Müller M, Bakos E, Welker E, Váradi A, Germann U A, Gottesman M M, Morse B S, Roninson I B, Sarkadi B
National Institute of Haematology, Blood Transfusion and Immunology, Budapest, Hungary.
J Biol Chem. 1996 Jan 26;271(4):1877-83. doi: 10.1074/jbc.271.4.1877.
The characteristics of P-glycoprotein (MDR1), an ATP-dependent drug extrusion pump responsible for the multidrug resistance of human cancer, were investigated in an in vitro expression system. The wild-type and several mutants of the human MDR1 cDNA were engineered into recombinant baculoviruses and the mutant proteins were expressed in Sf9 insect cells. In isolated cell membrane preparations of the virus-infected cells the MDR1-dependent drug-stimulated ATPase activity, and 8-azido-ATP binding to the MDR1 protein were studied. We found that when lysines 433 and/or 1076 were replaced by methionines in the ATP-binding domains, all these mutations abolished drug-stimulated ATPase activity independent of the MgATP concentrations applied. Photoaffinity labeling with 8-azido-ATP showed that the double lysine mutant had a decreased ATP-binding affinity. In the MDR1 mutant containing a Gly185 to Val replacement we found no significant alteration in the maximum activity of the MDR1-ATPase or in its activation by verapamil and vinblastine, and this mutation did not modify the MgATP affinity or the 8-azido-ATP binding of the transporter either. However, the Gly185 to Val mutation significantly increased the stimulation of the MDR1-ATPase by colchicine and etoposide, while slightly decreasing its stimulation by vincristine. These shifts closely correspond to the effects of this mutation on the drug-resistance profile, as observed in tumor cells. These data indicate that the Sf9-baculovirus expression system for MDR1 provides an efficient tool for examining structure-function relationships and molecular characteristics of this clinically important enzyme.
在体外表达系统中研究了负责人类癌症多药耐药性的ATP依赖性药物外排泵P-糖蛋白（MDR1）的特性。将人类MDR1 cDNA的野生型和几个突变体构建到重组杆状病毒中，并在Sf9昆虫细胞中表达突变蛋白。在病毒感染细胞的分离细胞膜制剂中，研究了MDR1依赖性药物刺激的ATP酶活性以及8-叠氮基-ATP与MDR1蛋白的结合。我们发现，当ATP结合结构域中的赖氨酸433和/或1076被甲硫氨酸取代时，所有这些突变都消除了药物刺激的ATP酶活性，且与所应用的MgATP浓度无关。用8-叠氮基-ATP进行光亲和标记表明，双赖氨酸突变体的ATP结合亲和力降低。在含有Gly185至Val取代的MDR1突变体中，我们发现MDR1-ATP酶的最大活性或其被维拉帕米和长春碱激活的情况没有显著改变，并且该突变也没有改变转运蛋白的MgATP亲和力或8-叠氮基-ATP结合。然而，Gly185至Val突变显著增加了秋水仙碱和依托泊苷对MDR1-ATP酶的刺激，同时略微降低了长春新碱对其的刺激。这些变化与该突变对肿瘤细胞中耐药谱的影响密切对应。这些数据表明，用于MDR1的Sf9-杆状病毒表达系统为研究这种临床重要酶的结构-功能关系和分子特性提供了一个有效的工具。
