Exposure to 2,3,7,8,-tetrachlorodibenzo-p-dioxin (TCDD, dioxin) and related compounds results in a wide variety of species- and tissue- specific toxic and biological effects, many of which appear to be mediated by the Ah receptor (AhR), an intracellular protein to which these chemicals bind with high affinity. The overall goal of our research is to understand the molecular mechanisms by which the AhR complex modulates dioxin action. The AhR is a ligand-dependent transcription factor that regulates gene expression via interaction with a specific DNA enhancer element, the dioxin responsive element (DRE), adjacent to TCDD- responsive genes. TCDD:AhR:DRB complex formation leads to activation of gene expression and is dependent upon at least one additional protein, the Ah receptor nuclear translocator (ARNT). Cloning of the AhR and ARNT genes has revealed that each contains domains are involved in protein dimerization and DNA binding. Recently, we demonstrated the existence of multiple heteromeric DNA binding forms of the AhR complex containing at least three distinct protein subunits. Given that the functional activity of protein transactivators is known to be dependent upon the specific protein(s) to which it is associated, we hypothesize that differences in AhR- and AhR complex-associated proteins could, result in an alteration of the functionality of the AhR complex which would therefore contribute to species- and tissue-specific differences in toxicity and gene expression. Here we propose to characterize the multiple DNA binding forms of the AhR complex as well as to isolate and characterize AhR- associated proteins. Utilizing AhR and ARNT antibodies we will determine the identity of the multiple proteins AhR complex subunits which bind (and can be crosslinked) to the DRE in a TCDD-dependent manner. Multiple DNA binding forms of the AhR complex will be isolated from guinea pig liver by a combination of conventional and magnetic DNA affinity binding techniques. The functional activity of each complex will he assessed by the ability of each to stimulate transcription in vitro and the DNA binding of each complex will be examined using gel shift, UV- crosslinking, DNA mutagenesis and DNA footprinting. In a complimentary approach, AhR-associated proteins will be isolated from mouse, guinea pig and human cell lines stably transfected with a recombinant AhR fusion protein expression vector. The presence of an affinity tag on the recombinant proteins will allow rapid and selective isolation of the recombinant AhR and its associated proteins. The biochemical and functional properties of the isolated complexes and their protein components will be examined in reconstitution experiments and identity of the associated proteins determined by sequence analysis. cDNA clones encoding AhR-associated proteins will be isolated from cDNA libraries using both the yeast two-hybrid system as well as probes derived from the amino acid sequence of purified proteins. The results of our studies will not only increase our understanding of the molecular mechanisms of dioxin action and provide insight into the diversity in TCDD-responsiveness in humans and animals but they will contribute to our knowledge of the inducible regulation of mammalian gene expression.
| Phelan, D M; Brackney, W R; Denison, M S (1998) The Ah receptor can bind ligand in the absence of receptor-associated heat-shock protein 90. Arch Biochem Biophys 353:47-54 |
