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

Abstract

The intracellular dioxin (aryl hydrocarbon) receptor is a ligand-activated transcription factor that mediates the adaptive and toxic responses to environmental pollutants such as 2,3,7,8-tetrachlorodibenzo-p-dioxin and structurally related congeners. Whereas the ligand-free receptor is characterized by its association with the molecular chaperone hsp90, exposure to ligand initiates a multistep activation process involving nuclear translocation, dissociation from the hsp90 complex, and dimerization with its partner protein Arnt. In this study, we have characterized a dioxin receptor deletion mutant lacking the minimal ligand-binding domain of the receptor. This mutant did not bind ligand and localized constitutively to the nucleus. However, this protein was functionally inert since it failed to dimerize with Arnt and to bind DNA. In contrast, a dioxin receptor deletion mutant lacking the minimal PAS B motif but maintaining the N-terminal half of the ligand-binding domain showed constitutive dimerization with Arnt, bound DNA, and activated transcription in a ligand-independent manner. Interestingly, this mutant showed a more potent functional activity than the dioxin-activated wild-type receptor in several different cell lines. In conclusion, the constitutively active dioxin receptor may provide an important mechanistic tool to investigate receptor-mediated regulatory pathways in closer detail.The intracellular dioxin receptor also known as the aryl hydrocarbon receptor, is a ligand-dependent transcription factor that mediates the biological effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 1 commonly known as dioxin (1). Although disruption of the dioxin receptor gene in mice has not yielded conclusive results, it remains a possible scenario that physiological mechanisms may exist for activation of receptor function, e.g. during critical stages of vertebrate development (2-5). However, an endogenous ligand for the receptor has not yet been identified, suggesting that alternative pathways for receptor activation may exist.In the absence of ligand, the dioxin receptor is generally found in the cytoplasm associated in high molecular weight complexes comprising a dimer of the molecular chaperone hsp90, an immunophilin homolog known as XAP2 (hepatitis B virus X-associated protein-2)/AIP (aryl hydrocarbon receptorinteracting protein)/ARA9 (aryl hydrocarbon receptor-associated protein-9), and the co-chaperone p23 (6 -11). In the presence of dioxin, the receptor is converted to a functional DNAbinding species in a multistep process involving nuclear translocation, dissociation from the hsp90 complex, and dimerization with its partner protein Arnt (Ah receptor nuclear translocator). Formation of the dioxin receptor/Arnt heterodimer is a prerequisite for DNA binding and promotes transcription of target genes including those encoding xenobioticmetabolizing enzymes such as CYP1A1 (1). Both the dioxin receptor and Arnt are members of a distinct subclass of the basic helix-loop-helix (bHLH) family of transcriptional regul...

Constitutively Active Aryl Hydrocarbon Receptor Expressed Specifically in T-Lineage Cells Causes Thymus Involution and Suppresses the Immunization-Induced Increase in Splenocytes

“…The AhR mutant constitutively localizes to the nucleus, heterodimerizes with ARNT, and activates transcription by binding XRE sequences in a ligand-independent manner (29,30). The results of the present study demonstrate that AhR activation in T-lineage cells alone directly induces the thymocyte changes.…”

Role of the Per/Arnt/Sim Domains in Ligand-dependent Transformation of the Aryl Hydrocarbon Receptor

“…1b)). The observed deficient Hsp90 binding to the AhR⌬PASB likely explains the previously observed constitutive activity of this construct in AhR transformation and nuclear translocation (17).…”

“…Deletion of the ligand binding PASB domain (amino acids 287-422) generates an active (ligand-independent) AhR that constitutively dimerizes with Arnt, binds to DNA, is capable of nuclear translocation, and activates gene transcription (17) despite the fact that it retains one Hsp90 binding site (in the bHLH domain). These observations raise the question whether the Hsp90-mediated inhibitory effect on AhR transformation is binding site dependent (e.g.…”

Hepatic Aryl Hydrocarbon Receptor Attenuates Fibroblast Growth Factor 21 Expression

“…3 In a related, separate study involving mice that express a constitutively active form of AHR in the liver, investigators also observed increased hepatic Fgf21 expression (13). However, this constitutively active form of AHR was previously determined to exhibit AHR activity similar to high-dose TCDD exposure (24). Data obtained using high-dose treatments are often hard to interpret due to acute toxicity and AHR-independent effects on metabolism (1,25).…”