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

Research output: Contribution to journalArticle

31 Citations (Scopus)

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

Fingerprint

Dioxins
Response Elements
Aryl Hydrocarbon Receptors
DNA
Ligands
Agarose Chromatography
Temperature
Electrophoretic Mobility Shift Assay
Cytoplasmic and Nuclear Receptors
Oligonucleotides
Sepharose
Transcriptional Activation
Genes
In Vitro Techniques
Rodentia
Liver
Polychlorinated Dibenzodioxins
Proteins

ASJC Scopus subject areas

  • Pharmacology

Cite this

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.

In vitro transformation of the human Ah receptor and its binding to a dioxin response element. / Harper, Patricia A.; Giannone, John V.; Okey, Allan B.; Denison, Michael S.

In: Molecular Pharmacology, Vol. 42, No. 4, 10.1992, p. 603-612.

Research output: Contribution to journalArticle

Harper, PA, Giannone, JV, Okey, AB & Denison, MS 1992, 'In vitro transformation of the human Ah receptor and its binding to a dioxin response element', Molecular Pharmacology, vol. 42, no. 4, pp. 603-612.
Harper, Patricia A. ; Giannone, John V. ; Okey, Allan B. ; Denison, Michael S. / In vitro transformation of the human Ah receptor and its binding to a dioxin response element. In: Molecular Pharmacology. 1992 ; Vol. 42, No. 4. pp. 603-612.
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