Different structural requirements of the ligand binding domain of the aryl hydrocarbon receptor for high- and low-affinity ligand binding and receptor activation
Different structural requirements of the ligand binding domain of the aryl hydrocarbon receptor for high- and low-affinity ligand binding and receptor activation
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DOI：
10.1124/mol.65.2.416
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发表时间：
2004-02-01
影响因子：
3.6
通讯作者：
Ingelman-Sundberg, M
中科院分区：
文献类型：
--
作者：
Backlund, M;Ingelman-Sundberg, M
芳香烃受体（AhR）作为一种配体激活的转录因子，负责调节几种反应基因，其中最具特征的是CYP 1A 1基因。本研究旨在阐明奥美拉唑（OME）、2-巯基-5-甲氧基苯并咪唑（MMB）和伯氨喹（PRQ）激活AhR的机制，这些化合物先前已报道可诱导CYP 1A 1表达，但不是典型的AhR配体。所有化合物在用Gal 4-AhR构建体和相应Gal 4-Luc报告基因转染的大鼠H4 IIE和人HepG 2肝癌细胞中引起荧光素酶活性的显著增加。此外，MMB和PRQ，但不是OME，能够转化细胞质AhR的DNA结合形式和取代AhR结合的[H-3] TCDD在大鼠肝细胞质中在体外。通过对Gal 4-AhR的配体结合结构域中的残基进行定点诱变，发现含有Y320 F取代的构建体对OME、MMB和PRQ的活化具有抗性，但对2，3，7，8-四氯二苯并-对-二恶英（TCDD）的活化没有抗性。[H-3] TCDD结合野生型和Y320 F突变Gal 4-蛋白，在人胚肾293细胞中表达的亲和力相当，在配体结合试验中获得。与此相反，受体结合的[H-3] TCDD的PRQ的竞争是不存在的Gal 4-Y320 F，但不是从Gal 4-AhR细胞提取物。本研究的结果证实，MMB和PRQ是低亲和力的配体的AhR，并建议，高和低亲和力的配体相互作用的AhR配体结合口袋的不同残基。此外，本文提供的数据表明Tyr（320）在AhR活化中起重要作用。
The aryl hydrocarbon receptor (AhR) functions as a ligand-activated transcription factor that is responsible for the regulation of several response genes, of which the best characterized is the CYP1A1 gene. The present study was undertaken to elucidate the mechanism of activation of the AhR by omeprazole (OME), 2-mercapto-5- methoxybenzimidazole (MMB), and primaquine (PRQ), compounds that have previously been reported to induce CYP1A1 expression but that are not typical AhR ligands. All compounds caused a significant increase in luciferase activity in rat H4IIE and human HepG2 hepatoma cells transfected with a Gal4-AhR construct and the corresponding Gal4-Luc reporter gene. Furthermore, MMB and PRQ, but not OME, were capable of transforming cytosolic AhR to a DNA-binding form and displacing AhR-bound [H-3] TCDD in rat hepatic cytosol in vitro. By performing site-directed mutagenesis of residues in the ligand-binding domain of the Gal4-AhR, a construct containing a Y320F substitution was found to be resistant to activation by OME, MMB, and PRQ, but not by 2,3,7,8-tetrachlorodibenzo-p-dioxin ( TCDD). Comparable affinities of [H-3] TCDD-binding to the wild-type and the Y320F mutant Gal4-proteins, expressed in human embryonic kidney 293 cells, were obtained in the ligand-binding assay. In contrast, the competition of receptor-bound [H-3] TCDD by PRQ was absent from Gal4-Y320F but not from Gal4-AhR cell extracts. The results of this study confirm that MMB and PRQ are low-affinity ligands for the AhR and suggest that high- and low-affinity ligands interact with different residues of the AhR ligand-binding pocket. In addition, the data presented here indicate that Tyr(320) plays an important role in AhR activation.