The photoaffinity labeling of the nuclear aryl
hydrocarbon (
Ah) receptor from mouse Hepa 1c1c7, rat
hepatoma H-4-II E, and human liver Hep G2 cells was investigated using two high affinity
ligands, namely 2,3,7,8-[3H]
tetrachlorodibenzo-p-dioxin (
TCDD) and 7-[125I]iodo-2,3,-
dibenzo-p-dioxin ([125I]DBDD). Irradiation of nuclear [3H]
TCDD-
Ah receptor complexes from the three cell lines for 5 min gave 47, 38, and 62% yields of
trichloroacetic acid-precipitable photoadducts from the Hepa 1c1c7, H-4-II E, and Hep G2 cell lines, respectively; denaturing
sodium dodecyl sulfate-
polyacrylamide gel electrophoresis separation followed by autoradiography gave one major
Ah receptor photoadduct for each cell line with apparent molecular masses at 97, 100, and 110 kDa, respectively. [125I]DBDD could also be used as a photoaffinity label for the nuclear
Ah receptor from the three cell lines; although the maximum net yield of photoaffinity labeled nuclear
Ah receptor from the rodent nuclear
Ah receptor preparations was relatively low (0.5-2.5%), a greater than 15% yield of photoadduct was obtained from the human Hep G2 cells. Both [3H]
TCDD and [125I]DBDD were utilized to photoaffinity label the nuclear
Ah receptor in Hepa 1c1c7 cells in
suspension and the net yield of photoadducts with these
ligands was 94.6 and 3.0%, respectively. The cytosolic
Ah receptor from the three cell lines was photolabeled with [125I]DBDD and the net yield of photoadducts varied from 3.3 to 14.7%. The functional activity of the photoaffinity-labeled nuclear
TCDD-
Ah receptor complexes from the cell lines was also determined by comparing relative binding affinities of the photolyzed and unphotolyzed complexes with a synthetic
dioxin-responsive
element (DRE) using a gel retardation assay. The photolyzed and unphotolyzed complexes from the three cell lines all bound with the DRE in the gel shift assay; however, the gel mobilities of the rodent and human
nuclear receptor-DRE complexes were different. Quantitative analysis of the DRE binding showed that there were no significant differences between the photolyzed and unphotolyzed
nuclear receptor complexes from the rodent cells, whereas there was a significant 27% decrease in the DRE binding of the photolyzed versus the unphotolyzed
nuclear receptor complex from the human Hep G2 cells. These studies demonstrate the utility of [3H]
TCDD and [125I]DBDD as
photoaffinity labels for the
Ah receptor and illustrate the structural and photochemical differences between the rodent and the human nuclear
Ah receptor complexes.