ABCB1 p.Gly341Cys
| Predicted by SNAP2: | A: N (53%), C: D (75%), D: D (85%), E: D (91%), F: D (80%), H: D (85%), I: D (85%), K: D (91%), L: D (85%), M: D (85%), N: D (80%), P: D (91%), Q: D (85%), R: D (91%), S: N (53%), T: D (80%), V: D (80%), W: D (91%), Y: D (91%), |
| Predicted by PROVEAN: | A: D, C: D, D: D, E: D, F: D, H: D, I: D, K: D, L: D, M: D, N: D, P: D, Q: D, R: D, S: D, T: D, V: D, W: D, Y: D, |
[switch to compact view]
Comments [show]
None has been submitted yet.
[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.
Comments [show]
None has been submitted yet.
No. Sentence Comment
189 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.
X
ABCB1 p.Gly341Cys 10506575:189:23
status: NEW205 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.
X
ABCB1 p.Gly341Cys 10506575:205:34
status: NEW[hide] Identification of residues in the drug-binding dom... J Biol Chem. 1999 Dec 10;274(50):35388-92. Loo TW, Clarke DM
Identification of residues in the drug-binding domain of human P-glycoprotein. Analysis of transmembrane segment 11 by cysteine-scanning mutagenesis and inhibition by dibromobimane.
J Biol Chem. 1999 Dec 10;274(50):35388-92., 1999-12-10 [PMID:10585407]
Abstract [show]
The drug-binding domain of the human multidrug resistance P-glycoprotein (P-gp) probably consists of residues from multiple transmembrane (TM) segments. In this study, we tested whether the amino acids in TM11 participate in binding drug substrates. Each residue in TM11 was initially altered by site-directed mutagenesis and assayed for drug-stimulated ATPase activity in the presence of verapamil, vinblastine, or colchicine. Mutants G939V, F942A, T945A, Q946A, A947L, Y953A, A954L, and G955V had altered drug-stimulated ATPase activities. Direct evidence for binding of drug substrate was then determined by cysteine-scanning mutagenesis of the residues in TM11 and inhibition of drug-stimulated ATPase activity by dibromobimane, a thiol-reactive substrate. Dibromobimane inhibited the drug-stimulated ATPase activities of two mutants, F942C and T945C, by more than 75%. These results suggest that residues Phe(942) and Thr(945) in TM11, together with residues previously identified in TM6 (Leu(339) and Ala(342)) and TM12 (Leu(975), Val(982), and Ala(985)) (Loo, T. W., and Clarke, D. M. (1997) J. Biol. Chem. 272, 31945-31948) form part of the drug-binding domain of P-gp.
Comments [show]
None has been submitted yet.
No. Sentence Comment
181 An example of an extreme case is mutant G341C (in TM6) that caused complete misfolding of P-gp such that the mutant P-gp was more susceptible to digestion in the first extracellular loop (50).
X
ABCB1 p.Gly341Cys 10585407:181:40
status: NEW[hide] Disulfide cross-linking analysis shows that transm... J Biol Chem. 2004 Feb 27;279(9):7692-7. Epub 2003 Dec 10. Loo TW, Bartlett MC, Clarke DM
Disulfide cross-linking analysis shows that transmembrane segments 5 and 8 of human P-glycoprotein are close together on the cytoplasmic side of the membrane.
J Biol Chem. 2004 Feb 27;279(9):7692-7. Epub 2003 Dec 10., 2004-02-27 [PMID:14670948]
Abstract [show]
Human P-glycoprotein (P-gp) transports a wide variety of structurally diverse compounds out of the cell. Knowledge about the packing of the transmembrane (TM) segments is essential for understanding the mechanism of drug recognition and transport. We used cysteine-scanning mutagenesis and disulfide cross-linking analysis to determine which TM segment in the COOH half of P-gp was close to TMs 5 and 6 since these segments in the NH(2) half are important for drug binding. An active Cys-less P-gp mutant cDNA was used to generate 240 double cysteine mutants that contained 1 cysteine in TMs 5 or 6 and another in TMs 7 or 8. The mutants were subjected to oxidative cross-linking analysis. No disulfide cross-linking was observed in the 140 TM6/TM7 or TM6/TM8 mutants. By contrast, cross-linking was detected in several P-gp TM5/TM8 mutants. At 4 degrees C, when thermal motion is low, P-gp mutants N296C(TM5)/G774C(TM8), I299C(TM5)/F770C(TM8), I299C(TM5)/G774C(TM8), and G300C(TM5)/F770C(TM8) showed extensive cross-linking with oxidant. These mutants retained drug-stimulated ATPase activity, but their activities were inhibited after treatment with oxidant. Similarly, disulfide cross-linking was inhibited by vanadate trapping of nucleotide. These results indicate that significant conformational changes must occur between TMs 5 and 8 during ATP hydrolysis. We revised the rotational symmetry model for TM packing based on our results and by comparison to the crystal structure of MsbA (Chang, G. (2003) J. Mol. Biol. 330, 419-430) such that TM5 is adjacent to TM8, TM2 is adjacent to TM11, and TMs 1 and 7 are next to TMs 6 and 12, respectively.
Comments [show]
None has been submitted yet.
No. Sentence Comment
227 We had shown that mutations in TM6 such as G341C promoted proteolytic cleavage of P-gp between residues Arg-113 and Tyr-114 in ECL1 in the endoplasmic reticulum (62).
X
ABCB1 p.Gly341Cys 14670948:227:43
status: NEW228 Mutant G341C was particularly sensitive to cleavage at R113 since no full-length protein was detected in cells expressing the mutant.
X
ABCB1 p.Gly341Cys 14670948:228:7
status: NEW[hide] The topography of transmembrane segment six is alt... J Biol Chem. 2004 Aug 13;279(33):34913-21. Epub 2004 Jun 10. Rothnie A, Storm J, Campbell J, Linton KJ, Kerr ID, Callaghan R
The topography of transmembrane segment six is altered during the catalytic cycle of P-glycoprotein.
J Biol Chem. 2004 Aug 13;279(33):34913-21. Epub 2004 Jun 10., 2004-08-13 [PMID:15192095]
Abstract [show]
Structural evidence has demonstrated that P-glycoprotein (P-gp) undergoes considerable conformational changes during catalysis, and these alterations are important in drug interaction. Knowledge of which regions in P-gp undergo conformational alterations will provide vital information to elucidate the locations of drug binding sites and the mechanism of coupling. A number of investigations have implicated transmembrane segment six (TM6) in drug-P-gp interactions, and a cysteine-scanning mutagenesis approach was directed to this segment. Introduction of cysteine residues into TM6 did not disturb basal or drug-stimulated ATPase activity per se. Under basal conditions the hydrophobic probe coumarin maleimide readily labeled all introduced cysteine residues, whereas the hydrophilic fluorescein maleimide only labeled residue Cys-343. The amphiphilic BODIPY-maleimide displayed a more complex labeling profile. The extent of labeling with coumarin maleimide did not vary during the catalytic cycle, whereas fluorescein maleimide labeling of F343C was lost after nucleotide binding or hydrolysis. BODIPY-maleimide labeling was markedly altered during the catalytic cycle and indicated that the adenosine 5'-(beta,gamma-imino)triphosphate-bound and ADP/vanadate-trapped intermediates were conformationally distinct. Our data are reconciled with a recent atomic scale model of P-gp and are consistent with a tilting of TM6 in response to nucleotide binding and ATP hydrolysis.
Comments [show]
None has been submitted yet.
No. Sentence Comment
130 Values refer to the mean Ϯ S.E. obtained from at least eight independent protein purification preparations. P-gp isoform Substrate affinity , Km Maximal activity, Vmax -Fold stimulationBasal Stimulated Basal Stimulated mM mol Pi min-1 mg protein-1 Cys-less 0.58 Ϯ 0.06 0.38 Ϯ 0.04 0.58 Ϯ 0.15 1.46 Ϯ 0.30 2.9 Ϯ 0.3 V331C 0.50 Ϯ 0.06 0.26 Ϯ 0.02 0.45 Ϯ 0.05 1.54 Ϯ 0.20 3.5 Ϯ 0.3 T333C 0.49 Ϯ 0.05 0.23 Ϯ 0.02 0.35 Ϯ 0.04 1.22 Ϯ 0.15 3.3 Ϯ 0.1 F335C 0.40 Ϯ 0.05 0.24 Ϯ 0.03 0.65 Ϯ 0.15 1.61 Ϯ 0.31 2.2 Ϯ 0.2 S337C 0.53 Ϯ 0.06 0.26 Ϯ 0.04 0.59 Ϯ 0.10 1.67 Ϯ 0.23 3.2 Ϯ 0.4 L339C 0.51 Ϯ 0.07 0.31 Ϯ 0.04 0.57 Ϯ 0.07 1.47 Ϯ 0.15 2.9 Ϯ 0.3 G341C 0.40 Ϯ 0.04 0.24 Ϯ 0.02 0.42 Ϯ 0.03 1.12 Ϯ 0.09 3.1 Ϯ 0.5 F343C 0.41 Ϯ 0.04 0.26 Ϯ 0.03 0.47 Ϯ 0.04 1.17 Ϯ 0.15 2.6 Ϯ 0.3 generate stable covalent bonds with thiol groups under physiological conditions.
X
ABCB1 p.Gly341Cys 15192095:130:829
status: NEW141 Typical time courses for labeling of T333C with FM and G341C with CM are shown in Fig 1, b and c, respectively.
X
ABCB1 p.Gly341Cys 15192095:141:55
status: NEW147 Panel c clearly demonstrates that the labeling of G341C reached significantly higher levels (lanes i-vii) when compared with denatured protein (lane viii), which in this case indicates 100% labeling.
X
ABCB1 p.Gly341Cys 15192095:147:50
status: NEW154 Isoforms V331C, T333C, F335C, S337C, L339C, and G341C displayed labeling extents in the range 7-12%, and none was significantly different from the Cys-less isoform (ANOVA).
X
ABCB1 p.Gly341Cys 15192095:154:48
status: NEW163 Isoforms T333C, F335C, S337C, and G341C did not label with BM since the Lext values (19-27%) were not signif- TABLE II Potency of drugs that affect the ATPase activity of purified reconstituted single cysteine mutants of P-gp Pure, reconstituted P-gp (0.3 g) was incubated in the presence of ATP (2 mM) and varying concentrations of nicardipine, vinblastine, or vanadate.
X
ABCB1 p.Gly341Cys 15192095:163:34
status: NEW166 Values refer to the mean Ϯ S.E. obtained from a minimum of three independent protein purification preparations. P-gp isoform Potency of drug effect Nicardipine, EC50 Vinblastine, EC50 Vanadate, IC50 M M M Cys-less 3.2 Ϯ 0.3 4.2 Ϯ 0.6 4.0 Ϯ 0.4 V331C 3.3 Ϯ 0.4 7.2 Ϯ 1.7 3.2 Ϯ 0.4 T333C 2.3 Ϯ 0.2 4.6 Ϯ 0.4 3.9 Ϯ 0.8 F335C 2.3 Ϯ 0.4 4.2 Ϯ 0.8 5.5 Ϯ 1.1 S337C 2.7 Ϯ 0.5 4.1 Ϯ 1.0 5.8 Ϯ 0.8 L339C 2.1 Ϯ 0.3 5.1 Ϯ 0.8 4.2 Ϯ 0.7 G341C 3.9 Ϯ 0.5 4.0 Ϯ 0.6 6.8 Ϯ 1.3 F343C 2.1 Ϯ 0.3 5.6 Ϯ 2.7 2.7 Ϯ 0.8 FIG. 1.
X
ABCB1 p.Gly341Cys 15192095:166:562
status: NEW168 Structures of maleimide-containing probes (a) and the fluorescence profiles of SDS-PAGE gels obtained for time courses of fluorescein maleimide labeling of T333C (b) and coumarin maleimide labeling of G341C (c).
X
ABCB1 p.Gly341Cys 15192095:168:201
status: NEW217 Two isoforms, F335C and G341C, could not be labeled by BM under any conditions and presumably lie in a region with low accessibility due to proximity of other structural elements.
X
ABCB1 p.Gly341Cys 15192095:217:24
status: NEW[hide] Drug-induced regulation of the MDR1 promoter in Ca... Antimicrob Agents Chemother. 2005 Jul;49(7):2785-92. Harry JB, Oliver BG, Song JL, Silver PM, Little JT, Choiniere J, White TC
Drug-induced regulation of the MDR1 promoter in Candida albicans.
Antimicrob Agents Chemother. 2005 Jul;49(7):2785-92., [PMID:15980350]
Abstract [show]
Resistance of Candida albicans to azole antifungal drugs is mediated by two types of efflux pumps, encoded by the MDR1 gene and the CDR gene family. MDR1 mRNA levels in a susceptible clinical isolate are induced by benomyl (BEN) but not by other drugs previously shown to induce MDR1. To monitor MDR1 expression under several conditions, the MDR1 promoter was fused to the Renilla reniformis luciferase reporter gene (RLUC). The promoter was monitored for its responses to four oxidizing agents, five toxic hydrophobic compounds, and an alkylating agent, all shown to induce major facilitator pumps in other organisms. Deletion constructs of the MDR1 promoter were used to analyze the basal transcription of the promoter and its responses to the toxic compound BEN and the oxidizing agent tert-butyl hydrogen peroxide (T-BHP). The cis-acting elements in the MDR1 promoter responsible for induction by BEN were localized between -399 and -299 upstream of the start codon. The cis-acting elements responsible for MDR1 induction by T-BHP were localized between -601 and -500 upstream of the start codon. The T-BHP induction region contains a sequence that resembles the YAP1-responsive element (YRE) in Saccharomyces cerevisiae. This Candida YRE was placed upstream of a noninducible promoter in the luciferase construct, resulting in an inducible promoter. Inversion or mutation of the 7-bp YRE eliminated induction. Many of the drugs used in this analysis induce the MDR1 promoter at concentrations that inhibit cell growth. These analyses define cis-acting elements responsible for drug induction of the MDR1 promoter.
Comments [show]
None has been submitted yet.
No. Sentence Comment
115 When the promoters from each clone were sequenced, 13 MDR1 sequence differences were identified between the clones produced from isolates 1 and 17 (A306G, G341C, T343A, A448T, T565G, T655C, A682G, G724C, A739G, C755T, AAACAC811-816CC, A832C, and G890A, where the number refers to a position upstream of the ATG start codon based on the genome sequence [16], the base[s] before the number is the sequence from isolate 1, and the base[s] after the number is from isolate 17).
X
ABCB1 p.Gly341Cys 15980350:115:155
status: NEW[hide] Identification of residues in the drug-binding sit... J Biol Chem. 1997 Dec 19;272(51):31945-8. Loo TW, Clarke DM
Identification of residues in the drug-binding site of human P-glycoprotein using a thiol-reactive substrate.
J Biol Chem. 1997 Dec 19;272(51):31945-8., 1997-12-19 [PMID:9405384]
Abstract [show]
We identified a thiol-reactive compound, dibromobimane (dBBn), that was a potent stimulator (8.2-fold) of the ATPase activity of Cys-less P-glycoprotein. We then used this compound together with cysteine-scanning mutagenesis to identify residues in transmembrane segment (TM) 6 and TM12 that are important for function. TM6 and TM12 lie close to each other in the tertiary structure and are postulated to be important for drug-protein interactions. The majority of P-glycoprotein mutants containing a single cysteine residue retained substantial amounts of drug-stimulated ATPase activity and were not inhibited by dBBn. The ATPase activities of mutants L339C, A342C, L975C, V982C, and A985C, however, were markedly inhibited (>60%) by dBBn. The drug substrates verapamil, vinblastine, and colchicine protected these mutants against inhibition by dBBn, suggesting that these residues are important for interaction of substrates with P-glycoprotein. We previously showed that residues Leu339, Ala342, Leu975, Val982, and Ala985 lie along the point of contact between helices TM6 and TM12, when both are aligned in a left-handed coiled coil (Loo, T. W., and Clarke, D. M. (1997) J. Biol. Chem. 272, 20986-20989). Taken together, these results suggest that the interface between TM6 and TM12 likely forms part of the potential drug-binding pocket in P-glycoprotein.
Comments [show]
None has been submitted yet.
No. Sentence Comment
83 There was no detectable activity with mutants S344C, G341C, and G984C, whereas mutants A342C, G346C, Q347C, A985C, G989C, and Q990C had much reduced activity (10-40%).
X
ABCB1 p.Gly341Cys 9405384:83:53
status: NEW87 Mutants G341C and G984C, however, appeared to be degraded quite rapidly.
X
ABCB1 p.Gly341Cys 9405384:87:8
status: NEW96 Mutants G341C, S344C, G346C, G984C, and G989C were not assayed because of their low or defective expression (Fig. 2B).
X
ABCB1 p.Gly341Cys 9405384:96:8
status: NEW[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.
Comments [show]
None has been submitted yet.
No. Sentence Comment
64 For some mutants such as G341C, no full-length protein could be detected.
X
ABCB1 p.Gly341Cys 9829963:64:25
status: NEW67 To inhibit or retard the degradation of mutant G341C, synthesis was carried out in the presence of proteasome inhibitors such as MG-132 or lactacystin, because the major cellular degradation pathway is mediated by proteasomes (17).
X
ABCB1 p.Gly341Cys 9829963:67:47
status: NEW68 Fig. 1 (lanes 8 and 9) shows that proteasomes are probably not involved because the 130-kDa protein was still the major product when mutant G341C was synthesized in the presence of proteasome inhibitors.
X
ABCB1 p.Gly341Cys 9829963:68:140
status: NEW69 In contrast to mutant G341C, the proteasome inhibitors had a dramatic effect on synthesis of the Cys-less parent.
X
ABCB1 p.Gly341Cys 9829963:69:22
status: NEW73 Therefore, the presence of a degradation product of 130 kDa in the Cys-less parent P-gp and in mutant G341C suggested that the protein had been cleaved at the NH2-terminal end.
X
ABCB1 p.Gly341Cys 9829963:73:102
status: NEW93 HEK 293 cells were transfected with Cys-less (C-less), mutant G341C (in Cys-less background), or glycosylation-deficient (N91A,N94A,N99A; -Glycos.)
X
ABCB1 p.Gly341Cys 9829963:93:62
status: NEW118 Mutant G341C appeared to have a greater folding defect than the G268V misprocessed mutant.
X
ABCB1 p.Gly341Cys 9829963:118:7
status: NEW122 The R113A mutation was then introduced to block cleavage of the G341C mutant.
X
ABCB1 p.Gly341Cys 9829963:122:64
status: NEW123 As shown in Fig. 5A (lane 3), the introduction of an R113A mutation significantly affected the synthesis of mutant G341C.
X
ABCB1 p.Gly341Cys 9829963:123:115
status: NEW126 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.
X
ABCB1 p.Gly341Cys 9829963:126:92
status: NEW129 Mutant (G341C ϩ R113A) exhibited verapamiland vinblastine-stimulated ATPase activities that were similar to the parent enzyme.
X
ABCB1 p.Gly341Cys 9829963:129:8
status: NEW130 These results show that blockage of the Arg113 cleavage site completely rescued the defects associated with the G341C mutation.
X
ABCB1 p.Gly341Cys 9829963:130:112
status: NEW131 We could not determine whether the 130-kDa product of mutant G341C was active because the histidine-tagged protein was not retained by the nickel column, a property also exhibited by the core-glycosylated intermediates of other P-gp processing mutants (10).
X
ABCB1 p.Gly341Cys 9829963:131:61
status: NEW132 The inability of the histidine-tagged 130-kDa product of mutant G341C to bind the nickel column was not due to loss of the histidine tag.
X
ABCB1 p.Gly341Cys 9829963:132:64
status: NEW137 FIG. 5. Effect of R113A mutation on maturation and drug-stimulated ATPase activity of mutant G341C.
X
ABCB1 p.Gly341Cys 9829963:137:93
status: NEW138 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.
X
ABCB1 p.Gly341Cys 9829963:138:45
status: NEWX
ABCB1 p.Gly341Cys 9829963:138:54
status: NEW139 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).
X
ABCB1 p.Gly341Cys 9829963:139:48
status: NEW146 This effect was enhanced in mutant G341C, such that the 130-kDa protein was the only product detected.
X
ABCB1 p.Gly341Cys 9829963:146:35
status: NEW147 Remarkably, the effects of the G341C mutation could be overcome by mutating out the hypersensitive protease cleavage site.
X
ABCB1 p.Gly341Cys 9829963:147:31
status: NEW148 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).
X
ABCB1 p.Gly341Cys 9829963:148:16
status: NEW158 By inhibiting digestion at Arg113 , it was possible to increase expression of P-gp mutants such as G341C.
X
ABCB1 p.Gly341Cys 9829963:158:99
status: NEW[hide] Single amino acid substitution of rat MRP2 results... Am J Physiol Gastrointest Liver Physiol. 2001 Oct;281(4):G1034-43. Ito K, Suzuki H, Sugiyama Y
Single amino acid substitution of rat MRP2 results in acquired transport activity for taurocholate.
Am J Physiol Gastrointest Liver Physiol. 2001 Oct;281(4):G1034-43., [PMID:11557524]
Abstract [show]
Multidrug resistance-associated protein 3 (MRP3), unlike other MRPs, transports taurocholate (TC). The difference in TC transport activity between rat MRP2 and MRP3 was studied, focusing on the cationic amino acids in the transmembrane domains. For analysis, transport into membrane vesicles from Sf9 cells expressing wild-type and mutated MRP2 was examined. Substitution of Arg at position 586 with Leu and Ile and substitution of Arg at position 1096 with Lys, Leu, and Met resulted in the acquisition of TC transport activity, while retaining transport activity for glutathione and glucuronide conjugates. Substitution of Leu at position 1084 of rat MRP3 (which corresponds to Arg-1096 in rat MRP2) with Lys, but not with Val or Met, resulted in the loss of transport activity for TC and glucuronide conjugates. These results suggest that the presence of the cationic charge at Arg-586 and Arg-1096 in rat MRP2 prevents the transport of TC, whereas the presence of neutral amino acids at the corresponding position of rat MRP3 is required for the transport of substrates.
Comments [show]
None has been submitted yet.
No. Sentence Comment
223 Although we do not have a satisfactory explanation to account for the production of a shorter band of ϳ80 kDa in L1084RMRP3, it is possible this mutant protein may have easy access to some type of protease(s) that may be produced due to the Arg substitution at position 1084. Loo and Clarke (25) demonstrated that the substitution of Gly with Cys at position 341, located in the middle of the TM6 of P-glycoprotein, resulted in cleavage of the extracellular loop located between TM1 and TM2 to produce a truncated protein product of 130 kDa.
X
ABCB1 p.Gly341Cys 11557524:223:340
status: NEW[hide] Mutational analysis of ABC proteins. Arch Biochem Biophys. 2008 Aug 1;476(1):51-64. Epub 2008 Mar 5. Loo TW, Clarke DM
Mutational analysis of ABC proteins.
Arch Biochem Biophys. 2008 Aug 1;476(1):51-64. Epub 2008 Mar 5., [PMID:18328253]
Abstract [show]
The 49 human members of the ATP-binding cassette (ABC) family of proteins are involved in a wide range of activities such as active transport of compounds across membranes, extraction of compounds out of membranes, functioning as ion channels, or regulators of channel activity. Mutations and/or overexpression of many of the proteins can have adverse effects on health. A goal in the study of ABC proteins is to understand their mechanisms of action. This review will focus on the mutational approaches that have been used to study the structure and mechanisms of some ABC proteins.
Comments [show]
None has been submitted yet.
No. Sentence Comment
315 Seven TM6 cysteine mutants (V331C, T333C, F335C, S337C, L339C, G341C, F343C) were labeled in the presence or absence of AMPÁPNP or after vanadate trapping of nucleotide.
X
ABCB1 p.Gly341Cys 18328253:315:63
status: NEW313 Seven TM6 cysteine mutants (V331C, T333C, F335C, S337C, L339C, G341C, F343C) were labeled in the presence or absence of AMPPNP or after vanadate trapping of nucleotide.
X
ABCB1 p.Gly341Cys 18328253:313:63
status: NEW[hide] On the origin of large flexibility of P-glycoprote... J Biol Chem. 2013 Jun 28;288(26):19211-20. doi: 10.1074/jbc.M113.450114. Epub 2013 May 8. Wen PC, Verhalen B, Wilkens S, Mchaourab HS, Tajkhorshid E
On the origin of large flexibility of P-glycoprotein in the inward-facing state.
J Biol Chem. 2013 Jun 28;288(26):19211-20. doi: 10.1074/jbc.M113.450114. Epub 2013 May 8., [PMID:23658020]
Abstract [show]
P-glycoprotein (Pgp) is one of the most biomedically relevant transporters in the ATP binding cassette (ABC) superfamily due to its involvement in developing multidrug resistance in cancer cells. Employing molecular dynamics simulations and double electron-electron resonance spectroscopy, we have investigated the structural dynamics of membrane-bound Pgp in the inward-facing state and found that Pgp adopts an unexpectedly wide range of conformations, highlighted by the degree of separation between the two nucleotide-binding domains (NBDs). The distance between the two NBDs in the equilibrium simulations covers a range of at least 20 A, including, both, more open and more closed NBD configurations than the crystal structure. The double electron-electron resonance measurements on spin-labeled Pgp mutants also show wide distributions covering both longer and shorter distances than those observed in the crystal structure. Based on structural and sequence analyses, we propose that the transmembrane domains of Pgp might be more flexible than other structurally known ABC exporters. The structural flexibility of Pgp demonstrated here is not only in close agreement with, but also helps rationalize, the reported high NBD fluctuations in several ABC exporters and possibly represents a fundamental difference in the transport mechanism between ABC exporters and ABC importers. In addition, during the simulations we have captured partial entrance of a lipid molecule from the bilayer into the lumen of Pgp, reaching the putative drug binding site. The location of the protruding lipid suggests a putative pathway for direct drug recruitment from the membrane.
Comments [show]
None has been submitted yet.
No. Sentence Comment
147 The suggested hinge role played by Gly-346 of human Pgp can also explain why the ATPase activity can only be significantly reduced by a mutation at this particular position but not elsewhere along the helix TM6 (73) even when other glycines in TM6 were targeted (G341C and G360C) (73).
X
ABCB1 p.Gly341Cys 23658020:147:263
status: NEW