SUBUNIT COMPOSITION OF THE HETEROMERIC CYTOSOLIC ARYL-HYDROCARBON RECEPTOR COMPLEX

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