Loe D W, Almquist K C, Cole S P, Deeley R G
Cancer Research Laboratories, Queen's University, Kingston, Ontario, Canada.
J Biol Chem. 1996 Apr 19;271(16):9683-9. doi: 10.1074/jbc.271.16.9683.
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, LTC4, 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 beta-estradiol 17-(beta-D-glucuronide) (E(2)17 beta G), was readily demonstrable in plasma membrane vesicles from populations of MRP-transfected HeLa cells (Vmax 1.4 nmol mg-1 min-1, K(m) 2.5 micron). 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 LTC4, was competitively inhibited by E(2)17 beta G (K(i(app)) 22 micron), despite the lack of structural similarity between these two substrates. Competitive inhibition of [3H]E(2)17 beta G transport was also observed with a number of other cholestatic conjugated steroids. All of these compounds prevented photolabeling of MRP with [3H]LTC4, 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 E(2)17 beta G transport indicated that daunorubicin had the highest relative potency of the drugs tested, whereas it was the least potent inhibitor of LTC4 transport. Non-cholestatic steroids glucuronidated at the 3 position of the steroid nucleus, such as 17 beta-estradiol 3-(beta-D-glucuronide), did not compete for transport of E(2)17 beta G by MRP, nor did they inhibit photolabeling of the protein with [3H]LTC4. These data identify MRP as a potential transporter of cholestatic conjugated estrogens and demonstrate site-specific requirements for glucuronidation of the steroid nucleus.
除了能够赋予对一系列天然产物类化疗药物的抗性外，多药耐药蛋白（MRP）还被证明能转运半胱氨酰白三烯LTC4以及其他几种谷胱甘肽（GSH）S-共轭物。我们现在证明其潜在生理底物的范围还包括胆汁淤积性葡糖醛酸化类固醇。在转染了MRP的HeLa细胞群体的质膜囊泡中，很容易证明天然存在的共轭雌激素17β-雌二醇17-（β-D-葡糖醛酸苷）（E(2)17βG）的ATP依赖性、渗透敏感性转运（Vmax 1.4 nmol mg-1 min-1，K(m) 2.5 μM）。通过证明一种针对该蛋白细胞内构象表位的单克隆抗体能完全抑制转运，证实了MRP的参与。尽管这两种底物缺乏结构相似性，但E(2)17βG竞争性抑制了MRP介导的LTC4转运（K(i(app)) 22 μM）。还观察到许多其他胆汁淤积性共轭类固醇对[3H]E(2)17βG转运有竞争性抑制作用。所有这些化合物都阻止了[3H]LTC4对MRP的光标记，表明胆汁淤积性类固醇和白三烯共轭物要么竞争该蛋白上相同的位点要么可能竞争重叠位点。与存在重叠但不同的位点一致的是, 使用化疗药物抑制MRP介导的E(2)17βG转运表明，柔红霉素在所测试的药物中相对效力最高，而它是LTC4转运的最无效抑制剂。类固醇核3位葡糖醛酸化的非胆汁淤积性类固醇, 如17β-雌二醇3-（β-D-葡糖醛酸苷），既不竞争MRP对E(2)17βG的转运，也不抑制[3H]LTC4对该蛋白的光标记。这些数据确定MRP是胆汁淤积性共轭雌激素潜在转运体，并证明了类固醇核葡糖醛酸化的位点特异性要求。
