Distinct Roles of the Molecular Chaperone hsp90 in Modulating Dioxin Receptor Function via the Basic Helix-Loop-Helix and PAS Domains

Abstract

The intracellular dioxin receptor mediates signal transduction by dioxin and functions as a ligand-activated transcription factor. It contains a basic helix-loop-helix (bHLH) motif contiguous with a Per-Arnt-Sim (PAS) homology region. In extracts from nonstimulated cells the receptor is recovered in an inducible cytoplasmic form associated with the 90-kDa heat shock protein (hsp90), a molecular chaperone. We have reconstituted ligand-dependent activation of the receptor to a DNA-binding form by using the dioxin receptor and its bHLH-PAS partner factor Arnt expressed by in vitro translation in reticulocyte lysate. Deletion of the PAS domain of the receptor resulted in constitutive dimerization with Arnt. In contrast, this receptor mutant showed low levels of xenobiotic response element-binding activity, indicating that the PAS domain 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 observations, reticulocyte lysate but not wheat germ lysate promoted the association 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, surprisingly, the bHLH domain. Whereas ligand-binding activity correlated with association with hsp90, bHLH-hsp90 interaction appeared to be important for DNA-binding activity but not for dimerization of the receptor. Several distinct roles for hsp90 in modulating dioxin receptor function are therefore likely: correct folding of the ligand-binding domain, interference with Arnt heterodimerization, and folding of a DNA-binding conformation of the bHLH domain. Thus, the dioxin receptor system provides a complex and interesting model of the regulation of transcription factors by hsp90.The intracellular dioxin receptor (also termed the aryl hydrocarbon receptor) is a ubiquitous basic helix-loop-helix (bHLH) factor (4, 10) that mediates signal transduction by the toxic environmental contaminant dioxin (2,3,7,8-tetrachlorodibenzo-p-dioxin) (for recent reviews, see references 33, 42, and 47). In the absence of ligand the receptor exists in an inducible cytoplasmic form. Dioxin induces nuclear translocation of the receptor (reference 34 and references therein) and regulates dimerization with the bHLH partner factor Arnt, enabling both proteins to specifically recognize cognate response elements (xenobiotic response elements [XREs]) within regulated genes (9,24,38,46). Individually, neither the receptor nor Arnt shows any detectable affinity for this target sequence (23, 46). Moreover, they do not appear to bind the twofold symmetric CACGTG or CAGCTG E box motifs (23, 46) that are recognized by the great majority of bHLH and bHLH-leucine zipper (bHLH-Zip) proteins, including lymphoid transcription factors, the oncoproteins Myc and Max, and factors involved in vertebrate myogenesis and Drosophila neurogenesis (for recent r...

Hsp90 Is Required for Pheromone Signaling in Yeast

“…Thus, Hsp90 may have a dual role: it ensures that receptors are kept inactive in the absence of hormone and helps them to respond specifically and efficiently to ligand. This view is also corroborated by pharmacological in vivo experiments with geldanamycin (Whitesell et al, 1994), a compound that interferes with certain Hsp90 functions such as the proper maturation of steroid receptor-Hsp90 complexes (Smith et al, 1995;Whitesell and Cook, 1996;Bamberger et al, 1997;Czar et al, 1997;Segnitz and Gehring, 1997).There is ample evidence for a role of Hsp90 in regulating the activity of several other signaling pathways, such as the xenobiotic response mediated by the dioxin receptor (see for example Pongratz et al, 1992;Carver et al, 1994;McGuire et al, 1994;Antonsson et al, 1995;Coumailleau et al, 1995;Whitelaw et al, 1995). Interaction of the dioxin receptor with Hsp90 is essential for ligand binding and for acquiring a DNAbinding conformation.…”

“…There is ample evidence for a role of Hsp90 in regulating the activity of several other signaling pathways, such as the xenobiotic response mediated by the dioxin receptor (see for example Pongratz et al, 1992;Carver et al, 1994;McGuire et al, 1994;Antonsson et al, 1995;Coumailleau et al, 1995;Whitelaw et al, 1995). Interaction of the dioxin receptor with Hsp90 is essential for ligand binding and for acquiring a DNAbinding conformation.…”

Definition of a Dioxin Receptor Mutant That Is a Constitutive Activator of Transcription

“…DNA Binding Assay-DNA binding experiments were performed with in vitro translated, unlabeled Arnt together with the wild-type dioxin receptor or the dioxin receptor deletion mutants DR⌬LBD and DR⌬PASB as described previously (31). Briefly, equivalent concentrations of the indicated in vitro translated proteins were incubated with 10 l of in vitro translated Arnt in the presence or absence of 10 nM TCDD for 2 h at 25°C.…”

“…In control experiments, in vitro translation of DR⌬LBD in rabbit reticulocyte lysate resulted in a protein that had lost the ability to bind ligand, consistent with deletion of the minimal domain required for high affinity ligand binding of the dioxin receptor (data not shown). Furthermore, in a specific co-immunoprecipitation assay using monoclonal anti-hsp90 antibodies, this protein showed only low levels of interaction with hsp90 (data not shown) that were attributed to non-LBD interactions via the bHLH motif (31).…”

The Aryl Hydrocarbon Receptor Complex and the Control of Gene Expression

“…HSP90 and ARNT both interact with the AhR via the receptors HLH and PAS domains, thereby making their association with the AhR mutually exclusive. 74,75,79,142 Similar to a number of other HSP90 substrates, the AhR interacts with HSP90 through the middle portion of the chaperone. 81 Upon ligand activation, the human and mouse AhR translocate into the nucleus still associated with HSP90, demonstrating that the cytoplasmic AhR complex does not have to shed its chaperone machinery before nuclear uptake.…”