title: Proteasome-mediated glucocorticoid receptor degradation restricts transcriptional signaling by glucocorticoids.
abstract: Ligand-dependent down-regulation of the glucocorticoid receptor (GR) has been shown to limit hormone responsiveness, but the mechanisms involved in this process are poorly understood. The glucocorticoid receptor is a phosphoprotein that upon ligand binding becomes hyperphosphorylated, and recent evidence indicates that phosphorylation status of the glucocorticoid receptor plays a prominent role in receptor protein turnover. Because phosphorylation is a key signal for ubiquitination and proteasomal catabolism of many proteins, we evaluated whether the ubiquitin-proteasomal pathway had a role in glucocorticoid receptor down-regulation and the subsequent transcriptional response to glucocorticoids. Pretreatment of COS-1 cells expressing mouse glucocorticoid receptor with the proteasome inhibitor MG-132 effectively blocks glucocorticoid receptor protein down-regulation by the glucocorticoid dexamethasone. Interestingly, both MG-132 and a second proteasome inhibitor beta-lactone significantly enhanced hormone response of transfected mouse glucocorticoid receptor toward transcriptional activation of glucocorticoid receptor-mediated reporter gene expression. The transcriptional activity of the endogenous human glucocorticoid receptor in HeLa cells was also enhanced by MG-132. Direct evidence for ubiquitination of the glucocorticoid receptor was obtained by immunoprecipitation of cellular extracts from proteasome-impaired cells. Examination of the primary sequence of mouse, human, and rat glucocorticoid receptor has identified a candidate PEST degradation motif. Mutation of Lys-426 within this PEST element both abrogated ligand-dependent down-regulation of glucocorticoid receptor protein and simultaneously enhanced glucocorticoid receptor-induced transcriptional activation of gene expression. Unlike wild type GR, proteasomal inhibition failed to enhance significantly transcriptional activity of K426A mutant GR. Together these findings suggest a major role of the ubiquitin-proteasome pathway in regulating glucocorticoid receptor protein turnover, thereby providing a mechanism to terminate glucocorticoid responses.
authors.affiliation: Molecular Endocrinology Group, Laboratory of Signal Transduction, NIEHS, National Institutes of Health, Research Triangle Park, North Carolina 27709, USA.
authors.forename: A D
authors.initials: AD
authors.lastname: Wallace
authors.forename: J A
authors.initials: JA
authors.lastname: Cidlowski
mesh_terms: D020816:Amino Acid Motifs; D000818:Animals; D015153:Blotting, Western; D019556:COS Cells; D015500:Chloramphenicol O-Acetyltransferase / Q000378:metabolism; D003546:Cysteine Endopeptidases / Q000737:chemistry / Q000378:metabolism; D003907:Dexamethasone / Q000494:pharmacology; D015536:Down-Regulation; D005938:Glucocorticoids / Q000737:chemistry*; D006367:HeLa Cells; D006801:Humans; D008024:Ligands; D008156:Luciferases / Q000378:metabolism; D008239:Lysine / Q000737:chemistry; D009097:Multienzyme Complexes / Q000737:chemistry / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010766:Phosphorylation; D010957:Plasmids / Q000378:metabolism; D011233:Precipitin Tests; D046988:Proteasome Endopeptidase Complex; D011965:Receptors, Glucocorticoid / Q000737:chemistry* / Q000378:metabolism*; D015398:Signal Transduction; D013997:Time Factors; D014158:Transcription, Genetic; D015533:Transcriptional Activation; D014162:Transfection
chemical_list: D005938:Glucocorticoids; D008024:Ligands; D009097:Multienzyme Complexes; D011965:Receptors, Glucocorticoid; D003907:Dexamethasone; D008156:Luciferases; D015500:Chloramphenicol O-Acetyltransferase; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; D008239:Lysine