Mouse glucocorticoid receptor phosphorylation status influences multiple functions of the receptor protein.
Although studies have shown that the mouse glucocorticoid receptor (mGR) contains eight phosphorylation sites (Bodwell, J. E., Ortí, E. , Coull, J. M., Pappin, D. J. C., Smith, L. I., and Swift, F. (1991) J. Biol. Chem. 266, 7549-7555), the effect of phosphorylation on receptor function is unclear. We have examined the consequences of single or multiple phosphorylation site mutations on several properties of mGR including receptor expression, ligand-dependent nuclear translocation, hormone-mediated transactivation, ligand-dependent down-regulation of mGR, and receptor protein half-life. Mutations had little effect on receptor expression, subcellular distribution, ligand-dependent nuclear translocation, or on the ability to activate hormone-mediated transcription from a complex (murine mammary tumor virus) promoter. In contrast, the phosphorylation status of the mGR had a profound effect on the ability to transactivate a minimal promoter containing simple glucocorticoid response elements after hormone administration. Similarly, ligand-dependent down-regulation by glucocorticoids of both receptor mRNA and protein was abrogated in mutants containing three or more phosphorylation site alterations. Finally, we show that the phosphorylation status of mGR has a profound effect on the stability of the glucocorticoid receptor protein. Receptors containing seven or eight mutated sites have a markedly extended half-life and do not show the ligand-dependent destabilization seen with wild type receptor. These data show that receptor phosphorylation may play a crucial role in regulating receptor levels and hence control receptor functions.
Webster
J C
JC
Molecular Endocrinology Group, the Laboratory of Signal Transduction, NIEHS, National Institutes of Health, Research Triangle Park, North Carolina 27709, USA.
Jewell
C M
CM
Bodwell
J E
JE
Munck
A
A
Sar
M
M
Cidlowski
J A
JA
D000818:Animals; D019556:COS Cells; D002467:Cell Nucleus / Q000378:metabolism; D005938:Glucocorticoids / Q000378:metabolism; D006207:Half-Life; D051379:Mice; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D012333:RNA, Messenger / Q000378:metabolism; D011965:Receptors, Glucocorticoid / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic; D014162:Transfection
D005938:Glucocorticoids; D012333:RNA, Messenger; D011965:Receptors, Glucocorticoid