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.

Figure 1. Analysis of N-terminal CFTR mutations.

(A) Comparison of amino acids 1–25 of human CFTR with those of other species. Residues conserved in all species are shaded black. Disease-causing missense mutations in CFTR identified from the CF mutation database are indicated above. (B and C) The indicated CFTR proteins were expressed in HEK293 or 16HBE140- cells and analyzed by Western blot. Untransfected cells (UNT) served as negative controls. (D) Data from HEK293 cells in C was quantitated using densitometry. For all CFTR proteins, the level of mature band C CFTR was normalized to that of band B CFTR. *P < 0.01, **P < 0.001 versus WT CFTR. n = 3.

Figure 2. The S13F mutation decreases the half-life of CFTR.

(A) The indicated CFTR proteins expressed in HEK293 cells were metabolically labeled and incubated in nonradioactive media for the indicated times. CFTR was immunoprecipitated and analyzed by SDS-PAGE/phosphorimager analysis. (B and C) Data were quantitated using ImageQuant software to examine CFTR maturation (B) and degradation (C). For each CFTR protein, data are expressed as percent of control (time 0 for B; band C at 4 hours for C). *P < 0.05 versus WT CFTR. n = 4.

Figure 3. FLNs interact with the CFTR N terminus.

(A) Coomassie-stained gel of proteins that copurified with CFTR_1–25 or CFTR_1–25/S13F from Calu-3 cell lysates. Bands identified as FLN-A and FLN-B are indicated by an asterisk. (B and C) FLN-A Western blots of CFTR peptide pulldowns from (B) Calu-3 lysates (100 μg) or (C) purified FLN-A (500 nM). Inputs are 5% of the total. S13A, CFTR_1–25/S13A. (D) Endogenous CFTR was immunoprecipitated from solubilized Calu-3 membranes and analyzed by Western blot for CFTR or FLN-A. (E) The indicated CFTR proteins were expressed in HEK293 cells at 37°C or 28°C to temperature-rescue ΔF508 (rΔF508). CFTR was immunoprecipitated, and samples were analyzed by Western blot for CFTR or FLN-A. Protein molecular weights in kDa are shown to the left of the blots.

Figure 4. FLN-A localizes to the subapical membrane of airway epithelia.

FLN-A localization was analyzed by immunofluorescence in (A) Calu-3 cells or (B) primary cultures of WD-HBEs as described in Methods. Arrowheads indicate the apical membrane. CFTR staining and purified, whole mouse IgG as a negative control are also shown in Calu-3 cells. Scale bars: 10 μm.

Figure 5. Surface expression of S13F CFTR is decreased.

The indicated HA-CFTR proteins were expressed in BHK cells. CFTR proteins were analyzed by (A) immunofluorescence or (B) surface ELISA in unpermeabilized cells (surface pool) or detergent permeabilized cells (total CFTR) using HA antibodies as described in Methods. Scale bars: 10 μm. *P < 0.05, **P < 0.005 versus WT CFTR. n = 36.

Figure 6. Perturbations in FLN-A binding decrease the surface expression of WT CFTR.

(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.

Figure 7. The membrane dynamics of S13F CFTR is altered.

WT and S13F CFTRs were analyzed by SPT in HeLa cells. Blue lines show trajectory of individual CFTR proteins labeled with gold particles during a 60-second recording. Red areas show regions of transient confinement. The diffusion coefficient (D) and number of TCZs in each CFTR are also indicated. Scale bars: 1 μM. n = 45.

Figure 8. S13F CFTR is internalized more rapidly than is WT CFTR.

WT or S13F CFTRs were transiently expressed in BHK cells. The cell surface pool was labeled with HA antibodies as described in Methods. Antibody-labeled CFTR proteins were allowed to internalize for the indicated times, and the disappearance of plasma membrane CFTR was monitored. *P < 0.05, **P < 0.005 versus WT CFTR. n = 3 with 24 replicates per experiment for each condition.

Figure 9. S13F CFTR prematurely accumulates in the lysosomes, where it is degraded.

(A) HA-CFTR proteins were expressed in BHK cells. The surface pool of CFTR was labeled with HA antibodies and allowed to internalize for the indicated times. CFTR was visualized using Alexa Fluor 488 antibodies, and lysosomes were labeled with lysotracker red. Both immunofluorescent and differential interference contrast (DIC) microscopy images are shown. Insets show colocalization of CFTR and the lysosomal marker. Magnification, ×63; insets, ×189. (B) WT CFTR and S13F CFTRs were analyzed by metabolic labeling in pulse-chase experiments in the presence of lysosomal protease inhibitors (Leupeptin) or with no treatment (No tx). Representative gels are shown for WT CFTR and S13F CFTR. (C) Quantitation of the data in B. *P < 0.05 versus control. n = 3.