Saatcioglu F, Perry D J, Pasco D S, Fagan J B
Molecular Biology Laboratory, Maharishi International University, Fairfield, Iowa 52556.
J Biol Chem. 1990 Jun 5;265(16):9251-8.
The aryl hydrocarbon (Ah) receptor, also called the xenobiotic or TCDD receptor, mediates transcriptional activation of the cytochrome P-450c (CYP1A1) gene by interacting with Ah or xenobiotic response elements. This paper presents evidence that a metal ion, probably Zn2+, is an essential cofactor for the Ah receptor. This paper also maps in detail the interactions between the Ah response element XRE1 and the Ah receptor from the rat hepatocyte-derived cell line LCS7. Interactions were mapped by three methods, 1) methylation interference footprinting, 2) mobility shift competition experiments, using a series of synthetic oligonucleotides with systematic alterations in the Ah response element core sequence, and 3) orthophenanthroline/Cu+ footprinting. These findings suggest the following consensus sequence for DNA recognition by the Ah receptor: CNA/TNA/TCACGCA/TA/T. The chelators 1,10-phenanthroline and oxalic acid inhibited the sequence-specific DNA-binding activity of the AH receptor in a concentration dependent manner, suggesting that the DNA-binding activity of the receptor requires divalent metal ions. Inhibition was due to metal-chelation, since: 1) inhibition was almost completely prevented by the presence of Zn2+, or other divalent metal ions having high affinity for the chelators used, while metal ions with low affinity did not protect; 2) the DNA-binding activity of the receptor could be restored by dialysis to remove 1,10-phenanthroline, but only in the presence of Zn2+, while dialysis in the absence of metal ions reversed inhibition by the nonchelating isomer 4,7-phenanthroline. The involvement of a divalent cation in receptor function, possibly bound via sulfhydryls, was also suggested by the finding that Cd2+ and Co2+ inhibited DNA-binding activity. Once bound to the XRE1 DNA sequence, the receptor could not be inhibited by 1,10-phenanthroline, suggesting that the essential metal ion must become inaccessible to chelation when the receptor binds DNA. The Zn2+ requirement of the Ah receptor is similar to that of the estrogen and the glucocorticoid receptors and is consistent with the hypothesis that the Ah receptor is a member of the steroid and thyroid hormone receptor superfamily.
芳基烃（Ah）受体，也称为外源性物质或四氯二苯并对二恶英受体，通过与Ah或外源性物质反应元件相互作用介导细胞色素P - 450c（CYP1A1）基因的转录激活。本文提供证据表明一种金属离子，可能是Zn2 +，是Ah受体的必需辅助因子。本文还详细绘制了来自大鼠肝细胞衍生细胞系LCS7的Ah反应元件XRE1与Ah受体之间的相互作用。通过三种方法绘制相互作用：1）甲基化干扰足迹法；2）迁移率变动竞争实验，使用一系列在Ah反应元件核心序列中有系统改变的合成寡核苷酸；3）邻菲罗啉/Cu +足迹法。这些发现提示Ah受体识别DNA的共有序列如下：CNA/TNA/TCACGCA/TA/T。螯合剂1,10 - 邻菲罗啉和草酸以浓度依赖的方式抑制Ah受体的序列特异性DNA结合活性，提示该受体的DNA结合活性需要二价金属离子。抑制是由于金属螯合，因为：1）Zn2 +或对所用螯合剂具有高亲和力的其他二价金属离子的存在几乎完全阻止了抑制，而低亲和力的金属离子则不能起到保护作用；2）受体的DNA结合活性可通过透析去除1,10 - 邻菲罗啉而恢复，但仅在有Zn2 +存在时，而在无金属离子的情况下透析则可逆转非螯合异构体4,7 - 邻菲罗啉的抑制作用。Cd2 +和Co2 +抑制DNA结合活性这一发现也提示二价阳离子参与受体功能，可能通过巯基结合。一旦与XRE1 DNA序列结合，受体就不能被1,10 - 邻菲罗啉抑制，提示当受体结合DNA时必需的金属离子必定无法被螯合。Ah受体对Zn2 +的需求与雌激素和糖皮质激素受体相似，并且与Ah受体是类固醇和甲状腺激素受体超家族成员的假说一致。
