Mutation of a single conserved tryptophan in multidrug resistance protein 1 (MRP1/ABCC1) results in loss of drug resistance and selective loss of organic anion transport
Mutation of a single conserved tryptophan in multidrug resistance protein 1 (MRP1/ABCC1) results in loss of drug resistance and selective loss of organic anion transport
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DOI：
10.1074/jbc.m011246200
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发表时间：
2001-05-11
影响因子：
4.8
通讯作者：
Cole, SPC
中科院分区：
文献类型：
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作者：
Ito, K;Olsen, SL;Cole, SPC
多药耐药蛋白1（MRP 1/ABCC 1）属于ATP结合盒转运蛋白超家族，能够对多种化疗药物产生耐药性，并转运结构多样的共轭有机阴离子。在本研究中，我们发现，用半胱氨酸取代1246位高度保守的色氨酸，MRP 1的最后一个跨膜片段（TM 17）中的W1246 C-MRP 1消除了17 β-雌二醇17-（β-D-葡糖苷酸）（E（2）17 β G）通过由瞬时转染的人胚肾细胞制备的膜囊泡转运，同时保留白三烯C-4-和维拉帕米刺激的谷胱甘肽转运。此外，与野生型MRP 1相反，W1246 C-MRP 1蛋白的白三烯C-4转运不再被E（2）17 β G所抑制，表明突变蛋白失去了结合葡糖苷酸的能力。当Trp（1246）被Ale、Phe和Tyr替换时，观察到类似的表型。表达Trp（1246）突变型MRP 1分子的细胞的共聚焦显微镜显示，它们被正确地引导到质膜，该突变型MRP 1分子在C末端与绿色荧光蛋白融合。除了E（2）17 β G转运的损失外，稳定转染W1246 C-MRP 1 cDNA的HeLa细胞对长春花生物碱长春新碱不耐药，[H-3]长春新碱的累积水平与载体对照转染细胞相当。表达W1246 C-MRP 1的细胞对阳离子蒽环类药物（多柔比星、柔红霉素）或电中性表鬼臼毒素VP-16也不耐药。相反，对亚砷酸钠的耐药性仅部分减弱，对酒石酸锑钾的耐药性与表达野生型MRP 1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不同。我们的研究结果提供了第一个例子，色氨酸残基是如此至关重要的底物特异性在真核ATP结合盒转运。
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 17 beta -estradiol 17-(beta -D-glucuronide) (E(2)17 betaG) 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)17 betaG, indicating that the mutant protein had lost the ability to bind the glucuronide. A similar phenotype was observed when Trp(1246) was replaced with Ale, 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)17 betaG transport, HeLa cells stably transfected with W1246C-MRP1 cDNA were not resistant to the Vinca alkaloid vincristine and accumulated levels of [H-3]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.