ABCMdb

PMID: 10506575

Loo TW, Clarke DM
The human multidrug resistance P-glycoprotein is inactive when its maturation is inhibited: potential for a role in cancer chemotherapy.
FASEB J. 1999 Oct;13(13):1724-32., [PubMed]

Sentences

No. Mutations Sentence Comment

42 ABCB1 p.Leu975Cys ABCB1 p.Leu332Cys The Cys-less mutant was also modified to contain mutations L332C in TM6 and L975C in TM12 (27). Login to comment 64 ABCB1 p.Tyr710Ala ABCB1 p.Gly269Val ABCB1 p.Gly268Val 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 ABCB1 p.Tyr710Ala ABCB1 p.Gly269Val ABCB1 p.Gly268Val 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 72 ABCB1 p.Tyr710Ala The mature enzyme of mutant Y710A had activities that were similar to wild-type P-gp. Login to comment 73 ABCB1 p.Gly269Val ABCB1 p.Gly268Val 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 ABCB1 p.Tyr710Ala ABCB1 p.Gly269Val ABCB1 p.Gly268Val 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 79 ABCB1 p.Arg113Ala A histidine-tagged Cys-less P-gp containing an R113A mutation and grown in the presence of 2 ␮M MG-132 was assayed for drug-stimulated ATPase activity. Login to comment 84 ABCB1 p.Tyr710Ala ABCB1 p.Gly269Val ABCB1 p.Gly268Val 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 ABCB1 p.Tyr710Ala ABCB1 p.Gly269Val ABCB1 p.Gly268Val 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 92 ABCB1 p.Arg113Ala The R113A mutation was included since it stabilizes P-gp against proteolytic digestion during biosynthesis, and it resulted in increased yield of enzyme (23). Login to comment 93 ABCB1 p.Arg113Ala Figure 3 shows that the major product of the Cys-less(R113A) mutant in the presence of increasing concentrations of MG-132 was the 150 kDa core-glycosylated intermediate. Login to comment 96 ABCB1 p.Arg113Ala Accordingly, the histidine-tagged Cys-less(R113A) P-gp grown with or without 2 ␮M MG-132 was isolated by nickel-chelate chromatography. Login to comment 97 ABCB1 p.Arg113Ala As shown in Fig. 4A (lane 4), the 150 kDa core glycosylated Cys-less(R113A) P-gp could be recovered by nickel-chelate chromatography when the mutant was grown with MG-132. Login to comment 99 ABCB1 p.Arg113Ala Equivalent amounts of the isolated Cys-less(R113A) P-gps were reconstituted with lipid and assayed for drug-stimulated ATPase activity. Login to comment 108 ABCB1 p.Arg113Ala HEK293 cells were transfected with Cys-less P-gp cDNA containing an R113A mutation. Login to comment 112 ABCB1 p.Arg113Ala Drug-stimulated ATPase activity of P-gp after expression in the presence or absence of MG-132. HEK293 cells were transfected with pMT21 vector (control) or histidine-tagged Cys-less(R113A) P-gp cDNA (P-gp). Login to comment 120 ABCB1 p.Leu975Cys ABCB1 p.Leu332Cys Cys-less P-gp containing the mutations L332C and L975C was expressed with or without MG-132 and cross-linked in the presence or absence of Mg⅐ATP. Login to comment 121 ABCB1 p.Leu975Cys ABCB1 p.Leu332Cys Figure 5 shows that ATP induces cross-linking in mutant L332C/L975C when expressed in the absence of MG-132. Login to comment 123 ABCB1 p.Leu975Cys ABCB1 p.Leu332Cys In the presence of MG-132, however, the 150 kDa core-glycosylated mutant L332C/L975C was not cross-linked by oxidant in the absence or presence of ATP (Fig. 5, lanes 6 and 8). Login to comment 139 ABCB1 p.Leu975Cys ABCB1 p.Leu332Cys HEK 293 cells were transfected with Cys-less P-gp cDNA containing mutations L332C/L975C and then incubated with (ϩMG-132) or without (-MG-132) MG-132 for 24 h. Membranes were then prepared and treated with (ϩ) or without (-) copper phenanthrolene (CuPhen) for 5 min at 37°C in the presence (ϩATP) or absence (-ATP) of 10 mM ATP. Login to comment 145 ABCB1 p.Arg113Ala Membranes were prepared from HEK293 cells expressing Cys-less(R113A) P-gp that were grown with or without MG-132. Login to comment 170 ABCB1 p.Arg113Ala Protease sensitivity of P-gp after expression in the presence or absence of MG-132. HEK293 cells expressing A52-epitope-tagged Cys-less(R113A) P-gp were treated with (ϩ) or without (-) 2 ␮M MG-132 for 24 h. Membranes were then prepared and treated with various concentrations of TPCK-trypsin. Login to comment 189 ABCB1 p.Gly341Cys ABCB1 p.Gln347Cys Some mutations such as G341C and Q347C (23, 46) expose a proteolytic site in the first extracellular loop that result in degradation of the protein during or immediately after synthesis. Login to comment 196 ABCB1 p.Gly268Val 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 205 ABCB1 p.Gly341Cys The presence of mutations such as G341C (1); processing mutations (2); or the presence of proteasome inhibitors, such as MG-132 (3), prevent maturation of P-gp and the P-gps are rapidly degraded. Login to comment