Myosin VI Regulates Endocytosis of the Cystic Fibrosis Transmembrane Conductance Regulator

Agnieszka Swiatecka-Urban,Cary Boyd,Bonita Coutermarsh,Katherine H Karlson,Roxanna Barnaby,Laura Aschenbrenner,George M Langford,Tama Hasson,Bruce A Stanton

doi:10.1074/jbc.m403141200

Abstract

The cystic fibrosis transmembrane conductance regulator (CFTR) is a cyclic AMP-regulated Cl(-) channel expressed in the apical plasma membrane in fluid-transporting epithelia. Although CFTR is rapidly endocytosed from the apical membrane of polarized epithelial cells and efficiently recycled back to the plasma membrane, little is known about the molecular mechanisms regulating CFTR endocytosis and endocytic recycling. Myosin VI, an actin-dependent, minus-end directed mechanoenzyme, has been implicated in clathrin-mediated endocytosis in epithelial cells. The goal of this study was to determine whether myosin VI regulates CFTR endocytosis. Endogenous, apical membrane CFTR in polarized human airway epithelial cells (Calu-3) formed a complex with myosin VI, the myosin VI adaptor protein Disabled 2 (Dab2), and clathrin. The tail domain of myosin VI, a dominant-negative recombinant fragment, displaced endogenous myosin VI from interacting with Dab2 and CFTR and increased the expression of CFTR in the plasma membrane by reducing CFTR endocytosis. However, the myosin VI tail fragment had no effect on the recycling of endocytosed CFTR or on fluid-phase endocytosis. CFTR endocytosis was decreased by cytochalasin D, an actin-filament depolymerizing agent. Taken together, these data indicate that myosin VI and Dab2 facilitate CFTR endocytosis by a mechanism that requires actin filaments.

Highlights

The cystic fibrosis transmembrane conductance regulator (CFTR) is a cyclic AMP-regulated Cl؊ channel expressed in the apical plasma membrane in fluid-transporting epithelia
CFTR is rapidly endocytosed from the apical membrane of polarized epithelial cells and efficiently recycled back to the plasma membrane, little is known about the molecular mechanisms regulating CFTR endocytosis and endocytic recycling
To provide direct evidence that CFTR forms a complex at the apical plasma membrane with myosin VI, Disabled 2 (Dab2), and clathrin, the apical membrane was selectively biotinylated, the biotinylated proteins were precipitated with streptavidin-agarose beads, biotinylated proteins were eluted from the beads, and the apical plasma membrane CFTR was immunoprecipitated with a monoclonal anti-CFTR antibody

Summary

Introduction

The cystic fibrosis transmembrane conductance regulator (CFTR) is a cyclic AMP-regulated Cl؊ channel expressed in the apical plasma membrane in fluid-transporting epithelia. CFTR is rapidly endocytosed from the apical membrane of polarized epithelial cells and efficiently recycled back to the plasma membrane, little is known about the molecular mechanisms regulating CFTR endocytosis and endocytic recycling. ⌬F508-CFTR has a greatly reduced half-life in the plasma membrane compared with wild type (wt)-CFTR (9 –11), most likely because of defects in endocytosis and/or endocytic recycling. Little is known about the mechanisms that regulate the half-life or number of CFTR channels in the plasma membrane and, the rate of transepithelial ClϪ secretion. Interaction between the ␮2 subunit of AP-2 and the tyrosine-based endo-

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