Modulation of endocytic trafficking and apical stability of CFTR in primary human airway epithelial cultures.
CFTR is a highly regulated apical chloride channel of epithelial cells that is mutated in cystic fibrosis (CF). In this study, we characterized the apical stability and intracellular trafficking of wild-type and mutant CFTR in its native environment, i.e., highly differentiated primary human airway epithelial (HAE) cultures. We labeled the apical pool of CFTR and subsequently visualized the protein in intracellular compartments. CFTR moved from the apical surface to endosomes and then efficiently recycled back to the surface. CFTR endocytosis occurred more slowly in polarized than in nonpolarized HAE cells or in a polarized epithelial cell line. The most common mutation in CF, DeltaF508 CFTR, was rescued from endoplasmic reticulum retention by low-temperature incubation but transited from the apical membrane to endocytic compartments more rapidly and recycled less efficiently than wild-type CFTR. Incubation with small-molecule correctors resulted in DeltaF508 CFTR at the apical membrane but did not restore apical stability. To stabilize the mutant protein at the apical membrane, we found that the dynamin inhibitor Dynasore and the cholesterol-extracting agent cyclodextrin dramatically reduced internalization of DeltaF508, whereas the proteasomal inhibitor MG-132 completely blocked endocytosis of DeltaF508. On examination of intrinsic properties of CFTR that may affect its apical stability, we found that N-linked oligosaccharides were not necessary for transport to the apical membrane but were required for efficient apical recycling and, therefore, influenced the turnover of surface CFTR. Thus apical stability of CFTR in its native environment is affected by properties of the protein and modulation of endocytic trafficking.
Cholon
Deborah M
DM
Cystic Fibrosis Research Center and Department of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7248, USA.
O'Neal
Wanda K
WK
Randell
Scott H
SH
Riordan
John R
JR
Gentzsch
Martina
M
D002454:Cell Differentiation; D002462:Cell Membrane; D016764:Cell Polarity; D002478:Cells, Cultured; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000378:metabolism*; D004705:Endocytosis; D004847:Epithelial Cells / Q000166:cytology* / Q000378:metabolism*; D006031:Glycosylation; D006801:Humans; D008168:Lung / Q000166:cytology*; D011134:Polysaccharides / Q000378:metabolism; D055550:Protein Stability; D021381:Protein Transport; D013347:Subcellular Fractions / Q000378:metabolism; D013499:Surface Properties; D013696:Temperature
D011134:Polysaccharides; C093349:cystic fibrosis transmembrane conductance regulator delta F508; D019005:Cystic Fibrosis Transmembrane Conductance Regulator