ABCMdb

ABCB1 p.Arg113Ala

Predicted by SNAP2:	A: N (53%), C: D (71%), D: D (63%), E: N (53%), F: D (80%), G: N (53%), H: N (61%), I: D (63%), K: N (82%), L: D (63%), M: N (61%), N: N (61%), P: D (71%), Q: N (72%), S: N (72%), T: N (72%), V: D (59%), W: D (80%), Y: D (75%),
Predicted by PROVEAN:	A: N, C: D, D: N, E: N, F: D, G: D, H: N, I: N, K: N, L: N, M: N, N: N, P: N, Q: N, S: N, T: N, V: N, W: D, Y: D,

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Publications
[hide] The human multidrug resistance P-glycoprotein is i... FASEB J. 1999 Oct;13(13):1724-32. 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., [PMID:10506575]

Abstract [show]
The human multidrug resistance P-glycoprotein (P-gp) contributes to the phenomenon of multidrug resistance during cancer and AIDS chemotherapy. A potential novel strategy to circumvent the effects of P-gp during chemotherapy is to prevent maturation of P-gp during biosynthesis so that the transporter does not reach the cell surface. Here we report that immature, core-glycosylated P-gp that is prevented from reaching the cell surface by processing mutations or by proteasome inhibitors such as lactacystin or MG-132 exhibited no detectable drug-stimulated ATPase activity. Disulfide cross-linking analysis also showed that the immature P-gp did not exhibit ATP-induced conformational changes as found in the mature enzyme. In addition, the immature P-gp was more sensitive to trypsin than the mature enzyme. These results suggest that P-gp is unlikely to be functional immediately after synthesis. These differences in the structural and enzymatic properties of the mature and core-glycosylated, immature P-gp could potentially be used during chemotherapy, and should result in the search for compounds that can specifically inhibit the maturation of P-gp.

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Sentences [show]
No. Sentence Comment
79 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
92 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 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 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 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 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 HEK293 cells were transfected with Cys-less P-gp cDNA containing an R113A mutation. Login to comment
112 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
145 Membranes were prepared from HEK293 cells expressing Cys-less(R113A) P-gp that were grown with or without MG-132. Login to comment
170 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

[hide] Quality control by proteases in the endoplasmic re... J Biol Chem. 1998 Dec 4;273(49):32373-6. Loo TW, Clarke DM
Quality control by proteases in the endoplasmic reticulum. Removal of a protease-sensitive site enhances expression of human P-glycoprotein.
J Biol Chem. 1998 Dec 4;273(49):32373-6., 1998-12-04 [PMID:9829963]

Abstract [show]
Human P-glycoprotein is synthesized in HEK 293 cells as two major products: the 150-kDa core-glycosylated intermediate and the 170-kDa mature proteins. The 150- and 170-kDa proteins were not detected in mutants such as G341C. The major protein in this mutant was a 130-kDa proteolytic degradation product. This result suggested that the mutant protein was misfolded and sensitive to proteolytic digestion during or immediately after synthesis. We found that mutation of Arg113, located in the first extracellular loop of P-glycoprotein and near the consensus glycosylation sites, to Ala, Lys, Glu, Met, or Cys blocked formation of the 130-kDa product. Introduction of R113A into mutant G341C resulted in the synthesis of a mature (170 kDa) and functional transporter. Similarly, when R113A was introduced into misprocessed mutants, there was increased synthesis of the 150-kDa core-glycosylated intermediate. Maturation of the core-glycosylated intermediate into the mature enzyme, however, was not observed. These results suggest that polytopic proteins are accessible to proteases in the lumen of the endoplasmic reticulum during biosynthesis and that proteases are important contributors to the quality control mechanism involved in protein folding. It is also shown that unstable proteins can be made more stable by removal of hypersensitive proteolytic sites.

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Sentences [show]
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
116 Fig. 4 shows that there was no detectable difference between the maturation of the core-glycosylated wild type enzyme and the (wild type ϩ R113A) mutant. 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
122 The R113A mutation was then introduced to block cleavage of the G341C mutant. Login to comment
123 As shown in Fig. 5A (lane 3), the introduction of an R113A mutation significantly affected the synthesis of mutant G341C. Login to comment
126 To determine whether the mature mutant protein was functional, the histidine-tagged mutant (G341C ϩ R113A) was expressed in HEK 293 cells and purified by nickel-chelate chromatography. Login to comment
129 Mutant (G341C ϩ R113A) exhibited verapamiland vinblastine-stimulated ATPase activities that were similar to the parent enzyme. 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
137 FIG. 5. Effect of R113A mutation on maturation and drug-stimulated ATPase activity of mutant G341C. Login to comment
138 A, HEK 293 cells expressing P-gp-A52 mutants G341C or G341C ϩ R113A in Cys-less background were grown with (ϩ) or without (-) 10 ␮M cyclosporin A for 24 h. Equivalent amounts of whole cell extracts were subjected to immunoblot analysis with monoclonal antibody A52. Login to comment
139 B, purified histidine-tagged Cys-less or mutant G341C ϩ R113A (in Cys-less background P-gp) were added to lipid and assayed for ATPase with or without verapamil (1 mM, shaded bars) or vinblastine (0.05 mM, unshaded bars). Login to comment
148 Apparently, the G341C mutation does not cause complete misfolding of the protein because introduction of an additional R113A mutation to prevent degradation converts the mutant to a functional enzyme with characteristics indistinguishable from parent enzyme (Fig. 5B). Login to comment