Localization of a substrate specificity domain in the multidrug resistanceprotein

Multidrug resistance protein (MRP) confers resistance to a number of natura l product chemotherapeutic agents. It is also a high affinity transporter o f some physiological conjugated organic anions such as cysteinyl leukotrien e C-4 and the cholestatic estrogen, 17 beta-estradiol 17(beta-D-glucuronide ) (E(2)17 beta G). We have shown that the murine orthologue of MRP (mrp), u nlike the human protein, does not confer resistance to common anthracycline s and is a relatively poor transporter of E(2)17 beta G. We have taken adva ntage of these functional differences to identify region(s) of MRP involved in mediating anthracycline resistance and E(2)17 beta G transport by gener ating mrp/MRP hybrid proteins. All hybrid proteins conferred resistance to the Vinca alkaloid, vincristine, when transfected into human embryonic kidn ey cells. However, only those in which the COOH-terminal third of mrp had b een replaced with the corresponding region of MRP-conferred resistance to t he anthracyclines, doxorubicin, and epirubicin. Exchange of smaller segment s of the COOH-terminal third of the mouse protein by replacement of either amino acids 959-1187 or 1188-1531 with those of MRP produced proteins capab le of conferring some level of resistance to the anthracyclines tested. All hybrid proteins transported cysteinyl leukotriene C-4 with similar efficie ncies. In contrast, only those containing the COOH-terminal third of MRP tr ansported E(2)17 beta G with an efficiency comparable with that of the inta ct human protein. The results demonstrate that differences in primary struc ture of the highly conserved COOH-terminal third of mrp and MRP are importa nt determinants of the inability of the murine protein to confer anthracycl ine resistance and its relatively poor ability to transport E(2)17 beta G.