Mutation of a single conserved tryptophan in multidrug resistance protein 1 (MRP1/ABCC1) results in loss of drug resistance and selective loss of organic anion transport

Multidrug resistance protein 1 (MRP1/ABCC1) belongs to the ATP-binding cass ette transporter superfamily and is capable of conferring resistance to a b road range of chemotherapeutic agents and transporting structurally diverse conjugated organic anions. In this study, we found that substitution of a highly conserved tryptophan at position 1246 with cysteine (W1246C-MRP1) in the putative last transmembrane segment (TM17) of MRP1 eliminated 17 beta -estradiol 17-(beta -D-glucuronide) (E(2)17 betaG) transport by membrane ve sicles prepared from transiently transfected human embryonic kidney cells w hile leaving the capacity for leukotriene C-4- and verapamil-stimulated glu tathione transport intact. In addition, in contrast to wild-type MRP1, leuk otriene C-4 transport by the W1246C-MRP1 protein was no longer inhibitable by E(2)17 betaG, indicating that the mutant protein had lost the ability to bind the glucuronide. A similar phenotype was observed when Trp(1246) was replaced with Ale, Phe, and Tyr. Confocal microscopy of cells expressing Tr p(1246) mutant MRP1 molecules fused at the C terminus with green fluorescen t protein showed that they were correctly routed to the plasma membrane. In addition to the loss of E(2)17 betaG transport, HeLa cells stably transfec ted with W1246C-MRP1 cDNA were not resistant to the Vinca alkaloid vincrist ine and accumulated levels of [H-3]vincristine comparable to those in vecto r control-transfected cells. Cells expressing W1246C-MRP1 were also not res istant to cationic anthracyclines (doxorubicin, daunorubicin) or the electr oneutral epipodophyllotoxin VP-16. In contrast, resistance to sodium arseni te was only partially diminished, and resistance to potassium antimony tart rate remained comparable to that of cells expressing wild-type MRP1. This s uggests that the structural determinants required for transport of heavy me tal oxyanions differ from those for chemotherapeutic agents. Our results pr ovide the first example of a tryptophan residue being so critically importa nt for substrate specificity in a eukaryotic ATP-binding cassette transport er.