Abstract
The glucocorticoid receptor (GR) is phosphorylated at multiple serine residues in a hormone-dependent manner, yet progress on elucidating the function of GR phosphorylation has been hindered by the lack of a simple assay to detect receptor phosphorylation in vivo. We have produced antibodies that specifically recognize phosphorylation sites within human GR at Ser(203) and Ser(211). In the absence of hormone, the level of GR phosphorylation at Ser(211) was low compared with phosphorylation at Ser(203). Phosphorylation of both residues increased upon treatment with the GR agonist dexamethasone. Using a battery of agonists and antagonists, we found that the transcriptional activity of GR correlated with the amount of phosphorylation at Ser(211), suggesting that Ser(211) phosphorylation is a biomarker for activated GR in vivo. Mechanistically, the kinetics of Ser(203) and Ser(211) phosphorylation in response to hormone differed, with Ser(211) displaying a more robust and sustained phosphorylation relative to Ser(203). Analysis of GR immunoprecipitates with phospho-GR-specific antibodies indicated that the receptor was phosphorylated heterogeneously at Ser(203) in the absence of hormone, whereas in the presence of hormone, a subpopulation of receptors was phosphorylated at both Ser(203) and Ser(211). Interestingly, biochemical fractionation studies following hormone treatment indicated that the Ser(203)-phosphorylated form of the receptor was predominantly cytoplasmic, whereas Ser(211)-phosphorylated GR was found in the nucleus. Likewise, by immunofluorescence, Ser(203)-phosphorylated GR was located in the cytoplasm and perinuclear regions of the cell, but not in the nucleoplasm, whereas strong phospho-Ser(211) staining was evident in the nucleoplasm of hormone-treated cells. Our results suggest that differentially phosphorylated receptor species are located in unique subcellular compartments, likely modulating distinct aspects of receptor function.
Highlights
The glucocorticoid receptor (GR)1 is a phosphoprotein that regulates a wide range of metabolic and developmental processes by controlling the expression of target genes in a hormonedependent and cell-specific manner [1, 2]
Using a battery of agonists and antagonists, we found that the transcriptional activity of GR correlated with the amount of phosphorylation at Ser211, suggesting that Ser211 phosphorylation is a biomarker for activated GR in vivo
Characterization of human GR (hGR) Phosphorylation Site-specific Antibodies—The polyclonal antibodies described in this study were raised against phosphopeptides LQDLEFSSGS(PO4)PGKE and GSPGKETNES(PO4)PWRS, corresponding to residues 194 –207 and 202– 215 of hGR, respectively (Fig. 1A)
Summary
The glucocorticoid receptor (GR)1 is a phosphoprotein that regulates a wide range of metabolic and developmental processes by controlling the expression of target genes in a hormonedependent and cell-specific manner [1, 2]. Characterization of hGR Phosphorylation Site-specific Antibodies—The polyclonal antibodies described in this study (antiphospho-Ser203 and anti-phospho-Ser211) were raised against phosphopeptides LQDLEFSSGS(PO4)PGKE and GSPGKETNES(PO4)PWRS, corresponding to residues 194 –207 and 202– 215 of hGR, respectively (Fig. 1A).
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