ABCMdb

ABCC7 p.Gly723*

None has been submitted yet.

Publications

PMID: 10933805 [PubMed] King SA et al: "R-domain interactions with distal regions of CFTR lead to phosphorylation and activation."

No. Sentence Comment

27 1 Abbreviations: CF, cystic fibrosis; CFTR, cystic fibrosis transmembrane conductance regulator; ABC, ATP binding cassette; NBD, nucleotide binding domain; TMD, transmembrane domain; R-domain, regulatory domain; PKA, cyclic AMP-dependent protein kinase A; His P, NBD of histidine permease; His Q and His M, TMDs of histidine permease; Mal K, NBD region of the maltose transport system; Mal F, integral membrane protein of the maltose transport system; AMP, adenosine monophosphate; ∆R-CFTR, CFTR lacking amino acids 708-835; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; DMEM, Dulbecco`s Modified Eagle`s Medium; FBS, fetal bovine serum; vTF7.3, vaccina virus encoding the T7 polymerase; MOI, multiplicity of infection; DOC, deoxycholic acid; PVDF, poly- (vinylidene difluoride); NBT, 4-nitroblue tetrazolium chloride; SPQ, 6-methoxy-N-(3-sulfopropyl)quinolonium; BCIP, 5-bromo-4-chloro-3-indolyl phosphate; -gal, -galactosidase; ∆1-836, carboxy hemi-CFTR beginning immediately after the R-domain; M837X, CFTR truncation that ends at CFTR position 836, after the R-domain; G723X, CFTR truncation ending at residue 722 within the R-domain; K593X, CFTR truncation ending immediately before the R-domain at position 592. Login to comment
53 The resulting product was inserted between the SphI and StuI sites of pTM-CFTR, to place a premature stop codon in place of CFTR amino acid 837. pTM-G723X was constructed using a similar strategy but with a stop codon in place of the glycine at residue 723. Login to comment
173 G723X (missing the 114 C-terminal amino acids of the R-domain, Table 1) was tested for the phosphorylation-dependent mobility shift following coexpression with ∆1-836. Login to comment
174 While G723X co-immunoprecipitated with ∆1-836 in a manner similar to that of M837X, this interaction did not result in a mobility shift of the G723X protein (Figure 7A, compare lanes 3 and 4). Login to comment
175 Furthermore, no molecular weight shift of G723X could be detected when cells were treated with forskolin prior to lysis (Figure 7B, lane 4). Login to comment
176 G723X and ∆1-836 expressed together produced constitutive (unregulated) activity (Figure 8A). Login to comment
179 K593X (CFTR truncated immediately before the R-domain, Table 1) co-immunoprecipitated with ∆1-836 in a manner similar to that of M837X or G723X (data not shown), but coexpression of K593X with ∆1-836 failed to produce enhanced halide efflux (Figure 8A). Login to comment
188 A shorter amino truncation (G723X) also tightly bound to ∆1-836. Login to comment
189 In contrast to M837X, G723X failed to exhibit a reduced mobility on SDS-PAGE after either coexpression with ∆1FIGURE 6: Halide permeability produced by M837X and ∆1-836 is PKA-dependent. Login to comment
197 FIGURE 7: G723X binds ∆1-836, but this interaction does not elicit a mobility shift of G723X. Login to comment
199 While both G723X and M837X bind ∆1-836 (lanes 3 and 4), only the interaction of M837X with ∆1-836 produced a higher-molecular weight phosphorylated protein (*, lane 3). Login to comment
200 (B) Cells expressing M837X or G723X were treated with forskolin prior to lysis (as described in the legend of Figure 4). Login to comment
202 However, forskolin treament of cells expressing G723X did not result in a higher-molecular weight protein (lane 4). Login to comment
211 G723X also produced high basal halide efflux when it was coexpressed with ∆1-836 (Figure 8A). Login to comment
228 FIGURE 8: G723X coexpression with ∆1-836 produces enhanced halide efflux, while K593X does not. Login to comment
229 (A) G723X and ∆1-836 (9) or K593X and ∆1-836 (b) were expressed in COS7 cells, and halide efflux was assayed as described in the legend of Figure 5. Login to comment
230 G723X produced high basal halide permeability when coexpressed with ∆1-836 (p < 0.01) similar to that of M837X with ∆1-836 (Figure 5). Login to comment
234 (B) M837X and ∆1-836 (0) or G723X and ∆1-836 (O) were expressed in COS7 cells and assayed for halide movement as described in the legend of Figure 6. Login to comment
235 Rp-8-CPT- cAMPS inhibited the enhanced halide permeability of M837X and ∆1-836 (9, p < 0.05), but not that of G723X and ∆1-836 (b). Login to comment