Ejendal Karin F K, Diop Ndeye Khady, Schweiger Linda C, Hrycyna Christine A
Department of Chemistry and Purdue Cancer Center, Purdue University, West Lafayette, IN 47907, USA.
Protein Sci. 2006 Jul;15(7):1597-607. doi: 10.1110/ps.051998406.
Several members of the ATP-binding cassette (ABC) transporter superfamily, including P-glycoprotein and the half-transporter ABCG2, can confer multidrug resistance to cancer cells in culture by functioning as ATP-dependent efflux pumps. ABCG2 variants harboring a mutation at arginine 482 have been cloned from several drug-resistant cell lines, and these variants differ in their substrate transport phenotype. In this study, we changed the wild-type arginine 482 in human ABCG2 to each one of the 19 other standard amino acids and expressed each one transiently in HeLa cells. Using the 5D3 antibody that recognizes a cell surface epitope of ABCG2, we observed that all the mutants were expressed at the cell surface. However, the mutant ABCG2 proteins differed markedly in transport activity. All of the variants were capable of transporting one or more of the substrates used in this study, with the exception of the R482K mutant, which is completely devoid of transport ability. Six of the mutants (R482G, R482H, R482K, R482P, R482T, and R482Y) and the wild-type protein (R482wt) were selected for studies of basal and stimulated ATPase activity and photoaffinity labeling with the substrate analog [125I]iodoarylazidoprazosin. Whereas these seven ABCG2 variants differed markedly in ATPase activity, all were able to specifically bind the substrate analog [125I]iodoarylazidoprazosin. These data suggest that residue 482 plays an important role in substrate transport and ATP turnover, but that the nature of this amino acid may not be important for substrate recognition and binding.
ATP结合盒(ABC)转运蛋白超家族的几个成员,包括P-糖蛋白和半转运蛋白ABCG2,可通过作为ATP依赖性外排泵发挥作用,赋予培养中的癌细胞多药耐药性。已从几种耐药细胞系中克隆出在精氨酸482处发生突变的ABCG2变体,这些变体在底物转运表型上有所不同。在本研究中,我们将人ABCG2中的野生型精氨酸482替换为其他19种标准氨基酸中的每一种,并在HeLa细胞中瞬时表达每一种。使用识别ABCG2细胞表面表位的5D3抗体,我们观察到所有突变体均在细胞表面表达。然而,突变的ABCG2蛋白在转运活性上有显著差异。除完全缺乏转运能力的R482K突变体外,所有变体都能够转运本研究中使用的一种或多种底物。选择六个突变体(R482G、R482H、R482K、R482P、R482T和R482Y)和野生型蛋白(R482wt)进行基础和刺激ATP酶活性研究以及用底物类似物[125I]碘芳叠氮基哌唑嗪进行光亲和标记。虽然这七种ABCG2变体在ATP酶活性上有显著差异,但它们都能够特异性结合底物类似物[125I]碘芳叠氮基哌唑嗪。这些数据表明,482位残基在底物转运和ATP周转中起重要作用,但该氨基酸的性质可能对底物识别和结合并不重要。