Structure and in Vitro Substrate Specificity of the Murine Multidrug Resistance-Associated Protein

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Abstract
MRP is a recently described ATP-binding cassette transporter that confers cellular resistance to natural product cytotoxic drugs. To examine the biochemical activity and cellular physiology of this transporter, we isolated the murine MRP homologue and analyzed its in vitro substrate specificity. Murine MRP transcript is widely expressed in tissues and encodes a protein of 1528 amino acids that is 88% identical to its human homologue. Hydropathy analysis indicated that murine and human MRP, the yeast cadmium resistance transporter and the sulfonylurea receptor share a conserved topology distinguished from P-glycoprotein and the cystic fibrosis conductance regulator by an N-terminal hydrophobic region that contains several potential transmembrane domains. Drug uptake assays performed with membrane vesicles prepared from NIH3T3 cells transfected with a murine MRP expression vector revealed ATP-dependent transport for the natural product cytotoxic drugs daunorubicin and vincristine, as well as for the glutathione S-conjugates leukotriene C-4 and azidophenacyl-S-glutathione. Drug transport was osmotically sensitive and saturable with regard to drug and ATP concentrations, with K-m values of 19 mu M, 19 mu M, 26 nM, 17 mu M, and 77 mu M for daunorubicin, vincristine, leukotriene C-4, APA-SG, and ATP, respectively. Consistent with broad substrate specificity, the drug glutathione conjugate APA-SG, oxidized glutathione, the LTD(4) antagonist MK571, arsenate, and genistein were competitive inhibitors of daunorubicin transport, with K-i values of 32 mu M, 25 mu M, 1.9 mu M, 108 mu M, and 23 mu M, respectively. This study demonstrates that the substrate specificity of murine MRP is quite broad and includes both the neutral or mildly cationic natural product cytotoxic drugs and the anionic products of glutathione conjugation. The widespread expression pattern of murine MRP in tissues, combined with its ability to transport both lipophilic xenobiotics and the products of phase II detoxification, indicates that it represents a widespread and versatile cellular defense mechanism.