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-Sim homology (bHLH/PAS) protein known to bind planar polycyclic ligands including polycyclic aromatic hydrocarbons, benzoflavones, heterocyclic 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 complex with a specific bHLH/PAS partner protein, Arnt. In analogy to the glucocorticoid receptor, the latent dioxin receptor is found associated with the molecular chaperone hsp90. We have defined and isolated a minimal ligand binding domain of the dioxin receptor from the central PAS region, comprising of amino acids 230 to 421, and found this domain to interact with hsp90 in vitro. Expression of the minimal ligand binding domain in wheat germ lysates or bacteria, systems which harbor hsp90 homologs unable to interact with the glucocorticoid or dioxin receptors, resulted in non-ligand binding forms of this minimal 230 to 421 fragment. Importantly, affinity of the minimal ligand binding domain for dioxin was similar to the affinity inherent in the full-length dioxin receptor, and a profile of ligand structures which specifically bound 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 qualitative aspects of ligand binding exhibited by the full-length receptor, implying 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 conformation.
Coumailleau
P
P
Department of Medical Nutrition, Karolinska Institute, Huddinge University Hospital F-60, Sweden.
Poellinger
L
L
Gustafsson
J A
JA
Whitelaw
M L
ML
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D018841:HSP90 Heat-Shock Proteins / Q000378:metabolism*; D018257:Helix-Loop-Helix Motifs; D008024:Ligands; D051379:Mice; D008969:Molecular Sequence Data; D011817:Rabbits; D018336:Receptors, Aryl Hydrocarbon / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D012680:Sensitivity and Specificity
D018841:HSP90 Heat-Shock Proteins; D008024:Ligands; D018336:Receptors, Aryl Hydrocarbon; D011994:Recombinant Proteins