title: Characterization of the Ah receptor for 2,3,7,8-tetrachlorodibenzo-p-dioxin: use of chemical crosslinking and a monoclonal antibody directed against a 59-kDa protein associated with steroid receptors.
abstract: The Ah receptor regulates induction of cytochrome P450IA1 (aryl hydrocarbon hydroxylase) by "3-methylcholanthrene-type" compounds and mediates the toxic effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin and related halogenated aromatic hydrocarbons. Hepatic Ah receptor from untreated rodents is localized in the cytosol and has an apparent molecular mass of 250 to 300 kDa. This large form can be dissociated into a smaller ligand-binding subunit upon exposure to high ionic strength. The Ah receptor displays many structural similarities to the receptors for steroid hormones. Two non-ligand-binding proteins have been identified to be associated with the cytosolic forms of the steroid hormone receptors. The first is a 90-kDa heat shock protein (hsp 90); the second is a 59-kDa protein (p59) of unknown function. The cytosolic Ah receptor ligand-binding subunit previously has been shown to be associated with hsp 90. In the present study, we used a monoclonal antibody, KN 382/EC1, generated against the 59-kDa protein which is associated with rabbit steroid receptors to determine if p59 also is a component of the large cytosolic Ah receptor complex. Cytosolic forms of rabbit progesterone receptor, glucocorticoid receptor, and Ah receptor were analyzed by velocity sedimentation on sucrose gradients under low-ionic-strength conditions and in the presence of molybdate. Progesterone receptor from rabbit uterine cytosol and glucocorticoid receptor from rabbit liver each had a sedimentation coefficient of approximately 9 S. In the presence of KN 382/EC1 antibody the progesterone receptor and the glucocorticoid receptor both underwent a shift in sedimentation to a value of approximately 11 S. The increase in sedimentation velocity is an indication that the receptor-protein complexes are interacting with the antibody. Under low-ionic-strength conditions the Ah receptors from rabbit uterine cytosol and liver cytosol had a sedimentation coefficient of approximately 9 S. However, in contrast to the steroid receptors, the Ah receptor showed no change in its sedimentation properties in either tissue in the presence of KN 382/EC1, indicating that the antibody is not interacting with the Ah receptor. Multimeric Ah receptor complexes that were chemically crosslinked still did not show any interaction with KN 382/EC1. These data indicate that the 59-kDa protein either is not associated with the Ah receptor or is present in an altered form which the antibody cannot recognize.
authors.affiliation: Department of Pediatrics, Hospital for Sick Children, Toronto, Ontario, Canada.
authors.forename: R D
authors.initials: RD
authors.lastname: Prokipcak
authors.forename: L E
authors.initials: LE
authors.lastname: Faber
authors.forename: A B
authors.initials: AB
authors.lastname: Okey
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal; D001666:Binding Sites, Antibody; D003432:Cross-Linking Reagents; D003600:Cytosol / Q000378:metabolism; D004147:Dioxins / Q000378:metabolism*; D005260:Female; D008099:Liver / Q000378:metabolism; D008970:Molecular Weight; D000072317:Polychlorinated Dibenzodioxins / Q000378:metabolism*; D011817:Rabbits; D018336:Receptors, Aryl Hydrocarbon; D011955:Receptors, Drug / Q000032:analysis* / Q000276:immunology; D011987:Receptors, Steroid / Q000276:immunology / Q000378:metabolism*; D014599:Uterus / Q000378:metabolism
chemical_list: D000911:Antibodies, Monoclonal; D003432:Cross-Linking Reagents; D004147:Dioxins; D000072317:Polychlorinated Dibenzodioxins; D018336:Receptors, Aryl Hydrocarbon; D011955:Receptors, Drug; D011987:Receptors, Steroid