Ito K, Olsen S L, Qiu W, Deeley R G, Cole S P
Cancer Research Laboratories, Queen's University, Kingston, Ontario K7L 3N6, Canada.
J Biol Chem. 2001 May 11;276(19):15616-24. doi: 10.1074/jbc.M011246200. Epub 2001 Feb 21.
Multidrug resistance protein 1 (MRP1/ABCC1) belongs to the ATP-binding cassette transporter superfamily and is capable of conferring resistance to a broad range of chemotherapeutic agents and transporting structurally diverse conjugated organic anions. In this study, we found that substitution of a highly conserved tryptophan at position 1246 with cysteine (W1246C-MRP1) in the putative last transmembrane segment (TM17) of MRP1 eliminated 17beta-estradiol 17-(beta-d-glucuronide) (E(2)17betaG) transport by membrane vesicles prepared from transiently transfected human embryonic kidney cells while leaving the capacity for leukotriene C(4)- and verapamil-stimulated glutathione transport intact. In addition, in contrast to wild-type MRP1, leukotriene C(4) transport by the W1246C-MRP1 protein was no longer inhibitable by E(2)17betaG, indicating that the mutant protein had lost the ability to bind the glucuronide. A similar phenotype was observed when Trp(1246) was replaced with Ala, Phe, and Tyr. Confocal microscopy of cells expressing Trp(1246) mutant MRP1 molecules fused at the C terminus with green fluorescent protein showed that they were correctly routed to the plasma membrane. In addition to the loss of E(2)17betaG transport, HeLa cells stably transfected with W1246C-MRP1 cDNA were not resistant to the Vinca alkaloid vincristine and accumulated levels of [(3)H]vincristine comparable to those in vector control-transfected cells. Cells expressing W1246C-MRP1 were also not resistant to cationic anthracyclines (doxorubicin, daunorubicin) or the electroneutral epipodophyllotoxin VP-16. In contrast, resistance to sodium arsenite was only partially diminished, and resistance to potassium antimony tartrate remained comparable to that of cells expressing wild-type MRP1. This suggests that the structural determinants required for transport of heavy metal oxyanions differ from those for chemotherapeutic agents. Our results provide the first example of a tryptophan residue being so critically important for substrate specificity in a eukaryotic ATP-binding cassette transporter.
多药耐药蛋白1（MRP1/ABCC1）属于ATP结合盒转运体超家族，能够赋予对多种化疗药物的抗性，并转运结构多样的共轭有机阴离子。在本研究中，我们发现，在MRP1假定的最后一个跨膜区段（TM17）中，将第1246位高度保守的色氨酸替换为半胱氨酸（W1246C-MRP1），消除了由瞬时转染的人胚肾细胞制备的膜囊泡对17β-雌二醇17-（β-D-葡萄糖醛酸苷）（E217βG）的转运，同时白三烯C4和维拉帕米刺激的谷胱甘肽转运能力保持完整。此外，与野生型MRP1不同，W1246C-MRP1蛋白对白三烯C4的转运不再受E217βG抑制，这表明突变蛋白已丧失结合葡萄糖醛酸苷的能力。当色氨酸（1246）被丙氨酸、苯丙氨酸和酪氨酸取代时，观察到类似的表型。对在C末端与绿色荧光蛋白融合的色氨酸（1246）突变型MRP1分子进行共聚焦显微镜观察，结果显示它们被正确转运至质膜。除了丧失E217βG转运能力外，稳定转染W1246C-MRP1 cDNA的HeLa细胞对长春花生物碱长春新碱不具有抗性，并且[3H]长春新碱的积累水平与载体对照转染细胞中的水平相当。表达W1246C-MRP1的细胞对阳离子蒽环类药物（阿霉素、柔红霉素）或电中性鬼臼毒素VP-16也不具有抗性。相反，对亚砷酸钠的抗性仅部分降低，对酒石酸锑钾的抗性仍与表达野生型MRP1的细胞相当。这表明重金属含氧阴离子转运所需的结构决定因素与化疗药物的不同。我们的结果提供了第一个色氨酸残基对真核ATP结合盒转运体底物特异性至关重要的例子。
