Estradiol 3-glucuronide is transported by the multidrug resistance-associated protein 2 but does not activate the allosteric site bound by estradiol 17-glucuronide

beta-Estradiol 17-(beta-D-glucuronide) (E(2)17G) is a well known cholestatic agent and substrate of multidrug resistance-associated protein 2 (Mrp2), whereas beta-estradiol 3-(beta-D-glucuronide) (E(2)3G) is a noncholestatic regioisomer of E(2)17G with unknown transport properties. The purpose of this study was to compare and contrast the Mrp2-mediated transport of E(2)17G and E(2)3G. The full coding region of rat Mrp2 was cloned into the baculovirus genome, the recombinant baculovirus used to infect Sf9 cells, and ATP-dependent transport of H-3-E(2)3G and H-3-E(2)17G in Sf9 cell membranes was characterized. Mrp2 transported E(2)3G into an osmotically sensitive space, requiring ATP, with S-50=55.7 muM, V-max=326 pmol.mg(-1).min(-1), and a Hill coefficient of 0.88. ATP-dependent Mrp2-mediated E(2)17G transport was markedly stimulated at high E(2)17G concentrations, consistent with positive cooperativity (Hill coefficient 1.5). E(2)17G (5-125 muM) increased S-50 but not V-max for E(2)3G transport, consistent with competitive inhibition. E(2)3G(0.4-400 muM) completely, potently (IC50=14.2 muM), and competitively inhibited E(2)17G transport, but E(2)17G (0.01-250 muM) inhibited only 53% of E(2)3G transport (IC50=33.4 muM). Estriol 16alpha-(beta-D-glucuronide) potently and completely inhibited transport of E(2)3G (IC50=2.23 muM), as did beta-estradiol 3-sulfate 17-(beta-D-glucuronide) (5-50 muM). In summary, E(2)17G binds not only to an Mrp2 transport site, but also to an allosteric site that activates Mrp2 with positive cooperativity, thus activating its own transport and potentially that of other Mrp2 substrates, such as E(2)3G. The noncholestatic E(2)3G is an Mrp2 substrate and competes with E(2)17G for transport, but does not activate the allosteric site.