DEFINITION OF A MINIMAL DOMAIN OF THE DIOXIN RECEPTOR THAT IS ASSOCIATED WITH HSP90 AND MAINTAINS WILD-TYPE LIGAND-BINDING AFFINITY AND SPECIFICITY

The dioxin receptor is a cytoplasmic basic helix-loop-helix/Per-Arnt-S im homology (bHLH/PAS) protein known to bind planar polycyclic ligands including polycyclic aromatic hydrocarbons, benzoflavones, heterocycl ic amines, and halogenated aromatic hydrocarbons, e.g. dioxins. Ligand -induced activation of the dioxin receptor initiates a process whereby the receptor is transformed into a nuclear transcription factor compl ex with a specific bHLH/PAS partner protein, Arnt. In analogy to the g lucocorticoid receptor, the latent dioxin receptor is found associated with the molecular chaperone hsp90. We have defined and isolated a mi nimal ligand binding domain of the dioxin receptor from the central PA S region, comprising of amino acids 230 to 421, and found this domain to interact with hsp90 in vitro. Expression of the minimal ligand bind ing domain in wheat germ lysates or bacteria, systems which harbor hsp 90 homologs unable to interact with the glucocorticoid or dioxin recep tors, resulted in non-ligand binding forms of this minimal 230 to 421 fragment. Importantly, affinity of the minimal ligand binding domain f or dioxin was similar to the affinity inherent in the full-length diox in receptor, and a profile of ligand structures which specifically bou nd the minimal ligand binding domain was found to be conserved between this domain and the native receptor. These experiments show that the minimal ligand binding domain maintains the quantitative and qualitati ve aspects of ligand binding exhibited by the full-length receptor, im plying that the central ligand binding pocket may exist to accommodate all classes of specific dioxin receptor ligands, and that this pocket is critically dependent upon hsp90 for its ligand binding conformatio n.