CHARACTERIZATION OF MULTIPLE FORMS OF THE AH RECEPTOR - RECOGNITION OF A DIOXIN-RESPONSIVE ENHANCER INVOLVES HETEROMER FORMATION

We have employed a combination of gel retardation, protein-DNA cross-linking, and protein-protein cross-linking techniques to further examine the 2,3,7,8-tetrachlorodibenzo-p-dioxin- (TCDD-) dependent changes in the Ah receptor that result in a DNA-binding conformation. Gel retardation analysis of DNA-Sepharose chromatographic fractions of rat hepatic cytosol indicated that TCDD-dependent and sequence-specific DNA binding coeluted with a 200-kDa form of the Ah receptor (peak 2) previously characterized as being multimeric and having high affinity for calf thymus DNA. The TCDD-bound, 100-kDa form of the receptor (peak 1) bound weakly to the DNA recognition motif. These results indicated that the DNA-binding form of the Ah receptor is a multimer. SDS-polyacrylamide gel electrophoresis of peak 2 cross-linked to a bromodeoxyuridine-substituted DNA recognition motif indicated that this form of the receptor present in rat hepatic cytosol is composed of at least two DNA-binding proteins of approximately 100 and 110 kDa. Using the chemical cross-linking agent dimethyl pimelimidate, we further established that the 100-kDa form of the receptor (peak 1) associates with a different protein to generate the receptor form (peak 2) that binds to the dioxin-responsive enhancer. Photoaffinity-labeling studies indicated that only the 100-kDa protein (peak 1), and not the 110-kDa protein, binds ligand. Together, these observations imply that the DNA-binding form of the Ah receptor exists as a heteromer.