A consecutive three alanine residue insertion mutant of human CAR: a novel CAR ligand screening system in HepG2 cells
A consecutive three alanine residue insertion mutant of human CAR: a novel CAR ligand screening system in HepG2 cells
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DOI：
10.2131/jts.35.515
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发表时间：
2010-08-01
影响因子：
2
通讯作者：
Inouye, Yoshio
中科院分区：
文献类型：
--
作者：
Kanno, Yuichiro;Inouye, Yoshio
在大多数培养的细胞系中，CAR基因的表达缺失，外源表达的CAR自发聚集在核室，在那里CAR具有结构性的活性。因此，对CAR依赖转录激活的评估必须使用动物模型或原代培养的肝细胞来评估。此外，由于CAR对个别化合物的物种特异性反应，用动物模型获得的结果不能直接适用于人类病例。我们建立了一种新的HCAR配体(包括激动剂和反向激动剂)的筛选系统，通过表达GAL4/DBD融合的HCAR/LBD，在LBD的螺旋11和螺旋12之间插入三个连续的丙氨酸残基，并在培养细胞系中获得由萤火虫荧光素酶基因组成的GAL4反应元件下游的质粒。雄烯醇(Andro)和克霉唑(CLT)都是HCAR的反向激动剂，在我们新的检测系统中，它们分别被归类为拮抗剂和弱激动剂。在DDT及其代谢物DDE和DDD中，只有DDT作为Hcar突变体的激动剂发挥作用，尽管它们都被登记为SXR的激动剂。使用该系统，可以在不牺牲动物的情况下进行Hcar配体的筛选。
The expression of the CAR gene is lost in most cultured cell lines, and exogenously expressed CAR accumulates spontaneously in the nuclear compartment, where CAR is constitutively active. Therefore, the assessment of CAR-dependent transcriptional activation has to be evaluated using either animal models or primary cultured hepatocytes. Furthermore, due to the species-specific response of CAR to individual compounds, the results obtained with animal models are not directly applicable to human cases. We established a novel screening system for hCAR ligands (including agonists and inverse agonists) by expressing GAL4/DBD-fused hCAR/LBD, in which three consecutive alanine residues were inserted between helix 11 and helix 12 of LBD, and a commercially obtained plasmid consisting of the firefly luciferase gene downstream of the GAL4 responsive element in a cultured cell line. Androstenol (Andro) and clotrimazole (CLT), which are both inverse agonists of hCAR, were classified as an antagonist and weak agonist, respectively, in our novel assay system. Among DDT and its metabolites DDE and DDD, only DDT worked as an agonist of the hCAR mutant, although all of them were enrolled as agonists of SXR. Using this system, the screening for hCAR ligands can be conducted without sacrificing animals.