ANALYSIS OF AH RECEPTOR LIGAND BINDING SPECIFICITY

芳烃受体（aryl hydrocarbon receptor，AhR）是一种配体依赖性转录因子，介导多种疏水性天然和合成化学物质的生物学和毒理学作用，包括环境污染物2，3，7，8-四氯二苯并-对-二恶英。最好的特点是高亲和力AhR配体包括各种有毒和致癌的卤代和多环芳烃;没有高亲和力内源性生理AhR配体已被确定。我们实验室的研究表明，AhR可以被多种结构不同的化学物质激活，并揭示了AhR配体结合特异性的物种差异。这些结果不仅表明AhR具有非常混杂的配体结合位点，而且还提出了关于可以在不同物种中结合和激活AhR的化学物质谱的重要问题。AhR配体结构的多样性的知识可以提供洞察其生理配体的身份。我们假设，AhR可以绑定和激活的各种各样的结构不同的内源性和外源性化学物质和AhR的激活是一个配体依赖性的事件，表现出物种特异性。该提案的总体目标是进一步表征来自不同物种的AhR的配体结合特异性，以确认AhR活化的配体依赖性，并鉴定AhR信号转导途径的新型内源性和外源性配体/活化剂。苯并咪唑，选定的农药和其他化学品激活AhR的机制，在一个“配体独立的方式”和一些内源性生理化学物质，即花生四烯酸衍生的脂质和色氨酸/吲哚衍生的产品，激活/增强AhR依赖性信号的能力，将使用配体结合和DNA结合和基因表达测定检查。将使用配体和DNA结合测定以及在用物种特异性AhR cDNA和AhR响应性荧光素酶报告质粒稳定转染的细胞中评估AhR的配体依赖性活化的物种特异性差异。基于AhR的生物测定也将与分析化学分馏程序结合使用，以纯化和阐明我们在商业产品的水和乙醇提取物中鉴定的新型AhR配体/活化剂的化学结构。总体而言，这些研究将定义可以结合并激活各种物种中AhR的化学物质的谱，并提供对内源性生理AhR配体的身份以及该受体在正常生理过程中的作用的见解。

The aryl hydrocarbon receptor (AhR) is a ligand-dependent transcription factor that mediates many of the biological and toxicological actions of a variety Of hydrophobic natural and synthetic chemicals, including the environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin. The best characterized high affinity AhR ligands include a variety of toxic and carcinogenic halogenated and polycyclic aromatic hydrocarbons; no high affinity endogenous physiological AhR ligand has been identified. Studies in our laboratory have indicated that the AhR can be activated by a variety of structurally dissimilar chemicals and have revealed the existence Of species differences in AhR ligand binding specificity. These results not only suggest that the AhR has a very promiscuous ligand binding site, but they raise significant questions as to the spectrum of chemicals which can bind and activate the AhR in different species. Knowledge of the diversity of AhR ligand structure may provide insight into the identity of its physiological ligand(s). We hypothesize that the AhR can bind and be activated by a wide variety of structurally dissimilar endogenous and exogenous chemicals and that the activation of the AhR is a ligand-dependent event which exhibits species specificity. The overall goals of this proposal are to further characterize the ligand binding specificity of the AhR from different species in order to both confirm the ligand-dependent nature of AhR activation and to identify novel endogenous and exogenous ligands/activators of the AhR signal transduction pathway. The mechanism by which benzimidazoles, selected pesticides and other chemicals activate the AhR in a "ligand-independent manner" and the ability of some endogenous physiological chemicals, namely arachidonic acid-derived lipids and tryptophan/indole-derived products, to activate/augment AhR-dependent signaling will be examined using ligand binding and DNA binding and gene expression assays. Species- specific differences in ligand-dependent activation of the AhR will be assessed using ligand and DNA binding assays and in cells stably transfected with species-specific AhR cDNAs and an AhR-responsive luciferase reporter plasmid. AhR-based bioassays will also be used in combination with analytical chemical fractionation procedures to purify and elucidate the chemical structure of novel AhR ligands/activators we have identified in water and ethanol extracts of commercial products. Overall, these studies will define the spectrum of chemicals which can bind to and activate the AhR in various species and provide insights into the identity of the endogenous physiological AhR ligand(s) and the role of this receptor in normal physiological processes.