ABCMdb

PMID: 15652236

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., [PubMed]

Sentences

No. Mutations Sentence Comment

41 ABCC8 p.Ser1237Tyr Binding of [3 H]glibenclamide to membranes expressing SUR1-S1237Y is abolished concomitantly with the loss of high-affinity tolbutamide block. Login to comment 59 ABCC1 p.Tyr1189Ala ABCC1 p.Tyr1190Ser ABCC1 p.Tyr1190Ala ABCC1 p.Tyr1189Ser 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 ). Login to comment 111 ABCC1 p.Tyr1189Ser ABCC1 p.Tyr1189Ser Kinetic analysis of [3 H]GSH transport Km and Vmax values of ATP-dependent GSH transport by membrane vesicles (20 mg protein) were determined by measuring uptake at eight different [3 H]GSH concentrations (10-2500 mM for wild-type MRP1 and 5010,000 mM for Y1189S mutant MRP1) for 20 min at 37 8C in 60 ml of transport buffer containing components as described above [16]. Login to comment 115 ABCC1 p.Tyr1189Ala ABCC1 p.Tyr1190Ala 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. Login to comment 119 ABCC1 p.Tyr1189Ala ABCC1 p.Tyr1190Ser ABCC1 p.Tyr1190Ala ABCC1 p.Tyr1189Ser 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. Login to comment 121 ABCC1 p.Tyr1189Ala ABCC1 p.Tyr1190Ser ABCC1 p.Tyr1190Ala ABCC1 p.Tyr1189Ser E217bG uptake by the Y1189A and Y1189S mutants was reduced by 30%, and by 55-65% for the Y1190A and Y1190S mutants. Login to comment 122 ABCC1 p.Tyr1189Ala ABCC1 p.Tyr1190Ser ABCC1 p.Tyr1190Ala ABCC1 p.Tyr1189Ser E13SO4 uptake was reduced by 35% in the case of Y1189S and by 60-65% for the Y1189A, Y1190A and Y1190S mutants. Login to comment 125 ABCC1 p.Tyr1189Phe ABCC1 p.Tyr1189Trp More conservatively substituted Y1189F and Y1189W mutant proteins were generated and these also exhibited levels of transport activity that were reduced by a similar extent (data not shown). Login to comment 129 ABCC1 p.Tyr1189Ala ABCC1 p.Tyr1190Ser ABCC1 p.Tyr1190Ala ABCC1 p.Tyr1189Ser As shown in Fig. 4B, Y1189A, Y1189S, Y1190A and Y1190S exhibited a moderate (10-30%) decrease in azido-ATP labeling at 4 8C. Login to comment 130 ABCC1 p.Tyr1189Ala ABCC1 p.Tyr1190Ser ABCC1 p.Tyr1190Ala ABCC1 p.Tyr1189Ser 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). Login to comment 133 ABCC1 p.Tyr1189Ala ABCC1 p.Tyr1190Ser ABCC1 p.Tyr1190Ala ABCC1 p.Tyr1189Ser (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. Login to comment 137 ABCC1 p.Tyr1189Ala Ala/Ser mutant MRP1 (Y1189A, ! Login to comment 138 ABCC1 p.Tyr1190Ser ABCC1 p.Tyr1190Ala ABCC1 p.Tyr1189Ser ; Y1189S, ~; Y1190A, *; Y1190S, *) cDNAs. Login to comment 143 ABCC1 p.Tyr1189Ala ABCC1 p.Tyr1190Ser ABCC1 p.Tyr1190Ala ABCC1 p.Tyr1189Ser 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. Login to comment 150 ABCC1 p.Tyr1189Ser Kinetic analysis of [3 H]GSH transport To better understand the basis for the markedly reduced levels of GSH uptake by the Tyr mutants, the kinetic parameters of apigenin-stimulated GSH uptake by the Y1189S mutant were determined and compared to those of wild-type MRP1 (Fig. 6). Login to comment 151 ABCC1 p.Tyr1189Ser Kinetic analysis of [3 H]GSH transport To better understand the basis for the markedly reduced levels of GSH uptake by the Tyr mutants, the kinetic parameters of apigenin-stimulated GSH uptake by the Y1189S mutant were determined and compared to those of wild-type MRP1 (Fig. 6). Login to comment 153 ABCC1 p.Tyr1189Ala ABCC1 p.Tyr1190Ser ABCC1 p.Tyr1190Ala ABCC1 p.Tyr1189Ser (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. Login to comment 154 ABCC1 p.Tyr1189Ala ABCC1 p.Tyr1190Ser ABCC1 p.Tyr1190Ala ABCC1 p.Tyr1189Ser (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. Login to comment 163 ABCC1 p.Tyr1189Ala ABCC1 p.Tyr1190Ser ABCC1 p.Tyr1190Ala ABCC1 p.Tyr1189Ser Ala/Ser MRP1 mutants (Y1189A, Y1189S, Y1190A, Y1190S). Login to comment 164 ABCC1 p.Tyr1189Ala ABCC1 p.Tyr1190Ser ABCC1 p.Tyr1190Ala ABCC1 p.Tyr1189Ser Ala/Ser MRP1 mutants (Y1189A, Y1189S, Y1190A, Y1190S). Login to comment 168 ABCC1 p.Tyr1189Ser that the Km (GSH) of Y1189S was increased 10-fold compared to that of wild-type MRP1 (1904 420 mM versus 162 29 mM). Login to comment 169 ABCC1 p.Tyr1189Ser ABCC1 p.Tyr1189Ser that the Km (GSH) of Y1189S was increased 10-fold compared to that of wild-type MRP1 (1904 Æ 420 mM versus 162 Æ 29 mM). Login to comment 170 ABCC1 p.Tyr1189Ser In addition, the Vmax of GSH transport was also moderately increased (8.9 Æ 0.4 versus 12.4 Æ 1.0 nmol mgÀ1 protein after 20 min, respectively, for wild-type MRP1 and Y1189S). Login to comment 171 ABCC1 p.Tyr1189Ser Ser mutation (Vmax/Km 103 = 54.9 versus 6.5 for wild-type MRP1 and the Y1189S mutant, respectively). Login to comment 172 ABCC1 p.Tyr1189Ser ABCC1 p.Tyr1189Ser Ser mutation (Vmax/Km Â 103 = 54.9 versus 6.5 for wild-type MRP1 and the Y1189S mutant, respectively). Login to comment 173 ABCC1 p.Tyr1189Ser These results indicate that the mutation Y1189S altered mainly the uptake affinity of MRP1 for GSH and to a lesser extent the transport capacity of MRP1 for this tripeptide. Login to comment 176 ABCC1 p.Tyr1189Ala ABCC1 p.Tyr1190Ser ABCC1 p.Tyr1190Ala ABCC1 p.Tyr1189Ser 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. Login to comment 177 ABCC1 p.Tyr1189Ala ABCC1 p.Tyr1190Ser ABCC1 p.Tyr1190Ala ABCC1 p.Tyr1189Ser 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. Login to comment 182 ABCC1 p.Tyr1189Ser Apigenin-stimulated GSH uptake by MRP1-enriched membrane vesicles was measured at different substrate concentrations (range 10-2500 mM for WT-MRP1 and 50-10,000 mM for Y1189S) in the presence of 30 mM apigenin. Login to comment 183 ABCC1 p.Tyr1189Ser Apigenin-stimulated GSH uptake by MRP1-enriched membrane vesicles was measured at different substrate concentrations (range 10-2500 mM for WT-MRP1 and 50-10,000 mM for Y1189S) in the presence of 30 mM apigenin. Login to comment 184 ABCC1 p.Tyr1189Ser For WT-MRP1, the Km and Vmax values for GSH uptake were 162 mM and 8.9 nmol mg1 20 min1 , respectively; for Y1189S, the Km and Vmax values were 1904 mM and 12.4 nmol mg1 20 min1 , respectively. Login to comment 185 ABCC1 p.Tyr1189Ser For WT-MRP1, the Km and Vmax values for GSH uptake were 162 mM and 8.9 nmol mgÀ1 20 minÀ1 , respectively; for Y1189S, the Km and Vmax values were 1904 mM and 12.4 nmol mgÀ1 20 minÀ1 , respectively. Login to comment 193 ABCC1 p.Tyr1190Ser ABCC1 p.Tyr1189Ser Effect of sulfonylurea drugs on [3 H]E217bG uptake To determine whether the Tyr to Ser mutations affected the ability of sulfonylureas to modulate the vesicular organic anion transport activity of MRP1, E217bG uptake by the Y1189S and Y1190S mutants in the presence of glibenclamide and tolbutamide was examined. Login to comment 194 ABCC1 p.Tyr1190Ser ABCC1 p.Tyr1189Ser Effect of sulfonylurea drugs on [3 H]E217bG uptake To determine whether the Tyr to Ser mutations affected the ability of sulfonylureas to modulate the vesicular organic anion transport activity of MRP1, E217bG uptake by the Y1189S and Y1190S mutants in the presence of glibenclamide and tolbutamide was examined. Login to comment 195 ABCC1 p.Tyr1190Ser ABCC1 p.Tyr1189Ser As summarized in Table 2, glibenclamide inhibited E217bG uptake by Y1189S and Y1190S by 26-36% at a concentration of 10 mM and by 78-88% at 100 mM. Login to comment 196 ABCC1 p.Tyr1190Ser ABCC1 p.Tyr1189Ser As summarized in Table 2, glibenclamide inhibited E217bG uptake by Y1189S and Y1190S by 26-36% at a concentration of 10 mM and by 78-88% at 100 mM. Login to comment 198 ABCC1 p.Tyr1190Ser ABCC1 p.Tyr1189Ser Probenecid (100 mM) inhibited E217bG transport by the Y1189S mutant by approximately 45%, the Y1190S mutant by 20%, and wild-type MRP1 by 61%. Login to comment 199 ABCC1 p.Tyr1190Ser ABCC1 p.Tyr1189Ser ABCC1 p.Tyr1189Ser Probenecid (100 mM) inhibited E217bG transport by the Y1189S mutant by approximately 45%, the Y1190S mutant by 20%, and wild-type MRP1 by 61%. Login to comment 200 ABCC1 p.Tyr1189Ser These results indicate that Ser substitution of Tyr1189 or Tyr1190 has no substantial effect on the sensitivity of MRP1 to sulfonylureas. Login to comment 212 ABCC1 p.Tyr1189Phe Conservatively substituted mutants Y1189F and Y1189Walso showed a comparable reduction in transport activity (not shown), suggesting that physical properties in addition to the aromaticity of Tyr1189 and Tyr1190 are important for their role in MRP1 transport activity. Login to comment 213 ABCC1 p.Tyr1189Phe Conservatively substituted mutants Y1189F and Y1189Walso showed a comparable reduction in transport activity (not shown), suggesting that physical properties in addition to the aromaticity of Tyr1189 and Tyr1190 are important for their role in MRP1 transport activity. Login to comment 218 ABCC1 p.Tyr1190Ser ABCC1 p.Tyr1189Ser ABCC1 p.Tyr1189Ser Indeed, the 10-fold difference in Km (GSH) observed for the Y1189S mutant compared to the wild-type protein indicates a substantial G. Conseil et al. / Biochemical Pharmacology 69 (2005) 451-461 458 Table 2 Effect of glibenclamide and tolbutamide on E217bG uptake by wild-type and Ser-substituted Tyr1189 and Tyr1190 mutant MRP1 proteins Drug Concentration (mM) %Inhibition of E217bG uptakea Wild-type Y1189S Y1190S Glibenclamide 10 54 7 36 11 26 16 Glibenclamide 100 67 10 78 10 88 10 Tolbutamide 100 5 7 0 14 0 7 Probenecid 100 61 6 45 9 20 10 a The values shown are means of triplicate determinations in a single experiment and are representative of results obtained in two independent experiments. Values have been corrected for different MRP1 expression levels according to the immunoblot shown Fig. 3A. decrease in the uptake affinity of the mutant MRP1 for this tripeptide. Login to comment 219 ABCC1 p.Tyr1190Ser ABCC1 p.Tyr1189Ser ABCC1 p.Tyr1189Ser Indeed, the 10-fold difference in Km (GSH) observed for the Y1189S mutant compared to the wild-type protein indicates a substantial G. Conseil et al. / Biochemical Pharmacology 69 (2005) 451-461458 Table 2 Effect of glibenclamide and tolbutamide on E217bG uptake by wild-type and Ser-substituted Tyr1189 and Tyr1190 mutant MRP1 proteins Drug Concentration (mM) %Inhibition of E217bG uptakea Wild-type Y1189S Y1190S Glibenclamide 10 54 Æ 7 36 Æ 11 26 Æ 16 Glibenclamide 100 67 Æ 10 78 Æ 10 88 Æ 10 Tolbutamide 100 5 Æ 7 0 Æ 14 0 Æ 7 Probenecid 100 61 Æ 6 45 Æ 9 20 Æ 10 a The values shown are means of triplicate determinations in a single experiment and are representative of results obtained in two independent experiments. Values have been corrected for different MRP1 expression levels according to the immunoblot shown Fig. 3A. decrease in the uptake affinity of the mutant MRP1 for this tripeptide. Login to comment