Ozvegy Csilla, Váradi András, Sarkadi Balázs
Institute of Enzymology, Biological Research Center, Hungarian Academy of Sciences and National Medical Center, Institute of Haematology and Immunology, Membrane Research Group of the Hungarian Academy of Sciences, H-1113 Budapest, Hungary.
J Biol Chem. 2002 Dec 13;277(50):47980-90. doi: 10.1074/jbc.M207857200. Epub 2002 Oct 8.
The overexpression of the human ATP-binding cassette half-transporter, ABCG2 (placenta-specific ABC transporter, mitoxantrone resistance-associated protein, breast cancer resistance protein), causes multidrug resistance in tumor cells. An altered drug resistance profile and substrate recognition were suggested for wild-type ABCG2 and its mutant variants (R482G and R482T); the mutations were found in drug-selected tumor cells. In order to characterize the different human ABCG2 transporters without possible endogenous dimerization partners, we expressed these proteins and a catalytic center mutant (K86M) in Sf9 insect cells. Transport activity was followed in intact cells, whereas the ATP binding and hydrolytic properties of ABCG2 were studied in isolated cell membranes. We found that the K86M mutant had no transport or ATP hydrolytic activity, although its ATP binding was retained. The wild-type ABCG2 and its variants, R482G and R482T, showed characteristically different drug and dye transport activities; mitoxantrone and Hoechst 33342 were transported by all transporters, whereas rhodamine 123 was only pumped by the R482G and R482T mutants. In each case, ABCG2-dependent transport was blocked by the specific inhibitor, fumitremorgin C. A relatively high basal ABCG2-ATPase, inhibited by fumitremorgin C, was observed in all active proteins, but specific drug stimulation could only be observed in the case of R482G and R482T mutants. We found that ABCG2 is capable of a vanadate-dependent adenine nucleotide trapping. Nucleotide trapping was stimulated by the transported compounds in the R482G and R482T variants but not in the wild-type ABCG2. These experiments document the applicability of the Sf9 expression system for parallel, quantitative examination of the specific transport and ATP hydrolytic properties of different ABCG2 proteins and demonstrate significant differences in their substrate interactions.
人类ATP结合盒半转运体ABCG2（胎盘特异性ABC转运体、米托蒽醌耐药相关蛋白、乳腺癌耐药蛋白）的过表达会导致肿瘤细胞产生多药耐药性。野生型ABCG2及其突变体变体（R482G和R482T）表现出改变的耐药谱和底物识别特性；这些突变在药物筛选的肿瘤细胞中被发现。为了在不存在可能的内源性二聚化伙伴的情况下表征不同的人类ABCG2转运体，我们在Sf9昆虫细胞中表达了这些蛋白以及一个催化中心突变体（K86M）。在完整细胞中监测转运活性，而在分离的细胞膜中研究ABCG2的ATP结合和水解特性。我们发现K86M突变体虽然保留了ATP结合能力，但没有转运或ATP水解活性。野生型ABCG2及其变体R482G和R482T表现出特征性不同的药物和染料转运活性；米托蒽醌和Hoechst 33342可被所有转运体转运，而罗丹明123仅由R482G和R482T突变体泵出。在每种情况下，ABCG2依赖性转运都被特异性抑制剂烟曲霉毒素C阻断。在所有活性蛋白中都观察到相对较高的基础ABCG2 - ATP酶活性，其被烟曲霉毒素C抑制，但只有在R482G和R482T突变体的情况下才能观察到特异性药物刺激。我们发现ABCG2能够进行钒酸盐依赖性腺嘌呤核苷酸捕获。在R482G和R482T变体中，转运化合物刺激了核苷酸捕获，但在野生型ABCG2中没有。这些实验证明了Sf9表达系统在平行、定量检测不同ABCG2蛋白的特异性转运和ATP水解特性方面的适用性，并证明了它们在底物相互作用方面的显著差异。
