Abstract

β-Estradiol 17-(β-d-glucuronide) (E₂17G) is a well known cholestatic agent and substrate of multidrug resistance-associated protein 2 (Mrp2), whereas β-estradiol 3-(β-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 μM, V_max = 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 μM) increased S₅₀ but not V_max for E₂3G transport, consistent with competitive inhibition. E₂3G (0.4-400 μM) completely, potently (IC₅₀ = 14.2 μM), and competitively inhibited E₂17G transport, but E₂17G (0.01-250 μM) inhibited only 53% of E₂3G transport (IC₅₀ = 33.4 μM). Estriol 16α-(β-d-glucuronide) potently and completely inhibited transport of E₂3G (IC₅₀ = 2.23 μM), as did β-estradiol 3-sulfate 17-(β-d-glucuronide) (5-50 μM). 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.