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) seen in rodents. The human population is constantly exposed to low levels of TCDD, and related compounds, the actual long term health effects 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 transcriptional factor. The multiple mechanisms of AhR regulation will be examined along with elucidation of the biochemical events from initial synthesis and assembly to translocation into the nucleus. Using an in vitro translation system the 90 kDa heat shock protein (hsp90) binding site on the AhR will be determined using a series of truncation and deletion cDNA mutants. The location of the binding site(s) will be compared with the DNA binding, dimerization, ligand binding, and transactivation domains to gain insight into the functional significance of this protein/protein interaction. Synthetic peptides will be used in an in vitro reconstitution system to precisely define the actual site of interaction The protein composition of the 9S cytosolic form of the AhR in Hepa 1 cells will be determined by immunoprecipitation with antibodies raised against specific AhR peptide sequences. Both chemically crosslinked and non-crosslinked AhR will be characterized on western blots using antibodies to the AhR, Arnt, and hsp90. The intracellular location of the unliganded and liganded. AhR in Hepa 1 cells will be determined using immunocytochemical techniques and one- and two-dimensional confocal microscopy. The protein and cofactor requirements (e.g. ATP, hsp70, Arnt) for nuclear localization/translocation will be determined using a permeabilized cell system. The role of phosphorylation in AhR 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. Another aspect of regulating the level of toxicity caused by exposure to a given amount of TCDD is how each cell type maintains a specific AhR concentration. The role of the cellular protease m-calpain in AhR turnover and ligand dependent down regulation will be examined by exposing Hep G2 cells to a calpain or calpastatin antisense oligonucleotides and determining their effect on AhR levels, both in the presence and absence of TCDD. Collectively, these studies will develop an understanding of the pathway of AhR action and the various points of regulation.
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