Down-regulation and phosphorylation of glucocorticoid receptors in cultured cells. Investigations with a monospecific antiserum against a bacterially expressed receptor fragment.
The expression of the glucocorticoid receptor (GR) gene and the phosphorylation of the GR protein has been studied as a function of time after hormone addition to NIH 3T3 cells. We detected a ligand-induced decrease of GR gene expression at both the level of RNA and protein. GR mRNA declined to 25% of the control within 3 h of dexamethasone treatment and remained at this level for at least 24 h. GR protein was analyzed with a monospecific antiserum directed against the DNA- and hormone-binding domains of the rat GR synthesized in Escherichia coli. Pulse-chase experiments revealed a decrease in GR half-life from 8 h in the absence of hormone to 3 h following hormone treatment. Both effects resulted in a reduction of total GR protein to 20% as determined by quantitative immunoblotting. The level of the GR protein returned to that of untreated cells within 24 h after withdrawal of hormone. Furthermore, dexamethasone treatment led to a 3-4-fold increase in GR phosphorylation within 60 min. The glucocorticoid antagonist 17 beta-hydroxy-11 beta-(4-dimethylamino-phenyl)-17 alpha-(1-propynyl)-oestra-4,9-dien-3-one (RU 486) did not change the phosphorylation state of the receptor, but a down-regulation of the GR was still observed. These results suggest that ligand-dependent receptor phosphorylation is not involved in down-regulation of the GR but may be important in the transcriptional activation of hormone responsive genes.
Hoeck
W
W
Friedrich Miescher-Institut, Basel, Switzerland.
Rusconi
S
S
Groner
B
B
D000818:Animals; D000937:Antigen-Antibody Reactions; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D003907:Dexamethasone / Q000037:antagonists & inhibitors / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D004963:Estrenes / Q000494:pharmacology; D005347:Fibroblasts / Q000378:metabolism*; D005822:Genetic Vectors; D006207:Half-Life; D007106:Immune Sera / Q000494:pharmacology; D051379:Mice; D015735:Mifepristone; D010766:Phosphorylation; D012333:RNA, Messenger / Q000378:metabolism; D011817:Rabbits; D011965:Receptors, Glucocorticoid / Q000187:drug effects / Q000276:immunology / Q000378:metabolism*
D004963:Estrenes; D007106:Immune Sera; D012333:RNA, Messenger; D011965:Receptors, Glucocorticoid; D015735:Mifepristone; D003907:Dexamethasone