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

非洲爪蟾体内的多个低亲和力芳烃受体

• 批准号: 7304018
• 负责人: WADE H POWELL
• 金额: $ 18.41万
• 依托单位: KENYON COLLEGE
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-09-01 至 2011-09-22
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7304018
• 关键词: Accounting; Affinity; African; Agonist; Amino Acid Sequence; Amino Acids; Animals; Aromatic Hydrocarbons; Aromatic Polycyclic Hydrocarbons; Aryl Hydrocarbon Receptor; Benzo(a)pyrene; Binding; Biological; Biological Assay; Biological Models; CYP1A1 gene; CYP1A6 gene; Carbazoles; Carboxylic Acids; Cells; Chemicals; Class; Data; Development; Dioxins; Dose; Elements; Embryo; Enhancers; Environmental Pollution; Esters; Exhibits; Fishes; Furans; Gene Expression; Gene Targeting; Genes; Genetic Transcription; Homology Modeling; Human; In Situ Hybridization; In Vitro; Individual; Indoles; Ligand Binding; Ligand Binding Domain; Ligands; Luciferases; Measures; Mediating; Mouse Strains; Mus; Mutate; Mutation; Pattern; Phenotype; Phylogenetic Analysis; Physiological; Play; Polychlorinated Biphenyls; Population; Positioning Attribute; Property; Protein Binding; Proteins; Rana; Range; Receptor Signaling; Relative (related person); Reporter Genes; Research; Risk Assessment; Role; Sampling; Scientist; Side; Signal Pathway; Signal Transduction Pathway; Site-Directed Mutagenesis; Small Interfering RNA; Structure; Structure-Activity Relationship; Tadpoles; Testing; Thiazoles; Toxic effect; Toxicity Tests; Toxicology; Variant; Vertebrates; Xenobiotics; Xenopus; Xenopus laevis; Xenopus sp.; activating transcription factor; aryl hydrocarbon receptor ligand; base; carbazole; chlorination; cholanthrene; comparative; environmental chemical; furan; human AHR protein; indole; interest; mouse Ahr protein; novel; paralogous gene; preference; promoter; receptor binding; receptor expression; research study; response; toxicant

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.

描述（由申请人提供）：芳烃受体（AHR）是一种配体激活的转录因子，介导多种结构多样的化学物质的生物和毒理学效应，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 表达水平和 AHR 对特定配体的亲和力，可能是不同配体的相对效力和不同动物群体的敏感性存在巨大差异的基础。然而，AHR 活性导致毒性的确切机制以及 AHR 信号传导的内源性非毒理学功能均不清楚。与大多数其他脊椎动物相比，青蛙对 TCDD 毒性极其不敏感。我们的小组已经从非洲爪蛙（Xenopus laevis）中鉴定出两个 AHR，最近复制的旁系同源物称为 AHR1？和AHR1？这两种蛋白与 TCDD 的结合亲和力至少比高度敏感小鼠品系的 AHR 低 25 倍，这可能是二恶英不敏感表型的原因。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测它们与一系列外源性和天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对比。我们提出了三个具体目标：（1）使用定点诱变使青蛙 AHR 更像“小鼠”，我们将测试假定配体结合域内一个或几个氨基酸的变化赋予低 TCDD 亲和力的假设。这种比较方法将极大地有助于鉴定 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。尽管非洲爪蟾 AHR 以低亲和力结合 TCDD，但它们可能仍然对结构不同的化合物，特别是推定的内源配体保持高度响应。我们将通过建立一系列候选配体的构效关系来检验这一假设，其中包括结合哺乳动物 AHR 的含吲哚化合物。 (3)我们将确定AHR1之间的功能差异？和 AHR1？，检查表达模式、增强子偏好以及个体旁系同源物介导的基因表达的广泛变化。这些研究将检验 AHR 旁系同源物表现出不同功能的假设，可能区分单个哺乳动物 AHR 的多种作用。总体而言，这种新型模型系统中的比较方法将提供有关所有脊椎动物 AHR 的结构和功能的重要基本信息。了解青蛙和人类 AHR 之间的差异也将有助于通过改进对 FETAX（青蛙胚胎致畸试验 - 非洲爪蟾）毒理学数据的解释以及使用青蛙胚胎的类似发育毒性测试来进行风险评估。该项目将研究芳基碳氢化合物受体（AHR），这是一种介导二恶英等环境污染物毒性作用的蛋白质。我们将对二恶英毒性不敏感的青蛙的 AHR 的结构和功能与来自小鼠和人类等更敏感的动物的 AHR 进行比较。这项比较研究将帮助科学家了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测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 原始非毒理学功能的基本信息。