ABCMdb

ABCB1 p.Gly268Val

None has been submitted yet.

Publications

PMID: 16196493 [PubMed] Loo TW et al: "Rescue of DeltaF508 and other misprocessed CFTR mutants by a novel quinazoline compound."

No. Sentence Comment

149 An explanation for the ability of CFcor-325 to promote folding and trafficking of misprocessed CFTR mutants is that it diffused into the ER and directly interacts with the mutant protein during biogenesis as observed with P-gp.31,32 Another explanation is that CFcor-325 has a nonspecific effect on protein folding similar to that observed when low temperature or osmolytes such as glycerol or trimethylamine N-oxide are used to rescue misfolded proteins.9,10 To test whether CFcor-325 had a direct or indirect effect on protein folding, we used the P-gp processing mutant G268V as a control.33 We used the G268V P-gp mutant, rather than mutant ∆Y490 P-gp (equivalent to ∆F508 in CFTR), as a control because maturation of the G268V mutant is not promoted by growth at low temperature (27 °C) or by osmolytes such as glycerol and trimethylamine N-oxide (unpublished observations). Login to comment
150 Maturation of mutant G268V to yield active enzyme at the cell surface, however, can be achieved by carrying out expression in the presence of drug substrates or modulators.34 Accordingly, BHK cells stably expressing P-gp mutant G268V were incubated with 0-10 µM CFcor-325 for 48 h. Login to comment
164 Effect of CFcor-325 on expression of misprocessed P-gp mutant G268V and on P-gp-mediated drug resistance. Login to comment
165 (A) BHK cells stably expressing P-gp mutant G268V were treated for 48 h with 0-10 µM CFcor-325. Whole cell extracts were then subjected to immunoblot analysis with rabbit polyclonal antibody against P-gp. Login to comment
170 The ability of CFcor-325 to rescue P-gp mutant G268V suggested that it was a substrate or modulator of P-gp. Login to comment
147 An explanation for the ability of CFcor-325 to promote folding and trafficking of misprocessed CFTR mutants is that it diffused into the ER and directly interacts with the mutant protein during biogenesis as observed with P-gp.31,32 Another explanation is that CFcor-325 has a nonspecific effect on protein folding similar to that observed when low temperature or osmolytes such as glycerol or trimethylamine N-oxide are used to rescue misfolded proteins.9,10 To test whether CFcor-325 had a direct or indirect effect on protein folding, we used the P-gp processing mutant G268V as a control.33 We used the G268V P-gp mutant, rather than mutant ∆Y490 P-gp (equivalent to ∆F508 in CFTR), as a control because maturation of the G268V mutant is not promoted by growth at low temperature (27 °C) or by osmolytes such as glycerol and trimethylamine N-oxide (unpublished observations). Login to comment
148 Maturation of mutant G268V to yield active enzyme at the cell surface, however, can be achieved by carrying out expression in the presence of drug substrates or modulators.34 Accordingly, BHK cells stably expressing P-gp mutant G268V were incubated with 0-10 µM CFcor-325 for 48 h. Login to comment
162 Effect of CFcor-325 on expression of misprocessed P-gp mutant G268V and on P-gp-mediated drug resistance. Login to comment
163 (A) BHK cells stably expressing P-gp mutant G268V were treated for 48 h with 0-10 µM CFcor-325. Whole cell extracts were then subjected to immunoblot analysis with rabbit polyclonal antibody against P-gp. Login to comment
168 The ability of CFcor-325 to rescue P-gp mutant G268V suggested that it was a substrate or modulator of P-gp. Login to comment

PMID: 18708637 [PubMed] Loo TW et al: "Processing mutations disrupt interactions between the nucleotide binding and transmembrane domains of P-glycoprotein and the cystic fibrosis transmembrane conductance regulator (CFTR)."

No. Sentence Comment

47 The G268V and L1260A processing mutations were introduced into the L443C(NBD1)/S909C(TMD2) mutant. Login to comment
108 The locations of processing mutations G268V and L1260A that were used to inhibit maturation of P-gp are indicated as squares. Login to comment
136 To test if NBD-TMD2 interactions differ in the mature and immature forms of P-gp, the G268V (26) or L1260A (38) processing mutations were introduced into mutant L443C(NBD1)/ S909C(TMD2) to inhibit its maturation. Login to comment
140 The G268V mutation is located in the second intracellular loop in TMD1 whereas the L1260A mutation is located at the COOH-end of NBD2. Login to comment
159 Mutants G268V/L443C(NBD1)/S909C(TMD2) and L1260A/L443C(NBD1)/S909C(TMD2) were expressed in HEK 293 cells in the presence of no drug (None) or 10 ␮m cyclosporin A (ϩ Cyclo). Login to comment
199 Processing mutations near the NH2- (G268V) or COOH-end (L1260A) of the molecule disrupted cross-linking between Cys-443(NBD1) and Cys-909(TMD2). Login to comment
5 The presence of a processing mutation (G268V/L443C(NBD1)/S909C(TMD2); L1260A/ A266C(TMD1)/F1086C(NBD2)) resulted in the synthesis of the immature (150 kDa) protein as the major product and the mutants could not be cross-linked with copper phenanthroline. Login to comment

PMID: 10506575 [PubMed] Loo TW et al: "The human multidrug resistance P-glycoprotein is inactive when its maturation is inhibited: potential for a role in cancer chemotherapy."

No. Sentence Comment

64 Accordingly, the histidine-tagged 150 kDa protein of mutants G268V, G269V, and Y710A were isolated (Fig. 1, lanes 1, 3, and 5). Login to comment
66 To convert the processing mutants G268V, G269V, and Y710A to the mature enzyme, the mutants were synthesized in the presence of 10 ␮M cyclosporin A to induce maturation (19). Login to comment
73 As reported previously (19), the mature enzyme of mutants G268V and G269V had ϳ50 to 75% of the activity of wild-type P-gp. Login to comment
76 ATPase activity of P-gp that is prevented from undergoing maturation It was possible that the 150 kDa core-glycosylated P-gp of the processing mutants G268V, G269V, and Y710A were inactive because the point mutations inherently caused misfolding of the protein and subsequent retention in the endoplasmic reticulum. Login to comment
84 Equivalent amounts of histidine-tagged mature and core-glycosylated P-gp of wild-type and mutants G268V, G269V, and Y710A, isolated by nickel-chelate chromatography, were reconstituted with lipid and assayed for verapamil- (1 mM) and vinblastine- (0.05 mM) stimulated ATPase activities. Login to comment
88 HEK293 cells expressing histidine-tagged P-gp mutants G268V, G269V or Y710A were incubated with (ϩ) or without (-) 10 ␮M cyclosporin A (cyclo) for 24 h. The cells were harvested and solubilized with n-dodecyl-beta-D-maltoside and the cell extracts were subjected to nickel-chelate chromatography with 20 mM imidazole in the wash buffers. Login to comment
196 Potential strategies would be to identify a pharmacological agent(s) that causes exposure of hypersensitive-protease sites such as R113 (Fig. 8, step 1), that can induce the same alterations as in the processing mutants (e.g., G268V, Fig. 8, step 2), or that blocks protein folding steps as seen with protease inhibitors (Fig. 8, step 3). Login to comment

PMID: 16691490 [PubMed] Loo TW et al: "Rescue of folding defects in ABC transporters using pharmacological chaperones."

No. Sentence Comment

76 The presence of a processing mutation in either half-molecule (N-half (G268V) or COOH-half (A841L)) resulted in the loss of interactions between the half-molecules. Login to comment

PMID: 8995353 [PubMed] Loo TW et al: "Correction of defective protein kinesis of human P-glycoprotein mutants by substrates and modulators."

No. Sentence Comment

51 RESULTS Effect of Drug Substrates on Processing of Misfolded Mutants-The effect of substrates and modulators of P-glycoprotein on the biosynthesis of two processing mutants were initially studied; G268V in the NH2-terminal transmembrane domain (16) and ⌬Y490 in the NH2-terminal nucleotide-binding domain (17). Login to comment
52 Mutant G268V is a temperature-insensitive processing mutant, whereas mutant ⌬Y490 contains a deletion at an equivalent position to the ⌬F508 mutation in CFTR. Login to comment
64 In addition to the mutants G268V and ⌬Y490, we were able to facilitate processing of P-glycoproteins with mutations in the predicted transmembrane segments (TM1, G54V; TM5, G300V; TM7, A718L; and TM9, A841L), in the extracellular loops between transmembrane segments (G854V), in the cytoplasmic loops (G251V and W803A), in the nucleotide-binding domains (G427C and S434C), and in the linker region connecting the two halves of the molecule (E707A) (data not shown). Login to comment
73 Fig. 2A shows that after 4 h in the presence of 15 ␮M cyclosporin A, about 50% of mutant G268V was present as the fully mature (170-kDa) form of the enzyme and that after 24 h, more than 80% of the mutant protein was present in the fully mature form. Login to comment
77 Fig. 2B shows that in the absence of cyclosporin A, the 150-kDa P-glycoprotein of mutant G268V was not processed to the mature enzyme. Login to comment
79 In the presence of cyclosporin A, however, the kinetics of maturation of the P-glycoprotein of mutant G268V was similar to that of wild-type enzyme. Login to comment
102 By contrast, most of the P-glycoprotein of mutants G268V and ⌬Y490 grown without drug substrate were recovered in the flow-through fractions during nickel-chelate chromatography. Login to comment
107 Similarly, drug-stimulated ATPase activity was detected in the mutant G268V after expression in the presence of cyclosporin A. Login to comment
108 The observation that mutant G268V exhibits reduced activity is consistent with previous observations that several glycine to valine mutations in the cytoplasmic loops of P-glycoprotein also alter the substrate specificity of the enzyme (16). Login to comment
110 Time-dependent appearance of the 170-kDa (mature) form of mutant P-glycoprotein-A52 (G268V). Login to comment
111 A, HEK 293 cells were transfected with A52-tagged mutant G268V cDNA and incubated for 24 h at 37 °C. Login to comment
114 B, HEK 293 cells were transfected with wild-type or mutant G268V P-glycoprotein-A52 cDNAs or vector alone (Control). Login to comment
140 Another interesting observation is that misfolded mutants that are temperatureand glycerol-insensitive, such as G251V, G268V, and E707A could also be rescued by these drug substrates when expressed in either HEK 293 or NIH 3T3 cells (data not shown). Login to comment

PMID: 9614062 [PubMed] Loo TW et al: "Superfolding of the partially unfolded core-glycosylated intermediate of human P-glycoprotein into the mature enzyme is promoted by substrate-induced transmembrane domain interactions."

No. Sentence Comment

52 When mutant G268V was expressed in HEK 293 cells (no drug, lane 1), the major product was a 150-kDa core-glycosylated protein that was sensitive to digestion with endoglycosidase H (data not shown). Login to comment
54 We attempted to purify the core-glycosylated intermediate of mutant G268V to determine whether it was still active. Login to comment
63 As shown in Fig. 1A (no drug), the 150-kDa protein of mutant G268V was quite sensitive to trypsin. Login to comment
74 To test this possibility, we expressed mutant G268V in the presence of a proteasome inhibitor (MG-132). Login to comment
76 As shown in Fig. 1C (lanes 3 and 4), incubation of mutant G268V for 24 h in the presence of MG-132 resulted in accumulation of the core-glycosylated intermediate and a 130-kDa digestion product. Login to comment
78 Increased accumulation of the core-glycosylated intermediate and the 130-kDa digestion product was also seen with the FIG. 1. Protease sensitivity of mutant G268V and wild-type P-glycoproteins. Login to comment
79 Membranes prepared from HEK 293 cells expressing mutant G268V (A) or wild-type (B) P-glycoproteins-A52 grown without (no drug) or with (ϩ drug) 10 ␮M CsA were treated with TPCK-trypsin followed by immunoblot analysis. Login to comment
81 C, HEK 293 cells transfected with wild-type or G268V cDNAs were incubated for 24 h in media containing 5 ␮M MG132 (ϩ) or no inhibitor (-), and equivalent amounts of whole cell extracts were immunoblotted with monoclonal antibody A52. Login to comment
89 We first introduced a processing mutation (G268V) into N-Half-His. Login to comment
90 Unlike the histidine-tagged full-length G268V P-glycoprotein, the mutant (G268V) N-Half-His could be recovered by nickel-chelate chromatography, even when grown with no CsA (Fig. 2A). Login to comment
91 Wild-type or G268V N-Half-His molecules were then coexpressed with the C-Half-A52 molecule. Login to comment
93 As shown in Fig. 2B, C-Half-A52 was not retained on the column when expressed alone or when coexpressed with mutant G268V N-Half- His in the absence of CsA. Login to comment
94 In contrast, CsA promoted interaction between C-Half-A52 and mutant G268V N-Half-His, such that most of the C-Half-A52 was now recovered by nickel-chelate chromatography. Login to comment
111 A, membranes from HEK 293 cells expressing histidine-tagged wild-type or G268V N-half molecules were treated for 24 h with (ϩ) or without (-) 10 ␮M CsA, solubilized with 1% (w/v) n-dodecyl-beta-D-maltoside, and the supernatants were subjected to nickel-chelate chromatography. Login to comment
113 B, detergent-solubilized HEK 293 membranes expressing only C-Half-A52, C-Half-A52, and wild-type N-Half-His, or both C-Half-A52 and G268V N-Half-His grown with (ϩ) or without (-) 10 ␮M CsA were subjected to nickel-chelate chromatography. Login to comment
120 B, wild-type or mutant (G268V) N-Half-A52 was coexpressed with wild-type C-Half-His in the presence of 10 ␮M CsA. Login to comment

PMID: 9829963 [PubMed] Loo TW et al: "Quality control by proteases in the endoplasmic reticulum. Removal of a protease-sensitive site enhances expression of human P-glycoprotein."

No. Sentence Comment

114 Accordingly, cells expressing a misprocessed mutant (G268V) with or without an additional R113A mutation were radiolabeled with [35 S]methionine and [35 S]cystine to monitor the fate of the mutant. Login to comment
115 Mutant G268V is a temperature-insensitive processing mutant (8) that yields mature enzyme (170 kDa) only when synthesized in the presence of drug substrates (7). Login to comment
117 By contrast, the core-glycosylated G268V protein failed to mature and was almost undetectable by 24 h. In the double mutant (G268V ϩ R113A), however, there was a 5-10-fold increase in the amount of P-gp at 0 h, but the mutant still failed to mature and was degraded by 24 h. A similar pattern was observed for the processing mutant G269V (data not shown). Login to comment
118 Mutant G341C appeared to have a greater folding defect than the G268V misprocessed mutant. Login to comment
134 FIG. 4. Effect of R113A mutation on maturation of wild type and mutant G268V P-gp. HEK 293 cells expressing wild type or mutant G268V P-gps-A52 and containing Arg113 or R113A were pulse-labeled for 20 min at 37 °C and then chased with fresh media for the various intervals (hr). Login to comment

PMID: 23733192 [PubMed] Loo TW et al: "Human P-glycoprotein contains a greasy ball-and-socket joint at the second transmission interface."

No. Sentence Comment

96 The I261S, V264S, F267A, G268V, and G269V mutations blocked maturation such that the 150-kDa immature protein was the major product (Fig. 1B). Login to comment

PMID: 25456855 [PubMed] Loo TW et al: "Tariquidar inhibits P-glycoprotein drug efflux but activates ATPase activity by blocking transition to an open conformation."

No. Sentence Comment

163 First, we tested if tariquidar could rescue the G268V processing mutant. Login to comment
164 The G268V mutant was selected because we previously found that it could be rescued with vinblastine, verapamil, or cyclosporine A [44]. Login to comment
174 processing mutations like G268V inhibit P-gp maturation because they trap P-gp in the endoplasmic reticulum as a partially folded immature 150 kDa core-glycosylated protein. Login to comment
177 Mutant G268V containing an A52 epitope tag was transiently expressed in HEK 293 cells in the presence of various concentrations of tariquidar for 18 h. Login to comment
183 The results show that tariquidar acted as a potent pharmacological chaperone to promote maturation of G268V P-gp. Login to comment
184 By comparison, 20-fold higher concentrations of high affinity drug substrates/modulators such as verapamil, vinblastine or cyclosporine A were required to promote maturation of G268V P-gp [44]. Login to comment
199 These concentrations of drug substrates were chosen because they maximally promoted maturation of the G268V mutant [44]. Login to comment
208 Does tariquidar also inhibit A80C/R741C cross-linking when A B G268V 0 12.5 25 50 100 200 400 800 [Tar] (nM) 170 kDa 150 kDa GAPDH C D 170 kDa 150 kDa GAPDH None Cyclo Tariq F804D 0 20 40 60 80 100 Percent Mature [Tariq] nM 0 12.5 25 50 100 200 400 800 * * * * * * None Cyclo Tariq 0 20 40 60 80 100 Percent Mature * Fig. 4. Login to comment