ABCMdb

PMID: 17132688

Wang Y, Loo TW, Bartlett MC, Clarke DM
Modulating the folding of P-glycoprotein and cystic fibrosis transmembrane conductance regulator truncation mutants with pharmacological chaperones.
Mol Pharmacol. 2007 Mar;71(3):751-8. Epub 2006 Nov 28., [PubMed]

Sentences

No. Mutations Sentence Comment

55 ABCC7 p.Gln1071Pro Cell surface labeling of ⌬NBD2/Q1071P CFTR was performed as described previously (Loo et al., 2006c). Login to comment 93 ABCC7 p.Gln1071Pro The position of the Q1071P mutation in ⌬NBD2 CFTR is indicated. Login to comment 139 ABCC7 p.Gln1071Pro Therefore, the Q1071P processing mutation was introduced into the ⌬NBD2 CFTR. Login to comment 140 ABCC7 p.Gln1071Pro The Q1071P processing mutation was selected because it is located in the fourth cytoplasmic loop (see Fig. 1B), and it was postulated to inhibit maturation because it disrupted NBD2-TMD2 interactions (Seibert et al., 1996). Login to comment 141 ABCC7 p.Gln1071Pro Because NBD2 is no longer present in ⌬NBD2 CFTR, we can now test whether the Q1071P mutation would still inhibit maturation. Login to comment 142 ABCC7 p.Gln1071Pro ABCC7 p.Gln1071Pro It was found that the processing mutant Q1071P could still inhibit maturation of CFTR lacking NBD2 (⌬NBD2/Q1071P, Fig. 5B). Login to comment 143 ABCC7 p.Gln1071Pro Therefore, the Q1071P mutation seems to interfere in the folding of TMD2. Login to comment 144 ABCC7 p.Gln1071Pro Maturation of the ⌬NBD2/Q1071P CFTR mutant could be promoted, however, when expression was carried out in the presence of VRT-325 or corr-4a (Fig. 5B). Login to comment 146 ABCC7 p.Gln1071Pro The conversion of ⌬NBD2/Q1071P CFTR to an endoglycosidase H-resistant protein after expression in the presence of correctors indicates that it reached the Golgi. Login to comment 147 ABCC7 p.Gln1071Pro To determine whether expression in the presence of correctors would increase the level of ⌬NBD2/Q1071P at the plasma membrane, cell surface labeling was carried out. Login to comment 148 ABCC7 p.Gln1071Pro HEK 293 cells transfected with ⌬NBD2/Q1071P cDNA containing an A52-epitope tag were expressed in the presence or absence of 10 ␮M VRT-325 or corr-4a. Login to comment 157 ABCC7 p.Gln1071Pro ABCC7 p.Gln1071Pro B, CFTR truncation mutants ⌬NBD2 CFTR or ⌬NBD2/Q1071P containing C-terminal A52 tags were expressed in the presence of 10 ␮M VRT-325 (325) or 10 ␮M corr-4a or no corrector (-) for 24 h. Whole-cell extracts were then subjected to immunoblot analysis with monoclonal antibody A52. C, the concentration-dependence of maturation of ⌬NBD2/Q1071P CFTR was determined by expressing the mutant in the indicated concentrations of corr-4a (4a) for 24 h followed by immunoblot analysis of whole-cell extracts. Login to comment 158 ABCC7 p.Gln1071Pro D, HEK 293 cells transfected with A52-tagged ⌬NBD2/Q1071P CFTR cDNA were grown in the presence (ϩ) or absence (-) of 10 ␮M VRT-325 (325) or 10 ␮M corr-4a (4a) for 24 h. Cell-surface labeling was then performed on whole cells using biotin-LC-hydrazide after oxidation of surface carbohydrates. Login to comment 164 ABCC7 p.Gln1071Pro Figure 5D shows that cell surface expression of ⌬NBD2/Q1071P was increased after expression in the presence of VRT-325 or corr-4a. Login to comment 165 ABCC7 p.Gln1071Pro A control immunoblot developed with monoclonal antibody A52 shows that similar levels of ⌬NBD2/Q1071P protein were recovered after immunoprecipitation (Fig. 5E). Login to comment 220 ABCC7 p.Gln1071Pro It is apparent, however, that NBD1-NBD2 or NBD2-TMD2 interactions are not essential for rescue with VRT-325 or corr-4a because both compounds could promote maturation of ⌬NBD2/Q1071P CFTR. Login to comment