Structure function studies of human multidrug resistance protein 1 (MRP1)

Murine multidrug resistance protein 1 (mMRP1), unlike human MRP1, does not confer resistance to anthracyclines and transports 17β-estradiol 17-(β-D-glucuronide) (E₂17βG) very poorly. Previous studies using hybrid murine/human proteins revealed that the COOH-terminal third of MRP1 contains amino acids important for the ability of the protein to confer anthracycline resistance and to transport E₂17βG efficiently. By systematically mutating non-conserved residues in mMRP1 to those present in MRP1, we found that an acidic amino acid residue, Glu, at position 1089 in the transmembrane (TM) helix 14 of MRP1 is critical for the ability of the protein to confer drug resistance particularly to the anthracyclines, but is not essential for its ability to transport conjugated organic anions such as LTC₄ and E₂17βG. We also identified a non-conserved amino acid residue, Thr, at position 1242 in TM17 of MRP1 that is important for mediating drug resistance. However, unlike Glu1089, Thr 1242 plays an important role in efficient transport of E₂17βG. Mutations of Thr1242 not only decreased the ability to confer resistance to all drugs tested but also dramatically decreased E₂17βG transport activity. Interestingly, the introduction of a second mutation based on the murine sequence to create MRP1E1089Q/T1242A restored resistance to vincristine and VP-16 without affecting the ability of MRP1 T1242A to transport LTC₄ and E₂17βG. These results demonstrate the strong functional interaction between the amino acid at position 1242 and the residue at position 1089 in determining the ability of MRP1 to confer resistance to both vincristine and VP-16. In addition to Thr1242, another hydrogen bonding amino acid in TM17, Trp 1246, was previously demonstrated to be important for drug resistance and E₂17βG transport. Of all the putative TMs of MRP1, TM17 is the most amphipathic. Thus, we further investigated the role of other residues capable of hydrogen bonding within TM17 in the overall function and substrate specificity of MRP1. Together with our previous results, we found that residues with side-chain hydrogen bonding potential, clustered in the cytoplasmic half of TM17, including Tyr1236, Thr1241, Thr 1242, Tyr1243 and Asn1245, may participate in the formation of a substrate binding site.