Pál A, Méhn D, Molnár E, Gedey S, Mészáros P, Nagy T, Glavinas H, Janáky T, von Richter O, Báthori G, Szente L, Krajcsi P
SOLVO Biotechnology, Central Hungarian Innovations Center, Gyár u. 2., H-2040 Budaörs, Hungary.
J Pharmacol Exp Ther. 2007 Jun;321(3):1085-94. doi: 10.1124/jpet.106.119289. Epub 2007 Mar 8.
ABCG2, a transporter of the ATP-binding cassette family, is known to play a prominent role in the absorption, distribution, metabolism, and excretion of xenobiotics. Drug-transporter interactions are commonly screened by high-throughput systems using transfected insect and/or human cell lines. The determination of ABCG2-ATPase activity is one method to identify ABCG2 substrate and inhibitors. We demonstrate that the ATPase activities of the human ABCG2 transfected Sf9 cell membranes (MXR-Sf9) and ABCG2-overexpressing human cell membranes (MXR-M) differ. Variation due to disparity in the glycosylation level of the protein had no effect on the transporter. The influence of cholesterol on ABCG2-ATPase activity was investigated because the lipid compositions of insect and human cells are largely different from each other. Differences in cholesterol content, shown by cholesterol loading and depletion experiments, conferred the difference in stimulation of basal ABCG2-ATPase of the two cell membranes. Basal ABCG2-ATPase activity could be stimulated by sulfasalazine, prazosin, and topotecan, known substrates of ABCG2 in cholesterol-loaded MXR-Sf9 and MXR-M cell membranes. In contrast, ABCG2-ATPase could not be stimulated in MXR-Sf9 or in cholesterol-depleted MXR-M membranes. Moreover, cholesterol loading significantly improved the drug transport into inside-out membrane vesicles prepared from MXR-Sf9 cells. MXR-M and cholesterol-loaded MXR-Sf9 cell membranes displayed similar ABCG2-ATPase activity and vesicular transport. Our study indicates an essential role of membrane cholesterol for the function of ABCG2.
ABCG2是一种ATP结合盒家族转运蛋白，已知在异生物的吸收、分布、代谢和排泄中起重要作用。药物转运体相互作用通常通过使用转染昆虫和/或人类细胞系的高通量系统进行筛选。测定ABCG2 - ATP酶活性是鉴定ABCG2底物和抑制剂的一种方法。我们证明，转染人ABCG2的Sf9细胞膜（MXR - Sf9）和过表达ABCG2的人细胞膜（MXR - M）的ATP酶活性不同。蛋白质糖基化水平差异引起的变化对转运体没有影响。由于昆虫和人类细胞的脂质组成差异很大，因此研究了胆固醇对ABCG2 - ATP酶活性的影响。胆固醇加载和耗竭实验显示的胆固醇含量差异导致了两种细胞膜基础ABCG2 - ATP酶刺激的差异。在加载胆固醇的MXR - Sf9和MXR - M细胞膜中，已知的ABCG2底物柳氮磺胺吡啶、哌唑嗪和拓扑替康可刺激基础ABCG2 - ATP酶活性。相比之下，在MXR - Sf9或胆固醇耗竭的MXR - M膜中，ABCG2 - ATP酶活性无法被刺激。此外，胆固醇加载显著改善了药物向由MXR - Sf9细胞制备的内翻膜囊泡内的转运。MXR - M和加载胆固醇的MXR - Sf9细胞膜显示出相似的ABCG2 - ATP酶活性和囊泡转运。我们的研究表明膜胆固醇对ABCG2功能具有重要作用。
