ABCMdb

PMID: 17235394

Thelin WR, Chen Y, Gentzsch M, Kreda SM, Sallee JL, Scarlett CO, Borchers CH, Jacobson K, Stutts MJ, Milgram SL
Direct interaction with filamins modulates the stability and plasma membrane expression of CFTR.
J Clin Invest. 2007 Feb;117(2):364-74. Epub 2007 Jan 18., [PubMed]

Sentences

No. Mutations Sentence Comment

4 ABCC7 p.Ser13Phe ABCC7 p.Ser13Phe Furthermore, we identified a disease-causing missense mutation in CFTR, serine 13 to phenylalanine (S13F), which disrupted this interaction. Login to comment 9 ABCC7 p.Ser13Phe In addition, we elucidate the molecular defect associated with the S13F mutation. Login to comment 30 ABCC7 p.Ser13Phe While characterizing these CFTR mutations, we discovered a protein-protein interaction between CFTR and filamin-A (FLN-A) that was disrupted by the S13F mutation. Login to comment 40 ABCC7 p.Ser13Phe Our data highlight what we believe to be a novel role for the CFTR N terminus and provide insights into the molecular mechanism underlying the defect associated with the disease-causing S13F mutation. Login to comment 42 ABCC7 p.Ser13Phe ABCC7 p.Ser13Phe ABCC7 p.Trp19Cys ABCC7 p.Trp19Cys ABCC7 p.Pro5Leu Using the CF mutations database (http://www.genet.sickkids.on.ca/cftr), we identified 3 previously described missense mutations in the extreme N terminus of CFTR: proline 5 to leucine (P5L), serine 13 to phenylalanine (S13F), and tryptophan 19 to cysteine (W19C) (31, 32). Login to comment 43 ABCC7 p.Ser13Phe ABCC7 p.Trp19Cys To determine whether these mutations would provide insights into the functional roles of the CFTR N terminus, we initially expressed P5L, S13F, and W19C CFTR in HEK293 cells and analyzed the proteins by Western blot. Login to comment 48 ABCC7 p.Trp19Cys Conversely, P5L and W19C were found only as the ER, core-glycosylated band B protein (Figure 1B). Login to comment 50 ABCC7 p.Ser13Phe Interestingly, however, S13F CFTR exhibited a clear pool of band C protein (Figure 1, B and C) with a significant 2.2-fold reduction in the band B/band C ratio relative to WT CFTR (Figure 1D). Login to comment 52 ABCC7 p.Ser13Phe ABCC7 p.Ser13Phe We observed similar decreases in the band C protein for S13F CFTR in an airway epithelial cell line, 16HBE140-, which demonstrates that the defect in S13F CFTR maturation is not cell type specific and is recapitulated in airway epithelial cells (Figure 1C). Login to comment 53 ABCC7 p.Ser13Phe Based on the band B/band C ratio, the S13F mutation appeared to be distinct from the majority of CFTR mutations identified to date. Login to comment 65 ABCC7 p.Ser13Phe Figure 2 The S13F mutation decreases the half-life of CFTR. Login to comment 71 ABCC7 p.Ser13Phe n = 4. 366 The Journal of Clinical Investigation http://www.jci.org Volume 117 Number 2 February 2007 The S13F mutation decreases the half-life of CFTR. Login to comment 72 ABCC7 p.Ser13Phe The decrease in the band B/band C ratio observed for S13F CFTR reflects either a defect in CFTR maturation and/or an increase in the degradation of the mature protein. Therefore, we monitored the maturation and degradation in metabolic pulse-chase experiments over a 24-hour period. Login to comment 73 ABCC7 p.Ser13Phe ABCC7 p.Ser13Ala WT, S13F, and S13A CFTRs were transiently expressed in HEK293 cells, labeled, immunoprecipitated, and analyzed by autoradiography (Figure 2A). Login to comment 75 ABCC7 p.Ser13Ala After 4 hours of chase, WT and S13A CFTRs were present almost exclusively as the mature band C, while no band C was observed for ΔF508 (Figure 2B). Login to comment 76 ABCC7 p.Ser13Phe ABCC7 p.Ser13Ala Like WT and S13A CFTR, S13F CFTR was clearly processed from the band B to band C protein during the first 4 hours of chase. Login to comment 77 ABCC7 p.Ser13Phe These data indicate that the major defect associated with the S13F mutation does not involve protein folding or ER exit. Login to comment 79 ABCC7 p.Ser13Ala The half-life was similar between WT and S13A CFTR (22.5 and 22.6 hours, respectively; Figure 2C). Login to comment 80 ABCC7 p.Ser13Phe However, the half-life of S13F CFTR was reduced by more than 50% (10.8 hours; P < 0.05). Login to comment 81 ABCC7 p.Ser13Phe Taken together, the results of our metabolic pulse-chase studies demonstrate that the S13F mutation decreases the stability of the mature band C CFTR. Login to comment 83 ABCC7 p.Ser13Phe We hypothesized that the N terminus of CFTR engages in protein-protein interactions that regulate its stability, which may be affected by the S13F mutation. Login to comment 85 ABCC7 p.Ser13Phe ABCC7 p.Ser13Phe CFTR peptides corresponding to residues 1-25 of either WT CFTR (CFTR1-25) or S13F CFTR (CFTR1-25/S13F) were used as affinity ligands to purify CFTR interacting proteins from Calu-3 airway epithelial cell lysates. Login to comment 87 ABCC7 p.Ser13Phe We observe several nonspecific bands associated with both CFTR1-25 and CFTR1-25/S13F. Login to comment 88 ABCC7 p.Ser13Phe However, 2 high-molecular weight bands specifically copurified with the CFTR1-25 but not the CFTR1-25/S13F peptides (Figure 3A). Login to comment 93 ABCC7 p.Ser13Phe The incorporation of the S13F mutation into the CFTR1-25 peptides nearly abolished the interaction with FLN-A in these assays. Login to comment 94 ABCC7 p.Ser13Ala However, CFTR1-25/S13A peptides bound to FLNs similarly to the CFTR1-25 peptides (Figure 3B). Login to comment 95 ABCC7 p.Ser13Phe Taken together, these data demonstrate that WT CFTR can directly interact with FLNs and that the S13F mutation disrupts this interaction. Login to comment 96 ABCC7 p.Ser13Phe ABCC7 p.Ser13Ala Thus, the reduction relative to WT CFTR in the band B/band C ratio observed for the S13F mutation, but not the S13A mutation, correlates with the ability to bind FLN-A in vitro. Login to comment 101 ABCC7 p.Ser13Phe ABCC7 p.Ser13Ala Furthermore, FLN-A coprecipitated with WT and S13A CFTR expressed in HEK293 cells, but not with S13F CFTR (Figure 3E). Login to comment 102 ABCC7 p.Ser13Phe Thus, the S13F mutation disrupted FLN-A binding to CFTR in vivo. Login to comment 108 ABCC7 p.Ser13Phe Importantly, the S13F mutation, which disrupted FLN binding in pulldown and immunoprecipitation assays, provided Figure 3 FLNs interact with the CFTR N terminus. Login to comment 109 ABCC7 p.Ser13Phe (A) Coomassie-stained gel of proteins that copurified with CFTR1-25 or CFTR1-25/S13F from Calu-3 cell lysates. Login to comment 113 ABCC7 p.Ser13Ala ABCC7 p.Ser13Ala S13A, CFTR1-25/S13A. Login to comment 125 ABCC7 p.Ser13Phe ABCC7 p.Ser13Ala The surface pool of CFTR was detected by immunofluorescence in unpermeabilized baby hamster kidney (BHK) cells expressing either WT, S13A, S13F, or ΔF508 HA-CFTR. Login to comment 126 ABCC7 p.Ser13Ala We observed significant amounts of WT and S13A CFTR at the cell surface, while no signal was observed for ΔF508 (Figure 5A). Login to comment 128 ABCC7 p.Ser13Phe ABCC7 p.Ser13Ala Unlike ΔF508 CFTR, we observed surface staining for S13F CFTR; however, the staining was greatly reduced compared with WT and S13A CFTR. Login to comment 130 ABCC7 p.Ser13Ala In agreement with our observations by immunofluorescence, a substantial pool of WT and S13A CFTR resided on the cell surface (41.2% and 37.8%, respectively), while no ΔF508 was observed (Figure 5B). Login to comment 131 ABCC7 p.Ser13Phe ABCC7 p.Ser13Phe Furthermore, the surface pool of S13F CFTR was significantly less than that of WT and S13F CFTR (8.7%; P < 0.01) but significantly greater Figure 4 FLN-A localizes to the subapical membrane of airway epithelia. Login to comment 135 ABCC7 p.Ser13Phe Scale bars: 10 μm. Figure 5 Surface expression of S13F CFTR is decreased. Login to comment 140 ABCC7 p.Ser13Phe Thus, in the absence of FLN binding, S13F CFTR localizes to the cell surface, albeit at reduced levels compared with WT CFTR. Login to comment 141 ABCC7 p.Ser13Phe The S13F mutation reflects a loss of FLN binding. Login to comment 149 ABCC7 p.Ser13Phe Importantly, this effect was not observed when the S13F peptide or the F(ab') fragments alone were transfected. Login to comment 169 ABCC7 p.Ser13Phe Trajectories of at least 50 different gold particles were analyzed from cells expressing WT or S13F CFTR. Login to comment 172 ABCC7 p.Ser13Phe To determine whether the association with FLNs is important for the transient confinement of CFTR, we examined S13F CFTR by SPT. Login to comment 173 ABCC7 p.Ser13Phe We found that S13F CFTR exhibited significantly less transient confinement than WT CFTR (Figure 7). Login to comment 174 ABCC7 p.Ser13Phe The confinement of S13F CFTR was reduced by more than 50% relative to WT, which reflects a decrease in both the number of confinements and the time spent in a single TCZ (Table 1). Login to comment 175 ABCC7 p.Ser13Phe Interestingly, the diffusion coefficients of WT and S13F were not greatly different (3.45 ± 0.41 versus 2.73 ± 0.71 × 10-10 cm2/s), suggesting that for CFTR, incorporation into TCZ does not alter the rate of diffusion in the membrane (Table 1). Login to comment 178 ABCC7 p.Ser13Phe S13F CFTR is rapidly cleared from the cell surface. Login to comment 180 ABCC7 p.Ser13Phe During the SPT experiments, we qualitatively observed many more S13F proteins internalized compared with WT CFTR. Login to comment 182 ABCC7 p.Ser13Phe HeLa cells transiently expressing either WT or S13F HA-CFTR were chilled to 4°C to block internalization and labeled with anti-HA antibodies. After removing excess antibodies, the cells were warmed to 37°C for 0, 5, 10, and 15 minutes and then fixed, labeled with IRDye- labeled secondary antibodies, and analyzed using a LI-COR Biosciences Odyssey instrument. Login to comment 184 ABCC7 p.Ser13Phe We found that the surface expression of S13F decreased significantly more rapidly than that of WT CFTR (Figure 8). Login to comment 185 ABCC7 p.Ser13Phe At the 5-minute time point, significantly more S13F CFTR was internalized compared with WT CFTR (25.4% versus 13.8%; P > 0.05). Login to comment 186 ABCC7 p.Ser13Phe This trend was more pronounced at the 15-minute time point, where 61.5% of S13F CFTR was cleared from the cell surface compared with 25.1% for WT CFTR. Login to comment 187 ABCC7 p.Ser13Phe Thus, the FLN-binding mutant S13F CFTR is less stable on the cell surface. Login to comment 188 ABCC7 p.Ser13Phe Importantly, the differences observed between WT and S13F CFTR at the 5-minute time point likely reflect changes in internalization. Login to comment 190 ABCC7 p.Ser13Phe Conversely, cell surface S13F CFTR continued to be lost between the 10-and 15-minute time points, consistent with defects in recycling kinetics. Login to comment 191 ABCC7 p.Ser13Phe The accelerated degradation of S13F CFTR is primarily mediated by lysosomes. Login to comment 193 ABCC7 p.Ser13Phe HeLa cells transiently expressing either WT or S13F HA-CFTR were chilled to 4°C to block internalization and labeled with anti-HA antibodies. After removing excess antibodies, the cells were warmed to 37°C for 2, 4, or 8 hours and then fixed, permeabilized, and labeled with fluorescent secondary antibodies. Login to comment 195 ABCC7 p.Ser13Phe In our experiments, both WT and S13F CFTR exhibited partial overlap with the early endosome marker EEA1 and internalized transferrin at the 2-hour time point (data not shown). Login to comment 196 ABCC7 p.Ser13Phe However, we observed striking dif- Figure 7 The membrane dynamics of S13F CFTR is altered. Login to comment 197 ABCC7 p.Ser13Phe WT and S13F CFTRs were analyzed by SPT in HeLa cells. Login to comment 202 ABCC7 p.Ser13Phe ABCC7 p.Ser13Phe Table 1 SPT data quantitation for WT and S13F CFTR proteins CFTR Diffusion coefficient Relative confinement TCZ dwell (× 10-10 cm2/s) time (%) time (s) WT 3.45 ± 0.41 8.41 ± 2.50 1.00 ± 0.32 S13F 2.73 ± 0.71 .45 ± 1.16A 0.30 ± 0.03A Values are mean ± SEM quantified from data in Figure 7. n = 45. Login to comment 203 ABCC7 p.Ser13Phe AP < 0.001. 370 The Journal of Clinical Investigation http://www.jci.org Volume 117 Number 2 February 2007 ferences between WT and S13F CFTR at the 24-hour time points (Figure 9A). Login to comment 205 ABCC7 p.Ser13Phe In contrast, we found some colocalization between S13F CFTR and lysosomes by 8 hours, which was more evident by 24 hours (Figure 9A). Login to comment 206 ABCC7 p.Ser13Phe In our initial studies of the CFTR N-terminal mutations, we found that the steady-state distribution of S13F CFTR displayed a decrease in the mature band C protein, which reflects increased degradation. Login to comment 207 ABCC7 p.Ser13Phe ABCC7 p.Ser13Phe ABCC7 p.Ser13Phe ABCC7 p.Ser13Ala Additionally, S13F was prematurely sorted to lysosomes, which may explain why mature S13F CFTR was degraded more rapidly than the WT protein. Therefore, we examined the half-life of WT, S13F, and S13A CFTR by pulse chase in the presence of the lysosomal protease inhibitor leupeptin. Login to comment 208 ABCC7 p.Ser13Phe Strikingly, leupeptin significantly increased the half-life of S13F CFTR from 13.4 hours to 18.3 hours (Figure 9, B and C). Login to comment 209 ABCC7 p.Ser13Phe Although leupeptin did Figure 8 S13F CFTR is internalized more rapidly than is WT CFTR. Login to comment 210 ABCC7 p.Ser13Phe WT or S13F CFTRs were transiently expressed in BHK cells. Login to comment 215 ABCC7 p.Ser13Phe Figure 9 S13F CFTR prematurely accumulates in the lysosomes, where it is degraded. Login to comment 222 ABCC7 p.Ser13Phe (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). Login to comment 223 ABCC7 p.Ser13Phe Representative gels are shown for WT CFTR and S13F CFTR. Login to comment 226 ABCC7 p.Ser13Phe ABCC7 p.Ser13Phe n = 3. 371 not fully rescue S13F to the 21.9-hour half-life of WT CFTR (Figure 9, B and C), it suggests that lysosomally mediated degradation accounts for the majority of S13F turnover. Login to comment 228 ABCC7 p.Ser13Phe 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. Login to comment 229 ABCC7 p.Ser13Phe To our knowledge, the disease-causing S13F mutation is the first missense mutation in CFTR found to disrupt a protein-protein interaction. Login to comment 234 ABCC7 p.Ser13Phe Likewise, we found an approximately 5-fold reduction in the cell surface pool of S13F CFTR relative to WT CFTR. Login to comment 236 ABCC7 p.Ser13Phe The decrease in the plasma membrane CFTR was greater for S13F in multiple cell types than for WT CFTR in the M2 cells. Login to comment 237 ABCC7 p.Ser13Phe 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. Login to comment 240 ABCC7 p.Ser13Phe For S13F CFTR, the partitioning of CFTR into confinement zones was significantly reduced (approximately 50%). Login to comment 244 ABCC7 p.Ser13Phe We predict that cytoskeletal interactions with the CFTR C terminus likely account for the residual membrane confinements observed for S13F CFTR. Login to comment 245 ABCC7 p.Ser13Phe In addition, we observed increased endocytosis of S13F CFTR from the plasma membrane relative to the WT protein. Login to comment 248 ABCC7 p.Ser13Phe 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. Login to comment 249 ABCC7 p.Ser13Phe ABCC7 p.Trp19Cys Unlike ΔF508, P5L, or W19C, S13F CFTR displayed a clear pool of band C protein in both heterologous expression systems and epithelial cells. Login to comment 251 ABCC7 p.Ser13Phe 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. Login to comment 258 ABCC7 p.Asn287Tyr ABCC7 p.Arg31Leu For example, the R31L or N287Y mutations may introduce a nonnative internalization motif in CFTR and result in increased plasma membrane internalization (8, 9). Login to comment 262 ABCC7 p.Ser13Phe ABCC7 p.Ser13Phe 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. Login to comment 263 ABCC7 p.Ser13Phe The half-life of S13F CFTR can be restored close to that of WT CFTR by inhibiting lysosomal proteases. Login to comment 264 ABCC7 p.Ser13Phe In addition, we found that S13F accumulated in a lysosomal compartment much more rapidly than did WT CFTR. Login to comment 265 ABCC7 p.Ser13Phe 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. Login to comment 267 ABCC7 p.Ser13Phe Thus, the decreased metabolic stability of mature S13F CFTR may reflect both its instability at the cell surface and defective endocytic trafficking. Login to comment 276 ABCC7 p.Ser13Phe Our observations are consistent with findings in CF patients, which suggest that S13F is a significant disease-causing mutation. Login to comment 278 ABCC7 p.Thr338Ile ABCC7 p.Ser13Phe In an individual with S13F paired with a known mild mutation, T338I, elevated sweat chloride was the only clinical manifestation (31). Login to comment 279 ABCC7 p.Ser13Phe 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). Login to comment 280 ABCC7 p.Ser13Phe 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). Login to comment 281 ABCC7 p.Ser13Phe However, more individuals with S13F should be examined to confirm the disease severity of this mutation. Login to comment 311 ABCC7 p.Ser13Phe The CFTR1-25 or CFTR1-25/S13F peptides were used to affinity purify CFTR-binding proteins as described by Thelin et al. (67), with the following exceptions. Login to comment 324 ABCC7 p.Ser13Phe ABCC7 p.Ser13Ala WT, S13F, S13A, and ΔF508 CFTRs were transiently expressed in HEK293 cells. Login to comment 352 ABCC7 p.Ser13Phe Specifically, nonbiotinylated CFTR1-25 or CFTR1-25/S13F peptides were introduced into BHK cells stably expressing HA-CFTR with the Pro-Ject transfection system (Pierce Biotechnology). Login to comment 365 ABCC7 p.Ser13Phe Acknowledgments We thank Michael Knowles (University of North Carolina), Jeffery Wine (Stanford University), Noreen Henig (Stanford University), and Gary Cutting (Johns Hopkins University) for helpful discussions regarding S13F patients; Gary Thomas (Oregon Health Sciences University) for providing M2 and A7 cells; John Riordan (University of North Carolina) for providing antibodies and plasmids; and Wendy Salmon (University of North Carolina) for help with microscopy. Login to comment 467 ABCC7 p.Ser13Phe Identification of a novel mutation (S13F) in the CFTR gene in a CF patient of Sardinian origin. Login to comment