The goal of this proposal is to characterize the Ah receptor - a soluble protein present in many tissues of vertebrate species and a presumed member of the erbA superfamily of receptors, which mediates the pleiotropic effects of halogenated aromatic hydrocarbons such as 2,3,7,8- tetrachlorodibenzo-p-dioxin. 1) We propose to clone and sequence the gene for the Ah receptor from cDNA libraries by using degenerate oligonucleotide probes and rabbit polyclonal antibodies to a synthetic peptide that corresponds to the N-terminal sequence of the receptor. We plan to analyze the structure-function analysis of this protein using mutagenesis and an expression assay. 2) We propose to characterize the four allelic variants of the Ah receptor among inbred mouse strains: a) in vitro by their binding kinetics K-D, B-MAX and ease of activation and b) in vivo by genetic analysis of the differences in functional phenotypes especially Ah(b-1)/Ah(d) vs. Ah(b-2)/Ah(d) and correlation of this with quantitative nuclear extract-gel retardation experiments. 3) We propose to expand on our photoaffinity labeling and immunologic methodology; a) to screen lower vertebrate and invertebrate species for the presence of the Ah receptor, b) to prepare new monoclonal and polyclonal antibodies to various regions of the Ah receptor, c) to prepare an immunoaffinity column to aid in rapid purification, and d) to examine murine tissues for the cellular localization of the Ah receptor using immunohistochemistry.
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