Subunit composition of the heteromeric cytosolic aryl hydrocarbon receptor complex

In a previous cross-linking study we have shown that the cytosolic aryl hydrocarbon receptor (AhR) complex has a heterotetrameric structure (Perdew, G. H. (1992) Biochem. Biophys. Res. Commun. 182, 55-62). In this report, both cross-linked and [35S]methionine-labeled Hepa 1c1c7 cytosol were used to characterize the subunit composition of the AhR complex by immunoprecipitation with an AhR polyclonal antibody followed by immunochemical analysis using antibodies against the AhR and 90-kDa heat shock protein (hsp90). Results indicated that the four subunits found in cross-linking experiments were composed of three species: the AhR ligand binding subunit, hsp90, and an unknown 43-kDa protein. The stoichiometry of hsp90 present in each AhR complex was determined in two separate experiments: 1) from cross-linking experiments, stoichiometry was determined by quantitative immunoblotting with anti-AhR and anti-hsp90 antibodies followed by quantitation with 125I-counterantibody on protein blots; 2) using 35S-labeled Hepa 1 cytosol, the hsp90/AhR stoichiometry was determined by immunopurifying receptor complexes, and the amount of 35S-labeled AhR and hsp90 was assessed. The stoichiometry values obtained were 2.4 and 1.72 mol of hsp90/mol of AhR using each experimental approach, respectively.