Characterization of the role of polar amino acid residues within predicted transmembrane helix 17 in determining the substrate specificity of multidrug resistance protein 3.
Human multidrug resistance protein (MRP) 3 is the most closely related homologue of MRP1. Like MRP1, MRP3 confers resistance to etoposide (VP-16) and actively transports 17 beta-estradiol 17-(beta-D-glucuronide) (E(2)17 beta G), cysteinyl leukotriene 4 (LTC(4)), and methotrexate, although with generally lower affinity. Unlike MRP1, MRP3 also transports monovalent bile salts. We have previously demonstrated that hydrogen-bonding residues predicted to be in the inner-leaflet spanning segment of transmembrane (TM) 17 of MRP1 are important for drug resistance and E(2)17 beta G transport. We have now examined the importance of the hydrogen-bonding potential of residues in TM17 of MRP3 on both substrate specificity and overall activity. Mutation S1229A reduced only methotrexate transport. Mutations S1231A and N1241A decreased resistance to VP-16 and transport of E(2)17 beta G and methotrexate but not taurocholate. Mutation Q1235A also reduced resistance to VP-16 and transport of E(2)17beta G but increased taurocholate transport without affecting transport of methotrexate. Mutations Y1232F and S1233A reduced resistance to VP-16 and the transport of all three substrates tested. In contrast, mutation T1237A markedly increased VP-16 resistance and transport of all substrates. On the basis of the substrates analyzed, residues Ser(1229), Ser(1231), Gln(1235), and Asn(1241) play an important role in determining the specificity of MRP3, while mutation of Tyr(1232), Ser(1233), and Thr(1237) affects overall activity. Unlike MRP1, the involvement of polar residues in determining substrate specificity extends throughout the TM helix. Furthermore, elimination of the hydrogen-bonding potential of a single amino acid, Thr(1237), markedly enhanced the ability of the protein to confer drug resistance and to transport all substrates examined.
Zhang
Da-Wei
DW
Division of Cancer Biology and Genetics, Cancer Research Institute, and Department of Pathology, Queen's University, Kingston, Ontario K7L 3N6, Canada.
Gu
Hong-Mei
HM
Vasa
Monika
M
Muredda
Mario
M
Cole
Susan P C
SP
Deeley
Roger G
RG
D018435:ATP Binding Cassette Transporter, Subfamily B / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018528:ATP-Binding Cassette Transporters / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D000972:Antineoplastic Agents, Phytogenic / Q000494:pharmacology*; D001692:Biological Transport; D002455:Cell Division / Q000187:drug effects; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D017931:DNA Primers; D018432:Drug Resistance, Multiple; D004791:Enzyme Inhibitors / Q000378:metabolism; D004958:Estradiol / Q000031:analogs & derivatives* / Q000378:metabolism*; D005047:Etoposide / Q000494:pharmacology*; D006801:Humans; D006860:Hydrogen Bonding; D007700:Kinetics; D017997:Leukotriene C4 / Q000378:metabolism; D008727:Methotrexate / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics; D016133:Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D013656:Taurocholic Acid / Q000378:metabolism; D014162:Transfection
D018435:ATP Binding Cassette Transporter, Subfamily B; D018528:ATP-Binding Cassette Transporters; D000972:Antineoplastic Agents, Phytogenic; D017931:DNA Primers; D004791:Enzyme Inhibitors; C025483:estradiol-17 beta-glucuronide; D017997:Leukotriene C4; D004958:Estradiol; D013656:Taurocholic Acid; D005047:Etoposide; C095122:multidrug resistance protein 3; D008727:Methotrexate