The Ah receptor (AhR) mediates the toxicity of 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) by its ability to bind DNA and modulate gene expression. Long-term goals are to understand what controls the functional activity of the AhR, what target genes are affected in sensitive tissues, and how the modulated expression of these genes leads to the toxic responses observed. In addition, determining what the normal biological function of the AhR is, represents a closely coupled issue. These also remain important goals for assessing human sensitivity to environmental levels of this group of xenobiotics. The studies proposed here will focus on those factors that control the functional activity of the AhR. Certain compounds inhibit the formation of an active AhR. Further studies will characterize the mechanisms by which these and certain other antagonists act, define the chemical characteristics determining antagonist activity, examine the structure-activity relationships for these actions, and design and synthesize chemicals that may be more potent antagonists. Previous data suggests that for the AhR to bind to DNA, it must interact with an additional protein. This interaction generates a heterodimer complex that specifically recognizes DNA elements responsible for enhancing the expression of certain genes. However, additional recent data indicate that more complex relationships exist. Using a purified AhR DNA-binding complex and a protein biochemical techniques, experiments will examine the nature of the DNA-binding AhR forms in different species and tissues, characterize their interactions with DNA, and determine if the AhR interacts with other proteins in a ligand-dependent manner. Indirect evidence suggests changes in the phosphorylation state of the AhR may determine its activity. Utilizing responsive cells in culture and methodology developed to characterize different AhR forms, studies will determine if phosphorylation of the AhR and/or associated proteins occurs during transformation to a DNA-binding state, at what step this occurs, and if this phosphorylation regulates AhR activity. Initial data indicates that certain cytokines regulate the activity and/or expression of the AhR in isolated cells. Experiments are designed to determine the cytokine specificity of these effects, the mechanisms by which this regulation occurs, and the tissue and cell specificity of these mechanisms. Preliminary results show that the subcellular distribution of the AhR is tissue-specific. Immunohistochemical studies will be performed to determine if this is indeed the case and examine if this distribution is species and developmentally regulated. Additional studies will examine the mechanisms contributing to these differences. Finally, some studies suggest that TCDD treatment causes down-regulation of the estrogen receptor (ER). Using a combination of in vivo and in vitro techniques, studies will examine the effect of TCDD on ER protein and mRNA to determine if down-regulation is occurring. Utilizing molecular biological techniques, experiments will identify the DNA regulatory sequences in the ER gene that are required for this effect, and characterize the nuclear proteins binding to these sequences. Appropriate vectors and expression systems will be constructed to verify the functional significance, of these interactions.
