The Ah-receptor (AHR) is a ligand activated transcription factor that belongs to the family of basic-helix-loop-helix/PER-ARNT-SIM (bHLH/PAS) proteins. AHR-mediated signaling has been extensively investigated in the C57BL/6J mouse model system in an attempt to define the components of the pathway, understand protein and DNA interactions and define specific changes in gene expression that likely impact human health. There has been less emphasis placed on the degradation of AHR protein especially as it relates turning the AHR signaling pathway off. Unfortunately, the proteins that modulate the degradation of the AHR and the domains of the AHR that are necessary for degradation remain to be identified. Thus, multifaceted approaches must be initiated to define candidate target enzymes involved in the degradation process and obtain """"""""gain of function"""""""" mutants of the AHR. Therefore, the central hypothesis of this proposal is that defects in protein degradation will be manifest as """"""""gain of function"""""""" mutants of the AHR. Three sets of studies are proposed to address this hypothesis and isolate the first ligand- activated gain of function mutants in the AHR pathway. First, studies will be carried out that utilize a bacterial two-hybrid screen to identify proteins interacting with the COOH-terminal domain of the AHR. Second, cell lines expressing a stably integrated GFP tagged AHR will be used to carry out a genetic screen for cells that do not degrade the AHR. Finally, yeast strains will be generated that express a functional AHR signaling pathway. Specific genes involved in protein degradation will be deleted from these strains to assess their role in AHR degradation. Thus, the proposed experiments utilize candidate gene approaches as well as genetic screens and employ mammalian cells, yeast and bacteria to address this hypothesis. The mechanism involved in turning off AHR-mediated signaling and the regulation of this pathway are especially critical with respect to halogenated aromatic compounds that are not readily metabolized or cleared from an organism. In addition, since modeling of AHR signaling and risk assessment in humans relies heavily on understanding the complete signaling pathway, it is essential to determine how the AHR pathway is attenuated. Statement of Relevance: Human health can be adversely affected by exposure to environmental contaminants. For chemicals that act through association with endogenous proteins and modulation of gene regulation, it is essential to understand how the pathways are regulated. Understanding how the Ah receptor protein is degraded is critical to risk assessment of chemicals typified by 2,3,7,8, tetrachlorodibezo-p-dioxin (Dioxin), and will be applicable to other toxicologically relevant receptors (i.e. steroid hormone receptors). ? ? ? ? ?
