ABCMdb

PMID: 20590134

Loo TW, Bartlett MC, Clarke DM
The V510D suppressor mutation stabilizes DeltaF508-CFTR at the cell surface.
Biochemistry. 2010 Aug 3;49(30):6352-7., 2010-08-03 [PubMed]

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No. Mutations Sentence Comment

0 ABCC7 p.Val510Asp pubs.acs.org/Biochemistry Published on Web 06/30/2010 r 2010 American Chemical Society 6352 Biochemistry 2010, 49, 6352-6357 DOI: 10.1021/bi100807h The V510D Suppressor Mutation Stabilizes ΔF508-CFTR at the Cell Surface† Tip W. Loo, M. Claire Bartlett, and David M. Clarke* Departments of Medicine and Biochemistry, University of Toronto, Toronto, Ontario M5S 1A8, Canada Received March 30, 2010; Revised Manuscript Received June 29, 2010 ABSTRACT: Deletion of Phe508 (ΔF508) in the first nucleotide-binding domain (NBD1) of CFTR causes cystic fibrosis. Login to comment 2 ABCC7 p.Val510Asp ABCC7 p.Val510Glu We found that replacement of Val510 with acidic residues (but not neutral or positive residues) promoted maturation of ΔF508-CFTR with V510D more efficiently than V510E. Login to comment 3 ABCC7 p.Val510Asp Promotion of ΔF508-CFTR maturation did not require NBD2 as introduction of V510D into a ΔNBD2/ΔF508-CFTR mutant restored maturation to levels similar to that of full-length protein. Login to comment 4 ABCC7 p.Val510Asp The V510D mutation increased the half-life of mature ΔF508-CFTR at the cell surface by about 5-fold to resemble the half-life of wild-type CFTR. Login to comment 5 ABCC7 p.Val510Asp ABCC7 p.Val510Arg ABCC7 p.Arg1070Ala ABCC7 p.Arg1070Asp It was also observed that introduction of the V510R/ R1070D mutations into ΔF508-CFTR also promoted maturation whereas the V510D/R1070A mutations did not. Login to comment 6 ABCC7 p.Val510Asp We propose that the V510D mutation in NBD1 promotes maturation and stabilizes ΔF508-CFTR at the cell surface through formation of a salt bridge with Arg1070 in TMD2. Login to comment 16 ABCC7 p.Val510Asp The V510D suppressor mutation is interesting because Val510 is predicted to reside at the domain-domain interface between NBD1 and TMD2 in close proximity to Phe508 (8). Login to comment 17 ABCC7 p.Val510Asp A negative charge appeared to be important because the V510D mutation promoted maturation of ΔF508-CFTR while mutation of Val510 to neutral amino acids (Cys, Gly, Ala, Ser, Asn, Pro, Thr, Tyr) did not (9). Login to comment 18 ABCC7 p.Val510Asp In this study, we tested whether the V510D mutation would reduce the turnover of ΔF508-CFTR at the cell surface. Login to comment 25 ABCC7 p.Val510Asp Generation of stable BHK cell lines expressing wild-type or mutant ΔF508/V510D-CFTRs was performed as described previously (9). Login to comment 27 ABCC7 p.Trp1145Cys ABCC7 p.Trp1145Cys ABCC7 p.Trp1145Cys ABCC7 p.Trp356Cys ABCC7 p.Trp356Cys ABCC7 p.Trp356Cys ABCC7 p.Val510Asp HEK 293 cells were transfected with W356C/W1145C-, ΔF508/W356C/W1145C-, or ΔF508/V510D/W356C/W1145C-CFTR cDNAs, and the cells were incubated for 4 h at 37 °C. Login to comment 40 ABCC7 p.Val510Asp clarke@utoronto.ca.1 Abbreviations: TM, transmembrane; NBD, nucleotide-binding domain; HEK, human embryonic kidney; BHK, baby hamster kidney. after oxidation of carbohydrate with sodium periodate (14) were performed using HEK 293 cells transfected with A52-tagged wild-type, ΔF508-, or ΔF508/V510D-CFTR cDNAs as described previously. Login to comment 45 ABCC7 p.Val510Asp Measurement of cAMP-stimulated iodide efflux was performed on baby hamster kidney (BHK) cells or BHK cells expressing wild-type or mutant ΔF508/V510D-CFTRs as described previously (9, 15). Login to comment 49 ABCC7 p.Val510Asp In a modeling study, it was predicted that the negative charge of the V510D mutation promoted maturation of ΔF508-CFTR be- causeitrestoresdefective NBD1-TMD2interactionsbyforming a salt bridge with Arg1070 (TMD2) (17). Login to comment 51 ABCC7 p.Val510Asp ABCC7 p.Val510Glu ABCC7 p.Val510Arg HEK 293 cells weretransfected withthe cDNAof wild-type CFTR (Figure 1E) or mutants ΔF508 (Figure 1, panels E-H), V510D/ΔF508 (Figure 1F), V510E/ΔF508 (Figure 1G), and V510R/ΔF508 (Figure 1H), and whole cell SDS extracts were subjected to immunoblot analysis 18 h after transfection. Login to comment 53 ABCC7 p.Val510Asp ABCC7 p.Val510Glu The V510E mutation also promoted maturation, but it was less efficient than the V510D mutation (12% and 35%, respectively) (Figure 1, panels F and G). Login to comment 54 ABCC7 p.Val510Arg Little mature CFTR was observed in mutant ΔF508 (Figure 1, panels E-H) or V510R/ΔF508 (Figure 1H). Login to comment 55 ABCC7 p.Val510Asp It was possible that the high level of expression of V510D/ ΔF508 in the transiently transfected HEK 293 cells was also a factor in enhancing maturation of the mutant. Login to comment 56 ABCC7 p.Val510Asp To examine the extent of maturation at lower levels of expression, HEK 293 cells were transfected with lower concentrations of plasmid containing V510D/ΔF508 cDNA and whole cell extracts subjected to immunoblot analysis 42 h after transfection. Login to comment 58 ABCC7 p.Val510Asp It was observed that expression of CFTR was reduced when cells were transfected with lower concentrations of plasmid(Figure1I) butthe relativesteady-state level of mature V510D/ΔF508-CFTR remained at about 50% of total CFTR (Figure 1J). Login to comment 59 ABCC7 p.Val510Asp To test if the mutant was active, BHK cell lines were generated that stably expressed wild-type or V510D/ΔF508-CFTRs for use in iodide efflux assays. Login to comment 60 ABCC7 p.Val510Asp The relative expression level of mature to total CFTR for mutant V510D/ΔF508 in BHK cells was also found to be about 50% (data not shown). Login to comment 62 ABCC7 p.Val510Asp Cells expressing wild-type or V510D/ΔF508-CFTRs exhibited iodide efflux upon addition of forskolin (Figure 2). Login to comment 64 ABCC7 p.Ile539Thr ABCC7 p.Gly550Glu ABCC7 p.Arg553Met ABCC7 p.Arg555Lys It was recently reported that rescue of ΔF508-CFTR byother suppressor mutations inNBD1(I539T,G550E,R553M, R555K) was drastically reduced in wild-type CFTR lacking NBD2 (ΔNBD2) (20). Login to comment 66 ABCC7 p.Val510Asp We introduced the V510D mutation into ΔF508-CFTR lacking NBD2 (truncated after residue 1196 and containing the epitope for monoclonal antibody A52 (Δ1197-1480)-A52) to test if it still promoted maturation. Login to comment 67 ABCC7 p.Val510Asp ABCC7 p.Val510Asp Immunoblot analysis of whole cell SDS extracts (Figure 3, left panel) showed that ΔF508/V510D/ΔNBD2-A52 (Δ1197-1480) had a maturation efficiency (about 50%) that was similar to that of the full-length ΔF508/V510D mutant (about 50%; Figure 1, panel F). Login to comment 71 ABCC7 p.Val510Glu ABCC7 p.Val510Arg Whole cell extracts of HEK 293 cells expressing full-length wild-type (E) or ΔF508-CFTRs (E-H) or ΔF508-CFTR containing the V510D(F), V510E (G), or V510R mutation (H) were subjected to immunoblot analysis 18 h after transfection. Login to comment 72 ABCC7 p.Val510Asp To test the maturation efficiency of ΔF508- and ΔF508/V510D-CFTRs at various levels of expression, HEK 293 cells were transfected with various levels of plasmid followed by immunoblot analysis and enhanced chemiluminescence 42 h after transfection (I). Login to comment 74 ABCC7 p.Val510Asp The positions of mature and immature CFTRs are These results suggest that the V510D suppressor mutation differs from other NBD1 suppressor mutations because the absence of NBD2 did not reduce its effect. Login to comment 77 ABCC7 p.Val510Asp To test if the position of the truncation affected maturation, mutant ΔF508/V510D/ΔNBD2-A52 (Δ1173-1480) was constructed and expressed in HEK293 cells. Login to comment 78 ABCC7 p.Val510Asp Immunoblot analysis of whole cell extracts showed that the V510D mutation promoted maturation of the truncation mutant but the relative level of mature was only about 20% of total CFTR protein. Login to comment 79 ABCC7 p.Val510Asp It was predicted in a modeling study (17) that the V510D mutation may promote NBD1-TMD2 interactions in ΔF508-CFTR by forming a salt bridge with positively charged residue Arg1070 (TMD2). Login to comment 80 ABCC7 p.Val510Asp ABCC7 p.Arg1070Ala We tested the contribution of Arg1070 by introducing the R1070A change intoΔF508/V510D. Login to comment 81 ABCC7 p.Val510Asp ABCC7 p.Arg1070Ala Asshownin Figure 4A, mature protein was not detectable in mutant ΔF508/ V510D/R1070A. Login to comment 82 ABCC7 p.Arg1070Ala ABCC7 p.Arg1070Ala To test if the R1070A mutation alone affected maturation of ΔF508, mutants ΔF508/R1070A and ΔF508 were expressed at 30 °C. Login to comment 84 ABCC7 p.Arg1070Ala Therefore, it was unlikely that the R1070A mutant caused further misfolding of ΔF508-CFTR. Login to comment 85 ABCC7 p.Arg1070Ala This is consistent with the observation that introduction of the R1070A mutation into wild-type CFTR also did not affect its maturation (data not shown). Login to comment 86 ABCC7 p.Val510Arg ABCC7 p.Arg1070Asp Mutant ΔF508/V510R/R1070D was constructed to reverse the positions of the charged residues. Login to comment 87 ABCC7 p.Val510Arg ABCC7 p.Arg1070Asp Figure 4C shows that the V510R/R1070D mutations promoted maturation of ΔF508-CFTR. Login to comment 88 ABCC7 p.Val510Arg ABCC7 p.Arg1070Asp Both mutations were required because the presence of V510R (Figure 1H) or R1070D (Figure 4D) alone did not promote maturation of ΔF508-CFTR. Login to comment 89 ABCC7 p.Val510Asp These results suggest that V510D may form a salt bridge with Arg1070 in ΔF508-CFTR. Login to comment 91 ABCC7 p.Val510Asp To test if the V510D mutation increased the stability of ΔF508-CFTR, a cross-linking assay was used to measure the half-life of mature CFTR at the cell surface. Login to comment 92 ABCC7 p.Trp1145Cys ABCC7 p.Trp356Cys We previously showed that cysteines introduced into TM segments 6 (W356C) and 12 (W1145C) could be cross-linked in mature CFTR when whole cells were treated with bifunctional thiol cross-linkers (14, 15). Login to comment 96 ABCC7 p.Trp1145Cys ABCC7 p.Trp356Cys ABCC7 p.Val510Asp Accordingly, the W356C and W1145C mutations were introduced into wild-type, ΔF508-, and ΔF508/V510D-CFTRs. Login to comment 100 ABCC7 p.Trp1145Cys ABCC7 p.Trp1145Cys ABCC7 p.Trp1145Cys ABCC7 p.Trp356Cys ABCC7 p.Trp356Cys ABCC7 p.Trp356Cys ABCC7 p.Val510Asp Panels A and B of Figure 5 show that the half-lives of cross-linked wild-type/W356C/W1145C-, ΔF508/ W356C/W1145C-, and ΔF508/V510D/W356C/W1145C-CFTRs were about 12,3,and 14 h, respectively. Login to comment 101 ABCC7 p.Val510Asp Theseresults indicatethat the stability of mutant V510D/ΔF508-CFTR resembled that of wild-type protein. Login to comment 103 ABCC7 p.Trp1145Cys ABCC7 p.Trp356Cys It was possible that cysteines W356C and W1145C may influence the stability of the protein. Login to comment 104 ABCC7 p.Val510Asp To examine the effect of the V510D mutation on turnover of ΔF508-CFTR without the introduced cysteines, a pulse-chase assay was performed. Login to comment 106 ABCC7 p.Val510Asp By contrast, the conversion of immature to mature form in mutant ΔF508/V510D was slower since there wasconsiderableamountofimmatureCFTRstillpresentat2-4h (Figure 6A, bottom panel). Login to comment 108 ABCC7 p.Val510Asp ABCC7 p.Val510Asp After an 8 h chase, the majority of labeled CFTR in wild-type and mutant ΔF508/V510D was present as the mature protein FIGURE 2: Iodide efflux activity of wild-type and ΔF508/V510D-CFTR. Login to comment 109 ABCC7 p.Val510Asp Iodide efflux assays were performed on BHK cells stably expressing wild-type CFTR, mutant ΔF508/V510D, or no CFTR (control). Login to comment 112 ABCC7 p.Val510Asp FIGURE 3: Effect of V510D on maturation of ΔF508/ΔNBD2 mutants. Login to comment 118 ABCC7 p.Val510Asp Therefore, we used this time point to compare the lifetimes of the mature protein in wild-type and mutant ΔF508/ V510D. Login to comment 120 ABCC7 p.Val510Asp The results indicate that V510D stabilizes ΔF508-CFTR. Login to comment 121 ABCC7 p.Val510Asp ABCC7 p.Val510Asp To test if V510D stabilizes ΔF508-CFTR at the cell surface, cells expressing wild-type, ΔF508-, or ΔF508/V510D-CFTRs werefirst incubated at 30°C for 18 h sothatΔF508-CFTR would be present at the cell surface. Login to comment 125 ABCC7 p.Val510Asp It was found that mature forms of wild-type or ΔF508/V510D-CFTRs had similar half-lives of about 12 h while ΔF508-CFTR had a short half-life of about 2 h (Figure 7). Login to comment 128 ABCC7 p.Val510Asp This study showed that the V510D mutation increased the cell surface stability of mature ΔF508-CFTR to levels that were similar to the wild-type protein (Figures 5-7). Login to comment 129 ABCC7 p.Ile539Thr ABCC7 p.Gly550Glu ABCC7 p.Arg553Met ABCC7 p.Arg555Lys A similar effect was observed when the combination of four NBD1 suppressormutations(I539T,G550E,R553M,R555K) was introduced into ΔF508-CFTR (20). Login to comment 131 ABCC7 p.Trp1145Cys ABCC7 p.Trp356Cys ABCC7 p.Val510Asp HEK 293 cells expressing wild-type, ΔF508-, or ΔF508/ V510D-CFTRs containing the W356C and W1145C cysteines were treated with the thiol cross-linker BMH. Login to comment 134 ABCC7 p.Val510Asp HEK 293 cells were transfected with A52-tagged wild-type, ΔF508, or ΔF508/V510D cDNAs. Login to comment 141 ABCC7 p.Val510Asp HEK 293 cells were transfected with the cDNAs of A52-tagged wild-type, ΔF508-, or ΔF508/V510D-CFTR. Login to comment 142 ABCC7 p.Val510Asp To compare the half-life of ΔF508-CFTR to wild-type or mutant ΔF508/V510D-CFTR, the cells were first incubated at 30 °C to promote maturation and increase the level of ΔF508-CFTR at the cell surface. Login to comment 148 ABCC7 p.Val510Asp The position of mature CFTR is had half-lives of about 12 h. Although the effects of these suppressor mutations on full-length CFTR were similar to that of V510D, there were different effects on NBD2 deletion mutants. Login to comment 150 ABCC7 p.Val510Asp The V510D mutation (present study) appeared to restore stability by promoting NBD1-TMD2 interactions in ΔF508-CFTR since deletion of NBD2 had little effect (Figure 3; Δ1197-1480). Login to comment 156 ABCC7 p.Val510Asp ABCC7 p.Val510Asp Other evidence suggesting that ΔF508/ΔNBD2 (Δ1173-1480) was more difficult to rescue was the observation that introduction of V510D into the mutant yielded lower levels of mature protein compared to the ΔF508/ΔNBD2/V510D (Δ1197-1480) construct (Figure 3). Login to comment 158 ABCC7 p.Val510Asp ABCC7 p.Val510Arg ABCC7 p.Arg1070Asp Mutation of Arg1070 to a neutral amino acid abolished V510D rescue of ΔF508-CFTR while V510R only rescued the mutant when the R1070D change was introduced (Figure 4C). Login to comment 160 ABCC7 p.Arg347Asp ABCC7 p.Asp924Arg It was found that mutation of Arg347 to neutral amino acids or Asp destabilized channel function but the D924R mutation complemented R347D to yield a channel that behaved like wild-type CFTR. Login to comment 173 ABCC7 p.Val510Asp ABCC7 p.Val510Asp In this study we found that the ΔF508/V510D mutant was active while we failed to detect activity when V510D was introduced into Cys-less CFTR (9). Login to comment 174 ABCC7 p.Val510Asp Although Cys-less/V510D-CFTR matured, its chloride channel activity was below the limits ofdetection inthe iodide efflux assay. Login to comment 178 ABCC7 p.Val510Cys ABCC7 p.Val510Asp ABCC7 p.Val510Thr ABCC7 p.Val510Gly While introduction of V510D into Cys-less or ΔF508-CFTRs promoted maturation, the Cys-less mutant was different because its maturation could also be promoted by changing Val510 to Thr, Cys, Gly, Ala, or Ser. Login to comment 180 ABCC7 p.Val510Asp In summary, the V510D mutation increases the stability of ΔF508-CFTR by promoting NBD1-TMD2 interactions likely through formation of a salt bridge with Arg1070 in ICL4. Login to comment