Studies on the Ah receptor signaling mechanism

描述（由申请人提供）：商业和自然产生的二恶英是氯化多环芳烃，是剧毒的环境污染物。已知这些物质是强效的啮齿动物致癌物和疑似的人类致癌物。这类制剂最著名的原型是3,4,7,8-四氯二苯并-对二恶英（TCDD）。有充分的证据表明，大多数（如果不是全部的话）TCDD效应是通过Ah受体介导的。因此，为了更好地理解二恶英的作用机制，我建议利用过表达的AhR和Arnt蛋白来研究AhR信号通路的分子机制。我的工作假设是：在配体结合后，AhR发生构象变化，导致一些AhR-蛋白相互作用的改变，从而导致一系列生物事件的发生。除了AhR蛋白的相互作用外，涉及Arnt和其他蛋白的其他相互作用也促进了由AhR介导的二恶英的作用。本研究旨在研究AhR信号通路中蛋白因子的作用机制。这些研究不仅为进一步研究AhR介导的PAH作用提供了重要的信息和试剂，而且还通过了解AhR信号通路中涉及的其他蛋白质，为AhR如何调节其靶基因提供了机制见解。提出了五个具体目标：目标1)确定兔网织红细胞裂解液（RRL）和Sf9可溶性提取物中负责AhR/Arnt/DRE复合物形成的蛋白质因子；目的2)鉴定AhR和Arnt的相互作用蛋白，并研究它们对AhR/Arnt/DRE复合物形成的影响；目的3)确定细胞系中是否存在不同形式的AhR/Arnt异源二聚体，这些异源二聚体有助于不同水平的配体依赖性交易激活；目的4)鉴定ara9介导的AhR/Arnt/DRE复合物形成的相互作用蛋白；目的5)确定cyp40o如何增强AhR/Arnt/DRE复合物的形成。

DESCRIPTION (provided by applicant): Dioxins, generated both commercially and naturally, are chlorinated polycyclic aromatic hydrocarbons that are highly toxic environmental contaminants. These agents are known to be potent rodent carcinogens and suspected human carcinogens. The best-known prototype of this group of agents is 3,4,7,8-tetrachlorodibenzo-p-dioxin (TCDD). It has been well documented that most, if not all, of the TCDD effects are mediated through the Ah receptor (AhR). Thus, in an effort to better understand the mechanism of dioxins action, I propose to investigate the molecular mechanism of the AhR signaling pathway using the overexpressed AhR and Arnt proteins. My working hypothesis is as follows: Upon ligand binding, the AhR undergoes conformational changes resulting in alteration of a number of AhR-protein interactions, which subsequently leads to a cascade of biologic events to occur. In addition to AhR-protein interactions, other interactions involving Arnt and other proteins also contribute to the action of dioxins mediated by the AhR. This proposal contains specific aims investigating the mechanism for protein factors involvement in the AhR signaling. Not only do these studies provide important information and reagents to further the study of the PAH action mediated by the AhR, but they also provide mechanistic insights on how the AhR regulates its target genes by understanding what other proteins are involved in the AhR signaling pathway. Five specific aims have been proposed: Aim 1) Identify the protein factors in rabbit reticulocyte lysate (RRL) and the Sf9 soluble extract which are responsible for the AhR/Arnt/DRE complex formation; Aim 2) Identity interacting proteins of the AhR and Arnt and examine their effect on the AhR/Arnt/DRE complex formation; Aim 3) Determine whether different forms of the AhR/Arnt heterodimer are present in cell lines which contribute to the different levels of ligand dependent transactivation; Aim 4) Identify ARA9-interacting proteins which are responsible for the ARA9-mediated AhR/Arnt/DRE complex formation and Aim 5) Determine how CyP4O enhances the AhR/Arnt/DRE complex formation.