In addition to its ability to confer resistance to a range of natural product type chemotherapeutic agents, multidrug resistance protein (MRP) has been shown to transport the cysteinyl leukotriene, LTC, and several other glutathione (GSH) S-conjugates. We now demonstrate that its range of potential physiological substrates also includes cholestatic glucuronidated steroids. ATP dependent, osmotically sensitive transport of the naturally occurring conjugated estrogen, 17-estradiol 17-(-D-glucuronide) (E17G), was readily demonstrable in plasma membrane vesicles from populations of MRP-transfected HeLa cells (V 1.4 nmol mg min, K 2.5 µM). The involvement of MRP was confirmed by demonstrating that transport was completely inhibited by a monoclonal antibody specific for an intracellular conformational epitope of the protein. MRP-mediated transport of LTC was competitively inhibited by E17G (K 22 µM), despite the lack of structural similarity between these two substrates. Competitive inhibition of [H]E17G transport was also observed with a number of other cholestatic conjugated steroids. All of these compounds prevented photolabeling of MRP with [H]LTC, demonstrating that the cholestatic steroid and leukotriene conjugates compete either for the same or possibly overlapping sites on the protein. Consistent with the presence of overlapping but non-identical sites, studies using chemotherapeutic drugs to inhibit MRP-mediated E17G transport indicated that daunorubicin had the highest relative potency of the drugs tested, whereas it was the least potent inhibitor of LTC transport. Non-cholestatic steroids glucuronidated at the 3 position of the steroid nucleus, such as 17-estradiol 3-(-D-glucuronide), did not compete for transport of E17G by MRP, nor did they inhibit photolabeling of the protein with [H]LTC. These data identify MRP as a potential transporter of cholestatic conjugated estrogens and demonstrate site-specific requirements for glucuronidation of the steroid nucleus.

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