Abstract
Human multidrug resistance protein 1 (MRP1) confers resistance to many natural product chemotherapeutic agents and actively transports structurally diverse organic anion conjugates. We previously demonstrated that two hydrogen-bonding amino acid residues in the predicted transmembrane 17 (TM17) of MRP1, Thr(1242) and Trp(1246), were important for drug resistance and 17beta-estradiol 17-(beta-d-glucuronide) (E(2)17betaG) transport. To determine whether other residues with hydrogen bonding potential within TM17 influence substrate specificity, we replaced Ser(1233), Ser(1235), Ser(1237), Gln(1239), Thr(1241), and Asn(1245) with Ala and Tyr(1236) and Tyr(1243) with Phe. Mutations S1233A, S1235A, S1237A, and Q1239A had no effect on any substrate tested. In contrast, mutations Y1236F and T1241A decreased resistance to vincristine but not to VP-16, doxorubicin, and epirubicin. Mutation Y1243F reduced resistance to all drugs tested by 2-3-fold. Replacement of Asn(1245) with Ala also decreased resistance to VP-16, doxorubicin, and epirubicin but increased resistance to vincristine. This mutation also decreased E(2)17betaG transport approximately 5-fold. Only mutation Y1243F altered the ability of MRP1 to transport both leukotriene 4 and E(2)17betaG. Together with our previous results, these findings suggest that residues with side chain hydrogen bonding potential, clustered in the cytoplasmic half of TM17, participate in the formation of a substrate binding site.
Highlights
Human multidrug resistance protein 1 (MRP1) confers resistance to many natural product chemotherapeutic agents and actively transports structurally diverse organic anion conjugates
We previously demonstrated that two hydrogen-bonding amino acid residues in the predicted transmembrane 17 (TM17) of MRP1, Thr1242 and Trp1246, were important for drug resistance and 17estradiol 17-(-D-glucuronide) (E217G) transport
We have shown that mutations of a nonconserved hydrophilic residue, Thr1242, within the putative TM17 of MRP1 dramatically decreases the ability of the protein to confer drug resistance and to transport E217G without significant effect on LTC4 transport [38]
Summary
Materials—Culture medium and fetal bovine serum were obtained from Invitrogen. [3H]LTC4 (38 Ci/mmol) was purchased from Amersham Biosciences, and [3H]E217G (44 Ci/mmol) was from PerkinElmer Life Sciences. Cell Lines and Tissue Culture—Stable transfection of HEK293 cells with the pCEBV7 vector containing the wild type MRP1 cDNAs has been described previously [37, 41]. Determination of Protein Levels in Transfected Cells—Plasma membrane vesicles were prepared by centrifugation through sucrose, as described previously [9, 10]. Vesicles (10 g of protein) were incubated at 23 °C in 100 l of transport buffer (50 mM Tris-HCl, 250 mM sucrose, 0.02% sodium azide, pH 7.4) containing ATP or AMP (4 mM), 10 mM MgCl2, and [3H]LTC4 (50 nM, 100 nCi). Km and Vmax values of ATP-dependent [3H]LTC4 uptake by membrane vesicles (2.5 g of protein) were measured at various LTC4 concentrations (0.01–1 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 the reaction was carried out at 37 °C
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
