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) (E217G) is a well known cholestatic agent and substrate of multidrug resistance-associated protein 2 (Mrp2), whereas beta-estradiol 3-(beta-D-glucuronide) (E23G) is a noncholestatic regioisomer of E217G with unknown transport properties. The purpose of this study was to compare and contrast the Mrp2-mediated transport of E217G and E23G. 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 3H-E23G and 3H-E217G in Sf9 cell membranes was characterized. Mrp2 transported E23G into an osmotically sensitive space, requiring ATP, with S50=55.7 microM, Vmax=326 pmol.mg(-1).min(-1), and a Hill coefficient of 0.88. ATP-dependent Mrp2-mediated E217G transport was markedly stimulated at high E217G concentrations, consistent with positive cooperativity (Hill coefficient 1.5). E217G (5-125 microM) increased S50 but not Vmax for E23G transport, consistent with competitive inhibition. E23G (0.4-400 microM) completely, potently (IC50=14.2 microM), and competitively inhibited E217G transport, but E217G (0.01-250 microM) inhibited only 53% of E23G transport (IC50=33.4 microM). Estriol 16alpha-(beta-D-glucuronide) potently and completely inhibited transport of E23G (IC50=2.23 microM), as did beta-estradiol 3-sulfate 17-(beta-D-glucuronide) (5-50 microM). In summary, E217G 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 E23G. The noncholestatic E23G is an Mrp2 substrate and competes with E217G for transport, but does not activate the allosteric site.