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Abstract
Murine multidrug resistance protein 1 (mrp1), differs from its human ortholog (MRP1) in that it fails to confer anthracycline resistance and transports the MRP1 substrate, 17beta-estradiol 17-(beta-d-glucuronide) (E(2)17betaG), very poorly. By mutating variant residues in mrp1 to those present in MRP1, we identified Glu(1089) of MRP1 as being critical for anthracycline resistance. However, Glu(1089) mutations had no effect on E(2)17betaG transport. 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 Thr, as in the corresponding position (1242) in MRP1, increased E(2)17betaG transport 3-fold. Any mutation of mrp1 Ala(1239), including substitution with Thr, decreased resistance to vincristine and VP-16 without altering anthracycline resistance. However, introduction of a second murine to human mutation, Q1086E, which alone selectively increases anthracycline resistance, into mrp1A1239T restored resistance to both vincristine and VP-16. To confirm the importance of MRP1 Thr(1242) for E(2)17betaG transport and drug resistance, we mutated this residue to Ala, Cys, Ser, Leu, and Lys. These mutations decreased E(2)17betaG transport 2-fold. Conversion to Asp eliminated transport of the estrogen conjugate and also decreased leukotriene C(4) transport approximately 2-fold. The mutations also reduced the ability of MRP1 to confer resistance to all drugs tested. As with mrp1, introduction of a second mutation based on the murine sequence to create MRP1E1089Q/T1242A restored resistance to vincristine and VP-16, but not anthracyclines, without affecting transport of leukotriene C(4) and E(2)17betaG. These results demonstrate the important role of Thr(1242) for E(2)17betaG transport. They also reveal a highly specific functional relationship between nonconserved amino acids in TM helices 14 and 17 of both mrp1 and MRP1 that enables both proteins to confer similar levels of resistance to vincristine and VP-16.
Highlights
Murine multidrug resistance protein 1, differs from its human ortholog (MRP1) in that it fails to confer anthracycline resistance and transports the MRP1 substrate, 17-estradiol 17-(-D-glucuronide) (E217G), very poorly
Studies using hybrid murine/human proteins revealed that the COOH-terminal third of MRP1 contains nonconserved amino acids that contribute to its ability to confer anthracycline resistance and to transport E217G efficiently [28]
Transport of [3H]leukotriene C4 (LTC4) and [3H]E217G by Wild Type and Mutant Murine mrp1—Previous studies demonstrated that the murine/human hybrid protein in which amino acids 1185–1528 of mrp1 were replaced with the corresponding region of MRP1 (mrp1/MRP1-(1188 –1531)) transported E217G more efficiently than wild type murine protein with no detectable change in the efficiency of LTC4 transport [28]
Summary
Materials—Culture medium and fetal bovine serum were obtained from Life Technologies, Inc. [3H]LTC4 (38 Ci/mmol) was purchased from Amersham Pharmacia Biotech, and [3H]E217G (44 Ci/mmol) was from PerkinElmer Life Sciences. All of the mutated MRP1 or mrp constructs were analyzed as stably transfected HEK293 cells grown in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum and 100 g/ml hygromycin B (Roche Molecular Biochemicals). Determination of Protein Levels in Transfected Cells—Plasma membrane vesicles were prepared as described previously [26, 29]. Km and Vmax values of ATP-dependent [3H]LTC4 uptake by membrane vesicles (2.5 g of protein) were measured at various LTC4 concentrations (0.01 to 2 M) for 1 min at 23 °C in 25 l of transport buffer containing 4 mM ATP and 10 mM MgCl2, followed by nonlinear regression analyses. Kinetic parameters of ATP-dependent [3H]E217G (0.1–16 M) uptake were determined as described for [3H]LTC4 except that 5 g of vesicle protein was used, and the reaction was carried out at 37 °C. Resistance factors were determined in three or more independent experiments
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