DISTINCT ROLES OF THE MOLECULAR CHAPERONE HSP90 IN MODULATING DIOXIN RECEPTOR FUNCTION VIA THE BASIC HELIX-LOOP-HELIX AND PAS DOMAINS

The intracellular dioxin receptor mediates signal transduction by diox in and functions as a ligand-activated transcription factor. It contai ns a basic helix-loop-helix (bHLH) motif contiguous with a Per-Amt-Sim (PAS) homology region. In extracts from nonstimulated cells the recep tor is recovered in an inducible cytoplasmic form associated with the 90-kDa heat shock protein (hsp90), a molecular chaperone, We have reco nstituted ligand dependent activation of the receptor to a DNA-binding form by using the dioxin receptor and its bHLH-PAS partner factor Amt expressed by in vitro translation in reticulocyte lysate. Deletion of the PAS domain of the receptor resulted in constitutive dimerization with Amt. In contrast, this receptor mutant showed low levels of xenob iotic response element-binding activity, indicating that the PAS domai n may be important for DNA-binding affinity and/or specificity of the receptor. It was not possible to reconstitute dioxin receptor function with proteins expressed in wheat germ lysate. In line with these obse rvations, reticulocyte lysate but not wheat germ lysate promoted the a ssociation of de novo synthesized dioxin receptor with hsp90. At least two distinct domains of the receptor mediated interaction with hsp90: the ligand-binding domain located within the PAS region and, surprisi ngly the bHLH domain. Whereas ligand-binding activity correlated ,vith association with hsp90, bHLH-hsp90 interaction appeared to be importa nt for DNA binding activity but not for dimerization of the receptor. Several distinct roles for hsp90 in modulating dioxin receptor functio n are therefore likely: correct folding of the ligand-binding domain, interference with Amt heterodimerization, and folding of a DNA-binding conformation of the bHLH domain. Thus, the dioxin receptor system pro vides a complex and interesting model of the regulation of transcripti on factors by hsp90.