The MRP gene encodes an ATP-dependent export pump for leukotriene C4 and structurally related conjugates

Abstract

The multidrug resistance-associated protein (MRP) is the product of an ATP-binding cassette transporter gene overexpressed in some tumor cells resistant to antineoplastic agents. We studied the transport function of MRP in membrane vesicles prepared from HeLa cells transfected with an MRP expression vector and overexpressing this 190-kDa membrane glycoprotein. ATP-dependent primary-active transport into the vesicles was demonstrated for leukotriene C4 (LTC4), LTD4, LTE4, and S-(2,4-dinitrophenyl)glutathione with relative rates, at a substrate concentration of 50 nM, of 1.0, 0.27, 0.14, and 0.16, respectively. The endogenous glutathione conjugate LTC4 had the highest affinity for this transporter with a Km of 97 nM. The Km for ATP was 19 microM. Direct photoaffinity labeling with [3H]LTC4 labeled a 190-kDa membrane protein predominantly in the MRP-transfected HeLa cells. ATP-dependent LTC4 transport was effectively inhibited by the LTD4 receptor antagonist MK 571, whereas cyclosporin A and, particularly, its analog PSC 833 were much less potent. The respective Ki values were 0.6, 5, and 27 microM, respectively. In addition, MK 571 preferentially inhibited photoaffinity labeling of the 190-kDa protein in the MRP transfectants. Our results provide direct evidence that the MRP gene encodes a primary-active ATP-dependent export pump for conjugates of lipophilic compounds with glutathione and several other anionic residues. We conclude that the biosynthetic release of LTC4 from cells is mediated by the 190-kDa product of the MRP gene.