RLIP76, a Novel Transporter Catalyzing ATP-Dependent Efflux of Xenobiotics

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Abstract
Transport of xenobiotics and their metabolites by ATP-binding cassette (ABC) transporters particularly P-glycoprotein (Pgp) and the multidrug resistance associated protein (MRP1) has been extensively studied during last decade. Our recent studies demonstrate that RLIP76, a previously known GTPase-activating protein catalyzes ATP-dependent, uphill transport of anionic glutathione conjugates as well as of weakly cationic anthracyclines including doxorubicin (Adriamycin), a widely used drug in cancer chemotherapy. RLIP76 has inherent ATPase activity, which is stimulated by doxorubicin and glutathione conjugates. RLIP76 does not meet the criteria for classical ABC proteins such as MRP1 or Pgp, but similar to ABC proteins, it has two ATP-binding sequences, 69GKKKGK74 and418GGIKDLSK425. Mutations in these sequences abrogate its ATP-binding, ATPase activity, and transport function. Purified RLIP76 when reconstituted in proteoliposomes mediates ATP-dependent saturable transport of doxorubicin and glutathione conjugates. Transfection of K562 cells with RLIP76 confers these cells resistance to doxorubicin and 4-hydroxynonenal. Cells enriched with RLIP76 also acquire resistance to radiation toxicity. RLIP76 also catalyzes the transport of physiologic ligands such as leukotrienes (LTC4) and the conjugate of 4-hydroxynonenal and glutathione. In some cells (e.g., erythrocytes and lung cancer cells), the majority of transport activity for Adriamycin and glutathione conjugates including LTC4 is accounted for by RLIP76. These studies strongly suggest that RLIP76-mediated transport of organic ions has physiological and toxicological relevance and that it may play an important role in the mechanism of drug resistance.