Here we report the characterization of what we believe to be a novel protein-protein interaction between the N terminus of CFTR and FLN proteins that is required for the surface expression and stability of CFTR. In addition, we found that the S13F mutation in CFTR disrupted the interaction with FLNs, resulting in a decrease in both metabolic and plasma membrane stability of CFTR. To our knowledge, the disease-causing S13F mutation is the first missense mutation in CFTR found to disrupt a protein-protein interaction.
FLNs are best appreciated as structural proteins that regulate cellular architecture. However, FLNs are now known to directly interact with membrane-spanning proteins including ion channels, adhesion molecules, and G protein–coupled receptors (36
). Based on these studies, it is clear that FLNs stabilize their binding partners at the cell surface. For example, the plasma membrane levels of the glycoprotein Ibα (GPIbα; ref. 37
), the D2- and D3-dopamine receptors (D2R and D3R, respectively; ref. 38
), the inwardly rectifying potassium channel 2.1 (Kir2.1; ref. 40
), furin (39
), and the calcium-sensing receptor (CaR; ref. 42
) are all significantly reduced in the absence of FLN-A binding. Likewise, we found an approximately 5-fold reduction in the cell surface pool of S13F CFTR relative to WT CFTR. This effect is specific to a loss of FLN binding, because decreased surface expression was observed for WT CFTR in the presence of a competitive peptide inhibitor or when expressed in cells lacking FLN-A (M2 cells). The decrease in the plasma membrane CFTR was greater for S13F in multiple cell types than for WT CFTR in the M2 cells. We hypothesize that this difference can be accounted for by the fact that the S13F mutation disrupted the interaction with both FLN-A and FLN-B, whereas the M2 cells expressed FLN-B, which may partially compensate for the loss of FLN-A.
The decrease in CFTR surface expression coincides with altered plasma membrane mobility and increased endocytosis. Consistent with the findings of Bates et al. (55
), we found that CFTR was highly mobile on the cell surface, where it underwent periods of diffusion followed by brief transient confinements. For S13F CFTR, the partitioning of CFTR into confinement zones was significantly reduced (approximately 50%). Previous studies have shown that transient confinement may represent either cytoskeletal anchorage and/or partitioning into lipid microdomains (56
). However, because cholesterol depletion has a minor effect on CFTR transient confinement (47
), we favor the hypothesis that FLN is involved in either directly tethering CFTR to the cytoskeleton and/or creating a cytoskeletal meshwork that confines gold-labeled CFTR by steric interactions with its cytoplasmic domain. Studies from our lab and others have shown that the C terminus of CFTR also interacts with the cytoskeleton via sodium-hydrogen exchanger regulatory factor (NHERF) proteins and ezrin (57
). We predict that cytoskeletal interactions with the CFTR C terminus likely account for the residual membrane confinements observed for S13F CFTR. In addition, we observed increased endocytosis of S13F CFTR from the plasma membrane relative to the WT protein. This result is consistent with the findings for other FLN-binding proteins such as furin (39
), the calcitonin receptor (41
), and the prostate-specific membrane antigen (PSMA; ref. 36
) that display accelerated internalization in cells lacking FLN-A. Although the mechanism is not clear, it is exciting to speculate that the transient confinement of CFTR in the plasma membrane delays its incorporation into endocytic vesicles.
We also found that the half-life of S13F was decreased compared with WT CFTR, suggesting a role for FLNs in the metabolic stability of CFTR. Unlike ΔF508, P5L, or W19C, S13F CFTR displayed a clear pool of band C protein in both heterologous expression systems and epithelial cells. However, this pool was substantially reduced (approximately 50%) relative to WT CFTR. These results, together with those of our pulse-chase studies, lead us to conclude that a primary defect associated with the S13F mutation is a decrease in the stability of the mature, complex glycoslyated protein. Likewise, the metabolic stability was altered for other FLN-binding proteins when FLN-A is absent or the interaction is disrupted. Feng et al. report that the interaction with FLN-A stabilizes nacent GPIbα in the ER, which is critical for both the metabolic stability and the surface expression of this protein (59
). Our findings are not consistent with this mechanism for CFTR, as the metabolic stability was decreased for the mature band C protein as opposed to band B CFTR. Furthermore, our observation that ER-retained ΔF508 did not interact with FLN-A as assessed by coimmunoprecipitation, but temperature-rescued ΔF508 CFTR did, suggests that the CFTR–FLN-A interaction likely takes place in a post-ER compartment.
An important consideration is the relationship between the loss of CFTR plasma membrane stability and the premature degradation of the channel. Previous studies have shown that increasing CFTR internalization or decreasing endocytic recycling do not necessarily affect the metabolic stability of mature CFTR. For example, the R31L or N287Y mutations may introduce a nonnative internalization motif in CFTR and result in increased plasma membrane internalization (8
). Additionally, the deletion of the C-terminal PDZ binding motif results in decreased apical surface expression, which reflects less efficient recycling and not a change in endocytosis rates (60
). However, the metabolic stability of mature CFTR was not different from that of WT in these studies. Sharma et al. demonstrated that CFTR proteins that are misfolded, however, escape the ER quality control (e.g., temperature-rescued ΔF508), are not stable on the cell surface, are rapidly internalized, and are degraded via the proteasome (61
). The accelerated degradation associated with the S13F mutation and loss of FLN binding is distinct from the mechanism proposed for temperature-rescued ΔF508 because the degradation of S13F is primarily mediated by the lysosomes. The half-life of S13F CFTR can be restored close to that of WT CFTR by inhibiting lysosomal proteases. In addition, we found that S13F accumulated in a lysosomal compartment much more rapidly than did WT CFTR. It is likely that the lysosomal targeting and degradation of S13F CFTR reflects alterations in endocytic trafficking as a result of the loss of FLN binding. Similarly, the membrane trafficking of other FLN-A–binding proteins including furin (39
), PSMA (36
), and the calcitonin receptor (41
) are altered in FLN-A–null cells, resulting in the mislocalization of these proteins to various endosomal compartments. Thus, the decreased metabolic stability of mature S13F CFTR may reflect both its instability at the cell surface and defective endocytic trafficking.
Although not tested here, the interaction with FLNs may also be important for regulating the channel activity of CFTR at the cell surface. The interaction with FLN-A results in clustering of the Kir2.1 channels and the hyperpolarization-activated cyclic nucleotide-gated channels (HCN1) at the cell surface, thereby increasing current density (40
). Additionally, FLNs can organize multi-protein complexes and compartmentalize regulatory factors with receptors and ion channels. Recent studies have shown a role for FLN in the organization of cAMP signaling machinery upstream of CFTR activation including the β2
-adrenergic receptor, Gαs
, and adenylate cyclases (62
). Furthermore, electrophysiological studies have shown that in the FLN-null M2 cells, both whole-cell and single-channel CFTR currents are reduced in response to cAMP/PKA activity (63
). While reductions in whole-cell currents in part reflect less plasma membrane CFTR, the decreased openings of single CFTR channels suggest that FLNs directly affect gating. Thus, FLNs may additionally compartmentalize CFTR with relevant signaling molecules.
It is therefore clear that FLNs regulate multiple aspects of CFTR biology. Our observations are consistent with findings in CF patients, which suggest that S13F is a significant disease-causing mutation. The clinical manifestations of CFTR mutations range from mild, resulting in elevated sweat chlorides, to severe, resulting in pulmonary defects and pancreatic insufficiency. In an individual with S13F paired with a known mild mutation, T338I, elevated sweat chloride was the only clinical manifestation (31
). However, a patient with the S13F mutation paired with a frame-shift mutation, 2185insA
, displayed symptoms of CF including elevated sweat chloride, Pseudomonas aeruginosa
lung infection, and pancreatic insufficiency (32
). In preliminary studies, nasal potential difference measurements from the individual with S13F/2185insA
were consistent with a functional loss of CFTR at the cell surface, as little to no CFTR activity was detected (M. Knowles, unpublished observations). However, more individuals with S13F should be examined to confirm the disease severity of this mutation.
CF results from the loss of CFTR from the cell surface of epithelial tissues. Because the most common disease-causing mutation in CFTR, ΔF508, is retained in the ER due to a folding defect, therapies that allow ΔF508 to escape the ER are being intensely investigated (11
). Importantly, multiple labs have demonstrated that rescued ΔF508 CFTR can reach the cell surface where it has chloride channel activity (4
). However, ΔF508 CFTR is cleared from the cell surface and degraded much more rapidly than WT CFTR (65
). Therefore, an understanding of the mechanisms that regulate the stability of mature CFTR will provide important insights into therapeutic strategies aimed at rescuing ΔF508 CFTR. It is clear from these previous results that the maintenance of the plasma membrane pool of CFTR involves a complex set of regulatory interactions that govern aspects of trafficking, anchoring, internalization, and endocytic recycling. Direct and indirect protein-protein interactions with PDZ protein, clathrin subunits, small GTPases, myosins, and syntaxins have all been shown to regulate these aspects of CFTR biology (reviewed in ref. 66
). Here we demonstrate that FLNs are a key element of this regulatory network by anchoring CFTR on the plasma membrane and stabilizing the mature protein. In future studies, it will be important to assess how these accessory proteins coordinately maintain the plasma membrane pool of CFTR.