Proteasome-mediated glucocorticoid receptor degradation restricts transcriptional signaling by glucocorticoids

Ligand-dependent down-regulation of the glucocorticoid receptor (GR) has be en shown to limit hormone responsiveness, but the mechanisms involved in th is process are poorly understood. The glucocorticoid receptor is a phosphop rotein that upon ligand binding becomes hyperphosphorylated, and recent evi dence indicates that phosphorylation status of the glucocorticoid receptor plays a prominent role in receptor protein turnover. Because phosphorylatio n is a key signal for ubiquitination and proteasomal catabolism of many pro teins, 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 bl ocks 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 glucocorticoi d receptor-mediated reporter gene expression. The transcriptional activity of the endogenous human glucocorticoid receptor in HeLa cells was also enha nced by MG-132. Direct evidence for ubiquitination of the glucocorticoid re ceptor was obtained by immunoprecipitation of cellular extracts from protea some-impaired cells. Examination of the primary sequence of mouse, hu man, 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 simultaneo usly enhanced glucocorticoid receptor-induced transcriptional activation of gene expression. Unlike wild type GR, proteasomal inhibition failed to enh ance significantly transcriptional activity of K426A mutant GR. Together th ese findings suggest a major role of the ubiquitin-proteasome pathway in re gulating glucocorticoid receptor protein turnover, thereby providing a mech anism to terminate glucocorticoid responses.