The Ah receptor plays a central role in the biological response to halogenated polycyclic aromatic hydrocarbons (HPAH), a major class of environmental contaminants, many of these components are known to be carcinogenic (e.g. dioxins). This receptor, upon binding a HPAH, is thought to translocate into the nucleus and induce the synthesis of enzymes involved in the metabolism of xenobiotics. The non-ligand binding protein composition of the Ah receptor complex has no been determined. Using 2-azido-3-(125I)7,8- dibromodibenzo-p-dioxin (a photoaffinity ligand), a monoclonal antibody to an Ah receptor, and cross-linking reagents, a series of experiments will be performed to determine the protein composition of the Ah receptor in the Hepa lcl cell line. Since phosphorylation is an important post-transnational modification involved in the regulation of receptor systems, and indirect evidence has suggested that the Ah receptor is phosphorylated, the studies outlined here will directly examine this possibility. Hepa 1 cells will be incubated in the presence of (32P) orthophosphate followed by the isolation of a cytosolic fraction. This preparation will be immunoprecipitated, subjected to two- dimensional gel electrophoresis, and the (32P)-labeled polypeptide pattern will be compared to photoaffinity-labeled control samples. In addition, studies will be performed to determine whether phosphorylation is required for Ah receptor ligand binding activity. The rate of Ah receptor turnover in Hepa 1 cells will be determined using a dense amino acid labeling method. The influence of ligand occupation on the rate of receptor turnover will also be determined. A number of compounds have been found to alter in vivo Ah receptor activity levels, studies will be performed to examine the ability of certain exogenous compounds to influence receptor levels in cell culture. Any differences seen can then be further examined by turnover experiments to determine if the rate of receptor synthesis has changed. The experiments proposed here will enable a better understanding of possible mechanisms of Ah receptor regulation.
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