ABCMdb

PMID: 12388549

Koike K, Oleschuk CJ, Haimeur A, Olsen SL, Deeley RG, Cole SP
Multiple membrane-associated tryptophan residues contribute to the transport activity and substrate specificity of the human multidrug resistance protein, MRP1.
J Biol Chem. 2002 Dec 20;277(51):49495-503. Epub 2002 Oct 17., 2002-12-20 [PubMed]

Sentences

No. Mutations Sentence Comment

48 ABCC1 p.Trp142Ala ABCC1 p.Trp445Phe ABCC1 p.Trp445Ala ABCC1 p.Trp142Phe ABCC1 p.Trp40Ala ABCC1 p.Trp94Ala ABCC1 p.Trp553Phe ABCC1 p.Trp553Ala ABCC1 p.Trp82Ala ABCC1 p.Trp361Ala ABCC1 p.Trp86Ala ABCC1 p.Trp553Tyr ABCC1 p.Trp459Ala ABCC1 p.Trp47Ala ABCC1 p.Trp1198Phe ABCC1 p.Trp445Tyr ABCC1 p.Trp1198Ala ABCC1 p.Trp1198Tyr Tryptophan substitutions were generated in the pGEM-3Z and pBluescriptSK(ϩ) plasmids above according to the manufacturer`s instructions with the following mutagenic primers (substituted nucleotides are underlined): MSD1 Trp mutants W40A (5Ј-G GTC CTC GTG GCC GTG CCT TG-3Ј, W47A (5Ј-GT TTT TAC CTC GCC GCC TGT TTC CCC-3Ј), W82A (5Ј-C TTG GGA TTT TTG CTG GCG ATC GTC TGC TGG GC-3Ј), W86A (5Ј-G CTG TGG ATC GTC TGC GCT GCA GAC CTC TTC TAC TC-3Ј), W94A (5Ј-C CTC TTC TAC TCT TTC GCG GAA AGA AGT CGG GGC-3Ј), W142A (5Ј-GGG ATC ATG CTC ACT TTC GCA CTG GTA GCC CTA ATG TG-3Ј), W142F (5Ј-G CTC ACT TTT TTC CTG GTA GCC C-3Ј); MSD2 Trp mutants W361A (5Ј-C ACG AAG GCC CCA GAT GCG CAG GGC TAC TTC TAC-3Ј), W445A (5Ј-G TAC ATT AAC ATG ATC GCG TCA GCC CCC CTG CAA G-3Ј), W445F (5Ј-CG TAC ATT AAC ATG ATC TTC TCA GCC CCC CTG CAA GTC-3Ј), W445Y (5Ј-CC ACG TAC ATT AAC ATG ATC TAC TCA GCG CCC CTG CAA GTC-3Ј), W459A (5Ј-GCT CTC TAC CTC CTG GCG CTG AAT CTG GGC CC-3Ј), W553A (5Ј-G GGC ACC TTC ACC GCG GTC TGC ACG CCC-3Ј), W553F (5Ј-G GGC ACC TTC ACC TTC GTC TGC ACG CCC-3Ј), W553Y (5Ј-GCC CTG GGC ACC TTC ACA TAT GTC TGC ACG CCC-3Ј; and MSD3 Trp mutants W1198A (5Ј-C GTG GCC AAC AGG GCG CTG GCC GTG CGG C-3Ј), W1198F (5Ј-GTG GCC AAC AGG TTC CTG GCC GTG CGG C-3Ј), W1198Y (5Ј-GTG GCC AAC AGG TAC CTG GCC GTG CGG C-3Ј). Login to comment 50 ABCC1 p.Trp445Ala ABCC1 p.Trp445Ala ABCC1 p.Trp553Ala ABCC1 p.Trp553Ala ABCC1 p.Trp1198Ala ABCC1 p.Trp1198Ala Construction of MRP1-GFP Fusion Proteins-Constructs encoding GFP fusion proteins of selected MRP1 Trp mutations were generated by exchanging the 1.3-kb ClaI/AflII fragment of a pcDNA3.1(-)-MRP1-GFP construct with the comparable fragments containing the W445A, W553A, and W1198A mutations generated above and designated pcDNA3.1-W445A/MRP1-GFP, pcDNA3.1-W553A/MRP1-GFP, and pcDNA3.1-W1198A/MRP1-GFP, respectively (39). Login to comment 94 ABCC1 p.Trp40Ala ABCC1 p.Trp94Ala ABCC1 p.Trp82Ala ABCC1 p.Trp86Ala ABCC1 p.Trp47Ala The levels of the W40A, W47A, W82A, W86A, and W94A mutant MRP1 proteins were comparable with that of wild-type MRP1 (range 75-100%). Login to comment 95 ABCC1 p.Trp142Ala In contrast, levels of the W142A MRP1 mutant were ϳ35% (range 30-40%) of wild-type MRP1 (Fig. 2A). Login to comment 96 ABCC1 p.Trp142Ala Reduced expression levels of the W142A-MRP1 mutant were observed in multiple different transfection assays using several independently derived cDNA clones. Login to comment 99 ABCC1 p.Trp142Ala ABCC1 p.Trp40Ala ABCC1 p.Trp94Ala ABCC1 p.Trp82Ala ABCC1 p.Trp86Ala ABCC1 p.Trp47Ala A, immunoblot of membrane vesicles prepared from HEK293T cells transfected with empty vector (pcDNA3.1(-)), wild type (WT-MRP1), and mutant (W40A, W47A, W82A, W86A, W94A, and W142A) MRP1 cDNAs. MRP1 proteins were detected with monoclonal antibody QCRL-1, and relative levels of expression shown under the blot were estimated by densitometry. Login to comment 108 ABCC1 p.Trp445Ala ABCC1 p.Trp553Ala ABCC1 p.Trp361Ala ABCC1 p.Trp459Ala All four mutants generated (W361A, W445A, W459A, and W553A) were expressed at levels 60-90% those of wild-type MRP1, indicating that none of the mutations had a major effect on the expression levels of the protein. Login to comment 110 ABCC1 p.Trp445Ala ABCC1 p.Trp553Ala ABCC1 p.Trp361Ala ABCC1 p.Trp459Ala After 1 min, ATP-dependent [3 H]LTC4 uptake by the W445A and W553A MRP1 mutants was reduced by ϳ75 and 50%, respectively, whereas uptake by the W361A and W459A mutants was comparable with wild-type MRP1 (Fig. 3B). Login to comment 112 ABCC1 p.Trp459Ala The ATP-dependent uptake rate of this substrate by the W459A MRP1 mutant, as for LTC4 uptake, was comparable with that of wild-type MRP1 (Fig. 3C). Login to comment 113 ABCC1 p.Trp445Ala ABCC1 p.Trp553Ala ABCC1 p.Trp361Ala In contrast, after 1 min, [3 H]E217betaG uptake by the W361A, W445A, and W553A MRP1 mutants was ϳ50, 25, and 10%, respectively, of wild-type MRP1 (Fig. 3D). Login to comment 117 ABCC1 p.Trp445Ala As shown in Fig. 4A, Ala substitution of Trp445 and Trp553 essentially eliminated apigenin-stimulated [3 H]GSH uptake by MRP1. Login to comment 119 ABCC1 p.Trp445Ala ABCC1 p.Trp553Ala ABCC1 p.Trp361Ala ABCC1 p.Trp459Ala Similarly, GSH-stimulated E13SO4 uptake levels by the W445A and W553A mutants were just 30 and Ͻ10% of wild-type MRP1 levels, respectively, whereas uptake by the W361A mutant was similar to wild-type MRP1, and uptake by W459A MRP1 was reduced by just 25% (Fig. 4B). Login to comment 121 ABCC1 p.Trp445Ala ABCC1 p.Trp553Ala ABCC1 p.Trp361Ala ABCC1 p.Trp459Ala A, ATP-dependent uptake of [3 H]LTC4 was measured in membrane vesicles prepared from HEK293T cells transfected with empty vector pcDNA3.1(-) (E) and vectors containing wild-type MRP1 (f) and MSD2 Trp-Ala mutant MRP1 cDNAs (W361A, Œ; W445A, ; W459A, ࡗ; and W553A, q). Login to comment 124 ABCC1 p.Trp445Ala ABCC1 p.Trp553Ala ABCC1 p.Trp361Ala ABCC1 p.Trp459Ala C, the time course of ATP-dependent uptake of [3 H]E217betaG by wild-type MRP1 and MSD2 mutants W361A, W445A, W459A, and W553A was measured as described for A. D, relative levels of [3 H]E217betaG uptake at 1 min are shown and were determined from the time course shown in C as described for B. Login to comment 126 ABCC1 p.Trp445Ala ABCC1 p.Trp553Ala Finally, [3 H]MTX uptake by the W445A and W553A MRP1 mutants, as observed for GSH and E13SO4 uptake, was dramatically reduced by more than 80% (Fig. 4C). Login to comment 127 ABCC1 p.Trp361Ala ABCC1 p.Trp459Ala MTX uptake by the W361A mutant was also reduced (ϳ40%), whereas uptake by the W459A mutant was similar to that of wild-type MRP1. Login to comment 128 ABCC1 p.Trp553Ala ABCC1 p.Trp361Ala Thus, substitution of either Trp445 or Trp553 by an Ala residue resulted in a general loss of MRP1 organic anion transport activity, whereas Ala substitution of Trp361 and Trp459 had a much more moderate and selective effect on the substrate specificity of the protein. Login to comment 132 ABCC1 p.Trp1198Ala When expressed in HEK cells, W1198A-MRP1 was found to be expressed at levels that were ϳ35% (range 30-40%) of those of wild-type MRP1, suggesting that MRP1 stability may be affected by this mutation. Login to comment 133 ABCC1 p.Trp1198Ala Time courses of [3 H]LTC4 uptake showed that transport of this GSH-conjugated substrate by the W1198A mutant was ϳ65% of that by wild-type MRP1 after correcting for differences in MRP1 expression levels (Fig. 5A). Login to comment 134 ABCC1 p.Trp1198Ala In contrast, ATP-dependent [3 H]E217betaG uptake by the W1198A mutant was not detectable (Fig. 5B). Login to comment 139 ABCC1 p.Trp445Phe ABCC1 p.Trp445Ala ABCC1 p.Trp445Tyr One mutant, W445Y, was reproducibly expressed at lower levels (approximately half) than the corresponding W445A and W445F mutants for reasons that are presently unclear. Login to comment 140 ABCC1 p.Trp445Ala ABCC1 p.Trp445Tyr The most conservatively substituted MRP1-Trp445 mutant, W445Y, showed significant transport activity with respect to all five MRP1 substrates compared with the W445A mutant after correcting for differences in MRP1 expression levels. Login to comment 141 ABCC1 p.Trp445Tyr Thus, the LTC4 and E13SO4 transport activities of W445Y MRP1 were similar to those of wild-type MRP1, whereas transport of E217betaG, GSH, and MTX was 30-60% of wild-type activity (Fig. 6, B-F). Login to comment 142 ABCC1 p.Trp445Phe ABCC1 p.Trp445Ala ABCC1 p.Trp445Tyr The Phe-substituted Trp445 mutant, W445F, showed levels of transport intermediate between those of the W445A and W445Y mutants. Login to comment 144 ABCC1 p.Trp1198Ala Transport activity of the W1198A MRP1 mutant was recovered to an even greater extent by Phe and Tyr substitutions. Login to comment 145 ABCC1 p.Trp1198Phe ABCC1 p.Trp1198Tyr Thus, the LTC4, GSH, and E13SO4 transport activities of the W1198F mutant were similar to those of wild-type MRP1, whereas transport of these substrates by the W1198Y mutant exceeded that of wild-type MRP1 (Fig. 6, B, D, and E). Login to comment 146 ABCC1 p.Trp1198Phe ABCC1 p.Trp1198Tyr Transport of E217betaG and MTX by the W1198F and W1198Y mutants was ϳ50-75% that of wild-type MRP1 when corrected for relative MRP1 expression levels (Fig. 6, C and F). Login to comment 147 ABCC1 p.Trp1198Tyr For all substrates, transport activity was restored more effectively when Trp1198 was mutated to Tyr compared with Phe, suggesting that, as observed for the Trp445 mutants, both the aromatic and polar properties of the Trp residue at position 1198 contribute to MRP1 transport activity. Login to comment 148 ABCC1 p.Trp445Ala ABCC1 p.Trp553Ala ABCC1 p.Trp1198Ala In contrast to the W445A and W1198A mutants, more conservative substitutions of the Ala-substituted Trp553 mutant W553A were much less effective in restoring MRP1 transport activity. Login to comment 150 ABCC1 p.Trp553Phe ABCC1 p.Trp553Tyr E217betaG transport by the W553F and W553Y mutants was 50 and 25% that of wild-type MRP1, respectively (Fig. 6C), and recovery of E13SO4 uptake by these mutants was modest (Ͻ30%) (Fig. 6E). Login to comment 153 ABCC1 p.Trp445Ala ABCC1 p.Trp553Ala ABCC1 p.Trp361Ala ABCC1 p.Trp459Ala Relative uptake levels of 3 H-labeled organic anions by membrane vesicles enriched for wild-type MRP1 (solid bar) and W361A, W445A, W459A, and W553A mutant MRP1 proteins (shaded bars) were determined as described under "Experimental Procedures." Login to comment 169 ABCC1 p.Trp142Ala Despite this unusually high frequency (4.5%), we found that none of the six singly Ala-substituted Trp mutants in the TM segments of this region showed significantly reduced E217betaG or LTC4 transport activity, and only one of them, W142A, was expressed at significantly lower levels than wild-type MRP1. Login to comment 171 ABCC1 p.Trp1198Ala C-E, relative uptake levels of 3 H-labeled organic anions by membrane vesicles enriched for wild-type MRP1 (solid bar) and Trp1198 3 Ala substituted MRP1 (W1198A; shaded bars) were determined as described under "Experimental Procedures." Login to comment 179 ABCC1 p.Trp361Ala Thus, the W361A mutant exhibited a 25-50% decrease in E217betaG, GSH, and MTX transport activity compared with wild-type MRP1, but LTC4 and E13SO4 transport activities were not affected. Login to comment 180 ABCC1 p.Trp459Ala The W459A mutant displayed a slightly different phenotype in that its LTC4, E217betaG, and MTX transport activities were similar to wild-type MRP1, but GSH and E13SO4 transport activities were reduced by 25-50%. Login to comment 182 ABCC1 p.Trp445Ala Thus, Ala substitution of Trp445 essentially eliminated the transport of all five MRP1 substrates tested. Login to comment 183 ABCC1 p.Trp553Ala Similarly, the transport of four MRP1 substrates by the W553A mutant was decreased by at least 80%, and transport of the fifth, LTC4, was reduced by ϳ50%. Login to comment 189 ABCC1 p.Trp445Phe ABCC1 p.Trp445Ala ABCC1 p.Trp553Phe ABCC1 p.Trp553Ala ABCC1 p.Trp553Tyr ABCC1 p.Trp1198Phe ABCC1 p.Trp445Tyr ABCC1 p.Trp1198Ala ABCC1 p.Trp1198Ala ABCC1 p.Trp1198Tyr Ala substitution of Trp1198 in MSD3 also resulted in a broad and profound decrease in MRP1 transport activity except for FIG. 6. ATP-dependent organic anion transport activity of wild-type and mutant MRP1 containing conservative Phe and Tyr substitutions of Trp1198 in TM16 of MSD3. A, immunoblot of membrane vesicles prepared from HEK293T cells transfected with empty vector (pcDNA3.1(-)), wild-type (WT-MRP1), and mutant (W445A, W445F, and W445Y; W553A, W553F and W553Y; W1198A, W1198F, and W1198Y) MRP1 cDNAs. MRP1 proteins were detected with monoclonal antibody QCRL-1, and relative levels of expression shown under the blot were estimated by densitometry. Login to comment 190 ABCC1 p.Trp445Phe ABCC1 p.Trp553Phe ABCC1 p.Trp553Tyr ABCC1 p.Trp1198Phe ABCC1 p.Trp445Tyr ABCC1 p.Trp1198Tyr B-F, relative levels of 3 H-labeled organic anion uptake by the membrane vesicles shown in A enriched for wild-type MRP1 (solid bar), Trp 3 Phe-substituted MRP1 mutants (W445F, W553F, and W1198F; hatched bars), and Trp 3 Tyr-substituted MRP1 mutants (W445Y, W553Y, and W1198Y; open bars) were determined as described under "Experimental Procedures." Login to comment 191 ABCC1 p.Trp445Ala ABCC1 p.Trp553Ala ABCC1 p.Trp1198Ala Ala-substituted mutants (W445A, W553A, and W1198A; shaded bars) were included for comparison. Login to comment 194 ABCC1 p.Trp553Ala ABCC1 p.Trp1198Ala The Ala-substituted Trp1246 mutant, like W553A and W1198A, also showed significantly reduced transport of multiple MRP1 substrates with the exception of LTC4 (39). Login to comment 196 ABCC1 p.Trp1246Ala ABCC1 p.Trp1198Ala However, the specific environment of the two Trp residues appears to differ, since Tyr and Phe substitutions restore the transport activity of the W1198A but not W1246A mutant (39). Login to comment 224 ABCC1 p.Trp445Ala ABCC1 p.Trp553Ala ABCC1 p.Trp1198Ala HEK293T cells were transfected with pcDNA3.1(-)MRP1K-GFP (WT-MRP1-GFP) (A), pcDNA3.1-W445A/MRP1-GFP (B), pcDNA3.1-W553A/MRP1-GFP (C), and pcDNA3.1-W1198A/MRP1-GFP (D), and 48 h later, cells were processed for confocal fluorescence microscopy. Login to comment