Zhang Da-Wei, Gu Hong-Mei, Vasa Monika, Muredda Mario, Cole Susan P C, Deeley Roger G
Division of Cancer Biology and Genetics, Cancer Research Institute, and Department of Pathology, Queen's University, Kingston, Ontario K7L 3N6, Canada.
Biochemistry. 2003 Aug 26;42(33):9989-10000. doi: 10.1021/bi034462b.
Human multidrug resistance protein (MRP) 3 is the most closely related homologue of MRP1. Like MRP1, MRP3 confers resistance to etoposide (VP-16) and actively transports 17 beta-estradiol 17-(beta-D-glucuronide) (E(2)17 beta G), cysteinyl leukotriene 4 (LTC(4)), and methotrexate, although with generally lower affinity. Unlike MRP1, MRP3 also transports monovalent bile salts. We have previously demonstrated that hydrogen-bonding residues predicted to be in the inner-leaflet spanning segment of transmembrane (TM) 17 of MRP1 are important for drug resistance and E(2)17 beta G transport. We have now examined the importance of the hydrogen-bonding potential of residues in TM17 of MRP3 on both substrate specificity and overall activity. Mutation S1229A reduced only methotrexate transport. Mutations S1231A and N1241A decreased resistance to VP-16 and transport of E(2)17 beta G and methotrexate but not taurocholate. Mutation Q1235A also reduced resistance to VP-16 and transport of E(2)17beta G but increased taurocholate transport without affecting transport of methotrexate. Mutations Y1232F and S1233A reduced resistance to VP-16 and the transport of all three substrates tested. In contrast, mutation T1237A markedly increased VP-16 resistance and transport of all substrates. On the basis of the substrates analyzed, residues Ser(1229), Ser(1231), Gln(1235), and Asn(1241) play an important role in determining the specificity of MRP3, while mutation of Tyr(1232), Ser(1233), and Thr(1237) affects overall activity. Unlike MRP1, the involvement of polar residues in determining substrate specificity extends throughout the TM helix. Furthermore, elimination of the hydrogen-bonding potential of a single amino acid, Thr(1237), markedly enhanced the ability of the protein to confer drug resistance and to transport all substrates examined.
人多药耐药蛋白（MRP）3是与MRP1关系最为密切的同源物。与MRP1一样，MRP3赋予对依托泊苷（VP - 16）的抗性，并能主动转运17β - 雌二醇17 - （β - D - 葡萄糖醛酸苷）（E₂17βG）、半胱氨酰白三烯4（LTC₄）和甲氨蝶呤，尽管其亲和力通常较低。与MRP1不同的是，MRP3还能转运单价胆汁盐。我们之前已经证明，预测位于MRP1跨膜（TM）17内小叶跨段中的氢键残基对于耐药性和E₂17βG转运很重要。我们现在研究了MRP3的TM17中残基的氢键潜力对底物特异性和整体活性的重要性。突变S1229A仅降低了甲氨蝶呤的转运。突变S1231A和N1241A降低了对VP - 16的抗性以及E₂17βG和甲氨蝶呤的转运，但不影响牛磺胆酸盐的转运。突变Q1235A也降低了对VP - 16的抗性和E₂17βG的转运，但增加了牛磺胆酸盐的转运，而不影响甲氨蝶呤的转运。突变Y1232F和S1233A降低了对VP - 16的抗性以及所有三种测试底物的转运。相反，突变T1237A显著增加了对VP - 16的抗性以及所有底物的转运。基于所分析的底物，Ser（1229）、Ser（1231）、Gln（1235）和Asn（1241）残基在决定MRP3的特异性方面起重要作用，而Tyr（1232）、Ser（1233）和Thr（1237）的突变影响整体活性。与MRP1不同，极性残基在决定底物特异性方面的作用贯穿整个TM螺旋。此外，消除单个氨基酸Thr（1237）的氢键潜力显著增强了该蛋白赋予耐药性和转运所有检测底物的能力。
