ABCMdb

ABCC7 p.Ser13Phe

ClinVar:	c.38C>T , p.Ser13Phe ? , not provided
CF databases:	c.38C>T , p.Ser13Phe (CFTR1) ? , This mutation was detected by DGGE and identified by direct sequencing in a CF patient of Sardinian origin.
Predicted by SNAP2:	A: D (95%), C: D (95%), D: D (95%), E: D (95%), F: D (63%), G: D (95%), H: D (95%), I: D (95%), K: D (95%), L: D (95%), M: D (95%), N: D (95%), P: D (95%), Q: D (95%), R: D (95%), T: D (95%), V: D (95%), W: D (95%), Y: D (95%),
Predicted by PROVEAN:	A: N, C: D, D: D, E: D, F: D, G: D, H: D, I: D, K: D, L: D, M: D, N: N, P: D, Q: D, R: D, T: N, V: D, W: D, Y: D,

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[hide] Comprehensive mutation screening in a cystic fibro... Pediatrics. 2001 Feb;107(2):280-6. Wine JJ, Kuo E, Hurlock G, Moss RB
Comprehensive mutation screening in a cystic fibrosis center.
Pediatrics. 2001 Feb;107(2):280-6., [PMID:11158459]

Abstract [show]
OBJECTIVES AND BACKGROUND: The identities of a cystic fibrosis (CF) patient's CFTR mutations can influence therapeutic strategies, but because >800 CFTR mutations exist, cost-effective, comprehensive screening requires a multistage approach. Single-strand conformation polymorphism and heteroduplex analysis (SSCP/HA) can be an important part of mutation detection, but must be calibrated within each laboratory. The sensitivity of a combined commercial-SSCP/HA approach to genotyping in a large, ethnically diverse US center CF population has not been established. STUDY DESIGN: We screened all 27 CFTR exons in 10 human participants who had an unequivocal CF diagnosis including a positive sweat chloride test and at least 1 unknown allele after commercial testing for the 70 most common mutations by SSCP/HA. These participants were compared with 7 participants who had negative sweat tests but at least 1 other CF-like symptom meriting complete genotyping. RESULTS: For the 10 CF participants, we detected 11 of 16 unknown alleles (69%) and all 4 of the known alleles (100%), for an overall rate of 75% inpatients not fully genotyped by conventional 70 mutation screen. For 7 participants with negative sweat tests, we confirmed 1 identified mutation in 14 alleles and detected 3 additional mutations. Mutations detected in both groups included 7 missense mutations (S13F, P67L, G98R, S492F, G970D, L1093P, N1303K) and 9 deletion, frameshift, nonsense or splicing mutations (R75X, G542X, DeltaF508, 451-458Delta8 bp, 5T, 663DeltaT, exon 13 frameshift, 1261+1G-->A and 3272-26A-->G). Three of these mutations were novel (G970D, L1093P, and 451-458Delta8 bp(1)). Thirteen other changes were detected, including the novel changes 1812-3 ins T, 4096-278 ins T, 4096-265 ins TG, and 4096-180 T-->G. CONCLUSION: When combined with the 70 mutation Genzyme test, SSCP/HA analysis allows for detection of >95% of the mutations in an ethnically heterogeneous CF center population. We discuss 5 possible explanations that could account for the few remaining undetected mutations.

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16 Mutations detected in both groups included 7 missense mutations (S13F, P67L, G98R, S492F, G970D, L1093P, N1303K) and 9 deletion, frameshift, nonsense or splicing mutations (R75X, G542X, ⌬F508, 451-458⌬8 bp, 5T, 663⌬T, exon 13 frameshift, 1261؉1G3A and 3272-26A3G). Login to comment

[hide] Pancreatitis among patients with cystic fibrosis: ... Pediatrics. 2005 Apr;115(4):e463-9. Epub 2005 Mar 16. De Boeck K, Weren M, Proesmans M, Kerem E
Pancreatitis among patients with cystic fibrosis: correlation with pancreatic status and genotype.
Pediatrics. 2005 Apr;115(4):e463-9. Epub 2005 Mar 16., [PMID:15772171]

Abstract [show]
OBJECTIVE: Pancreatitis is an infrequent complication among patients with cystic fibrosis (CF). It has mainly been reported for patients with pancreatic sufficiency (PS). Previous studies involved only a small number of patients because they contained data from single centers. The aim of this study was to evaluate the incidence of pancreatitis in a large heterogeneous CF population, to determine the relationship with pancreatic function, and to assess whether pancreatitis is associated with specific CFTR mutations. METHODS: Physicians caring for patients with CF were approached through the CF Thematic Network or through the European Cystic Fibrosis Foundation newsletter. They were asked to provide data on their current patient cohort through a standardized questionnaire and to report how many patients they had ever diagnosed as having pancreatitis. A detailed questionnaire was then sent, to be filled out for all of their patients for whom pancreatitis had ever occurred. We defined pancreatitis as an episode of acute abdominal pain associated with serum amylase levels elevated above the ranges established by each participating center's laboratory. General clinical data included age, genotype, age at diagnosis of CF, sweat chloride concentrations, pancreatic status, biometric findings, and respiratory status. CFTR mutations were also reported according to the functional classification of classes I to V. Patients were categorized as having PS, pancreatic insufficiency (PI), or PI after an initial period of PS. PI was defined as a 72-hour stool fat loss of >7 g/day, fat absorption of <93%, or fecal elastase levels of <200 microg/g feces. Clinical data on pancreatitis included age at the first episode, amylase and lipase levels, possible triggers, and occurrence of relapses or complications. RESULTS: A total of 10071 patients with CF, from 29 different countries, who were undergoing follow-up monitoring in 2002 were surveyed. Among this group, pancreatitis had ever been diagnosed for 125 patients (1.24%; 95% confidence interval [CI]: 1.02-1.46%). There was variability in the reported rates of pancreatitis for different countries. Twenty-six centers in 15 different countries sent detailed clinical data on their patients with pancreatitis and on their whole CF clinic. This involved 3306 patients with CF and 61 cases of pancreatitis, leading to a prevalence of 1.84% (95% CI: 1.39-2.30%). The mean age of the patients with pancreatitis ever was 24.4 years (SD: 10.8 years). The first episode of pancreatitis occurred at a mean age of 19.9 years (SD: 9.6 years). The median serum amylase level at the time of pancreatitis was 746 IU/L (interquartile range: 319-1630 IU/L), and the median lipase level was 577 IU/L (interquartile range: 229-1650 IU/L). The majority of patients had PS (34 of 61 patients, 56%; 95% CI: 43-68%). Pancreatitis occurred for 15 patients with PI (25%; 95% CI: 14-35%). Eight patients developed PI after initial PS. The occurrence of pancreatitis among patients with PS was 34 cases per 331 patients, ie, 10.27% (95% CI: 7.00-13.55%); the occurrence of pancreatitis among patients with PI was 15 cases per 2971 patients, ie, 0.5% (95% CI: 0.25-0.76%). The mean age (in 2002) of the CF cohort with pancreatitis did not differ between the PS and PI subgroups. The forced expiratory volume in 1 second was significantly lower among the patients with PI than among the patients with PS, ie, 65% (SEM: 7%) vs 79% (SEM: 4%). The mean age at the occurrence of pancreatitis and the amylase and lipase levels during pancreatitis were not different for patients with pancreatitis and PI versus PS. In the group with PS, 31 of 34 patients carried at least 1 class IV or V CFTR mutation. In the groups with PI and PI after PS, 5 of 15 patients and 3 of 8 patients, respectively, carried 2 class I, II, or III CFTR mutations. Relapses and/or evolution to chronic pancreatitis occurred for 42 patients. Pancreatitis preceded the diagnosis of CF in 18 of 61 cases. These patients were significantly older than the rest of the cohort, ie, age of 28.4 years (SEM: 3.4 years) vs 22.7 years (SEM: 1.3 years). Their median age at the diagnosis of CF was also significantly greater, ie, 21.5 years (interquartile range: 11.9-31 years) vs 7.6 years (interquartile range: 0.4-17.0 years). However, the ages at the occurrence of pancreatitis were similar, ie, 21.0 years (SEM: 3.0 years) vs 19.5 years (SEM: 1.2 years). CONCLUSIONS: This study of 10071 patients with CF from 29 different countries revealed an estimated overall occurrence of pancreatitis among patients with CF of 1.24% (95% CI: 1.02-1.46%). The incidence of pancreatitis was much higher among patients with PS. However, pancreatitis was also reported for 15 patients with PI from 11 centers in 9 different countries. A correct diagnosis of pancreatitis for the reported patients with PI was supported by amylase and lipase levels increased above 500 IU/L, similar to those for patients with PS and pancreatitis. A correct diagnosis of PI for these patients with pancreatitis was supported by the adequacy of the methods used. We chose the cutoff values used to distinguish between patients with PI and control subjects without gastrointestinal disease. For one half of the patients, the diagnosis of PI was established on the basis of low levels of stool elastase (mean: 97 mug/g stool). With a cutoff value of 200 microg/g stool, this noninvasive test has high sensitivity (>95%) and high specificity (>90%) to differentiate patients with PI from control subjects with normal pancreatic function. For the other one half of the patients with PI in the cohort, the pancreatic status was determined on the basis of the 3-day fecal fat balance, with the widely used cutoff value of >7 g of fat loss per day. The most likely reason for pancreatitis occurring among patients with PI is that some residual pancreatic tissue is present among these patients. Pancreatitis is a rare complication among patients with CF. It occurred for 1.24% (95% CI: 1.02-1.46%) of a large CF cohort. Pancreatitis occurs mainly during adolescence and young adulthood. It is much more common among patients with CF and PS (10.3%), but it can occur among patients with PI (0.5%). Pancreatitis can be the first manifestation of CF. Pancreatitis was reported for patients carrying a wide range of mutations.

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136 Class IV and V mutations reported among the patients with PI included D1152H (n ϭ 2), A455E (n ϭ 2), R1066H (n ϭ 1), S13F (n ϭ 1), and 1898ϩ3AϾG (n ϭ 1). Login to comment

[hide] Direct interaction with filamins modulates the sta... J Clin Invest. 2007 Feb;117(2):364-74. Epub 2007 Jan 18. 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., [PMID:17235394]

Abstract [show]
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.

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4 Furthermore, we identified a disease-causing missense mutation in CFTR, serine 13 to phenylalanine (S13F), which disrupted this interaction. Login to comment
9 In addition, we elucidate the molecular defect associated with the S13F mutation. Login to comment
30 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 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 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 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
50 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 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 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 Figure 2 The S13F mutation decreases the half-life of CFTR. Login to comment
71 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 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 WT, S13F, and S13A CFTRs were transiently expressed in HEK293 cells, labeled, immunoprecipitated, and analyzed by autoradiography (Figure 2A). Login to comment
76 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 These data indicate that the major defect associated with the S13F mutation does not involve protein folding or ER exit. Login to comment
80 However, the half-life of S13F CFTR was reduced by more than 50% (10.8 hours; P < 0.05). Login to comment
81 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 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 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 We observe several nonspecific bands associated with both CFTR1-25 and CFTR1-25/S13F. Login to comment
88 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 The incorporation of the S13F mutation into the CFTR1-25 peptides nearly abolished the interaction with FLN-A in these assays. Login to comment
95 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 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 Furthermore, FLN-A coprecipitated with WT and S13A CFTR expressed in HEK293 cells, but not with S13F CFTR (Figure 3E). Login to comment
102 Thus, the S13F mutation disrupted FLN-A binding to CFTR in vivo. Login to comment
108 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 (A) Coomassie-stained gel of proteins that copurified with CFTR1-25 or CFTR1-25/S13F from Calu-3 cell lysates. Login to comment
125 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
128 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
131 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 Scale bars: 10 μm. Figure 5 Surface expression of S13F CFTR is decreased. Login to comment
140 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 The S13F mutation reflects a loss of FLN binding. Login to comment
149 Importantly, this effect was not observed when the S13F peptide or the F(ab') fragments alone were transfected. Login to comment
169 Trajectories of at least 50 different gold particles were analyzed from cells expressing WT or S13F CFTR. Login to comment
172 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 We found that S13F CFTR exhibited significantly less transient confinement than WT CFTR (Figure 7). Login to comment
174 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 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 S13F CFTR is rapidly cleared from the cell surface. Login to comment
180 During the SPT experiments, we qualitatively observed many more S13F proteins internalized compared with WT CFTR. Login to comment
182 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 We found that the surface expression of S13F decreased significantly more rapidly than that of WT CFTR (Figure 8). Login to comment
185 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 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 Thus, the FLN-binding mutant S13F CFTR is less stable on the cell surface. Login to comment
188 Importantly, the differences observed between WT and S13F CFTR at the 5-minute time point likely reflect changes in internalization. Login to comment
190 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 The accelerated degradation of S13F CFTR is primarily mediated by lysosomes. Login to comment
193 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 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 However, we observed striking dif- Figure 7 The membrane dynamics of S13F CFTR is altered. Login to comment
197 WT and S13F CFTRs were analyzed by SPT in HeLa cells. Login to comment
202 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 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 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 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 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 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 Although leupeptin did Figure 8 S13F CFTR is internalized more rapidly than is WT CFTR. Login to comment
210 WT or S13F CFTRs were transiently expressed in BHK cells. Login to comment
215 Figure 9 S13F CFTR prematurely accumulates in the lysosomes, where it is degraded. Login to comment
222 (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 Representative gels are shown for WT CFTR and S13F CFTR. Login to comment
226 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 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 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 Likewise, we found an approximately 5-fold reduction in the cell surface pool of S13F CFTR relative to WT CFTR. Login to comment
236 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 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 For S13F CFTR, the partitioning of CFTR into confinement zones was significantly reduced (approximately 50%). Login to comment
244 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 In addition, we observed increased endocytosis of S13F CFTR from the plasma membrane relative to the WT protein. Login to comment
248 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 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 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
262 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 The half-life of S13F CFTR can be restored close to that of WT CFTR by inhibiting lysosomal proteases. Login to comment
264 In addition, we found that S13F accumulated in a lysosomal compartment much more rapidly than did WT CFTR. Login to comment
265 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 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 Our observations are consistent with findings in CF patients, which suggest that S13F is a significant disease-causing mutation. Login to comment
278 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 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 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 However, more individuals with S13F should be examined to confirm the disease severity of this mutation. Login to comment
311 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 WT, S13F, S13A, and ΔF508 CFTRs were transiently expressed in HEK293 cells. Login to comment
352 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 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 Identification of a novel mutation (S13F) in the CFTR gene in a CF patient of Sardinian origin. Login to comment

[hide] Cystic fibrosis transmembrane conductance regulato... J Biol Chem. 2010 May 28;285(22):17156-65. Epub 2010 Mar 29. Playford MP, Nurminen E, Pentikainen OT, Milgram SL, Hartwig JH, Stossel TP, Nakamura F
Cystic fibrosis transmembrane conductance regulator interacts with multiple immunoglobulin domains of filamin A.
J Biol Chem. 2010 May 28;285(22):17156-65. Epub 2010 Mar 29., 2010-05-28 [PMID:20351098]

Abstract [show]
Mutations of the chloride channel cystic fibrosis transmembrane conductance regulator (CFTR) that impair its apical localization and function cause cystic fibrosis. A previous report has shown that filamin A (FLNa), an actin-cross-linking and -scaffolding protein, interacts directly with the cytoplasmic N terminus of CFTR and that this interaction is necessary for stability and confinement of the channel to apical membranes. Here, we report that the CFTR N terminus has sequence similarity to known FLNa-binding partner-binding sites. FLNa has 24 Ig (IgFLNa) repeats, and a CFTR peptide pulled down repeats 9, 12, 17, 19, 21, and 23, which share sequence similarity yet differ from the other FLNa Ig domains. Using known structures of IgFLNa.partner complexes as templates, we generated in silico models of IgFLNa.CFTR peptide complexes. Point and deletion mutants of IgFLNa and CFTR informed by the models, including disease-causing mutations L15P and W19C, disrupted the binding interaction. The model predicted that a P5L CFTR mutation should not affect binding, but a synthetic P5L mutant peptide had reduced solubility, suggesting a different disease-causing mechanism. Taken together with the fact that FLNa dimers are elongated ( approximately 160 nm) strands, whereas CFTR is compact (6 approximately 8 nm), we propose that a single FLNa molecule can scaffold multiple CFTR partners. Unlike previously defined dimeric FLNa.partner complexes, the FLNa-monomeric CFTR interaction is relatively weak, presumptively facilitating dynamic clustering of CFTR at cell membranes. Finally, we show that deletion of all CFTR interacting domains from FLNa suppresses the surface expression of CFTR on baby hamster kidney cells.

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21 The CF-causing S13F mutation that disrupts this interaction in vivo leads to a reduced pool of CFTR at apical membrane sites and is prematurely delivered to lysosomes and degraded (11). Login to comment
68 CFTR Peptide Pulldown Assay-Various concentration of FLNa constructs were incubated with increasing concentrations of wild-type or S13F mutant biotin⅐CFTR1-25 peptides FIGURE 1. Login to comment
72 Biotinylated wild-type or S13F mutant CFTR peptides (1.0 ␮M) were incubated with the His-tagged FLNa fragments (0.1 ␮M). Login to comment
97 Biotinylated wild-type or S13F mutant CFTR peptides (1.0 ␮M) were incubated with the GST-tagged FLNa fragments (0.1␮M). Login to comment
102 Structure of Filamin A⅐CFTR Complex 17158 S13F mutant CFTR peptide (Fig. 1B), consistent with previous results (11). Login to comment
113 Amino acid changes reflecting known CF mutations within this peptide (S13F, L15P, or W19C) prevented FLNa binding (Fig. 5C). Login to comment
118 B, His-16-24 and deletion mutant (⌬17,19,21,23 deletion of IgFLNa17, 19, 21, and 23) were pulled down with increasing amounts of the wild-type (WT) or S13F mutant CFTR1-25 peptide. Login to comment
125 Red and blue amino acids indicate residues mutated in CF patients (P5L, S13F, L15P, and W19C). Login to comment
133 In this model, the S13F mutation of CFTR is incapable of forming hydrogen-bonding interactions with the main-chain carbonyl oxygen atom of Val-2472 (Fig. 5A), consistent with in vitro binding experiments (Figs. Login to comment
169 As anticipated, Myc-FLNa wild-type protein bound to wild-type, but not the S13F mutant CFTR peptide. Login to comment
185 Lysates from these cells were incubated with N-terminal wild type (wt) (1-25) or mutant (1-25 S13F) CFTR peptides. Login to comment
192 FLNa Repeats 9, 12, 17, 19, 21, and 23 Are Necessary for Optimal Surface Expression of CFTR-We previously reported that an interaction with FLNa is important for the expression of CFTR on the cell surface; the surface expression of the CFTR mutant S13F, which is defective in FLNa binding, is dramatically reduced. Login to comment
203 As expected, co-expression of wild-type FLNa had no effect on the surface localization of the FLNa binding-defective CFTR Structure of Filamin A⅐CFTR Complex 17162 mutant S13F. Login to comment
227 Structural Basis for Mutations That Impair the FLNa-CFTR Interaction-Three missense mutations in the FLNa-binding site of CFTR have been reported: S13F, L15P, and W19C. Login to comment
228 The S13F mutation eliminates hydrogen bonding between the main-chain carbonyl oxygen atom of Val or Ala (Val-2472: IgFLNa23 numbering) in IgFLNa repeats 9, 12, 17, 19, 21, and 23, and the bulky Phe residue cannot stack with Val or Ala. Login to comment
232 However, unlike the S13F mutant of CFTR, which is still expressed, albeit reduced ϳ50% compared with wild type, on the cell surface in a mature glycosylated form, the W19C mutant of CFTR remains immature and is retained in the endoplasmic reticulum (ER) (11), suggesting that it causes the disease without disrupting the interaction with FLNa. Login to comment

[hide] Biochemical basis of the interaction between cysti... J Biol Chem. 2010 May 28;285(22):17166-76. Epub 2010 Mar 29. Smith L, Page RC, Xu Z, Kohli E, Litman P, Nix JC, Ithychanda SS, Liu J, Qin J, Misra S, Liedtke CM
Biochemical basis of the interaction between cystic fibrosis transmembrane conductance regulator and immunoglobulin-like repeats of filamin.
J Biol Chem. 2010 May 28;285(22):17166-76. Epub 2010 Mar 29., 2010-05-28 [PMID:20351101]

Abstract [show]
Mutations in the chloride channel cystic fibrosis transmembrane regulator (CFTR) cause cystic fibrosis, a genetic disorder characterized by defects in CFTR biosynthesis, localization to the cell surface, or activation by regulatory factors. It was discovered recently that surface localization of CFTR is stabilized by an interaction between the CFTR N terminus and the multidomain cytoskeletal protein filamin. The details of the CFTR-filamin interaction, however, are unclear. Using x-ray crystallography, we show how the CFTR N terminus binds to immunoglobulin-like repeat 21 of filamin A (FlnA-Ig21). CFTR binds to beta-strands C and D of FlnA-Ig21 using backbone-backbone hydrogen bonds, a linchpin serine residue, and hydrophobic side-chain packing. We use NMR to determine that the CFTR N terminus also binds to several other immunoglobulin-like repeats from filamin A in vitro. Our structural data explain why the cystic fibrosis-causing S13F mutation disrupts CFTR-filamin interaction. We show that FlnA-Ig repeats transfected into cultured Calu-3 cells disrupt CFTR-filamin interaction and reduce surface levels of CFTR. Our findings suggest that filamin A stabilizes surface CFTR by anchoring it to the actin cytoskeleton through interactions with multiple filamin Ig repeats. Such an interaction mode may allow filamins to cluster multiple CFTR molecules and to promote colocalization of CFTR and other filamin-binding proteins in the apical plasma membrane of epithelial cells.

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No. Sentence Comment
6 Our structural data explain why the cystic fibrosis-causing S13F mutation disrupts CFTR-filamin interaction. Login to comment
48 A CF-associated mutation, S13F, disrupts the interaction between the N terminus of CFTR and FlnA or FlnB (15). Login to comment
50 Unlike wild-type CFTR, the internalized S13F CFTR is targeted preferentially to lysosomes rather than being recycled to the plasma membrane (15). Login to comment
55 Our results explain why the S13F mutation disrupts the interaction between CFTR and filamins. Login to comment
91 Peptide binding titrations were initially pursued using CFTR4-22 or CFTR4-22/S13F. Login to comment
94 15 N-Ig repeats were titrated with CFTR7-20/F16E (EKASVVSKLEFSWT) and CFTR7-20/S13F/F16E (EKASVVFKLEFSWT) at ratios ranging from 1:0 up to 1:100 (FlnA:peptide). Login to comment
171 Importantly, our crystal structure readily explains the basis of the defect in the CF-causing mutation S13F, which was previously shown to eliminate binding between CFTR and filamins (15). Login to comment
179 Because the S13F mutation eliminates CFTR-filamin interactions in vivo (15), we conclude that the contacts between CFTR4-22 and Ig21B are a crystallographic artifact and do not represent a physiologically relevant interaction (see below). Login to comment
183 In contrast, addition of CFTR7-20-S13F/F16E had no effect (Fig. 3A; supplemental Fig. 3). Login to comment
184 The CFTR7-20-F17E and CFTR7-20- S13F/F17E peptides behaved similarly to their F16E counterparts (data not shown). Login to comment
185 If the Ig21B-CFTR4-22 interaction mode observed in our crystal structure is also present in solution, this interaction should be insensitive to the S13F mutation. Login to comment
186 We observe, however, that the S13F mutation eliminates all binding of CFTR N-terminal peptides to Ig21 in solution. Login to comment
195 In contrast, the corresponding S13F peptide does not bind to any of these repeats (supplemental Fig. 5). Login to comment
232 CSPs from 1 H/15 N HSQC spectra of selected 15 N-FlnA Ig repeats upon addition of 20-fold excess CFTR7-20-F16E or CFTR7-20-S13F/ F16E peptides. Login to comment
260 S13F-CFTR, which is unable to bind to filamins, exhibits reduced surface levels compared with wild-type CFTR. Login to comment
283 Substitution of Ser13 with phenylalanine disrupts the interaction between CFTR N-terminal peptides and all of the filamin Ig-like repeats that we tested because of steric clashes with the CD loop of the Ig repeats (supplemental Fig. 2). Login to comment
284 The S13F mutation is one of several mutations in the CFTR N terminus that are listed in the Cystic Fibrosis Mutation Database. Login to comment
287 Unlike the S13F mutation, the CF-causing defect in these CFTR mutants is probably not primarily due to disruption of filamin- CFTR interactions. Login to comment

[hide] Reduced PDZ interactions of rescued DeltaF508CFTR ... J Biol Chem. 2012 Dec 21;287(52):43630-8. doi: 10.1074/jbc.M112.421172. Epub 2012 Oct 31. Valentine CD, Lukacs GL, Verkman AS, Haggie PM
Reduced PDZ interactions of rescued DeltaF508CFTR increases its cell surface mobility.
J Biol Chem. 2012 Dec 21;287(52):43630-8. doi: 10.1074/jbc.M112.421172. Epub 2012 Oct 31., [PMID:23115232]

Abstract [show]
Deletion of phenylalanine 508 (DeltaF508) in the cystic fibrosis transmembrane conductance regulator (CFTR) plasma membrane chloride channel is the most common cause of cystic fibrosis (CF). Though several maneuvers can rescue endoplasmic reticulum-retained DeltaF508CFTR and promote its trafficking to the plasma membrane, rescued DeltaF508CFTR remains susceptible to quality control mechanisms that lead to accelerated endocytosis, ubiquitination, and lysosomal degradation. To investigate the role of scaffold protein interactions in rescued DeltaF508CFTR surface instability, the plasma membrane mobility of DeltaF508CFTR was measured in live cells by quantum dot single particle tracking. Following rescue by low temperature, chemical correctors, thapsigargin, or overexpression of GRASP55, DeltaF508CFTR diffusion was more rapid than that of wild-type CFTR because of reduced interactions with PDZ domain-containing scaffold proteins. Knock-down of the plasma membrane quality control proteins CHIP and Hsc70 partially restored DeltaF508CFTR-scaffold association. Quantitative comparisons of CFTR cell surface diffusion and endocytosis kinetics suggested an association between reduced scaffold binding and CFTR internalization. Our surface diffusion measurements in live cells indicate defective scaffold interactions of rescued DeltaF508CFTR at the cell surface, which may contribute to its defective peripheral processing.

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No. Sentence Comment
224 For CFTR, Thelin et al. (41) reported that the S13F mutation increased CFTR diffusion coefficient by only b03;20%, but increased internalization by b03;2-fold. Login to comment