Multiple low-affinity aryl hydrocarbon receptors in the frog Xenopus laevis

描述(申请人提供)：芳烃受体(AHR)是一种配体激活的转录因子，介导一系列结构不同的化学品的生物学和毒理学效应，包括2，3，7，8四氯二苯并对二恶英(TCDD)。AHR信号通路的固有特性，包括AHR的表达水平和对特定配体的亲和力，可能导致不同配体的相对效力和不同动物群体的敏感性存在很大差异。然而，无论是AHR活性导致毒性的确切机制，还是AHR信号的内源性、非毒理学功能都不是很清楚。与大多数其他脊椎动物相比，青蛙对TCDD的毒性极其不敏感。我们的团队已经从非洲爪蛙(非洲爪蛙)中鉴定出两个AHR，最近复制的并列动物称为AHR1？那么AHR1呢？这两种蛋白与TCDD的结合亲和力至少比来自高度敏感品系的小鼠的AHR低25倍，这可能是二恶英不敏感的表型的原因。拟议的项目将利用青蛙AHR独特的氨基酸序列、功能特性和系统发育位置来探索它们与一系列外来和自然存在的配体的结构相互作用，并将它们的功能与TCDD敏感物种的AHR以及彼此之间的功能进行比较。我们提出了三个具体目标：(1)使用定点突变使青蛙AHR更像“老鼠”，我们将检验假设，即假设配体结合域中的一个或几个氨基酸的变化会导致TCDD亲和力较低。这种比较方法将有助于确定AHR配体结合口袋的重要结构特征。(2)我们将确定结构不同的候选配体的相对效力。虽然X.laevis AHR以低亲和力结合TCDD，但它们可能对结构不同的化合物，特别是假定的内源性配体保持高度响应。我们将通过建立一系列候选配体的结构-活性关系来检验这一假设，包括与哺乳动物AHR结合的含有吲哚的化合物。(3)我们将确定AHR1？和AHR1？，研究表达模式、增强子偏好以及由单个副基因介导的基因表达的广泛变化。这些研究将检验这样一种假设，即AHR旁系动物表现出不同的功能，可能划分了单一哺乳动物AHR的多种角色。总体而言，在一个新的模型系统中的这种比较方法将提供关于所有脊椎动物AHR的结构和功能的重要基本信息。了解青蛙和人类AHR之间的差异也将通过完善对FETAX(青蛙胚胎致畸试验)和类似的使用青蛙胚胎的发育毒性测试得出的毒理学数据的解释来帮助风险评估。该项目将研究芳烃受体(AHR)，这是一种介导环境污染物(如二恶英)毒性效应的蛋白质。我们将比较青蛙AHR的结构和功能，这些AHR对二恶英毒性不敏感，与更敏感的动物如老鼠和人类的AHR进行比较。这项比较研究将帮助科学家理解青蛙和人类之间的重要差异，因为他们使用青蛙胚胎作为衡量化学品和环境样本影响的模型系统来解释毒理学研究。它还将提供有关AHR最初的无毒功能的基本信息。

DESCRIPTION (provided by applicant): The aryl hydrocarbon receptor (AHR) is a ligand activated transcription factor that mediates the biological and toxicological effects of a broad range of structurally diverse chemicals, including 2,3,7,8 tetrachlorodibenzo-p-dioxin (TCDD). Inherent properties of the AHR signaling pathway, including AHR expression levels and the affinity of AHR for specific ligands, can underlie large variations in the relative potency of different ligands and the sensitivity of different animal groups. However, neither the exact mechanisms by which AHR activity leads to toxicity nor the endogenous, non-toxicological functions of AHR signaling are well understood. Relative to most other vertebrates, frogs are extremely insensitive to TCDD toxicity. Our group has identified two AHRs from the African clawed frog (Xenopus laevis), recently duplicated paralogs called AHR1? and AHR1?. Both proteins bind TCDD with at least 25-fold lower affinity than the AHR from a highly sensitive strain of mouse, likely accounting for the dioxin-insensitive phenotype. The proposed project will take advantage of the unique amino acid sequence, functional properties, and phylogenetic position of the frog AHRs to probe their structural interactions with a range of xenobiotic and naturally occurring ligands and to contrast their function with AHRs from TCDD-sensitive species and with each other. We propose three specific aims: (1) Using site-directed mutagenesis to make the frog AHRs more "mouse-like," we will test the hypothesis that changes in one or a few amino acids within the putative ligand binding domain confer low TCDD affinity. This comparative approach will contribute significantly to the identification of important structural features of AHR's ligand binding pocket. (2) We will determine the relative potency of structurally diverse candidate ligands. Although X. laevis AHRs bind TCDD with low affinity, they may remain highly responsive to structurally distinct compounds, especially putative endogenous ligands. We will test this hypothesis by establishing structure-activity relationships for a range of candidate ligands, including indole-containing compounds that bind mammalian AHRs. (3) We will determine the functional differences between AHR1? and AHR1?, examining expression patterns, enhancer preferences, and broad-based changes in gene expression mediated by individual paralogs. These studies will test the hypothesis that the AHR paralogs exhibit distinct functions, possibly partitioning multiple roles of the single mammalian AHR. Overall, this comparative approach in a novel model system will provide important basic information about the structure and function of all vertebrate AHRs. Understanding the differences between frog and human AHRs will also aid risk assessment by refining interpretation of toxicological data derived from FETAX (Frog Embryo Teratogenesis Assay-Xenopus) and similar developmental toxicity tests that employ frog embryos. This project will study the aryl hydrocarbon receptor (AHR), a protein that mediates the toxic effects of environmental contaminants such as dioxin. We will compare the structure and function of the AHR from frogs, which are insensitive to dioxin toxicity, with AHRs from more sensitive animals like mice and humans. This comparative research will help scientists understand important differences between frogs and humans as they interpret toxicology studies using frog embryos as a model system for measuring the effects of chemicals and environmental samples. It will also provide basic information about the original, non-toxicological function of the AHR.