MOLECULAR MECHANISMS OF ENDOCYTIC CFTR RETRIEVAL

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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.