ABCC1 p.Tyr1189Ala
| Predicted by SNAP2: | A: D (63%), C: N (57%), D: D (85%), E: D (80%), F: N (66%), G: D (75%), H: D (63%), I: D (59%), K: D (75%), L: D (63%), M: D (63%), N: D (71%), P: D (85%), Q: D (66%), R: D (71%), S: D (63%), T: D (66%), V: D (53%), W: D (71%), |
| Predicted by PROVEAN: | A: D, C: D, D: D, E: D, F: D, G: 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, |
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[hide] Transport of glutathione and glutathione conjugate... Trends Pharmacol Sci. 2006 Aug;27(8):438-46. Epub 2006 Jul 3. Cole SP, Deeley RG
Transport of glutathione and glutathione conjugates by MRP1.
Trends Pharmacol Sci. 2006 Aug;27(8):438-46. Epub 2006 Jul 3., [PMID:16820223]
Abstract [show]
Glutathione (GSH)-conjugated xenobiotics and GSH-conjugated metabolites (e.g. the cysteinyl leukotriene C4) must be exported from the cells in which they are formed before they can be eliminated from the body or act on their cellular targets. This efflux is often mediated by the multidrug resistance protein 1 (MRP1) transporter, which also confers drug resistance to tumour cells and can protect normal cells from toxic insults. In addition to drugs and GSH conjugates, MRP1 exports GSH and GSH disulfide, and might thus have a role in cellular responses to oxidative stress. The transport of several drugs and conjugated organic anions by MRP1 requires the presence of GSH, but it is not well understood how GSH (and its analogues) enhances transport. Site-directed mutagenesis studies and biophysical analyses have provided important insights into the structural determinants of MRP1 that influence GSH and GSH conjugate binding and transport.
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No. Sentence Comment
59 Also indicated are the predicted locations of the amino acid residues identified by site-directed mutagenesis studies to be selectively important for GSH conjugation (LTC4) and/or GSH transport [TM6-Lys332, TM6-His335, TM9-Pro478 (P478A), TM11-Phe594 (F594Y), TM15-Arg1138 (R1138K), CL-Tyr1189, Tyr1190 (Y1189A/S, Y1190A/S) and TM16-Glu1204 (E1204D)] [33,38,40,42,43,75,76].
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ABCC1 p.Tyr1189Ala 16820223:59:304
status: NEW[hide] Role of two adjacent cytoplasmic tyrosine residues... Biochem Pharmacol. 2005 Feb 1;69(3):451-61. Epub 2004 Dec 16. Conseil G, Deeley RG, Cole SP
Role of two adjacent cytoplasmic tyrosine residues in MRP1 (ABCC1) transport activity and sensitivity to sulfonylureas.
Biochem Pharmacol. 2005 Feb 1;69(3):451-61. Epub 2004 Dec 16., [PMID:15652236]
Abstract [show]
The human ATP-binding cassette (ABC) protein MRP1 causes resistance to many anticancer drugs and is also a primary active transporter of conjugated metabolites and endogenous organic anions, including leukotriene C(4) (LTC(4)) and glutathione (GSH). The sulfonylurea receptors SUR1 and SUR2 are related ABC proteins with the same domain structure as MRP1, but serve as regulators of the K(+) channel Kir6.2. Despite their functional differences, the activity of both SUR1/2 and MRP1 can be blocked by glibenclamide, a sulfonylurea used to treat diabetes. Residues in the cytoplasmic loop connecting transmembrane helices 15 and 16 of the SUR proteins have been implicated as molecular determinants of their sensitivity to glibenclamide and other sulfonylureas. We have now investigated the effect of mutating Tyr(1189) and Tyr(1190) in the comparable region of MRP1 on its transport activity and sulfonylurea sensitivity. Ala and Ser substitutions of Tyr(1189) and Tyr(1190) caused a > or =50% decrease in the ability of MRP1 to transport different organic anions, and a decrease in LTC(4) photolabeling. Kinetic analyses showed the decrease in GSH transport was attributable primarily to a 10-fold increase in K(m). In contrast, mutations of these Tyr residues had no major effect on the catalytic activity of MRP1. Furthermore, the mutant proteins showed no substantial differences in their sensitivity to glibenclamide and tolbutamide. We conclude that MRP1 Tyr(1189) and Tyr(1190), unlike the corresponding residues in SUR1, are not involved in its differential sensitivity to sulfonylureas, but nevertheless, may be involved in the transport activity of MRP1, especially with respect to GSH.
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No. Sentence Comment
59 Tyr substitutions were generated in the pGEM-3Z plasmids according to the manufacturer`s instructions with the following mutagenic primers (substituted nucleotides are in bold) obtained from Integrated DNA Technologies: Y1189A (50 -GAT GCT GGG GAT CGC GGC CTT CTG GTT CTC-30 ), Y1189S (50 -GAT GCT GGG GTA ACT GGC CTT CTG G-30 ), Y1190A (50 -C CAC GAT GCT CGG GGC ATA GGC CTT C-30 ), Y1190S (50 -C GAT GCT GGG ACT ATA GGC CTT C30 ).
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ABCC1 p.Tyr1189Ala 15652236:59:220
status: NEW115 Expression levels and organic anion uptake by Tyr1189 and Tyr1190 mutant MRP1 proteins Replacement of Tyr1189 and Tyr1190 with Ala and Ser was done by site-directed mutagenesis and then HEK293T cells were transfected to express the mutant and wild-type MRP1 proteins.
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ABCC1 p.Tyr1189Ala 15652236:115:102
status: NEW119 As shown in Fig. 3B, the ability of the mutants to support LTC4 uptake after 3 min was reduced by approximately 55 and 25% for Y1189A and Y1189S, respectively, and by approximately 40 and 50% for Y1190A and Y1190S, compared to wild-type MRP1.
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ABCC1 p.Tyr1189Ala 15652236:119:127
status: NEW121 E217bG uptake by the Y1189A and Y1189S mutants was reduced by 30%, and by 55-65% for the Y1190A and Y1190S mutants.
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ABCC1 p.Tyr1189Ala 15652236:121:21
status: NEW122 E13SO4 uptake was reduced by 35% in the case of Y1189S and by 60-65% for the Y1189A, Y1190A and Y1190S mutants.
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ABCC1 p.Tyr1189Ala 15652236:122:77
status: NEW129 As shown in Fig. 4B, Y1189A, Y1189S, Y1190A and Y1190S exhibited a moderate (10-30%) decrease in azido-ATP labeling at 4 8C.
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ABCC1 p.Tyr1189Ala 15652236:129:21
status: NEW130 When the amount of azido-ADP trapped by vanadate under hydrolysis conditions at 37 8C was measured, the Ala-substituted mutants Y1189A and Y1190A exhibited a reduction of 30-40% compared to wild-type MRP1, whereas little or no ( 10%) decrease in trapping was observed for the Ser-substituted Y1189S and Y1190S mutants (Fig. 4C).
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ABCC1 p.Tyr1189Ala 15652236:130:128
status: NEW133 (A) Immunodot blot of membrane vesicles (0.5 and 1 mg protein) prepared from HEK293T cells transfected with wild-type (WT-MRP1) and mutant (Y1189A, Y1189S, Y1190A, Y1190S) MRP1 cDNAs.
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ABCC1 p.Tyr1189Ala 15652236:133:140
status: NEW137 Ala/Ser mutant MRP1 (Y1189A, !
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ABCC1 p.Tyr1189Ala 15652236:137:21
status: NEW143 Table 1 Summary of transport activities for Tyr1189 and Tyr1190 mutants of MRP1 Mutant %Wild-type MRP1 uptake activitya LTC4 b E217bG E13SO4 MTX GSH Y1189A 45 70 40 35 10 Y1189S 75 70 65 35 30 Y1190A 60 45 40 35 20 Y1190S 50 35 35 25 25 a The values shown are means of triplicate determinations in a single experiment and are representative of results obtained in two to three independent experiments. Values have been corrected for different MRP1 expression levels according to the immunoblot shown Fig. 3A.
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ABCC1 p.Tyr1189Ala 15652236:143:149
status: NEW154 (A) Immunoblot of membrane vesicles (1 mg protein) prepared from HEK293T cells transfected with wild-type (WT-MRP1a, b, c), and mutant (Y1189A, Y1189S, Y1190A, Y1190S) MRP1 cDNAs as in Fig 3A. (B) Membrane vesicles (20 mg protein) were incubated with 5 mM [a-32 P]-8N3-ATP at 4 8C for 5 min in transport buffer containing 5 mM MgCl2. Samples were photo-crosslinked, resolved by SDS-PAGE and exposed to film.
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ABCC1 p.Tyr1189Ala 15652236:154:136
status: NEW164 Ala/Ser MRP1 mutants (Y1189A, Y1189S, Y1190A, Y1190S).
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ABCC1 p.Tyr1189Ala 15652236:164:22
status: NEW177 Membrane vesicles (2.5 mg protein) prepared from transfected HEK293T cells expressing wild-type MRP1 (WT-MRP1) (A), Tyr1189 mutants Y1189A (B), and Y1189S (C), and Tyr1190 mutants Y1190A (D) and Y1190S (E) were incubated for 3 min at 37 8C with [3 H]E13SO4 (300 nM, 100 nCi) and ATP or AMP.
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ABCC1 p.Tyr1189Ala 15652236:177:132
status: NEW153 (A) Immunoblot of membrane vesicles (1 mg protein) prepared from HEK293T cells transfected with wild-type (WT-MRP1a, b, c), and mutant (Y1189A, Y1189S, Y1190A, Y1190S) MRP1 cDNAs as in Fig 3A. (B) Membrane vesicles (20 mg protein) were incubated with 5 mM [a-32 P]-8N3-ATP at 4 8C for 5 min in transport buffer containing 5 mM MgCl2. Samples were photo-crosslinked, resolved by SDS-PAGE and exposed to film.
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ABCC1 p.Tyr1189Ala 15652236:153:136
status: NEW163 Ala/Ser MRP1 mutants (Y1189A, Y1189S, Y1190A, Y1190S).
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ABCC1 p.Tyr1189Ala 15652236:163:22
status: NEW176 Membrane vesicles (2.5 mg protein) prepared from transfected HEK293T cells expressing wild-type MRP1 (WT-MRP1) (A), Tyr1189 mutants Y1189A (B), and Y1189S (C), and Tyr1190 mutants Y1190A (D) and Y1190S (E) were incubated for 3 min at 37 8C with [3 H]E13SO4 (300 nM, 100 nCi) and ATP or AMP.
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ABCC1 p.Tyr1189Ala 15652236:176:132
status: NEW