TRANSFORMATION OF THE ARYL-HYDROCARBON RECEPTOR TO A DNA-BINDING FORMIS ACCOMPANIED BY RELEASE OF THE 90-KDA HEAT-SHOCK PROTEIN AND INCREASED AFFINITY FOR 2,3,7,8-TETRACHLORODIBENZO-P-DIOXIN

The binding of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) to the aryl hydrocarbon receptor (AhR) elicits a sequence of poorly defined molecu lar events that ultimately yield a heteromeric transformed AhR that is active as a transcription factor. We have previously developed a mode l of the ligand-initiated transformation of the AhR to the DNA-binding state based on characterization of several forms of the AhR with resp ect to their physicochemical properties and DNA-binding affinities. Th e present studies were designed to determine whether, and at what stag e, this process of transformation alters the receptor's affinity for T CDD. In rat hepatic cytosol, approx. 10% of the TCDD specifically boun d to the AhR rapidly dissociated (t1/2 approximately 1 h), while the r emainder was only slowly dissociable (t1/2 approximately 70 h). The is olated DNA-binding forms of the receptor (monomeric and transformed) b ound TCDD very tightly (t1/2 > 100 h), whereas TCDD was dissociable fr om the non-DNA-binding receptor form(s). A lower incubation temperatur e (0-4-degrees-C) and the presence of molybdate partially stabilized t he non-DNA-binding fraction of the TCDD.receptor complex and also enha nced TCDD dissociation in crude cytosol. Immunoprecipitation of the di fferent AhR forms with an anti-AhR antibody and immunoblotting with an tibody to the 90 kDa heat-shock protein (hsp90) demonstrated that hsp9 0 was associated with the unoccupied receptor complex as well as with a fraction of the non-DNA-binding TCDD- receptor complex; isolated DNA -binding forms did not contain detectable hsp90. We conclude that whil e hsp90 remains associated with the AhR, TCDD is readily dissociable; following release of hsp90, however, TCDD becomes very tightly bound, and remains so upon completion of transformation.