Localization of a Substrate Specificity Domain in the Multidrug Resistance Protein

Abstract

Multidrug resistance protein (MRP) confers resistance to a number of natural product chemotherapeutic agents. It is also a high affinity transporter of some physiological conjugated organic anions such as cysteinyl leukotriene C 4 and the cholestatic estrogen, 17␤-estradiol 17(␤-D-glucuronide) (E 2 17␤G). We have shown that the murine orthologue of MRP (mrp), unlike the human protein, does not confer resistance to common anthracyclines and is a relatively poor transporter of E 2 17␤G. We have taken advantage of these functional differences to identify region(s) of MRP involved in mediating anthracycline resistance and E 2 17␤G transport by generating mrp/MRP hybrid proteins. All hybrid proteins conferred resistance to the Vinca alkaloid, vincristine, when transfected into human embryonic kidney cells. However, only those in which the COOH-terminal third of mrp had been replaced with the corresponding region of MRP-conferred resistance to the anthracyclines, doxorubicin, and epirubicin. Exchange of smaller segments 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 capable of conferring some level of resistance to the anthracyclines tested. All hybrid proteins transported cysteinyl leukotriene C 4 with similar efficiencies. In contrast, only those containing the COOH-terminal third of MRP transported E 2 17␤G with an efficiency comparable with that of the intact human protein. The results demonstrate that differences in primary structure of the highly conserved COOH-terminal third of mrp and MRP are important determinants of the inability of the murine protein to confer anthracycline resistance and its relatively poor ability to transport E 2 17␤G.

Transport of the β-O-Glucuronide Conjugate of the Tobacco-specific Carcinogen 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) by the Multidrug Resistance Protein 1 (MRP1)

“…While this may hold functionally, it is now clear that although binding of many substrates may be mutually exclusive, each substrate/inhibitor forms its own individual set of atomic contacts within a multipartite binding pocket(s) of the protein (14,15,21,32,34,48,49). Such a model has been proposed for the transcriptional regulator of the Bacillus subtilis multidrug transporter Bmr, which binds multiple structurally dissimilar hydrophobic cations, and is supported by considerable biochemical and structural data (50,51).…”

Identification of a Nonconserved Amino Acid Residue in Multidrug Resistance Protein 1 Important for Determining Substrate Specificity

“…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 E 2 17␤G more efficiently than wild type murine protein with no detectable change in the efficiency of LTC 4 transport (28). To further localize residues in this region responsible for enhancing E 2 17␤G transport activity, amino acids 1351-1528 of mrp1 were replaced with the corresponding part of MRP1.…”

“…Resistance Profiles of Wild Type and TM17 Mutant Murine/ Human Proteins-In addition to the differences in E 2 17␤G transport activity, the hybrid protein mrp1/MRP1-(1188 -1531) has been shown to increase the resistance to anthracyclines ϳ2-fold relative to wild type mrp1 (28 4 or E 2 17␤G transport were determined by nonlinear regression analysis of the combined data using GraphPad software and are shown in Table I. amined the drug resistance profiles of transfectants expressing mrp1 mutations A1239T, A1239S, and A1239C. The results are summarized as relative resistance factors in Table II.…”

“…However, transport of the estrogen conjugate was increased in a mrp1/MRP1 hybrid containing a relatively highly conserved region of the human protein extending from amino acid 1188 to the COOH terminus (28). In the present study, we examined the consequences of replacing amino acid residues in this region that differ between the human and murine proteins.…”

“…In addition, despite the fact that MRP1 and mrp1 transport LTC 4 with similar efficiency, the murine protein transports E 2 17␤G very poorly (26). 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 E 2 17␤G efficiently (28).…”