Glucocorticoid receptor identified on nuclear envelopes of male rat livers by affinity labeling and immunochemistry.
To exert their action at the genome, steroids must traverse the nuclear envelope, either alone or complexed to their receptor. Our previous studies identified two classes of dexamethasone-binding sites on male rat liver nuclear envelopes: a low capacity, high affinity site and a high capacity, low affinity site. The affinity reagent, [3H]dexamethasone mesylate, labeled peptides at 35-85 kDa, which may be the low affinity glucocorticoid-binding peptides, as these peptides showed the same response to hormonal manipulation as the low affinity [3H]dexamethasone-binding sites previously characterized. With dexamethasone mesylate and a monoclonal antibody against the glucocorticoid receptor, we have confirmed that the high affinity binding site on the nuclear envelope is the glucocorticoid receptor. Affinity labeling revealed the presence of a doublet of peptides at 85 and 110 kDa, in the same mol wt range as that reported for the glucocorticoid receptor. Furthermore, these affinity-labeled peptides responded to hormonal manipulation like nuclear glucocorticoid receptors. The monoclonal antibody identified a doublet of peptides, a major component of 92-94 kDa and a minor component of 98 kDa. Again, both peptides responded to hormonal manipulation like nuclear glucocorticoid receptors. The nuclear envelope-associated glucocorticoid receptor is not extracted by 0.1 M NaCl or 1% Triton X-100. These results show that glucocorticoid hormone interacts with the nuclear envelope via binding to the transformed glucocorticoid receptor, lending support to the two-step model of steroid hormone action.
Howell
G M
GM
Department of Medicine, University of Ottawa, Moses and Rose Loeb Research Institute, Ottawa Civic Hospital, Canada.
Gustafsson
J A
JA
Lefebvre
Y A
YA
D000345:Affinity Labels; D000818:Animals; D001667:Binding, Competitive; D003907:Dexamethasone / Q000031:analogs & derivatives* / Q000378:metabolism; D004229:Dithiothreitol / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D015151:Immunoblotting; D008099:Liver / Q000648:ultrastructure*; D008297:Male; D008970:Molecular Weight; D009685:Nuclear Envelope / Q000187:drug effects / Q000378:metabolism*; D017830:Octoxynol; D011092:Polyethylene Glycols / Q000494:pharmacology; D051381:Rats; D011919:Rats, Inbred Strains; D011965:Receptors, Glucocorticoid / Q000378:metabolism*; D012965:Sodium Chloride / Q000494:pharmacology
D000345:Affinity Labels; D011965:Receptors, Glucocorticoid; D011092:Polyethylene Glycols; D012965:Sodium Chloride; D003907:Dexamethasone; D017830:Octoxynol; C031710:dexamethasone 21-methanesulfonate; D004229:Dithiothreitol