Cystic fibrosis transmembrane conductance regulator (CFTR) functions as an anion channel, conducting chloride and bicarbonate, and as a regulator of several membrane ion channels and transporters. Several proteins have been reported to bind to the carboxyl-terminus of CFTR and affect its cell surface expression and channel gating. However, these regulatory proteins identified to date do not account for all the known functions of the CFTR C-terminus, suggesting that yet unidentified proteins bind to the CFTR C-terminus for regulating its function. To find such proteins, our lab performed a yeast two-hybrid screen and identified vimentin, a type III intermediate filament protein, as a CFTR C-terminus-binding protein. The interaction was further verified by GST pull down assays in...[ Read more ]

Cystic fibrosis transmembrane conductance regulator (CFTR) functions as an anion channel, conducting chloride and bicarbonate, and as a regulator of several membrane ion channels and transporters. Several proteins have been reported to bind to the carboxyl-terminus of CFTR and affect its cell surface expression and channel gating. However, these regulatory proteins identified to date do not account for all the known functions of the CFTR C-terminus, suggesting that yet unidentified proteins bind to the CFTR C-terminus for regulating its function. To find such proteins, our lab performed a yeast two-hybrid screen and identified vimentin, a type III intermediate filament protein, as a CFTR C-terminus-binding protein. The interaction was further verified by GST pull down assays in fibroblast cell line HEK293T cells and co-immunoprecipitation assays in airway epithelial Calu-3 cells. In addition, immunostaining results showed a partial co-localization of endogenous CFTR and vimentin in Calu-3 cells. We also found that vimentin expression suppressed the surface expression of CFTR. Furthermore, knocking down vimentin led to a significant increase in surface CFTR expression in both Calu-3 and HEK293T cells. More importantly, knocking down vimentin in Calu-3 cells led to a significant increase in forskolin-induced short circuit current, a readout of cell surface CFTR function, further supporting that vimentin knocking down up-regulated CFTR surface expression in Calu-3 cells. In addition, we found that the mutation of the hydrophobic patch (F₁₄₁₃LVI) of CFTR C-terminus to alanines (CFTR-M1) abolished the interaction of CFTR with vimentin and the expression of this mutant CFTR was not affected by the co-expression of vimentin as the wild type CFTR was, indicating that vimentin exerts its effect on CFTR expression through physical interaction via F₁₄₁₃LVI motif. The deletion of vimentin C-terminus abolished its interaction with CFTR C-terminus, suggesting that vimentin physically interacts with CFTR via its C-terminus. Although vimentin knockdown significantly increased CFTR’s half-life, it had little effect on the rates of CFTR biosynthesis and maturation.

These findings shed new light on the regulation of CFTR and broadened our understanding on the function of cytoskeletal protein vimentin.