AH RECEPTOR ANATOMY--IMPLICATIONS FOR DIOXIN TOXICITY

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Inconclusive epidemiological studies on the risks of environmental contaminants, exemplified by the human health concerns over 2,3,7,8- tetrachlorodibenzo-p-dioxin (TCDD, dioxin), emphasizes the need for a mechanistic approach to these toxicological issues. Most, if not all the biological effects of TCDD are mediated through the aromatic hydrocarbon (Ah) receptor. This research proposal centers on the premise that a mechanistic understanding of TCDD toxicity, is contingent upon identifying and characterizing all the components involved in Ah receptor function. Following binding by TCDD, the Ah receptor binds to specific DNA enhancer elements to regulate gene expression. Target genes include those encoding drug metabolizing enzymes and growth factors important in normal cell growth and differentiation. The Ah receptor-DNA interaction involves the Ah receptor nuclear translocator (Arnt) protein, a DNA-binding partner. We previously identified a 110 kDa as part of the Ah receptor DNA-binding complex. Here, our preliminary research identifies a distinct 110 kDa DNA- binding protein that copurifies with both the rat Ah receptor and Arnt protein. Peptide sequencing of 110 kDa protein fragments reveals no homology to known proteins. Our data suggests that the 110 kDa protein represents an additional component of the Ah receptor DNA-binding complex. Functional, genetic and biochemical evidence from others provides further support for our hypothesis that Ah receptor DNA binding and function involves association with factors other than the Arnt protein. Therefore, this proposal seeks to define the role of the 110 kDa protein in Ah receptor DNA-binding and function, in the context of our long range objective of understanding TCDD-induced intracellular signaling. We propose four specific aims to test the hypothesis: 1. cDNA cloning the rat 110 kDa protein. cDNA clones will be isolated as a first step towards learning the function of the 110 kDa protein. Amino acid microsequencing on several peptide fragments from the purified 110 kDa protein permits design of degenerate oligonucleotides intended for use in either of two cDNA cloning strategies. 2. The isolation of cDNA clones for the human homolog of the rat 110 kDa protein. In order to study the role of the 110 kDa protein in human Ah receptor function, cDNA clones for the human protein will be isolated using the rat clones as probes, and used in expression studies. 3. Studies using the yeast two-hybrid system. The two-hybrid system represents a third alternative for isolating 110 kDa protein clones, that is distinct from the two strategies using the degenerate oligonucleotides. The two-hybrid system also permits an in vivo examination of functionally important protein-protein interactions between the 110 kDa protein, Ah receptor and Arnt protein. 4. Expression studies to examine the role of the 110 kDa protein in Ah receptor DNA binding and function. Studies will focus on identifying the important protein domains i the 110 kDa protein and their impact on Ah receptor and Arnt protein DNA binding and function.