Lysine 419 targets human glucocorticoid receptor for proteasomal degradation.
Glucocorticoid receptors (GRs) are members of a highly conserved family of ligand dependent transcription factors which following hormone binding undergo homologous down-regulation reducing the levels of receptor protein. This decline in human GR (hGR) is due in part to a decrease in protein receptor stability that may limit cellular responsiveness to ligand. To examine the role of the proteasome protein degradation pathway in steroid-dependent hGR responsiveness, we utilized the proteasomal inhibitors MG-132, beta-lactone, and epoxomicin. HeLa cells and COS cells were treated with proteasome inhibitors in the presence of the GR agonist dexamethasone (Dex), or were pretreated with proteasomal inhibitor and then Dex. Dexamethasone induced glucocorticoid responsive reporter activity significantly over untreated controls, whereas cells treated with proteasomal inhibitors and Dex together showed 2-3-fold increase in activity. Protein sequence analysis of the hGR protein identified several candidate protein degradation motifs including a PEST element. Mutagenesis of this element at lysine 419 was done and mutant K419A hGR failed to undergo ligand dependent down-regulation. Mutant K419A hGR displayed 2-3-fold greater glucocorticoid responsive reporter activity in the presence of Dex than wild type hGR. These differences in transcriptional activity were not due to altered subcellular localization, since when the mutant K419A hGR was fused with the green fluorescent protein (GFP) it was found to move in and out of the nucleus similarly to wild type hGR. Together these results suggest that the proteasome and the identified PEST degradation motif limit steroid-dependent human glucocorticoid receptor signaling.
Wallace
Andrew D
AD
Department of Environmental & Molecular Toxicology, Campus Box 7633, North Carolina State University, Raleigh, NC 27695-7633, USA. adwallac@unity.ncsu.edu
Cao
Yan
Y
Chandramouleeswaran
Sindhu
S
Cidlowski
John A
JA
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D003907:Dexamethasone / Q000494:pharmacology; D005938:Glucocorticoids / Q000494:pharmacology; D006801:Humans; D042541:Intracellular Space / Q000378:metabolism; D008239:Lysine; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011401:Promoter Regions, Genetic / Q000235:genetics; D011480:Protease Inhibitors / Q000494:pharmacology; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism*; D061988:Proteasome Inhibitors; D021381:Protein Transport; D051381:Rats; D011965:Receptors, Glucocorticoid / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D020218:Response Elements / Q000235:genetics; D015398:Signal Transduction / Q000187:drug effects
D005938:Glucocorticoids; D011480:Protease Inhibitors; D061988:Proteasome Inhibitors; D011965:Receptors, Glucocorticoid; D003907:Dexamethasone; D046988:Proteasome Endopeptidase Complex; D008239:Lysine