An analysis of autologous glucocorticoid receptor protein regulation in AtT-20 cells also reveals differential specificity of the BuGR2 monoclonal antibody.
When the anti-glucocorticoid receptor monoclonal antibody (BuGR2) was initially incorporated either into a new immunoassay strategy or into a traditional sedimentation analysis technique, both methods failed to reveal any change in the cellular content or distribution of BuGR2-reactive antigen following glucocorticoid treatment of AtT-20 cells. Furthermore, the immunoassay also generated strong positive signals with cytosol and nuclear extracts from a receptor-negative cell line (E8.2) derived from L929 cells. However, when the BuGR2 antibody was incorporated into a combined immunoprecipitation/Western blot analysis of AtT-20 cell extracts, only the glucocorticoid receptor protein produced a signal on the Western blot, even though other proteins had been specifically immunoprecipitated by BuGR2 antibody and were clearly present on the Western blot membrane. Applying the latter approach to AtT-20 cells chronically treated with glucocorticoid, we observed not only that the receptor protein rapidly and persistently (1-96 h) accumulated in the nucleus, but also that its total cellular content was first depleted (24 h) and then was progressively replenished (48-96 h). From these studies in AtT-20 cells we conclude: (i), the BuGR2 antibody can exhibit differential immunospecificity dependent upon whether antigen mixtures are denatured or not; (ii), glucocorticoid receptor protein resided almost exclusively in the nucleus during four days of glucocorticoid treatment and (iii), the same treatment regimen resulted in total receptor protein levels being regulated in a biphasic pattern. Together, these results suggest that receptor regulation in AtT-20 cells is a complex event, and that, since steroid was constantly present during our experiments, other factors are involved in regulation of receptor levels.
Hendry
W J
WJ
Department of Medicine, University of Arkansas for Medical Sciences, Little Rock.
Hakkak
R
R
Harrison
R W
RW
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000918:Antibody Specificity; D015153:Blotting, Western; D002499:Centrifugation, Density Gradient; D003907:Dexamethasone / Q000494:pharmacology; D015536:Down-Regulation; D004591:Electrophoresis, Polyacrylamide Gel; D007118:Immunoassay; D051379:Mice; D011233:Precipitin Tests; D011965:Receptors, Glucocorticoid / Q000032:analysis / Q000187:drug effects / Q000378:metabolism*; D013347:Subcellular Fractions / Q000378:metabolism; D014222:Triamcinolone Acetonide / Q000494:pharmacology; D014407:Tumor Cells, Cultured
D000911:Antibodies, Monoclonal; D011965:Receptors, Glucocorticoid; D003907:Dexamethasone; D014222:Triamcinolone Acetonide