Abstract
The dioxin (aryl hydrocarbon) receptor is a ligand-dependent transcription factor that induces expression of a number of genes encoding drug metabolizing enzymes. In the absence of ligand the dioxin receptor is present in the cytoplasmic compartment of the cell associated with the molecular chaperone hsp90, which has been implicated in regulating the correct folding of the ligand binding domain of the receptor. In this study we have examined a potential role of the hsp90-associated p23 protein in the activation process of the dioxin receptor to a DNA binding form. In an in vitro model we show that addition of ligand alone to the dioxin receptor fails to induce release of hsp90 from the dioxin receptor. In the presence of ligand, this release was, however, induced upon addition of purified preparations of Arnt. Interestingly, p23 was also found to be associated with the nonactivated form of the dioxin receptor. Following fractionation on sucrose gradients p23 was dissociated from the receptor-hsp90 complex generating a receptor form, which showed ligand-independent release of hsp90 by Arnt and, consequently, ligand-independent activation of the DNA binding activity of the dioxin receptor. Ligand dependence was reconstituted in the presence of molybdate, a transition metal ion known to stabilize the interaction between the molecular chaperone hsp90 and p23. Taken together these experiments suggest a role of p23 in modulating ligand responsiveness in the activation process of the dioxin receptor.
Highlights
The bHLH-PAS dioxin1 receptor [1, 2] is a member of a growing family of transcription factors that includes, among others, a number of circadian rhythmicity regulatory proteins [3, 4] and proteins such as the hypoxia-inducible factor HIF-1␣ [5], its endothelial cell-specific homologue E-PAS/HLF [6, 7], and Arnt [8], which is a partner factor both of the dioxin receptor and of hypoxia-inducible factors
Consistent with the model that ligand alone is not sufficient to induce release of hsp90 but requires functional Arnt [29], ligand treatment did not induce any change in the sedimentation profile of the dioxin receptor from 9 S to the 5– 6 S region of the gradient, corresponding to the sedimentation position of hsp90-free receptor forms [13]
Given the loss of ligand dependence to induce DNA binding activity by the dioxin receptor, we examined the effect of Arnt on ligand binding activity by sucrose gradient-fractionated receptor preparations
Summary
The bHLH-PAS dioxin (aryl hydrocarbon) receptor [1, 2] is a member of a growing family of transcription factors that includes, among others, a number of circadian rhythmicity regulatory proteins [3, 4] and proteins such as the hypoxia-inducible factor HIF-1␣ [5], its endothelial cell-specific homologue E-PAS/HLF [6, 7], and Arnt [8], which is a partner factor both of the dioxin receptor and of hypoxia-inducible factors. The hepatitis virus X protein-associated protein (XAP-2) known as ARA 9 or AIP of 38 kDa has been shown to interact with the latent form of the dioxin receptor (19 –21) This protein has been reported to increase the transcriptional activity of the dioxin receptor, the mechanism of action has not been elucidated [22]. In the present study, fractionation of cellular extracts through sucrose density gradients yielded an hsp90-associated form of the dioxin receptor, which did not require ligand to generate the DNA binding complex with Arnt. This loss of ligand dependence correlated with dissociation of p23 from the dioxin receptor-hsp complex.
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