title: In situ and in vitro photoaffinity labeling of the nuclear aryl hydrocarbon receptor from transformed rodent and human cell lines.
abstract: 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.
authors.affiliation: Department of Veterinary Physiology and Pharmacology, Texas A&M University, College Station 77843-4466.
authors.forename: X
authors.initials: X
authors.lastname: Wang
authors.forename: T R
authors.initials: TR
authors.lastname: Narasimhan
authors.forename: V
authors.initials: V
authors.lastname: Morrison
authors.forename: S
authors.initials: S
authors.lastname: Safe
mesh_terms: D000345:Affinity Labels; D000818:Animals; D001483:Base Sequence; D002455:Cell Division; D002461:Cell Line, Transformed; D002467:Cell Nucleus / Q000737:chemistry*; D004247:DNA; D004147:Dioxins; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D006838:Hydrocarbons; D007700:Kinetics; D008969:Molecular Sequence Data; D010782:Photolysis; D000072317:Polychlorinated Dibenzodioxins; D018336:Receptors, Aryl Hydrocarbon; D011955:Receptors, Drug / Q000032:analysis*; D012377:Rodentia
chemical_list: D000345:Affinity Labels; D004147:Dioxins; D006838:Hydrocarbons; D000072317:Polychlorinated Dibenzodioxins; D018336:Receptors, Aryl Hydrocarbon; D011955:Receptors, Drug; C056902:2-iodo-7,8-dibromodibenzo-1,4-dioxin; D004247:DNA