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Abstract
Multidrug resistance protein 1 (MRP1/ABCC1) belongs to the ATP-binding cassette transporter superfamily and is capable of conferring resistance to a broad 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 17beta-estradiol 17-(beta-d-glucuronide) (E(2)17betaG) transport by membrane vesicles prepared from transiently transfected human embryonic kidney cells while leaving the capacity for leukotriene C(4)- and verapamil-stimulated glutathione transport intact. In addition, in contrast to wild-type MRP1, leukotriene C(4) transport by the W1246C-MRP1 protein was no longer inhibitable by E(2)17betaG, indicating that the mutant protein had lost the ability to bind the glucuronide. A similar phenotype was observed when Trp(1246) was replaced with Ala, Phe, and Tyr. Confocal microscopy of cells expressing Trp(1246) mutant MRP1 molecules fused at the C terminus with green fluorescent protein showed that they were correctly routed to the plasma membrane. In addition to the loss of E(2)17betaG transport, HeLa cells stably transfected with W1246C-MRP1 cDNA were not resistant to the Vinca alkaloid vincristine and accumulated levels of [(3)H]vincristine comparable to those in vector control-transfected cells. Cells expressing W1246C-MRP1 were also not resistant to cationic anthracyclines (doxorubicin, daunorubicin) or the electroneutral epipodophyllotoxin VP-16. In contrast, resistance to sodium arsenite was only partially diminished, and resistance to potassium antimony tartrate remained comparable to that of cells expressing wild-type MRP1. This suggests that the structural determinants required for transport of heavy metal oxyanions differ from those for chemotherapeutic agents. Our results provide the first example of a tryptophan residue being so critically important for substrate specificity in a eukaryotic ATP-binding cassette transporter.
Highlights
The ability of multidrug resistance protein 1 (MRP1) to confer drug resistance and transport conjugated organic anions is shared by at least two other proteins belonging to the ABCC subfamily, MRP2 (ABCC2) and MRP3 (ABCC3) [5, 6]
We found that substitution of a highly conserved tryptophan at position 1246 with cysteine (W1246CMRP1) in the putative last transmembrane segment (TM17) of MRP1 eliminated 17-estradiol 17-(-D-glucuronide) (E217G) transport by membrane vesicles prepared from transiently transfected human embryonic kidney cells while leaving the capacity for leukotriene C4- and verapamil-stimulated glutathione transport intact
Acid in MRP1—During the course of a mutational analysis of proline residues in MRP1, we generated an MRP1 mutant in which proline at position 196 was replaced with alanine (P196A)
Summary
The ability of MRP1 to confer drug resistance and transport conjugated organic anions is shared by at least two other proteins belonging to the ABCC subfamily, MRP2 (ABCC2) and MRP3 (ABCC3) [5, 6]. When stably expressed in HeLa cells, the ability of this mutant to transport organic anions was reduced, and it no longer conferred resistance to natural product drugs.
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