ABCMdb

PMID: 20667826

Thibodeau PH, Richardson JM 3rd, Wang W, Millen L, Watson J, Mendoza JL, Du K, Fischman S, Senderowitz H, Lukacs GL, Kirk K, Thomas PJ
The cystic fibrosis-causing mutation deltaF508 affects multiple steps in cystic fibrosis transmembrane conductance regulator biogenesis.
J Biol Chem. 2010 Nov 12;285(46):35825-35. Epub 2010 Jul 28., 2010-11-12 [PubMed]

Sentences

No. Mutations Sentence Comment

19 ABCC7 p.Arg553Gln A single mutation, R553Q, was first identified in a patient homozygous for the ⌬F508 allele but having only a mild CF phenotype (18). Login to comment 20 ABCC7 p.Arg553Gln ABCC7 p.Ile539Thr Subsequently, in a screen for suppressor mutations of the ⌬F508 defect, the original R553Q suppressor mutation was identified as were I539T, * Thisworkwassupported,inwholeorinpart,byNationalInstitutesofHealth NIDDK Grants 49835 (to P. J. T.) and 75302 (to G. L. L.). Login to comment 34 ABCC7 p.Arg553Gln ABCC7 p.Gly550Glu ABCC7 p.Arg555Lys Printed in the U.S.A. NOVEMBER 12, 2010•VOLUME 285•NUMBER 46 JOURNAL OF BIOLOGICAL CHEMISTRY 35825 G550E, R553Q, and R555K (19-21). Login to comment 61 ABCC7 p.Phe508Lys In addition, the ability of a novel second-site suppressor, located within TMD2, to rescue the ⌬F508 and F508K mutants demonstrates that proper domain-domain assembly is critical to CFTR maturation. Login to comment 104 ABCC7 p.Arg553Gln ABCC7 p.Gly550Glu ABCC7 p.Arg553Met ABCC7 p.Arg555Lys The introduction of the single mutations, G550E, R553M or R553Q, and R555K, has previously been shown to partially rescue the ⌬F508 trafficking defect in CFTR and restore channel activity at the plasma membrane (Fig. 1A) (19-21). Login to comment 114 ABCC7 p.Phe508Asp ABCC7 p.Phe508Lys The inclusion of the -3M mutations failed to significantly rescue the folding of the F508D and F508K mutants, suggesting that the -3M suppressors do not directly influence the interaction between NBD1 and other domains of CFTR (Fig. 1B). Login to comment 115 ABCC7 p.Phe508Cys ABCC7 p.Phe508Ala Consistent with this result, the introduction of the -3M mutations onto F508A and F508C had little effect on protein maturation. Login to comment 116 ABCC7 p.Phe508Pro Interestingly, the -3M mutations rescued the folding and maturation of the F508P protein, ⌬F508 Perturbs Multiple Steps in CFTR Biogenesis NOVEMBER 12, 2010•VOLUME 285•NUMBER 46 JOURNAL OF BIOLOGICAL CHEMISTRY 35827 which has previously been reported to be refractory to low temperature rescue (12). Login to comment 131 ABCC7 p.Arg555Lys Disruption of the RXR by substitution of Arg-555 with lysine showed no discernible effects on wild type CFTR maturation. Login to comment 142 ABCC7 p.Gly550Glu ABCC7 p.Arg553Met ABCC7 p.Arg555Lys The introduction of the -3M mutations (G550E, R553M, R555K) rescues the trafficking defects associated with the ⌬F508 mutation and restores near wild type function. Login to comment 178 ABCC7 p.Arg555Lys ABCC7 p.Arg555Thr ABCC7 p.Arg555Gly ABCC7 p.Arg555Ala The substitution of R555A, R555G, and R555T resulted in a marked reduction in the formation of band C CFTR, whereas the R555K, as measured by Western blotting of transiently transfected HEK-293 cells displays near wild type CFTR maturation. Login to comment 200 ABCC7 p.Lys1250Ala ABCC7 p.Lys464Ala Mutations of the Walker A lysine (K464A and K1250A in NBD1 and NBD2, respectively) have been shown to dramatically decrease ATP affinity (40). Login to comment 201 ABCC7 p.Glu1371Gln Conversely, mutation of the catalytic glutamate to glutamine in NBD2, E1371Q, has previously been shown to stabilize NBD dimers by trapping ATP at the NBD-NBD interface (39). Login to comment 204 ABCC7 p.Glu1371Gln Stabilization of the putative NBD1-NBD2 dimer via the E1371Q mutation did not facilitate the trafficking of the ⌬F508 protein and had no discernible effect on the maturation of the wild type protein. Login to comment 205 ABCC7 p.Lys1250Ala Similarly, the introduction of the K1250A mutation had minimal effects on the maturation of wild type CFTR and failed to rescue the ⌬F508 CFTR protein. Login to comment 206 ABCC7 p.Lys464Ala The NBD1 K464A mutation also failed to rescue ⌬F508 trafficking. Login to comment 207 ABCC7 p.Lys464Ala However, when introduced into the wild type background, the K464A reduced CFTR maturation, as evidenced by a decrease in band C. Login to comment 228 ABCC7 p.Arg1070Trp The substitution of R1070W had little effect on the maturation of wild type CFTR but measurably promoted trafficking of ⌬F508 CFTR (Fig. 6B). Login to comment 230 ABCC7 p.Arg1070Trp ABCC7 p.Phe508Lys To evaluate the potential mechanisms by which the R1070W mutation rescued ⌬F508, this mutation was also introduced into the ⌬F508-3M and F508K backgrounds (Fig. 6, C and D). Login to comment 231 ABCC7 p.Phe508Lys F508K is expected to disrupt the interdomain interaction, as it interferes with maturation but does not affect the isolated NBD1 (26). Login to comment 232 ABCC7 p.Arg1070Trp ABCC7 p.Arg1070Trp The combination of the -3M mutations with the R1070W mutation increased ⌬F508 Band C production, as compared with ⌬F508-3M and ⌬F508/ R1070W alone. Login to comment 234 ABCC7 p.Arg1070Trp ABCC7 p.Phe508Lys In this regard, the R1070W mutation induced the formation of Band C in the F508K mutant predicted to disrupt the interdomain interaction, an effect not seen for low temperature or with the -3M mutations (Fig. 6D). Login to comment 250 ABCC7 p.Phe508Asp ABCC7 p.Phe508Pro ABCC7 p.Phe508Lys The suppression of the ⌬F508 and F508P substitutions, but not the F508D and F508K mutants, indicates that these mutations alter CFTR folding by discrete mechanisms or are of differing severities. Login to comment 254 ABCC7 p.Phe508Ser ABCC7 p.Phe508Ser ABCC7 p.Phe508Arg ABCC7 p.Phe508Arg Consistent with this, structures of NBD1 F508S and F508R and trafficking of F508S and F508R full-length CFTR demonstrate that the severity of physicochemical alterations at the 508 position correlate with protein trafficking (12, 26). Login to comment 280 ABCC7 p.Lys464Ala B, mutation of the composite ATP-binding site in NBD1, K464A, adversely affects the trafficking of wild type CFTR. Login to comment 281 ABCC7 p.Lys1250Ala Mutation of the equivalent position in NBD2, K1250A, has minimal effect on CFTR maturation. Login to comment 282 ABCC7 p.Glu1371Gln The NBD-dimer stabilizing mutation, E1371Q, does not dramatically alter the trafficking of the wild type or ⌬F508 CFTR proteins when expressed transiently in HEK-293 cells. Login to comment 293 ABCC7 p.Arg555Ala The decrease in wild type CFTR trafficking seen with the R555A/ G/T demonstrates that the basic side chain at the 555 locus is required for proper trafficking. Login to comment 294 ABCC7 p.Arg555Ala The loss of CFTR trafficking with the R555A/G/T mutations suggests this site defines more than a simple signal motif. Login to comment 295 ABCC7 p.Arg555Gly ABCC7 p.Arg555Gly ABCC7 p.Tyr1307* Consistent with these data, the R555G mutation has previously been identified in a heterozygous CF patient (R555G/Y1307X). Login to comment 305 ABCC7 p.Glu1371Gln Stabilization of the NBD heterodimer by the E1371Q mutation had no discernible effect on wild type or ⌬F508 maturation (Fig. 5B). Login to comment 306 ABCC7 p.Glu1371Gln The failure of the E1371Q mutant to rescue ⌬F508 CFTR is consistent with ⌬F508 influence on the early step of NBD1 folding and that FIGURE 6. Login to comment 308 ABCC7 p.Phe508Lys Mutations in ICL4 at position 1070 were evaluated for effects on the trafficking of wild type, ⌬F508, and F508K CFTR, transiently expressed in HEK293 cells. Login to comment 313 ABCC7 p.Phe508Lys Two views, rotated by 90 degrees are shown. B, Western blots show the effects of the ICL4 Arg-1070 mutations on the trafficking of wild type, ⌬F508, and F508K CFTR. Login to comment 314 ABCC7 p.Arg1070Trp C, the R1070W and -3M suppressor mutations were evaluated for their ability to rescue the ⌬F508 mutation either independently or in combination. Login to comment 315 ABCC7 p.Arg1070Trp The inclusion of the -3M and R1070W mutations in combination rescued more ⌬F508 CFTRthaneithersuppressorsetalone.Cellswereculturedat37 °CandevaluatedbyWesternblottingusingthe M3A7 antibody. Login to comment 316 ABCC7 p.Arg1070Trp ABCC7 p.Phe508Lys D, trafficking of the F508K missense protein was evaluated with the R1070W mutation. Login to comment 317 ABCC7 p.Arg1070Trp ABCC7 p.Phe508Lys Trafficking of F508K was partially rescued by the R1070W mutation. Login to comment 325 ABCC7 p.Arg1070Trp The R1070W mutant in TMD2 suppresses ⌬F508 by promoting the interactions between NBD1 and ICL4 as required for maturation. Login to comment 326 ABCC7 p.Arg1070Trp By relieving the QC-NBD interactions, the -3M and R1070W mutations promote CFTR maturation; upper pathway. Login to comment 329 ABCC7 p.Lys1250Ala As well, disruption of the composite ATP-binding site in NBD2 by the K1250A mutant had no discernible effect on CFTR maturation. Login to comment 330 ABCC7 p.Lys464Ala In contrast, the K464A NBD1 ATP-binding mutant decreased wild type CFTR maturation. Login to comment 339 ABCC7 p.Arg1070Trp Although both the Arg-1070 and -3M suppressors rescue ⌬F508, suppression by the R1070W mutation is likely accomplished by independent mechanisms. Login to comment 341 ABCC7 p.Arg1070Trp Thus, the R1070W mutation putatively promotes appropriate domain-domain associations by increasing hydrophobic interactions (affinity) at the NBD1-ICL4 domain-domain interaction surface. Login to comment 342 ABCC7 p.Arg1070Trp ABCC7 p.Arg1070Trp The relatively hydrophobic surface proximal to the Phe508 position could potentially accommodate the hydrophobicity and volume of the R1070W substitution. Login to comment 343 ABCC7 p.Arg1070Lys Decreases in side-chain volume (Ala/Gly/Thr substitution) and the presence of charge (Arg-1070 and R1070K) fail to facilitate ⌬F508 trafficking. Login to comment 344 ABCC7 p.Arg1070Trp ABCC7 p.Phe508Lys Maturation of the F508K CFTR molecule was potentially facilitated by interactions between the indole side chain from R1070W and the NBD1 surface. Login to comment 346 ABCC7 p.Arg1070Trp Intriguingly, the R1070W mutation, which rescues ⌬F508 CFTR, has been identified in patients with mild disease (congenital bilateral absence of the vas deferens, pancreatic sufficient CF). Login to comment 347 ABCC7 p.Arg1070Trp Previous studies suggest that in the wild type background the R1070W mutation alters protein expression, localization, and function (44). Login to comment 355 ABCC7 p.Glu1371Gln Acknowledgments-We thank David Gadsby for suggesting the use of the NBD dimer stabilizing E1371Q mutation and members of the Thomas laboratory for critical comments and helpful suggestions. Login to comment