Determinants of the substrate specificity of multidrug resistance protein 1: role of amino acid residues with hydrogen bonding potential in predicted transmembrane helix 17.
Human multidrug resistance protein 1 (MRP1) confers resistance to many natural product chemotherapeutic agents and actively transports structurally diverse organic anion conjugates. We previously demonstrated that two hydrogen-bonding amino acid residues in the predicted transmembrane 17 (TM17) of MRP1, Thr(1242) and Trp(1246), were important for drug resistance and 17beta-estradiol 17-(beta-d-glucuronide) (E(2)17betaG) transport. To determine whether other residues with hydrogen bonding potential within TM17 influence substrate specificity, we replaced Ser(1233), Ser(1235), Ser(1237), Gln(1239), Thr(1241), and Asn(1245) with Ala and Tyr(1236) and Tyr(1243) with Phe. Mutations S1233A, S1235A, S1237A, and Q1239A had no effect on any substrate tested. In contrast, mutations Y1236F and T1241A decreased resistance to vincristine but not to VP-16, doxorubicin, and epirubicin. Mutation Y1243F reduced resistance to all drugs tested by 2-3-fold. Replacement of Asn(1245) with Ala also decreased resistance to VP-16, doxorubicin, and epirubicin but increased resistance to vincristine. This mutation also decreased E(2)17betaG transport approximately 5-fold. Only mutation Y1243F altered the ability of MRP1 to transport both leukotriene 4 and E(2)17betaG. Together with our previous results, these findings suggest that residues with side chain hydrogen bonding potential, clustered in the cytoplasmic half of TM17, participate in the formation of a substrate binding site.
Zhang
Da-Wei
DW
Cancer Research Laboratories and Department of Pathology, Queen's University, Kingston, Ontario K7L 3N6, Canada.
Cole
Susan P C
SP
Deeley
Roger G
RG
D001483:Base Sequence; D001692:Biological Transport; D002460:Cell Line; D017931:DNA Primers; D018432:Drug Resistance, Multiple; D004958:Estradiol / Q000031:analogs & derivatives* / Q000378:metabolism; D006801:Humans; D006860:Hydrogen Bonding; D007700:Kinetics; D017997:Leukotriene C4 / Q000378:metabolism; D018613:Microscopy, Confocal; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D013379:Substrate Specificity; D014162:Transfection
D017931:DNA Primers; D004958:Estradiol; D017997:Leukotriene C4; D027425:ATP-Binding Cassette, Sub-Family C Proteins; C025483:estradiol-17 beta-glucuronide; C421109:multidrug resistance-associated protein 1