ABCMdb

PMID: 11500505

Ito K, Oleschuk CJ, Westlake C, Vasa MZ, Deeley RG, Cole SP
Mutation of Trp1254 in the multispecific organic anion transporter, multidrug resistance protein 2 (MRP2) (ABCC2), alters substrate specificity and results in loss of methotrexate transport activity.
J Biol Chem. 2001 Oct 12;276(41):38108-14. Epub 2001 Aug 10., [PubMed]

Sentences

No. Mutations Sentence Comment

6 ABCC2 p.Trp1254Tyr In addition, only the most conservatively substituted mutant (W1254Y) transported [3 H]leukotriene C4, whereas all other substitutions eliminated transport of this substrate. Login to comment 47 ABCC2 p.Trp1254Tyr ABCC2 p.Trp1254Ala ABCC2 p.Trp1254Phe ABCC2 p.Trp1254Cys Mutagenesis was carried out using the TransformerTM kit (CLONTECH, Palo Alto, CA) according to the manufacturer`s instructions with the following sense mutagenic primers (substituted nucleotides are underlined): W1254A (5Ј-C- ACAAACCCTGAACGCTTCTGGTGAGGATGAC-3Ј), W1254C (5Ј-CAC- AAACCCTGAACTGTCTGGTGAGGATGAC-3Ј), W1254F (5Ј-CACAAA- CCCTGAACTTTCTGGTGAGGATGAC-3Ј), and W1254Y (5Ј-CACAAA- CCCTGAACTATCTGGTGAGGATGAC-3Ј). Login to comment 74 ABCC2 p.Trp1254Tyr ABCC2 p.Trp1254Ala ABCC2 p.Trp1254Phe ABCC2 p.Trp1254Cys These included nonconservative substitutions with a nonaromatic nonpolar amino acid (Ala; W1254A-MRP2) and a nonaromatic polar amino acid (Cys; W1254C-MRP2), as well as conservative substitutions with polar (Tyr; W1254Y-MRP2) and nonpolar (Phe; W1254F-MRP2) aromatic amino acids. Login to comment 76 ABCC2 p.Trp1254Tyr ABCC2 p.Trp1254Ala ABCC2 p.Trp1254Phe ABCC2 p.Trp1254Cys As shown in Fig. 1B, wild-type MRP2 and its four mutants (W1254A, W1254C, W1254Y, and W1254F) were all expressed in the HEK293T cells at comparable levels. Login to comment 77 ABCC2 p.Trp1254Tyr ABCC2 p.Trp1254Ala ABCC2 p.Trp1254Phe ABCC2 p.Trp1254Cys Mean values of expression levels from four to six independent transfections relative to wild-type MRP2 were W1254A, 1.1; W1254C, 0.9; W1254F, 0.9; W1254Y, 1.0, respectively (Fig. 1B). Login to comment 80 ABCC2 p.Trp1254Ala ABCC2 p.Trp1254Cys Like the nonconservatively substituted MRP1-Trp1246 mutants, the W1254A-MRP2 and W1254C-MRP2 mutants either did not transport or only very poorly transported [3 H]E217betaG (Fig. 2B). Login to comment 81 ABCC2 p.Trp1254Tyr ABCC2 p.Trp1254Phe However, E217betaG transport by the W1254Y-MRP2 and W1254F-MRP2 mutants was similar to that of wild-type MRP2, in marked contrast to the comparable conservatively substituted MRP1-Trp1246 mutants, which were inactive for transport of this substrate (40). Login to comment 83 ABCC2 p.Trp1254Tyr The [3 H]LTC4 transport activity of the fourth and most conservatively substituted mutant, W1254Y-MRP2, was ϳ40% of wild-type MRP2. Login to comment 84 ABCC2 p.Trp1254Phe [3 H]E217betaG Transport by W1254F-MRP2 Is No Longer Inhibited by LTC4-It has been shown previously that LTC4 inhibits MRP2-mediated E217betaG transport (39, 44). Login to comment 85 ABCC2 p.Trp1254Phe Consequently, to determine whether E217betaG transport by a mutant MRP2 protein that no longer transports LTC4 could still be inhibited by this cysteinyl leukotriene, [3 H]E217betaG transport by the W1254F-MRP2 mutant was examined. Login to comment 87 ABCC2 p.Trp1254Phe In contrast, LTC4 had no significant effect on [3 H]E217betaG transport by W1254F-MRP2, indicating that the loss of LTC4 transport by this mutant is associated with a loss of binding of this substrate. Login to comment 97 ABCC2 p.Trp1254Tyr ABCC2 p.Trp1254Ala ABCC2 p.Trp1254Phe ABCC2 p.Trp1254Cys B, immunoblot of membrane vesicles prepared from HEK293T cells transfected with empty vector (pcDNA3.1(-)), wild-type (WT-MRP2) and mutant (W1254A, W1254C, W1254F, and W1254Y) MRP2 cDNAs. Login to comment 103 ABCC2 p.Trp1254Tyr ABCC2 p.Trp1254Ala ABCC2 p.Trp1254Phe ABCC2 p.Trp1254Cys A, [3 H]E217betaG uptake was measured in membrane vesicles prepared from HEK293T cells transfected with empty vector (pcDNA3.1(-); open bar), wild-type MRP2 (WT-MRP2; solid bar), and mutant (W1254A-MRP2, W1254C-MRP2, W1254F-MRP2, and W1254Y-MRP2; shaded bars) cDNA expression vectors. Login to comment 106 ABCC2 p.Trp1254Tyr ABCC2 p.Trp1254Ala ABCC2 p.Trp1254Phe ABCC2 p.Trp1254Cys The results shown are the means Ϯ S.D. of triplicate determinations in a single experiment, and similar results were obtained in a second experiment. relative levels of [3 H]MTX transport by the W1254A, W1254C, W1254F, and W1254Y mutants were 14, 14, 11, and 1%, respectively, of wild-type MRP2. Login to comment 108 ABCC2 p.Trp1254Phe [3 H]E217betaG Transport by Wild-type MRP2, but Not W1254F-MRP2, Is Inhibited by MTX-It has previously been shown that MTX inhibits MRP2-mediated transport of the GSH-conjugated substrates, dinitrophenyl-glutathione and N-ethylma- leimide-glutathione (21, 24). Login to comment 111 ABCC2 p.Trp1254Tyr ABCC2 p.Trp1254Phe To determine whether E217betaG transport by mutant MRP2 proteins that no longer transport MTX remained inhibitable by this antifolate, [3 H]E217betaG transport in membrane vesicles prepared from cells expressing W1254F-MRP2 and W1254Y-MRP2 was examined. Login to comment 112 ABCC2 p.Trp1254Tyr ABCC2 p.Trp1254Phe In contrast to wild-type MRP2, MTX had no significant effect on [3 H]E217betaG transport by either W1254F-MRP2 or W1254Y-MRP2 at concentrations of either 500 or 1000 ␮M MTX (Fig. 4B). Login to comment 116 ABCC2 p.Trp1254Tyr ABCC2 p.Trp1254Ala ABCC2 p.Trp1254Phe ABCC2 p.Trp1254Cys In addition, we observed that [3 H]E217betaG transport by the MRP2 mutants W1254A, W1254C, W1254F, and W1254Y was enhanced 9-, 6-, 9-, and 10-fold, respectively, by sulfinpyrazone compared with 4-fold for wild-type MRP2. Login to comment 129 ABCC2 p.Trp1254Phe Membrane vesicles prepared from HEK293T cells expressing wild-type (WT-MRP2) and mutant W1254F-MRP2 were incubated with [3 H]E217betaG in the absence (open bars) and presence (solid and shaded bars) of 1 ␮M LTC4 for 5 min as described under "Experimental Procedures." Login to comment 133 ABCC2 p.Trp1254Tyr ABCC2 p.Trp1254Phe B, membrane vesicles (8 ␮g of protein) from cells expressing WT-MRP2 and mutants W1254F-MRP2 and W1254Y-MRP2 were incubated at 37 °C with 400 nM [3 H]E217betaG in transport buffer and other components for 5 min in the absence (open bars) or the presence of MTX (500 ␮M, shaded bar; 1000 ␮M, solid bar) as described under "Experimental Procedures." Login to comment 138 ABCC2 p.Trp1254Ala ABCC2 p.Trp1254Cys Aromatic interactions also appear critical for LTC4 transport activity by MRP2, again in contrast to MRP1, because transport of this conjugated organic anion was eliminated when Trp1254 was replaced with Ala and Cys. Login to comment 139 ABCC2 p.Trp1254Phe However, LTC4 transport by MRP2 was also markedly reduced when Trp1254 was replaced with Phe but less so when it was replaced with Tyr. Login to comment 141 ABCC2 p.Trp1254Phe The selective loss of LTC4 transport activity of the W1254F-MRP2 mutant was matched by the inability of the cysteinyl leukotriene to inhibit [3 H]E217betaG transport by this mutant, suggesting that loss of LTC4 transport is caused by a loss of binding of this substrate to the protein. Login to comment 151 ABCC2 p.Trp1254Tyr ABCC2 p.Trp1254Phe However, although [3 H]E217betaG transport by the MRP2- W1254F and MRP2-W1254Y mutants appeared unchanged despite their inability to transport MTX, transport of the conjugated estrogen was no longer inhibited by the antifolate agent. Login to comment 156 ABCC2 p.Trp1254Tyr ABCC2 p.Trp1254Ala ABCC2 p.Trp1254Phe ABCC2 p.Trp1254Cys Of the three MRP2 substrates examined in the present study, none were transported by the nonconservatively substituted W1254C and W1254A mutants, one was transported by the W1254F mutant, and two were transported by the W1254Y mutant. Login to comment 159 ABCC2 p.Trp1254Tyr ABCC2 p.Trp1254Ala ABCC2 p.Trp1254Phe ABCC2 p.Trp1254Cys Effect of sulfinpyrazone on [3 H]E217betaG uptake and [3 H]MTX uptake by wild-type and Trp1254 mutant MRP2 proteins. A, [3 H]E217betaG uptake was measured in the absence (open bars) and the presence (solid and shaded bars) of sulfinpyrazone (100 ␮M) in membrane vesicles prepared from cells expressing wild-type (WT-MRP2) and mutant (W1254A, W1254C, W1254F, and W1254Y) MRP2 proteins as described under "Experimental Procedures." Login to comment