Direct demonstration of glucocorticoid receptor phosphorylation by intact L-cells.
Glucocorticoid-sensitive L-cells were cultured for 18 h in the presence of [32P]orthophosphate and steroid-binding proteins of cytosol were separated by affinity chromatography and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and isoelectric focusing. Cytosol contains a major phosphoprotein of Mr = 92,000 and a minor phosphoprotein of Mr = 100,000, both of which bind glucocorticoids in a stereospecific, high affinity manner and have the same Mr as glucocorticoid receptor species that have been covalently labeled with the site-specific affinity ligand [3H] 9 alpha-fluoro-16-methyl-11 beta,17 alpha,21-trihydroxypregna-1, 4-diene-3,20-dione 21-mesylate. Cytosol from 32P-labeled, glucocorticoid-resistant L-cells possessing 5% of the steroid-binding capacity of sensitive cells contains very little of the Mr = 92,000 phosphoprotein and none of the Mr = 100,000 phosphoprotein. These observations provide strong evidence that the glucocorticoid receptor is phosphorylated by intact L-cells. The Mr = 92,000 protein is phosphorylated on serine and it can be resolved into two species using isoelectric focusing, consistent with the proposal that there is more than 1 phosphorylated serine/steroid-binding unit. The glucocorticoid-resistant L-cell line produces a unique phosphoprotein of Mr = 104,000 that is recovered in variable amounts after affinity chromatography. It is not known whether this phosphoprotein is a separate gene product or whether it represents a precursor with weak steroid-binding activity that is not cleaved in the resistant cell to the high affinity, Mr = 92,000 mature receptor form.
D000818:Animals; D003907:Dexamethasone / Q000031:analogs & derivatives / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D007739:L Cells / Q000378:metabolism; D051379:Mice; D008970:Molecular Weight; D010710:Phosphates / Q000378:metabolism; D010761:Phosphorus Radioisotopes; D010766:Phosphorylation; D011965:Receptors, Glucocorticoid / Q000302:isolation & purification / Q000378:metabolism*; D011987:Receptors, Steroid / Q000378:metabolism*; D014222:Triamcinolone Acetonide / Q000378:metabolism
D010710:Phosphates; D010761:Phosphorus Radioisotopes; D011965:Receptors, Glucocorticoid; D011987:Receptors, Steroid; D003907:Dexamethasone; D014222:Triamcinolone Acetonide; C031710:dexamethasone 21-methanesulfonate