Transport of methotrexate, methotrexate polyglutamates, and 17β-estradiol 17-(β-D-glucuronide) by ABCG2: Effects of acquired mutations at R482 on methotrexate transport

ABCG2 is a plasma membrane efflux pump that is able to confer resistance to several anticancer agents, including mitoxantrone, camptothecins, anthracyclines, and flavopiridol. The antimetabolite methotrexate (MTX) was inferred recently to be an additional substrate of the pump based on the analysis of ABCG2-overexpressing cell lines. However, the transport characteristics of the pump with regard to this agent have not been determined. In addition, physiological substrates of ABCG2 have not been identified. Here we examine the in vitro transport properties of the pump using membrane vesicles prepared from HEK293 cells transfected with ABCG2 expression vector. In so doing it is shown that MTX is a high capacity low affinity substrate of the pump, with K_m and V_max values of 1.34 ± 0.18 mM and 687 ± 87 pmol/mg/min, respectively. Unlike previously characterized multidrug resistance protein family members, ABCG2 is also able to transport MTX diglutamate and MTX triglutamate. However, addition of even one more glutamyl residue is sufficient to completely abrogate ABCG2-mediated transport. By contrast with the wild-type protein (ABCG2-R482), two ABCG2 variants that have been identified in drug selected cell lines, R482T and R482G, were unable to transport MTX to any extent. Similarly, folic acid was subject to efflux by the wild-type protein but not by the two mutants. However, transport of the reduced folate leucovorin was not detected for either the wild-type or the mutant proteins. Finally, it is shown that ABCG2 is capable of transporting E₂17βG with K_m and V_max values of 44.2 ± 4.3 μM and 103 ± 17 pmol/mg/min, respectively. These results indicate that ABCG2 is a component of the energy-dependent efflux system for certain folates and antifolates, but that its transport characteristics with respect to polyglutamates and reduced folates are not identical to those of multidrug resistance protein family members. In addition, it is demonstrated that R482 mutations observed in drug-resistant cell lines have profound effects on the in vitro transport properties of the pump.