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. The human population is exposed to low levels of TCDD and related compounds, and 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 transcription factor. It is the investigator's underlying hypothesis that interspecies differences in toxicity result 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: 1) Purify and clone the p43 protein that is part of the unactivated Ah receptor complex. Determine its role in Ah receptor function. 2) The role of phosphorylation in AhR and ARNT function will be examined by first mapping the location of the phosphoamino acid residues, followed by examination of the functional significance of individual phosphoamino acids using site-directed mutagenesis and cell transfection techniques. 3) Identify and clone co-activator proteins that are capable of enhancing AhR/ARNT gene transcription. Determine whether these proteins are capable of enhancing/repressing AhR/ARNT-mediated gene transcription, and 4) Identify the AhR nuclear localization signal(s) and relate this information to other aspects of AhR activation and overall regulation. The Investigator will utilize a variety of techniques including the use of flag/AhR and flag/ARNT constructs, transients transfections techniques, radiolabeling of proteins in culture, a far-western blotting system, 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 the developmental-, tissue-, and species-specific differences in toxicity.
Showing the most recent 10 out of 52 publications
