Single amino acid (482) variants of the ABCG2 multidrug transporter: major differences in transport capacity and substrate recognition |
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Authors: | Csilla Özvegy-Laczka Gabriella Köblös András Váradi |
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Institution: | a National Medical Center, Institute of Haematology and Immunology, Membrane Research Group of the Hungarian Academy of Sciences, Diószegi út 64., H-1113 Budapest, Hungary b Institute of Enzymology, Hungarian Academy of Sciences, Karolina út 29., H-1113 Budapest, Hungary |
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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. |
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Keywords: | ABC ATP binding cassette ABCP placenta specific ABC transporter BCRP breast cancer resistance protein β-gal β-galactosidase Hst Hoechst 33342 MDR1 human multidrug resistance protein (P-glycoprotein ABCB1) MRP human multidrug resistance associated protein ABCC1 MXR mitoxantrone resistance protein MTX methotrexate MX mitoxantrone R123 rhodamine 123 Sf9 cells Spodoptera frugiperda ovarian cells SN-38 7-ethyl-10-hydroxy-camptothecin TM transmembrane wt wild-type |
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