Received for publication April 21, 2004.
Revised July 12, 2004.
Accepted for publication July 15, 2004.

Estradiol 3-glucuronide is transported by the multidrug resistance-associated protein 2 (Mrp2) but does not activate the allosteric site bound by estradiol 17- glucuronide

Abstract

-Estradiol 17-(-D-glucuronide) (E₂17G) is a well-known cholestatic agent and substrate of Mrp2, while -estradiol 3-(-D-glucuronide) (E₂3G) is a non-cholestatic 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-E₂17Gin Sf9 cell membranes characterized. Mrp2 transported E₂3G into an osmotically sensitive space, required ATP, with S₅₀=55.7 µM, 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 µM) increased S₅₀ but not Vmax 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 16-(-D-glucuronide) potently and completely inhibited transport of E₂3G (IC₅₀=2.23 µM), as did -estradiol 3- sulfate 17-(-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 non-cholestatic E₂3G is an Mrp2 substrate and competes with E₂17G for transport, but does not activate the allosteric site.

Key words: ABC transporters, active transport, allosterism, drug efflux, drug transport, female reproductive system toxicology, hepatic transport, hepatobiliary transport, isolated membranes, MRP

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