Analysis of the Hsp90-aryl hydrocarbon receptor interactions

The aryl hydrocarbon receptor (AhR) mediates toxic and biological effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and related compounds. Ligand binding converts AhR into its high affinity DNA binding form in a process called transformation. In this process, AhR dissociated from heat shock protein 90 (Hsp90) to which it is bound in the inactive state and dimerizes with a homologous nuclear protein Arnt. This study characterized a hypothetical mechanism of AhR transformation, in which ligand binding in the ligand binding PASB domain of the AhR results in exposure of dimerization interface and initiation of dimerization with Arnt followed by displacement of Hsp90 from the AhR complex. These results suggest masking by Hsp90 of dimerization domains in the inactive AhR complex and ligand-dependent release of such inhibition. Consistent with this inhibitory role of Hsp90 in the AhR transformation, the relative strength of Hsp90 association with mouse AhR was found to affect the maximal level of AhR transformation and explained the observed limited level of transformation efficiency in the mouse AhR compared to other species. In addition, the transformation process was controlled kinetically by dimerization with Arnt, suggesting a unique kinetic mechanism of AhR transformation. Moreover, due to their direct effect on limited AhR transformation efficiency, Hsp90 and Arnt appear as possible targets to study interspecies differences in responsiveness to TCDD and related compounds. The proposed transformation mechanism was further utilized to develop a novel approach to analyze Hsp90 binding to the AhR PASB domain. It was found that although the PASB region encompassing amino acids 288-327 contained Hsp90 binding determinants, Hsp90 binding was impaired with deletion of other PASB regions in the context of the full length AhR. Intricate relationship was established between such loss and constitutive functional activation in AhR variants, suggesting active involvement of Hsp90 binding in AhR transformation and a dynamic nature of this binding. These results will contribute to better understanding of the ligand-driven AhR transformation and its linkage to the biological and toxic mechanisms of TCDD and related compounds.