PDZ Domain Interaction Controls the Endocytic Recycling of the Cystic Fibrosis Transmembrane Conductance Regulator

Abstract

The C terminus of CFTR contains a PDZ interacting domain that is required for the polarized expression of cystic fibrosis transmembrane conductance regulator (CFTR) in the apical plasma membrane of polarized epithelial cells. To elucidate the mechanism whereby the PDZ interacting domain mediates the polarized expression of CFTR, Madin-Darby canine kidney cells were stably transfected with wild type (wt-CFTR) or C-terminally truncated human CFTR (CFTR-⌬TRL). We tested the hypothesis that the PDZ interacting domain regulates sorting of CFTR from the Golgi to the apical plasma membrane. Pulse-chase studies in combination with domain-selective cell surface biotinylation revealed that newly synthesized wt-CFTR and CFTR-⌬TRL were targeted equally to the apical and basolateral membranes in a nonpolarized fashion. Thus, the PDZ interacting domain is not an apical sorting motif. Deletion of the PDZ interacting domain reduced the half-life of CFTR in the apical membrane from ϳ24 to ϳ13 h but had no effect on the half-life of CFTR in the basolateral membrane. Thus, the PDZ interacting domain is an apical membrane retention motif. Next, we examined the hypothesis that the PDZ interacting domain affects the apical membrane half-life of CFTR by altering its endocytosis and/or endocytic recycling. Endocytosis of wt-CFTR and CFTR-⌬TRL did not differ. However, endocytic recycling of CFTR-⌬TRL was decreased when compared with wt-CFTR. Thus, deletion of the PDZ interacting domain reduced the half-life of CFTR in the apical membrane by decreasing CFTR endocytic recycling. Our results identify a new role for PDZ proteins in regulating the endocytic recycling of CFTR in polarized epithelial cells.

Myosin VI Regulates Endocytosis of the Cystic Fibrosis Transmembrane Conductance Regulator

“…Immunoprecipitation and Immunoblotting-CFTR and myosin VI were immunoprecipitated from Calu-3 and HEK293 cell lysates by methods described previously (54,55). Briefly, cells were solubilized in lysis buffer containing 150 mM NaCl, 50 mM Tris, pH 8.0, 1% IGEPAL (Sigma), and Complete Protease Inhibitor mixture (Roche Molecular Biochemicals, Indianapolis, IN).…”

“…Cell Surface Biotinylation, Endocytic Assay, and Endocytic Recycling Assay-Cell surface biotinylation, endocytic assays, and endocytic recycling assays were performed on HEK293 cells as described previously (55).…”

The Role of the C Terminus and Na+/H+ Exchanger Regulatory Factor in the Functional Expression of Cystic Fibrosis Transmembrane Conductance Regulator in Nonpolarized Cells and Epithelia

“…The reported metabolic destabilization of the ⌬3 and ⌬26 CFTR in MDCK II cells (34,35), contrasting with the WT-like expression level of ⌬6, ⌬26, ⌬40, and ⌬50 in BHK and COS-1 cells, has several plausible explanations. First, NHERF binding may have a dual role in nonpolarized cells.…”

“…4B). Considering that the complex-glycosylated form stability reflects the overall characteristic of post-Golgi, cell surface, and endosomal CFTR pools, and NHERF may facilitate recycling of CFTR as observed for G-protein-coupled receptors (24,26,35,50), next we evaluated the effect of C-terminal truncation on the apical localization and channel function of CFTR.…”

“…NHERF and the C terminus tail were proposed to have a central role in the apical expression of CFTR (32)(33)(34)(35). This hypothesis was supported by the observation that deletion of the last 26 amino acids of CFTR provoked the accumulation of the channel at the lateral membrane in MDCK II and bronchial epithelia (32,34) and caused almost a complete (Ͼ97%) loss of the PKA-activated CFTR function (34).…”