The Ah (aryl hydrocarbon) receptor (AhR) mediates the toxicity of 2,3,7,8- tetrachloro-dibenzo-p-dioxin (TCDD) by means of its ability to bind to DNA in a sequence-specific manner and modulate gene expression. For these events to occur, the ligand-bound protein (AhR) must interact with an additional protein, the """"""""AhR transforming"""""""" or ART protein. This interaction generates a heterodimer complex that specifically recognizes the DNA elements responsible for enhancing the expression of certain genes. The regulated inter-action of the AhR with ART protein may play a role in the species-and tissue-specific gene expression and toxicity observed in animals following TCDD exposure. By an understanding of these processes, we may ultimately begin to determine the genes affected in humans and the significance of environmental levels of TCDD to the health of both human and non-human animals. Further studies will focus on the properties of the ART protein and its ability to interact with the AhR to produce a complex that recognizes specific sequences of DNA. The role of metal ions in this interaction will be determined using a combination of chelating agents and assaying for the ability of the complex to bind to a [32p]-labeled dioxin- responsive element (DRE) located upstream of the structural gene for cytochrome P450IA1. Using this assay and a combination of precipitation, chromatography, and electrophoretic techniques, we will purify the ART protein. Monoclonal antibodies against the purified ART protein will be prepared and characterized. Fragments of the purified ART protein will be sequenced and the respective cDNA will be cloned. From the full-length DNA sequence,the amino acid sequence of the ART protein will be determined and compared to known proteins. Using the antibodies and cDNA probes developed, the subcellular localization, and tissue and developmental expression of the ART protein will be determined. The ability of known TCDD antagonists to inhibit formation of the heterodimer complex will be examined. These studies will be useful in determining the mechanism of these antagonists, and defining the TCDD-dependent conformational changes which occur in the AhR prior to its interaction with ART protein. The properties of the ART protein in different tissues and species will be examined and compared. Finally, the possibility that the ART protein may represent only one of a family of proteins having the ability to interact with the AhR in a TCDD-dependent manner to form a gene regulatory complex will be examined.
