The Ah receptor (AhR) has been shown to be largely responsible for the toxic and tumor promotional properties of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), especially, in rodents. Although the human population is exposed to low levels of TCDD, and related compounds, the actual long term health effect(s) remain to be elucidated. Little is known about the biochemical processes involved in the activation and regulation of this ligand-activated helix-loop helix/basic region transcriptional factor. It is our underlying hypothesis that interspecies differences in toxicity results from differences in the biochemical and transcriptional regulatory pathways for the AhR and Ah receptor nuclear translocator protein (ARNT). In this application the multiple mechanisms of AhR regulation will be examined including the following aims; la) Determine the role of XAP2 in the Ali receptor complex, 1b) Complete the mapping of the phosphorylation sites on XAP2 and determine their possible functional role in regulating AhR activity and levels, 2) Complete mapping of the human AhR and ARNT phosphoamino acid sites and determine their possible role in regulating function, 3a) Determine which domains or subdomains of the Ah receptor transactivation domain are key to the transactivation potential of the AhR and what type of coactivators are recruited to this domain, 3b) Identify and clone unique coactivator proteins that are capable of enhancing or repressing AhR/ARNT gene transcription. We will utilize a variety of techniques, including; AhR/FLAG, XAP2/FLAG, and ARNT/FLAG constructs, transient transfection techniques, radiolabeling of proteins in culture, phosphopeptide mapping, site-directed mutagenesis, and various PCR and library screening/cloning techniques. Collectively, these studies will develop an understanding of the pathway of AhR action and the various points of regulation. This information can then be used to explore developmental-, tissue-, and species-specific differences in toxicity.
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