ABCMdb

PMID: 9790686

Clancy JP, Hong JS, Bebok Z, King SA, Demolombe S, Bedwell DM, Sorscher EJ
Cystic fibrosis transmembrane conductance regulator (CFTR) nucleotide-binding domain 1 (NBD-1) and CFTR truncated within NBD-1 target to the epithelial plasma membrane and increase anion permeability.
Biochemistry. 1998 Oct 27;37(43):15222-30., 1998-10-27 [PubMed]

Sentences

No. Mutations Sentence Comment

4 ABCC7 p.Arg553* ABCC7 p.Gly542* Furthermore, when clinically relevant CFTR proteins truncated within NBD-1 (R553X or G542X) are expressed, surface localization and enhanced halide permeability are again established. Login to comment 32 ABCC7 p.Arg553* ABCC7 p.Gly542* Expression of CFTR containing the clinically relevant mutations R553X or G542X, which each contain >70% of the NBD-1 motif, produce polypeptides that also target the cell membrane and enhance halide permeability in a manner similar to isolated NBD-1. Login to comment 45 ABCC7 p.Arg553* ABCC7 p.Gly542* Recombinant vaccinia viruses containing wtCFTR and CFTR containing a premature stop codon at position 542 (G542X) or 553 (R553X) under the regulatory control of the T7 promoter was generated from constructs in the pTM1 vector (15) using standard techniques (16). Login to comment 75 ABCC7 p.Arg553* ABCC7 p.Gly542* Immunoprecipitation of NBD-1, G542X, R553X, and wtCFTR. Login to comment 87 ABCC7 p.Arg553* ABCC7 p.Gly542* Digital Confocal Immunofluorescent Microscopy of COS-7 Cells Expressing wt or ∆F508 CFTR, G542X, or R553X. Login to comment 157 ABCC7 p.Arg553* ABCC7 p.Gly542* Expression of R553X and G542X CFTR in Mammalian Epithelial Cell Lines. Login to comment 160 ABCC7 p.Arg553* ABCC7 p.Gly542* We chose to study two clinically relevant mutant CFTR molecules possessing premature stop codons, G542X and R553X. Login to comment 161 ABCC7 p.Arg553* ABCC7 p.Gly542* These mutations are found in approximately 5% of the CF population (34) and include 72% (G542X) to 79% (R553X) of the NBD-1 (aa 432-586). Login to comment 162 ABCC7 p.Arg553* ABCC7 p.Gly542* Figure 6 shows detection of 35 S-labeled truncated proteins [~53 kDa (G542X); ~55 kDa (R553X)] from COS-7 cell lysates following vaccinia-based expression. Login to comment 176 ABCC7 p.Arg553* ABCC7 p.Gly542* This antibody recognizes an epitope in the first predicted extracellular loop of CFTR TM-1, which is shared by wtCFTR, R553X, and G542X. Login to comment 179 ABCC7 p.Arg553* ABCC7 p.Gly542* Figure 7 indicates a plasma membrane staining pattern in cells without detergent permeabilization following wtCFTR, R553X, and G542X expression, but not ∆F508 CFTR. Login to comment 181 ABCC7 p.Arg553* ABCC7 p.Gly542* These studies provide evidence of plasma membrane targeting by CFTR possessing either the G542X or R553X mutations. Login to comment 182 ABCC7 p.Arg553* ABCC7 p.Gly542* To determine whether either R553X or G542X CFTR had positive effects on basal cellular halide permeability, we performed SPQ analysis in COS-7 cells expressing these mutant cDNAs (Figure 8). Login to comment 185 ABCC7 p.Arg553* ABCC7 p.Gly542* The effects of R553X, G542X, and NBD-1 on COS-7 cell basal halide permeability were qualitatively and quantitatively similar (compare with Figure 5B). Login to comment 199 ABCC7 p.Arg553* ABCC7 p.Arg553* ABCC7 p.Gly542* ABCC7 p.Gly542* FIGURE 6: Immunoblot of wt, R553X, and G542X CFTR in COS-7 cells. Cells were labeled with translabel for 20 min at 12 h after infection and immunoprecipitated with anti-NBD-1 antibody as described in the Experimental Procedures. Lane A ) wtCFTR, lane B ) G542X, lane C ) R553X. Login to comment 202 ABCC7 p.Arg553* ABCC7 p.Gly542* Expression of truncated CFTR (R553X CFTR and G542X CFTR, Figures 6, 7, and 8) produces a protein which also targets the eukaryotic plasma membrane, enhancing cellular basal halide permeability. Login to comment 203 ABCC7 p.Arg553* ABCC7 p.Gly542* Neither R553X nor G542X produces cAMP-regulated halide permeability, and no full-length CFTR could be detected by immunoprecipitation, indicating that suppression of the premature termination codons is not responsible for the ion transport results shown here (39, 40). Login to comment 204 ABCC7 p.Arg553* ABCC7 p.Gly542* The enhanced basal halide permeability conferred by R553X and G542X is qualitatively and quantitatively similar to that produced by NBD-1 (Figure 5). Login to comment 205 ABCC7 p.Arg553* ABCC7 p.Gly542* We suggest that the truncated CFTR proteins G542X and R553X contain adequate domains and the appropriate cellular signals to fold into functional peptides and localize to the cell membrane. Login to comment 206 ABCC7 p.Arg553* ABCC7 p.Gly542* The common halide permeability enhancing effects produced by G542X, R553X, and isolated NBD-1 suggest that these three polypeptides share common amino acids (422-542 of the complete CFTR molecule) which may be able to activate halide permeability in several epithelial cell types. Login to comment 207 ABCC7 p.Arg553* ABCC7 p.Gly542* On the basis of the findings that R553X and G542X retain surface localizing and residual halide transport function, we recently studied five patients possessing at least one of these alleles, but found no evidence of residual Cl-secretion during a nasal potential difference protocol (25). Login to comment 211 ABCC7 p.Arg553* ABCC7 p.Gly542* In either case, our results clearly establish that, unlike class II CFTR mutations (34), R553X and G542X CFTR are capable of escaping intracellular degradation, targetting the plasma membrane, and forming functional proteins that maintain some residual halide transport function. Login to comment 213 ABCC7 p.Arg553* ABCC7 p.Gly542* FIGURE 7: Localization of wtCFTR, R553X, and G542X to the cell surface in COS-7 cells by confocal immunocytochemistry. Login to comment 217 ABCC7 p.Arg553* ABCC7 p.Gly542* Lower panels from left to right are (D) G542X without permeabilization, (E) R553X without permeabilization, and (F) vT7 controls with permeabilization. Login to comment 218 ABCC7 p.Arg553* ABCC7 p.Gly542* FIGURE 8: R553X or G542X expression increases halide permeability in COS-7 cells. Cells grown on glass coverslips were studied by SPQ as in Figure 5. Login to comment 221 ABCC7 p.Gly542* (A) Dequench of the G542X expressing cells was significantly elevated above control cells (P < 0.0001 compared with vT7 or LacZ conditions). Login to comment 222 ABCC7 p.Arg553* (B) Dequench of the R553X expressing cells was significantly elevated above controls (P < 0.0001 compared with vT7 expressing cells). Login to comment 227 ABCC7 p.Asp836* On the other hand, Sheppard et al. have demonstrated that a truncated CFTR molecule containing TM-1, NBD-1, and the R domain (D836X) remains capable of forming anion-selective channels with phosphorylation-dependent regulation (49). Login to comment