Mechanism of conditional regulation of the bHLH/PAS dioxin receptor

Abstract

The intracellular dioxin receptor is a bHLH/PAS protein which mediates signal transduction by dioxin and upon heterodimerisation with the bHLH/PAS partner factor Arnt, functions as a ligand-activated transcription factor by binding to specific xenobiotic response elements in promoters of target genes. Although the physiological function of the dioxin receptor and the nature of a possible physiological ligand are presently not known, targeted disruption of the mouse dioxin receptor gene has indicated that the receptor may play an important role in hepatic development. In the absence of ligand, the dioxin receptor is recovered in a latent, non-DNA binding heteromeric complex with the molecular chaperone, heat shock protein hsp90.

Conversion of the receptor to a high affinity DNA binding species is a multi-step process involving ligand binding, release of hsp90 and dimerisation with Arnt. Using a specific co-immunoprecipitation assay, we have demonstrated that in vitro expressed dioxin receptor but not Arnt, is stably associated with hsp90. Surprisingly, however, the receptor-hsp90 interaction appeared to be very stable as exposure to ligand failed to dissociate hsp90 from the receptor. Using specific fractions from extracts of the dioxin responsive Hepa lclc7 cell line, we have identified a stimulatory factor that facilitates the bHLH mediated ligand-dependent dissociation of hsp90, which appears to represent the bHLH/PAS partner factor Arnt. In analogy to the dioxin receptor, we have also demonstrated that the distinct Drosophila neurodevelopmental bHLH/PAS factor Sim, also forms a stable association with hsp90. Furthermore, dimerisation with Arnt very efficiently disrupted the hsp90-Sim association.

To investigate the role of hsp90 in dioxin receptor signalling, we have studied receptor function in a yeast strain in which the levels of hsp90 can be reduced to ~5% of the wild type level. In this way, we have demonstrated that low levels of hsp90 result in severe impairment of dioxin receptor responsiveness, thus suggesting that hsp90 is a critical determinant of the conditional regulation of the dioxin receptor in vivo. Finally, based on our initial observation that dimerisation with Arnt facilitates the ligand dependent release of hsp90, we have demonstrated that an overexpression of Arnt promotes derepression of dioxin receptor function in the absence of ligand thereby representing an alternative mechanism of activation that is distinct from activation by xenobiotic ligands and thus may be of physiological importance. In conclusion, these studies demonstrate a critical role for hsp90 in determining ligand responsiveness of the dioxin receptor and the importance of Arnt for inducing release of hsp90 and derepression of dioxin receptor function.