Chemical cross-linking of the cytosolic and nuclear forms of the Ah receptor in hepatoma cell line 1c1c7.
Both cytosolic and high salt nuclear extracts were isolated from Hepa 1c1c7 cells incubated with 2-azido-3[125I]iodo-7,8-dibromo-dibenzo-p-dioxin ([125I]N3Br2DpD). The [125I]N3Br2DpD-labeled cytosolic fraction was subjected to chemical cross-linking with dimethyl pimelimidate and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Chemical cross-linking of the cytosolic form of the AhR revealed monomeric (97 kDa), dimeric (185 kDa), trimeric (281 kDa), and tetrameric (327 kDa) complexes. In a time course of exposure to the cross-linking reagent, the largest form given above became the predominant AhR form observed in the cytosolic extracts. The 327 kDa cytosolic species apparently consists of a 97 kDa AhR, an approximately 88 kDa protein, an approximately 96 kDa protein, and an approximately 46 kDa protein. Nuclear extracts from [125I]N3Br2DpD-labeled Hepa 1c1c7 cells were applied to sucrose density gradients. The 6 S nuclear receptor peak fractions were pooled and subjected to chemical cross-linking. Analysis by SDS-PAGE revealed a monomeric (97 kDa) ligand binding protein and a dimeric (182 kDa) complex. This would suggest that the nuclear 6 S AhR consists of a 97 kDa AhR and an approximately 85 kDa protein. These findings would indicate that the AhR exists in cytosol as a tetrameric species, while in the nucleus the AhR exists as a heterodimer.
Perdew
G H
GH
Department of Foods and Nutrition, Purdue University, West Lafayette, Indiana 47907.
D006528:Carcinoma, Hepatocellular; D002458:Cell Fractionation; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism*; D002499:Centrifugation, Density Gradient; D003432:Cross-Linking Reagents; D003600:Cytosol / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D008113:Liver Neoplasms; D046911:Macromolecular Substances; D008970:Molecular Weight; D000072317:Polychlorinated Dibenzodioxins / Q000378:metabolism; D011090:Polyenes / Q000494:pharmacology; D018336:Receptors, Aryl Hydrocarbon; D011955:Receptors, Drug / Q000302:isolation & purification / Q000378:metabolism*
D003432:Cross-Linking Reagents; D046911:Macromolecular Substances; D000072317:Polychlorinated Dibenzodioxins; D011090:Polyenes; D018336:Receptors, Aryl Hydrocarbon; D011955:Receptors, Drug; C100114:hamycin