ANALYSIS OF AH RECEPTOR LIGAND BINDING SPECIFICITY

芳烃受体(AhR)是一种配体依赖的转录因子，介导了包括环境污染物2，3，7，8-四氯二苯并-对二恶英在内的多种疏水性天然和合成化学品的许多生物学和毒理作用。最具特点的高亲和力AhR配体包括各种有毒和致癌的卤代和多环芳烃；尚未鉴定出高亲和力的内源性生理性AhR配体。我们实验室的研究表明，AhR可以被各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有一个非常杂乱的配体结合部位，而且它们提出了关于在不同物种中可以结合和激活AhR的化学物质的光谱的重要问题。了解AhR配体结构的多样性可能有助于深入了解其生理配体的身份(S)。我们假设AhR可以与多种结构不同的内源和外源化学物质结合并被激活，并且AhR的激活是一个配体依赖的事件，表现出物种特异性。该建议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，以便既证实AhR激活的配体依赖性质，又识别AhR信号转导途径的新的内源和外源配体/激活剂。苯并咪唑、选定的农药和其他化学物质以“非配体依赖的方式”激活AhR的机制，以及一些内源生理化学物质，即花生四烯酸衍生的脂类和色氨酸/吲哚衍生的产物，激活/增强AhR依赖的信号的能力，将通过配体结合、DNA结合和基因表达分析来研究。将使用配体和DNA结合分析，以及稳定转染物种特异性AhR cDNA和AhR反应荧光素酶报告质粒的细胞中，评估不同物种对AhR依赖的配体激活的差异。基于AHR的生物测定也将与分析化学分级程序结合使用，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/激活剂的化学结构。总之，这些研究将确定不同物种中能够与AhR结合并激活AhR的化学物质的谱，并为深入了解内源性生理AhR配体(S)的身份以及该受体在正常生理过程中的作用提供深入的见解。

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.