Direct interaction with filamins modulates the stability and plasma membrane expression of CFTR
William R. Thelin, Yun Chen, Martina Gentzsch, Silvia M. Kreda, Jennifer L. Sallee, Cameron O. Scarlett, Christoph H. Borchers, Ken Jacobson, M. Jackson Stutts, Sharon L. Milgram
William R. Thelin, Yun Chen, Martina Gentzsch, Silvia M. Kreda, Jennifer L. Sallee, Cameron O. Scarlett, Christoph H. Borchers, Ken Jacobson, M. Jackson Stutts, Sharon L. Milgram
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Research Article Pulmonology

Direct interaction with filamins modulates the stability and plasma membrane expression of CFTR

Abstract

The role of the cystic fibrosis transmembrane conductance regulator (CFTR) as a cAMP-dependent chloride channel on the apical membrane of epithelia is well established. However, the processes by which CFTR is regulated on the cell surface are not clear. Here we report the identification of a protein-protein interaction between CFTR and the cytoskeletal filamin proteins. Using proteomic approaches, we identified filamins as proteins that associate with the extreme CFTR N terminus. Furthermore, we identified a disease-causing missense mutation in CFTR, serine 13 to phenylalanine (S13F), which disrupted this interaction. In cells, filamins tethered plasma membrane CFTR to the underlying actin network. This interaction stabilized CFTR at the cell surface and regulated the plasma membrane dynamics and confinement of the channel. In the absence of filamin binding, CFTR was internalized from the cell surface, where it prematurely accumulated in lysosomes and was ultimately degraded. Our data demonstrate what we believe to be a previously unrecognized role for the CFTR N terminus in the regulation of the plasma membrane stability and metabolic stability of CFTR. In addition, we elucidate the molecular defect associated with the S13F mutation.

Authors

William R. Thelin, Yun Chen, Martina Gentzsch, Silvia M. Kreda, Jennifer L. Sallee, Cameron O. Scarlett, Christoph H. Borchers, Ken Jacobson, M. Jackson Stutts, Sharon L. Milgram

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Figure 6

Perturbations in FLN-A binding decrease the surface expression of WT CFTR.

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Perturbations in FLN-A binding decrease the surface expression of WT CFT...
(A) The indicated CFTR peptides were introduced into BHK cells stably expressing WT HA-CFTR using the Pro-Ject delivery system. Cell surface CFTR was assessed by labeling unpermeabilized cells with HA antibodies followed by Alexa Fluor 594–labeled secondary antibodies. Transfected cells were identified by the uptake of fluorescently labeled F(ab′) fragments included in the transfection complexes. (B) Peptide-transfected cells were assessed for CFTR surface expression as described in Methods. *P < 0.05. n = 4 with 50 transfected cells counted per individual experiment. (C and D) HA-CFTR or GFP was expressed using adenovirus in FLN-A–replete cells (M2) or FLN-A–reexpressing cells (A7). The surface expression of CFTR was analyzed by immunofluorescence (C) and surface ELISA (D) as described in Figure 5. Scale bars: 10 μm.