Multiple membrane-associated tryptophan residues contribute to the transport activity and substrate specificity of the human multidrug resistance protein, MRP1
Multiple membrane-associated tryptophan residues contribute to the transport activity and substrate specificity of the human multidrug resistance protein, MRP1
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DOI：
10.1074/jbc.m206896200
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发表时间：
2002-12-20
影响因子：
4.8
通讯作者：
Cole, SPC
中科院分区：
文献类型：
--
作者：
Koike, K;Oleschuk, CJ;Cole, SPC
多药耐药蛋白MRP1是临床上重要的atp结合盒转运体，其中包含三个跨膜结构域（MSDs），其中包含多达17个跨膜（TM）螺旋，以及两个核苷酸结合结构域（NEDs） MSD1-MSD2-NIBD1-MSD3-NBD2。在肿瘤细胞中，MRP1赋予对多种药物的抗性，但在正常细胞中，它作为有机阴离子（如白三烯C-4和17 β -雌二醇17 β -（d -葡萄糖醛酸盐））的主要活性转运体。我们之前的研究表明，TM17-Trp（1246）突变消除了MRP1带来的17β -雌二醇17p-（d -葡糖苷）转运和耐药性，而白三烯C4转运完好无损。通过突变MRP1预测TM片段中剩余的11个Trp残基，我们现在已经确定其中5个也是MRP1功能的主要决定因素。其中三个残基，即Trp(445) （TMS）、Trp(553) （TM10）和Trp(1198) （TM16）的Ala取代，消除或大大降低了MRP1的五种有机阴离子底物的运输水平。相比之下，Trp(361) （TM7）和Trp(459) （TM9）的Ala取代导致MRP1功能更温和和底物选择性降低。TrP445、Trp553和Trp（1198）突变体更保守的替换（Tyr和Phe）导致某些情况下（Trp（445）和Trp(1198)）的底物选择性转运保留，而其他情况（Trp553）则没有。我们的研究结果表明，这五个Trp残基的极性芳香吲哚侧链对MRP1的转运活性和底物特异性有重要影响。
The multidrug resistance protein, MRP1, is a clinically important ATP-binding cassette transporter in which the three membrane-spanning domains (MSDs), which contain up to 17 transmembrane (TM) helices, and two nucleotide binding domains (NEDs) are configured MSD1-MSD2-NIBD1-MSD3-NBD2. In tumor cells, MRP1 confers resistance to a broad spectrum of drugs, but in normal cells, it functions as a primary active transporter of organic anions such as leukotriene C-4 and 17beta-estradiol 17beta-(D-glucuronide). We have previously shown that mutation of TM17-Trp(1246) eliminates 17beta-estradiol 17p-(D-glucuronide) transport and drug resistance conferred by MRP1 while leaving leukotriene C4 transport intact. By mutating the 11 remaining Trp residues that are in predicted TM segments of MRP1, we have now determined that five of them are also major determinants of MRP1 function. Ala substitution of three of these residues, Trp(445) (TMS), Trp(553) (TM10), and Trp(1198) (TM16), eliminated or substantially reduced transport levels of five organic anion substrates of MRP1. In contrast, Ala substitutions of Trp(361) (TM7) and Trp(459) (TM9) caused a more moderate and substrate-selective reduction in MRP1 function. More conservative substitutions (Tyr and Phe) of the TrP445, Trp 553, and Trp(1198) mutants resulted in substrate selective retention of transport in some cases (Trp(445) and Trp(1198)) but not others (Trp553). Our findings suggest that the bulky polar aromatic indole side chain of each of these five Trp residues contributes significantly to the transport activity and substrate specificity of MRP1.