Dioxins, generated both commercially and naturally, are chlorinated polycyclic aromatic hydrocarbons that are highly toxic environmental contaminants. These agents are known to be potent rodent carcinogens and suspected human carcinogens. The best-known prototype of this group of agents is 3,4,7,8-tetrachlorodibenzo-p-dioxin (TCDD). It has been well documented that most, if not all, of the TCDD effects are mediated through the Ah receptor (AhR). Thus, in an effort to better understand the mechanism of dioxins action, I propose to investigate the molecular mechanism of the AhR signaling pathway using the overexpressed AhR and Arnt proteins. My working hypothesis is as follows: Upon ligand binding, the AhR undergoes conformational changes resulting in alteration of a number of AhR-protein interactions, which subsequently leads to a cascade of biologic events to occur. In addition to AhR-protein interactions, other interactions involving Arnt and other proteins also contribute to the action of dioxins mediated by the AhR. This proposal contains specific aims investigating the mechanism for protein factors involvement in the AhR signaling. Not only do these studies provide important information and reagents to further the study of the PAH action mediated by the AhR, but they also provide mechanistic insights on how the AhR regulates its target genes by understanding what other proteins are involved in the AhR signaling pathway.
Five specific aims have been proposed:
Aim 1) Identify the protein factors in rabbit reticulocyte lysate (RRL) and the Sf9 soluble extract which are responsible for the AhR/Arnt/DRE complex formation;
Aim 2) Identity interacting proteins of the AhR and Arnt and examine their effect on the AhR/Arnt/DRE complex formation;
Aim 3) Determine whether different forms of the AhR/Arnt heterodimer are present in cell lines which contribute to the different levels of ligand dependent transactivation;
Aim 4) Identify ARA9-interacting proteins which are responsible for the ARA9-mediated AhR/Arnt/DRE complex formation and Aim 5) Determine how CyP4O enhances the AhR/Arnt/DRE complex formation.
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