Zeng H, Liu G, Rea P A, Kruh G D
Medical Sciences Division, Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111, USA.
Cancer Res. 2000 Sep 1;60(17):4779-84.
The multidrug resistance-associated protein 1 (MRP1) and the canalicular multispecific organic anion transporter (cMOAT or MRP2) are ATP-binding cassette transporters that confer resistance to some anticancer drugs and efflux glutathione and glucuronate conjugates from the cell. The MRP subfamily of ABC transporters, however, contains at least four other members of which MRP3 (MOAT-D) bears the closest structural resemblance to MRP1. Although transfection studies have established that human MRP3 confers increased resistance to several anticancer agents, neither the substrate selectivity nor physiological functions of this transporter have been determined. Here we report the results of investigations of the in vitro transport properties of cloned human MRP3 using membrane vesicles prepared from MRP3-transfected HEK293 cells. It is shown that the expression of MRP3 is specifically associated with enhancement of the MgATP-dependent transport into membrane vesicles of the glucuronide estradiol 17-beta-D-glucuronide (E(2)17betaG), the glutathione conjugates 2,4-dinitrophenyl S-glutathione (DNP-SG) and leukotriene C4 (LTC4), the antimetabolite methotrexate, and the bile acid glycocholate. DNP-SG, LTC4, and E(2)17betaG are transported at moderate affinity and low capacity with Km and Vmax values of 5.7 +/- 1.7 microM and 3.8 +/- 0.1 pmol/mg/min, 5.3 +/- 2.6 microM and 20.2 +/- 5.9 pmol/mg/min, and 25.6 +/- 5.4 microM and 75.6 +/- 5.9 pmol/mg/min, respectively. Methotrexate and glycocholate are transported at low affinity and high capacity with Km and Vmax values of 776 +/- 319 microM and 288 +/- 54 pmol/mg/min and 248 +/- 113 microM and 183 +/- 34 pmol/mg/min, respectively. On the basis of these findings, the osmotic dependence of the transport measured and its inability to transport taurocholate, MRP3, like MRP1 and cMOAT, is concluded to be competent in the transport of glutathione S-conjugates, glucuronides, and methotrexate, albeit at low to moderate affinity. In contrast to MRP1, cMOAT, and all other characterized mammalian ABC transporters, however, MRP3 is active in the transport of the monoanionic human bile constituent glycocholate.
多药耐药相关蛋白1（MRP1）和小管多特异性有机阴离子转运体（cMOAT或MRP2）是ATP结合盒转运蛋白，可赋予细胞对某些抗癌药物的抗性，并使谷胱甘肽和葡萄糖醛酸共轭物从细胞中流出。然而，ABC转运蛋白的MRP亚家族至少还包含其他四个成员，其中MRP3（MOAT-D）与MRP1的结构最为相似。虽然转染研究已证实人MRP3可增强对几种抗癌药物的抗性，但该转运蛋白的底物选择性和生理功能均未确定。在此，我们报告了使用从转染MRP3的HEK293细胞制备的膜囊泡对克隆的人MRP3的体外转运特性进行研究的结果。结果表明，MRP3的表达与以下物质向膜囊泡的MgATP依赖性转运增强特异性相关：葡萄糖醛酸雌二醇17-β-D-葡萄糖醛酸（E(2)17βG）、谷胱甘肽共轭物2,4-二硝基苯基S-谷胱甘肽（DNP-SG）和白三烯C4（LTC4）、抗代谢物甲氨蝶呤以及胆汁酸甘氨胆酸。DNP-SG、LTC4和E(2)17βG以中等亲和力和低容量进行转运，Km和Vmax值分别为5.7±1.7μM和3.8±0.1 pmol/mg/min、5.3±2.6μM和20.2±5.9 pmol/mg/min以及25.6±5.4μM和75.6±5.9 pmol/mg/min。甲氨蝶呤和甘氨胆酸以低亲和力和高容量进行转运，Km和Vmax值分别为776±319μM和288±54 pmol/mg/min以及248±113μM和183±34 pmol/mg/min。基于这些发现，所测量的转运的渗透压依赖性及其不能转运牛磺胆酸盐，得出结论：与MRP1和cMOAT一样，MRP3能够转运谷胱甘肽S-共轭物、葡萄糖醛酸共轭物和甲氨蝶呤，尽管亲和力较低至中等。然而，与MRP1、cMOAT和所有其他已表征的哺乳动物ABC转运蛋白不同，MRP3在单阴离子人胆汁成分甘氨胆酸的转运中具有活性。
