A cellular factor stimulates ligand-dependent release of hsp90 from the basic helix-loop-helix dioxin receptor.

Abstract

In response to dioxin, the nuclear basic helix-loop-helix (bHLH) dioxin receptor forms a complex with the bHLH partner factor Arnt that regulates target gene transcription by binding to dioxin-responsive sequence motifs. Previously, we have demonstrated that the latent form of dioxin receptor present in extracts from untreated cells is stably associated with molecular chaperone protein hsp9O, and Arnt is not a component of this complex. Here, we used a coimmunoprecipitation assay to demonstrate that the in vitro-translated dioxin receptor, but not Arnt, is stably associated with hsp9O. Although it showed ligand-binding activity, the in vitro-translated dioxin receptor failed to dissociate from hsp9O upon exposure to ligand. Addition of a specific fraction from wild-type hepatoma cells, however, to the in vitro-expressed receptor promoted dioxin-dependent release of hsp9O. This stimulatory elfect was mediated via the bHLH dimerization and DNA-binding motif of the receptor. Moreover, ligand-dependent release of hsp9O from the receptor was not promoted by fractionated cytosolic extracts from mutant hepatoma cells which are deficient in the function of bHLH dioxin receptor partner factor Arnt. Thus, our results provide a novel model for regulation of bHLH factor activity and suggest that derepression of the dioxin receptor by ligand-induced release of hsp9O may require bHLH-mediated concomitant recruitment of an additional cellular factor, possibly the structurally related bHLH dimerization partner factor Arnt. In support of this model, addition of in vitro-expressed wild-type Arnt, but not a mutated form of Arnt lacking the bHLH motif, promoted release of hsp9O from the dioxin receptor in the presence of dioxin.The nuclear dioxin receptor (also termed the aryl hydrocarbon receptor) mediates signal transduction by dioxin (2,3,7,8-tetrachlorodibenzo-p-dioxin). Like other nuclear receptors, the dioxin receptor is a ligand-inducible transcriptional regulator that directly binds to cognate response elements within regulated genes (for a recent review, see reference 37). In contrast to members of the steroid receptor superfamily, however, the dioxin receptor harbors a basic helix-loop-helix (bHLH) motif (5, 17) that represents a dimerization and DNA-binding surface (18) of a broad class of gene regulatory proteins including Myc, its positive and negative regulators Max and Mad (1 and references therein), lymphoid transcription factors, and muscle-differentiating factors such as MyoD and myogenin (for a recent review, see reference 27).Dioxin and other receptor ligands (i.e., structurally related environmental pollutants) strictly regulate dioxin receptor function by modulating its DNA-binding activity in vivo (14,19,24,32) and in vitro (6). A physiological ligand, if any, of the dioxin receptor has not been identified (for a review, see reference 37). Although the detailed mechanism of ligandinduced activation of the dioxin receptor into a functional form remains unclear, this process involves several distinct steps. Mos...

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.…”

Host cell factor requirement for hepatitis C virus enzyme maturation

“…The monoclonal IgM antibodies, 3G3 (anti-HSP90, Affinity Bioreagents) and TEPC-183 (control, Sigma), have been described for use in immunoprecipitation of HSP90 (19). Luciferase RNA was obtained from Promega.…”

Trypanosoma bruceimetabolite indolepyruvate decreases HIF-1α and glycolysis in macrophages as a mechanism of innate immune evasion

“…Several studies suggest tryptophan catabolites, including breakdown products of indolepyruvate, act as endogenous ligands for the aryl hydrocarbon receptor (AhR), a ligand-activated transcription factor (35,36). AhR was of particular interest because AhR dimerizes with aryl hydrocarbon receptor nuclear translocator (ARNT) (36,37), the binding partner of HIF-1α; thus, an increase in AhR activation could cause a decrease in HIF-1α-ARNT complexes, reducing the production of HIF-1α-dependent target genes. Indolepyruvate was still able to inhibit LPS-induced pro-IL-1β production in BMDM from AhR knockout mice (Fig.…”