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The aryl hydrocarbon receptor (AHR) plays pleiotropic roles in the development and physiology of vertebrates in conjunction with xenobiotic and endogenous ligands. It is best known for mediating the toxic effects of dioxin-like pollutants such as 2,3,7,8-tetracholordibenzo-p-dioxin (TCDD). While most vertebrates possess at least one AHR that binds TCDD tightly, amphibian AHRs bind TCDD with very low affinity. Previous analyses of AHRs fromXenopus laevis(a frog; order Anura) andAmbystoma mexicanum(a salamander; order Urodela) identified three amino acid residues in the ligand-binding domain (LBD) that underlie low-affinity binding. InX. laevisAHR1β, these are A354, A370, and N325. Here we extend the analysis of amphibian AHRs to the caecilianGymnopis multiplicata, representing the remaining extant amphibian order, Apoda.G. multiplicataAHR groups with the monophyletic vertebrate AHR/AHR1 clade. The LBD includes all three signature residues of low TCDD affinity, and a structural homology model suggests that its architecture closely resembles those of other amphibians. In transactivation assays, the EC50 for reporter gene induction by TCDD was 17.17 nM, comparable toX. laevisAhR1β (26.23 nM) andAmbystomaAHR (34.09 nM) and dramatically higher than mouse AhR (0.13 nM), a trend generally reflected in direct measures of TCDD binding. These shared properties distinguish amphibian AHRs from the high-affinity proteins typical of both more ancient vertebrate groups (teleost fish) and those that appeared more recently (tetrapods). We suggest that AHRs with low TCDD affinity represent a basal characteristic that evolved in a common ancestor of all three extant amphibian groups.Research HighlightsA caecilian aryl hydrocarbon receptor exhibits low dioxin binding and sensitivity.The protein’s ligand-binding domain resembles frog and salamander AHRs in structure and function.AHR with low dioxin affinity likely evolved in a common ancestor of all three extant amphibian groups.Graphical Abstract

© 2018 by the authors. Licensee MDPI, Basel, Switzerland. The aryl hydrocarbon receptor (AhR) is a ligand-dependent transcription factor that modulates gene expression following its binding and activation by structurally diverse chemicals. Species differences in AhR functionality have been observed, with the mouse AhR (mAhR) and human AhR (hAhR) exhibiting significant differences in ligand binding, coactivator recruitment, gene expression and response. While the AhR agonist indirubin (IR) is a more potent activator of hAhR-dependent gene expression than the prototypical ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), it is a significantly less potent activator of the mAhR. DNA binding analysis confirmed the greater potency/efficacy of IR in stimulating transformation/DNA binding of the hAhR in vitro and domain-swapping experiments demonstrated that the enhanced response to IR was primarily due to the hAhR ligand binding domain (LBD). Site-directed mutagenesis and functional analysis studies revealed that mutation of H326 and A349 in the mAhR LBD to the corresponding residues in the hAhR LBD significantly increased the potency of IR. Since these mutations had no significant effect on ligand binding, these residues likely contribute to an enhanced efficiency of transformation/DNA binding by IR-bound hAhR. Molecular docking to mAhR LBD homology models further elucidated the different roles of the A375V mutation in TCDD and IR binding, as revealed by [3H]TCDD competitive binding results. These results demonstrate the differential binding of structurally diverse ligands within the LBD of a given AhR and confirm that amino acid differences within the LBD of AhRs contribute to significant species differences in ligand response.

Modern Molecular Modeling methods are at the core of mechanistic and computational toxicology, indeed these methods are able to analyze 3D molecular structures and their interactions in order to better understand the mechanism by which toxic compounds or drugs execute their action. Toxic effects of a molecule can be explained by the Adverse Outcome Pathway model (AOP); the molecular initiating event (MIE) can be the interaction of the molecule with a biological target, a protein for example. After the initial interaction many Key Events can link the MIE to the Adverse Outcome. It is useful to describe also the Key Event Relationship to assess the causal nature between two measurable biological events. Our focus of study is the Aryl hydrocarbon Receptor (AhR), a ligand-dependent transcription factor that responds to exogenous and endogenous chemicals with the induction of gene expression and production of diverse biological and toxic effects. Well known ligands of AhR are polychlorinated-dibenzodioxins (among them the 2,3,7,8-TCDD), dibenzofurans and biphenyls (PCB), and polycyclic aromatic hydrocarbons (PAH). The analysis of specific binding interactions of ligands within the AhR Ligand Binding Domain (LBD) would allow a deeper comprehension of the key molecular events regulating the mechanism of ligand-dependent and ligand-specific AhR activation. Aim of our current studies is to combine molecular modeling methods with experimental approaches to gain insights into the binding specificity of AhR ligands with different structures and properties