, i.e., via caveolae. Secondly, we will investigate the hypothesis that CFTR is endocytically retrieved into CCV by clustering in clathrin coated pits. Thus, we will determine the ability of CFTR to interact with plasma membrane adaptor complexes, and determine whether binding efficiency is dependent upon the phosphorylation status of CFTR, i.e., whether clustering in coated pits is dependent upon the secretory status of the cell. The results of these studies will provide important insights into the cellular processes and mechanisms that underlie CFTR turnover and trafficking in normal cells. These results will provide the basis for future studies on the turnover and trafficking of wtCFTR transfected into affected epithelial of CF patients, which will be necessary to provide a full rationale for genetic therapeutic intervention. Understanding the molecular mechanisms underlying the maintenance of an appropriate number of CFTR molecules in the plasma membrane may also provide an additional means of therapeutic intervention in CF patients.