In vitro transformation of the human Ah receptor and its binding to a dioxin response element

Patricia A. Harper, John V. Giannone, Allan B. Okey, Michael S. Denison

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34 Scopus citations

Abstract

Many biological effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, dioxin) are mediated by a soluble intracellular protein, the Ah receptor (AhR). After binding of TCDD to the cytoplasmic AhR there occurs a poorly understood "transformation" step, wherein the TCDD-AhR complex is converted to a form that can bind to DNA with high affinity. The binding of transformed AhR to a specific dioxin-responsive element (DRE) upstream of a given gene stimulates transcriptional activation of that gene. Using a gel retardation assay we examined the interaction of transformed human cytosolic TCDD-AhR complexes with a synthetic DNA oligonucleotide containing a single DRE site. Transformation and DNA binding of human AhR in vitro was ligand dependent and specific for DRE-containing DNA. Unlike rodent hepatic AhR, in vitro transformation of human AhR was completely temperature dependent. Although at 4° AhR binds ligand, no transformation of human TCDD-AhR complex was observed at 4° even after 24 h; however, rapid transformation as measured by DNA binding was detectable as early as 10 min after warming to 22°, with maximal binding by about 60 min. Calf thymus DNA-Sepharose or DRE-Sepharose column chromatography showed that transformed human cytosolic AhR interacts with DNA as a single species. The absolute temperature dependency of human AhR transformation mimics that observed in vivo and provides a useful system to study the mechanism of AhR transformation in detail.

Original languageEnglish (US)
Pages (from-to)603-612
Number of pages10
JournalMolecular Pharmacology
Volume42
Issue number4
StatePublished - Oct 1992
Externally publishedYes

ASJC Scopus subject areas

  • Pharmacology

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    Harper, P. A., Giannone, J. V., Okey, A. B., & Denison, M. S. (1992). In vitro transformation of the human Ah receptor and its binding to a dioxin response element. Molecular Pharmacology, 42(4), 603-612.