PT - JOURNAL ARTICLE AU - P A Harper AU - J V Giannone AU - A B Okey AU - M S Denison TI - In vitro transformation of the human Ah receptor and its binding to a dioxin response element. DP - 1992 Oct 01 TA - Molecular Pharmacology PG - 603--612 VI - 42 IP - 4 4099 - http://molpharm.aspetjournals.org/content/42/4/603.short 4100 - http://molpharm.aspetjournals.org/content/42/4/603.full SO - Mol Pharmacol1992 Oct 01; 42 AB - 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 degrees AhR binds ligand, no transformation of human TCDD-AhR complex was observed at 4 degrees even after 24 h; however, rapid transformation as measured by DNA binding was detectable as early as 10 min after warming to 22 degrees, 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.