Identification of a Nonconserved Amino Acid Residue in Multidrug Resistance Protein 1 Important for Determining Substrate Specificity
Identification of a Nonconserved Amino Acid Residue in Multidrug Resistance Protein 1 Important for Determining Substrate Specificity
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多药耐药蛋白 1 中非保守氨基酸残基的鉴定对于确定底物特异性很重要
DOI：
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发表时间：
2001
影响因子：
4.8
通讯作者：
R. Deeley
中科院分区：
文献类型：
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作者：
Da;S. Cole;R. Deeley
鼠多药耐药蛋白 1 (mrp1) 与其人类直系同源蛋白 (MRP1) 不同，它不能赋予蒽环类药物耐药性，并且转运 MRP1 底物 17β-雌二醇 17-(β-d-葡萄糖醛酸) (E217βG) 的能力非常差。通过将 mrp1 中的变异残基突变为 MRP1 中存在的残基，我们确定 MRP1 的 Glu1089 对蒽环类药物耐药性至关重要。然而，Glu1089 突变对 E217βG 转运没有影响。我们现已在 MRP1/mrp1 高度保守的 COOH 近端跨膜螺旋内鉴定出一个非保守氨基酸，该氨基酸对于结合雌激素的转运非常重要。将 mrp1 中的 Ala1239 转换为 Thr，如 MRP1 中的相应位置 (1242) 所示，使 E217βG 转运增加 3 倍。 mrp1 Ala1239 的任何突变（包括用 Thr 取代）都会降低对长春新碱和 VP-16 的耐药性，但不会改变蒽环类药物耐药性。然而，将第二种鼠类突变为人类突变 Q1086E（单独选择性增加蒽环类抗生素耐药性）引入 mrp1A1239T 中，恢复了对长春新碱和 VP-16 的耐药性。为了确认 MRP1 Thr1242 对于 E217βG 转运和耐药性的重要性，我们将该残基突变为 Ala、Cys、Ser、Leu 和 Lys。这些突变使 E217βG 转运减少了 2 倍。转化为天冬氨酸消除了雌激素结合物的运输，并且还减少了白三烯 C4 运输约 2 倍。这些突变还降低了 MRP1 对所有测试药物产生耐药性的能力。与 mrp1 一样，引入基于鼠序列的第二个突变以创建 MRP1E1089Q/T1242A，恢复了对长春新碱和 VP-16 的耐药性，但不恢复对蒽环类药物的耐药性，且不影响白三烯 C4 和 E217βG 的转运。这些结果证明了 Thr1242 对于 E217βG 转运的重要作用。他们还揭示了 mrp1 和 MRP1 TM 螺旋 14 和 17 中非保守氨基酸之间高度特异性的功能关系，使这两种蛋白质能够对长春新碱和 VP-16 产生相似水平的耐药性。
Murine multidrug resistance protein 1 (mrp1), differs from its human ortholog (MRP1) in that it fails to confer anthracycline resistance and transports the MRP1 substrate, 17β-estradiol 17-(β-d-glucuronide) (E217βG), very poorly. By mutating variant residues in mrp1 to those present in MRP1, we identified Glu1089of MRP1 as being critical for anthracycline resistance. However, Glu1089 mutations had no effect on E217βG transport. We have now identified a nonconserved amino acid within the highly conserved COOH-proximal transmembrane helix of MRP1/mrp1 that is important for transport of the conjugated estrogen. Converting Ala1239 in mrp1 to Thr, as in the corresponding position (1242) in MRP1, increased E217βG transport 3-fold. Any mutation of mrp1 Ala1239, including substitution with Thr, decreased resistance to vincristine and VP-16 without altering anthracycline resistance. However, introduction of a second murine to human mutation, Q1086E, which alone selectively increases anthracycline resistance, into mrp1A1239T restored resistance to both vincristine and VP-16. To confirm the importance of MRP1 Thr1242 for E217βG transport and drug resistance, we mutated this residue to Ala, Cys, Ser, Leu, and Lys. These mutations decreased E217βG transport 2-fold. Conversion to Asp eliminated transport of the estrogen conjugate and also decreased leukotriene C4 transport ∼2-fold. The mutations also reduced the ability of MRP1 to confer resistance to all drugs tested. As with mrp1, introduction of a second mutation based on the murine sequence to create MRP1E1089Q/T1242A restored resistance to vincristine and VP-16, but not anthracyclines, without affecting transport of leukotriene C4 and E217βG. These results demonstrate the important role of Thr1242 for E217βG transport. They also reveal a highly specific functional relationship between nonconserved amino acids in TM helices 14 and 17 of both mrp1 and MRP1 that enables both proteins to confer similar levels of resistance to vincristine and VP-16.
多药耐药相关蛋白 (MRP) 介导道诺霉素和白三烯 C4 (LTC4) 在分离质膜囊泡中的转运。
DOI：
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发表时间：
1999
期刊：
Anticancer research.
影响因子：
--
作者：
Ding,GY;Shen,T;Center,MS
通讯作者：
Center,MS