Identification of a nonconserved amino acid residue in multidrug resistance protein 1 important for determining substrate specificity - Evidence for functional interaction between transmembrane helices 14 and 17

Murine multidrug resistance protein 1 (mrp1), differs from its human orthol og (MRP1) in that it fails to confer anthracycline resistance and transport s the MRP1 substrate, 17 beta -estradiol 17-(beta -D-glucuronide) (E(2)17 b etaG), very poorly. By mutating variant residues in mrp1 to those present i n MRP1, we identified Glu(1089) of MRP1 as being critical for anthracycline resistance. However, Glu(1089) mutations had no effect on E(2)17 betaG tra nsport. We have now identified a nonconserved amino acid within the highly conserved COOH-proximal transmembrane helix of MRP1/mrp1 that is important for transport of the conjugated estrogen. Converting Ala(1239) in mrp1 to T hr, as in the corresponding position (1242) in MRP1, increased E(2)17 betaG transport 3-fold. Any mutation of mrp1 Ala(1239), including substitution w ith Thr, decreased resistance to vincristine and VP-16 without altering ant hracycline resistance. However, introduction of a second murine to human mu tation, Q1086E, which alone selectively increases anthracycline resistance, into mrp1A1239T restored resistance to both vincristine and VP-16. To conf irm the importance of MRP1 Thr(1242) for E(2)17 betaG transport and drug re sistance, we mutated this residue to Ala, Cys, Ser, Leu, and Lys. These mut ations decreased E(2)17 betaG transport 2-fold. Conversion to Asp eliminate d transport of the estrogen conjugate and also decreased leukotriene C-4 tr ansport similar to2-fold. The mutations also reduced the ability of MRP1 to confer resistance to all drugs tested. As with mrp1, introduction of a sec ond mutation based on the murine sequence to create MRP1E1089Q/ T1242A rest ored resistance to vincristine and VP-16, but not anthracyclines, without a ffecting transport of leukotriene C-4 and E(2)17 betaG. These results demon strate the important role of Thr(1242) for E(2)17 betaG transport. They als o reveal a highly specific functional relationship between nonconserved ami no acids in TM helices 14 and 17 of both mrp1 and MRP1 that enables both pr oteins to confer similar levels of resistance to vincristine and VP-16.