title: Wild-type breast cancer resistance protein (BCRP/ABCG2) is a methotrexate polyglutamate transporter.
abstract: The existence of an ATP-dependent methotrexate (MTX) efflux mechanism has long been postulated; however, until recently, the molecular components were largely unknown. We have previously demonstrated a role for the ATP-binding cassette transporter breast cancer resistance protein (BCRP) in MTX resistance (Volk et al., Cancer Res., 62: 5035-5040, 2002). Resistance to this antifolate directly correlated with BCRP expression, and was reversible by the BCRP inhibitors fumitremorgin C and GF120918. Here, we provide evidence for BCRP as a MTX-transporter using an in vitro membrane vesicle system. Inside-out membrane vesicles were generated from both drug-selected and stably transfected cell lines expressing either wild-type (Arg482) or mutant (Gly482) variants of BCRP. In the presence of the wild-type variant of BCRP, transport of MTX into vesicles was ATP-dependent, osmotically sensitive, and inhibited by fumitremorgin C. In contrast, no transport was observed in vesicles containing the mutant form of BCRP. Wild-type BCRP appeared to have low affinity, but high capacity, for the transport of MTX, with an estimated K(m) of 680 micro M and a V(max) of 2400 pmol/mg/min. MTX accumulation was greatly decreased by mitoxantrone, a known BCRP substrate, suggesting competition for transport. Furthermore, and in contrast to the multidrug resistance-associated proteins, BCRP also transported significant amounts of polyglutamylated MTX. Although transport gradually decreased as the polyglutamate chain length increased, both MTX-Glu(2) and MTX-Glu(3) were substrates for BCRP. Together, these data demonstrate that BCRP is a MTX and MTX-polyglutamate transporter and reveal a possible mechanism by which it confers resistance.
mesh_terms: D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D001667:Binding, Competitive; D001693:Biological Transport, Active; D001943:Breast Neoplasms / Q000378:metabolism*; D002352:Carrier Proteins / Q000378:metabolism*; D022162:Cytoplasmic Vesicles / Q000378:metabolism; D006801:Humans; D007425:Intracellular Membranes / Q000378:metabolism; D008727:Methotrexate / Q000031:analogs & derivatives* / Q000493:pharmacokinetics*; D008942:Mitoxantrone / Q000493:pharmacokinetics; D009363:Neoplasm Proteins; D009995:Osmosis; D011099:Polyglutamic Acid / Q000031:analogs & derivatives* / Q000493:pharmacokinetics*; D014407:Tumor Cells, Cultured
chemical_list: C116614:ABCG2 protein, human; D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters; D002352:Carrier Proteins; D009363:Neoplasm Proteins; D011099:Polyglutamic Acid; C014085:methotrexate polyglutamate; D008942:Mitoxantrone; D008727:Methotrexate