CHARACTERIZATION OF THE MOLECULAR AND STRUCTURAL-PROPERTIES OF THE TRANSFORMED AND NUCLEAR ARYL-HYDROCARBON (AH) RECEPTOR COMPLEXES BY PROTEOLYTIC DIGESTION

Ligand-dependent differences in the molecular properties of the transf ormed cytosolic and nuclear aryl hydrocarbon receptor (AhR) were inves tigated using the proteolytic clipping band shift assay, AhR complexes were incubated with [(32)]dioxin responsive element (DRE) (26-mer) or bromodeoxyuridine (BrdU)-DRE and the resulting protein-DNA or crossli nked protein-DNA complexes were treated with trypsin or V8 protease an d analyzed by electrophoresis. The results showed that for several dif ferent AhR ligands including 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD ), 2,3,7,8-tetrachlorodibenzofuran, 1,2,7,8-tetrachlorodibenzofuran an d alpha-naphthoflavone, the pattern of degraded protein-DNA products w ere similar using transformed cytosolic or nuclear AhR complexes. In c ontrast, the proteolytic clipping band shift assay showed that there w ere significant differences in the pattern of degraded protein-DNA pro ducts using nuclear AhR complexes derived from mouse Hepa 1c1c7 cells treated with TCDD or 6-methyl-1,3,8-trichlorodibenzofuran (MCDF). The differences detected in this in vitro assay parallel the in vivo and i n vitro activities of these compounds in which TCDD is a potent AhR ag onist whereas MCDF is a partial AhR agonist and antagonist.