Identification of amino acid residues in the Ah receptor involved in ligand binding
Identification of amino acid residues in the Ah receptor involved in ligand binding
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DOI：
10.1016/j.bbrc.2006.12.227
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发表时间：
2007-03-09
影响因子：
3.1
通讯作者：
Sogawa, Kazuhiro
中科院分区：
文献类型：
--
作者：
Goryo, Kenji;Suzuki, Ai;Sogawa, Kazuhiro
AhR是一种配体激活的转录因子。根据它们的进化保守性和能与外源配体相互作用的芳香性，选择了5个氨基酸作为配体结合域内或附近与配体结合所必需的候选氨基酸。这些氨基酸被改变为ALA，突变的AHRs在HeLa细胞中进行反式激活活性测试。Phe318的突变完全失去了活性，而其他突变只是微弱地削弱了活性。亮氨酸取代突变体AhR(Phe318Leu)在3-甲基胆蒽(MC)处理的细胞中激活了与野生型相当的荧光素酶活性，而在β-萘黄酮(β-NF)处理下则完全不激活。用[H-3]MC体外检测突变体的配体结合活性。AHR(Phe318Ala)不能与[H-3]MC结合。[H-3]与AhR(Phe318Leu)结合的MC与未标记的MC竞争，但不与β-NF竞争。构建了配体结合域的结构模型。(C)2007 Elsevier Inc.保留所有权利。
The Ah receptor (AhR) is a ligand-activated transcription factor. Five amino acids as candidate amino acids necessary for ligand binding within or near the ligand-binding domain were selected based on their evolutional conservation and their aromatic nature that could interact with xenobiotic ligands. These amino acids were changed to Ala, and the mutated AhRs were subjected to a test of their transactivation activity in HeLa cells. Mutation of Phe318 completely lost its activity whereas other mutations only weakly impaired activity. The Leu-substituted mutant, AhR(Phe318Leu), activated the luciferase activity to the level comparable to wild type in the cells treated with 3-methylcholanthrene (MC) but not at all with beta-naphthoflavone (beta-NF). Ligand-binding activity of mutants was examined with [H-3]MC in vitro. AhR(Phe318Ala) could not bind to [H-3]MC. [H-3]MC bound by AhR(Phe318Leu) was competed with unlabeled MC but not with beta-NF. A structural model of the ligand-binding domain was constructed. (c) 2007 Elsevier Inc. All rights reserved.