Definition of a novel ligand binding domain of a nuclear bHLH receptor: Co-localization of ligand and hsp90 binding activities within the regulable inactivation domain of the dioxin receptor

M. L. Whitelaw, M. Gottlicher, J. A. Gustafsson, L. Poellinger

Research output: Contribution to journalArticle

119 Scopus citations

Abstract

The dioxin receptor mediates signal transduction by dioxin (2,3,7,8-tetrachlorodibenzo-p-dioxin) and binds to DNA target sequences as a heterodimer of the ∼100 kDa ligand binding receptor and the ∼85 kDa auxiliary factor, Arnt. Both of these factors encompass an N-terminal basic helix-loop-helix (bHLH) motif required for DNA binding and dimerization. In this study we describe the construction of glucocorticoid/dioxin receptor fusion proteins which allow the regulation of glucocorticoid receptor activity by dioxin in transient transfections of CHO and hepatoma cells. Thus, in the absence of dioxin, chimeric receptor constructs which contain large 500-720 amino acid C-terminal dioxin receptor fragments, but lack the N-terminal bHLH motif, confer repression upon the transcriptional activity of a glucocorticoid receptor derivative, τDBD, containing its N-terminal strong transactivating signal (τ) and its DNA binding domain (DBD). In the presence of dioxin, this repression is reversed. Importantly, these chimeric receptors did not require the bHLH Arnt co-factor for function. A considerably smaller region of the dioxin receptor, located between amino acids 230 and 421, showed specific dioxin binding activity in vitro. Moreover, dioxin binding in vitro correlated with the ability of receptor fragments to form stable complexes in vitro with the molecular chaperone hsp90. These findings support the notion that hsp90 may be important for folding of a dioxin binding configuration of the receptor. Finally, τDBD activity was constitutively repressed in a dioxin non-responsive manner by dioxin receptor fragments which failed to bind ligand but also failed to bind hsp90 in vitro, indicating that alternative mechanisms in addition to hsp90 binding may contribute to the inactivation function. In summary, the dioxin receptor system provides a novel and complex model of regulation of bHLH factors that may also give important insights into the mechanism of action of ligand-activated nuclear receptors.

Original languageEnglish (US)
Pages (from-to)4169-4179
Number of pages11
JournalEMBO Journal
Volume12
Issue number11
DOIs
StatePublished - 1993

Keywords

  • Basic helix-loop-helix factors
  • Dioxin receptor
  • Glucocorticoid receptor
  • Ligand binding domain
  • Negative regulation

ASJC Scopus subject areas

  • Cell Biology
  • Genetics

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