The Ah receptor (AhR) is a ligand-dependent transcription factor known to regulate the toxic and biological effects of a variety of exogenous chemicals and these effects result from AhR-dependent gene expression. The AhR is also involved in endogenous developmental and physiological processes, although the responsible endogenous ligand(s) is unknown. While toxic halogenated aromatic hydrocarbons are the prototypical and highest affinity ligands, the AhR can bind and be activated by a diverse range of structurally dissimilar compounds and these ligands can produce distinctly different AhR-dependent responses, both in magnitude and specificity of gene induction. Site-directed mutagenesis and functional analysis studies based on our homology model of the AhR ligand binding domain (LBD) revealed significant differences in the interactions of these diverse ligands with residues within the AhR LBD, providing new insights into how ligands can activate the AhR. It is currently assumed that the diversity in AhR-dependent gene expression response of diverse ligands results from ligand-specific differences in structure and function of the AhR and/or ARNT that leads to differential coactivator recuitment, but this has not been demonstrated. While the classical AhR mechanism involves AhR dimerization with ARNT and binding of this complex to DNA to regulate gene expression, the AhR can also heterodimerize with other proteins (KLF6 and RelB), bind to distinctly different DNA sequences and regulate other genes. Thus, a given ligand can differentially regulate AhR-dependent gene expression in a given cell or tissue by multiple mechanisms. We hypothesize that the structure of the AhR complex can be altered in a ligand-selective manner, resulting in distinct differences in AhR functionality when bound by structurally diverse ligands and this can contribute to the diversity in AhR response. We propose to examine whether differential AhR binding and activation by structurally diverse ligands alters the structure and function of the AhR and/or its heterodimeric partners (ARNT, KLF6 or RelB), as measured by ligand and DNA binding, limited proteolysis and gene expression analysis and interpreted by Molecular Docking. Ligand-selective differences in AhR heterodimer-specific coactivator recruitment to the CYP1A1, PAI2 or IL8 gene promoters in human and mouse hepatoma cells will be determined using chromatin immunoprecipitation and will link ligand- induced alterations in receptor structure to changes in functional analysis in intact cells. The newly developed homology model of the AhR:ARNT bHLH-PASA-PASB dimer will provide an avenue to examine both the molecular mechanisms involved in ligand-dependent transformation/AhR:ARNT dimerization as well as the effect of diverse ligands on this process by Molecular Dynamics methods. The studies proposed here will provide detailed analysis of the molecular mechanisms by which structurally diverse ligands can differentially affect the AhR and its associated factors and will yield insights into the mechanisms of ligand-dependent AhR transformation, the influence of ligand structure on these processes and the diversity of AhR responsiveness.
Little is known about the molecular details and mechanisms by which structurally diverse exogenous and endogenous chemicals can to bind to and activate/inhibit the Ah (dioxin) receptor (AhR), a chemical-responsive cellular protein responsible for mediating many of the toxic, biological, developmental and reproductive effects produced by these substances. The work proposed in this application will increase our understanding of the structure of the AhR and the basic molecular mechanisms by which structually diverse chemicals can activate or inhibit the function of the AhR and its interaction with alternate dimerization partners. It will also lead to the identification and development of AhR activators and inhibitors with therapeutic potential (i.e. as anticancer agents and immune modulators) and provide new avenues in which to gain insights into the normal physiological role(s) of this poorly understood receptor.
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