Title : Modulation of endocytic trafficking and apical stability of
CFTR in primary human airway epithelial cultures
Abstract :
- 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
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