Zhang D W, Cole S P, Deeley R G
Cancer Research Laboratories and Department of Pathology, Queen's University, Kingston, Ontario K7L 3N6, Canada.
J Biol Chem. 2001 Apr 20;276(16):13231-9. doi: 10.1074/jbc.M010008200. Epub 2001 Jan 23.
Murine multidrug resistance protein 1 (mrp1), unlike human MRP1, does not confer resistance to anthracyclines. Previously, we have shown that a human/murine hybrid protein containing amino acids 959-1187 of MRP1 can confer resistance to these drugs. We have now examined the functional characteristics of mutant proteins in which we have converted individual amino acids in the comparable region of mrp1 to those present at the respective locations in MRP1. These mutations had no effect on the drug resistance profile conferred by mrp1 with the exception of converting glutamine 1086 to glutamate, as it is in the corresponding position (1089) in MRP1. This mutation created a protein that conferred resistance to doxorubicin without affecting vincristine resistance, or the ability of mrp1 to transport leukotriene C(4) (LTC(4)) and 17beta-estradiol 17-(beta-d-glucuronide) (E(2)17betaG). Furthermore, mutation Q1086D conferred the same phenotype as mutation Q1086E while the mutation Q1086N did not detectably alter the drug resistance profile of mrp1, suggesting that an anionic side chain was required for anthracycline resistance. To confirm the importance of MRP1 E1089 for conferring resistance to anthracyclines, we mutated this residue to Gln, Asp, Ala, Leu, and Lys in the human protein. The mutation E1089D showed the same phenotype as MRP1, while the E1089Q substitution markedly decreased resistance to anthracyclines without affecting LTC(4) and E(2)17betaG transport. Conversion of Glu-1089 to Asn, Ala, or Leu had a similar effect on resistance to anthracyclines, while conversion to a positive amino acid, Lys, completely eliminated resistance to anthracyclines and vincristine without affecting transport of LTC(4), E(2)17betaG, and the GSH-dependent substrate, estrone-3-sulfate. These results demonstrate that an acidic amino acid residue at position 1089 in predicted TM14 of MRP1 is critical for the ability of the protein to confer drug resistance particularly to the anthracyclines, but is not essential for its ability to transport conjugated organic anions such as LTC(4) and E(2)17betaG.
与人类多药耐药蛋白1（MRP1）不同，小鼠多药耐药蛋白1（mrp1）对蒽环类药物不具有耐药性。此前，我们已表明，一种包含MRP1第959 - 1187位氨基酸的人/鼠杂交蛋白可对这些药物产生耐药性。我们现在研究了突变蛋白的功能特性，在这些突变蛋白中，我们将mrp1可比区域中的单个氨基酸转换为MRP1相应位置上的氨基酸。这些突变对mrp1赋予的耐药谱没有影响，但将谷氨酰胺1086转换为谷氨酸除外，因为它在MRP1中的相应位置（1089）。这种突变产生了一种对阿霉素具有耐药性的蛋白，而不影响长春新碱耐药性，也不影响mrp1转运白三烯C4（LTC4）和17β - 雌二醇17 - （β - D - 葡萄糖醛酸苷）（E217βG）的能力。此外，Q1086D突变赋予的表型与Q1086E突变相同，而Q1086N突变未明显改变mrp1的耐药谱，这表明蒽环类药物耐药性需要一个阴离子侧链。为了证实MRP1的E1089对于赋予蒽环类药物耐药性的重要性，我们将该残基在人蛋白中突变为谷氨酰胺、天冬氨酸、丙氨酸、亮氨酸和赖氨酸。E1089D突变表现出与MRP1相同的表型，而E1089Q替代显著降低了对蒽环类药物的耐药性，同时不影响LTC4和E217βG的转运。将Glu - 1089转换为天冬酰胺、丙氨酸或亮氨酸对蒽环类药物耐药性有类似影响，而转换为带正电荷的氨基酸赖氨酸则完全消除了对蒽环类药物和长春新碱的耐药性，同时不影响LTC4、E217βG以及谷胱甘肽依赖性底物硫酸雌酮的转运。这些结果表明，MRP1预测的第14跨膜区（TM14）中第1089位的酸性氨基酸残基对于该蛋白赋予耐药性的能力至关重要，尤其是对蒽环类药物，但对于其转运诸如LTC4和E217βG等共轭有机阴离子的能力并非必需。
