title: Single amino acid (482) variants of the ABCG2 multidrug transporter: major differences in transport capacity and substrate recognition.
abstract: The human ABCG2 protein is an ATP binding cassette half-transporter, which protects our cells and tissues against various xenobiotics, while overexpression of ABCG2 in tumor cells confers multidrug resistance. It has been documented that single amino acid changes at position 482 resulted in altered drug resistance and transport capacity. In this study, we have generated nine Arg-482 mutants (G, I, M, S, T, D, N, K, Y) of ABCG2, and expressed them in insect cells. All ABCG2 variants showed cell surface expression and, in isolated membranes, an ABCG2-specific ATPase activity. When methotrexate accumulation was measured in inside-out membrane vesicles, this transport was supported only by the wild-type ABCG2. In intact cells, mitoxantrone was transported by all ABCG2 variants, except by R482K. Rhodamine 123 was extruded by most of the mutants, except by R482K, Y and by wild-type ABCG2. Hoechst 33342 was pumped out from cells expressing the wild-type and all Arg-482 variants, but not from those expressing R482K and Y. Our study demonstrates that the substrate specificity of the Arg (wild-type) form is unique and that amino acid replacements at position 482 induce major alterations in both the transport activity and substrate specificity of this protein.
authors.affiliation: National Medical Center, Institute of Haematology and Immunology, Membrane Research Group of the Hungarian Academy of Sciences, Diószegi út 64., H-1113 Budapest, Hungary.
authors.forename: Csilla
authors.initials: C
authors.lastname: Ozvegy-Laczka
authors.forename: Gabriella
authors.initials: G
authors.lastname: Köblös
authors.forename: Balázs
authors.initials: B
authors.lastname: Sarkadi
authors.forename: András
authors.initials: A
authors.lastname: Váradi
mesh_terms: D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters / Q000235:genetics* / Q000378:metabolism*; D000251:Adenosine Triphosphatases / Q000378:metabolism; D019943:Amino Acid Substitution / Q000235:genetics*; D000818:Animals; D001120:Arginine / Q000235:genetics / Q000378:metabolism; D001562:Benzimidazoles / Q000494:pharmacology; D001692:Biological Transport; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D005434:Flow Cytometry; D005786:Gene Expression Regulation; D006801:Humans; D008727:Methotrexate / Q000493:pharmacokinetics; D009154:Mutation / Q000235:genetics; D009363:Neoplasm Proteins / Q000235:genetics* / Q000378:metabolism*; D020112:Rhodamine 123 / Q000494:pharmacology; D018411:Spodoptera; D013379:Substrate Specificity
chemical_list: C116614:ABCG2 protein, human; D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters; D001562:Benzimidazoles; D009363:Neoplasm Proteins; D020112:Rhodamine 123; D001120:Arginine; D000251:Adenosine Triphosphatases; C017807:bisbenzimide ethoxide trihydrochloride; D008727:Methotrexate