Characterization of the Activated Form of the Aryl Hydrocarbon Receptor in the Nucleus of HeLa Cells in the Absence of Exogenous Ligand

Abstract

The Aryl hydrocarbon receptor (AhR) is known to mediate 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced toxic effects. Immunocytochemical studies revealed that AhR in HeLa cells is localized throughout the cell. Upon TCDD treatment most of the cytoplasmic receptor is translocated into the nucleus in a time-dependent manner. A significant amount of AhR was found to be tightly associated with the nuclear fraction of untreated HeLa cells. The level of receptor in the nuclear fraction was approximately 16% of the total cellular receptor pool. Further characterization of AhR heterocomplex from the HeLa nuclear fraction by sucrose density gradient analysis revealed that the AhR was present in the 6 S form, and that the nuclear AhR could be coimmunoprecipitated using anti-Arnt mAb. The ability of the AhR to specifically interact with dioxin-responsive elements (DRE) was demonstrated utilizing wild-type and two mutant DREs in gel shift assays. These results would suggest that, in HeLa cells, the AhR–Arnt heterodimer is associated with the nuclear fraction under normal culture conditions. Therefore, HeLa cells can be used as a model system to study the biochemical and molecular function of the Ah receptor and the process that leads to activation of the AhR in the absence of exogenous ligand.