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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Environmental Health Sciences (NIEHS)
Type
Academic Research Enhancement Awards (AREA) (R15)
Project #
3R15ES011130-03S1
Application #
7902975
Study Section
Xenobiotic and Nutrient Disposition and Action Study Section (XNDA)
Program Officer
Humble, Michael C
Project Start
2009-08-15
Project End
2011-08-14
Budget Start
2009-08-15
Budget End
2011-08-14
Support Year
3
Fiscal Year
2009
Total Cost
$25,000
Indirect Cost
Name
Kenyon College
Department
Biology
Type
Schools of Arts and Sciences
DUNS #
063580369
City
Gambier
State
OH
Country
United States
Zip Code
43022
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Freeburg, Scott H; Engelbrecht, Eric; Powell, Wade H (2017) Subfunctionalization of Paralogous Aryl Hydrocarbon Receptors from the Frog Xenopus Laevis: Distinct Target Genes and Differential Responses to Specific Agonists in a Single Cell Type. Toxicol Sci 155:337-347
Shoots, Jenny; Fraccalvieri, Domenico; Franks, Diana G et al. (2015) An Aryl Hydrocarbon Receptor from the Salamander Ambystoma mexicanum Exhibits Low Sensitivity to 2,3,7,8-Tetrachlorodibenzo-p-dioxin. Environ Sci Technol 49:6993-7001
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Philips, Blythe H; Susman, Thomas C; Powell, Wade H (2006) Developmental differences in elimination of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) during Xenopus laevis development. Mar Environ Res 62 Suppl:S34-7
Lavine, Jeremy A; Rowatt, Ashley J; Klimova, Tatyana et al. (2005) Aryl hydrocarbon receptors in the frog Xenopus laevis: two AhR1 paralogs exhibit low affinity for 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Toxicol Sci 88:60-72
Rowatt, Ashley J; DePowell, John J; Powell, Wade H (2003) ARNT gene multiplicity in amphibians: characterization of ARNT2 from the frog Xenopus laevis. J Exp Zool B Mol Dev Evol 300:48-57