Abstract
Immunoprecipitation experiments performed on cytosolic extracts of the mouse hepatoma cell line Hepa-1c1c7 (Hepa-1) confirm that the 9-S, unliganded, cytosolic aryl hydrocarbon (Ah) receptor complex contains the 90-kDa heat shock protein and the Ah receptor protein but reveal that it does not contain the Ah receptor nuclear translocator (ARNT) protein. These experiments confirm that the 6-S liganded form of the receptor identified in nuclear extracts of cells treated with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) contains the Ah receptor protein and ARNT but not the 90-kDa heat shock protein. The 6-S liganded Ah receptor complex activates transcription of the CYP1A1 gene via its binding to upstream xenobiotic-responsive elements (XREs). Treatment of cytosolic extracts of Hepa-1 cells with TCDD in vitro transforms the Ah receptor complex to the XRE-binding state. No such transformation occurs in a C- mutant deficient in ARNT activity. When in vitro synthesized ARNT was added concomitantly with TCDD to C- cytosolic extracts, it associated with the Ah receptor and restored Ah receptor-dependent XRE-binding activity to the extracts. Covalent cross-linking experiments in nuclear extracts of Hepa-1 and human LS180 cells treated with TCDD in vivo demonstrate that both ARNT and the Ah receptor bind directly to the XRE core sequence.
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