Background:In humans, glucocorticoids (GCs) regulate a broad spectrum of physiologic functions and exert both genomic and non-genomic actions through their ubiquitously expressed glucocorticoid receptor (hGR).The rapid non-genomic actions of GCs are likely to be mediated by membrane hGRs that transduce the glucocorticoid signal via activation of kinases.S-palmitoylation plays an important role in plasma membrane (PM) localization and occurs through a highly conserved 9 amino acid motif in the ligand-binding domain (LBD) of steroid receptors.A highly homologous sequence is present in the LBD of the hGRα protein, suggesting that the hGR might also undergo S-palmitoylation.Objective and hypotheses: To determine the role of S-palmitoylation of hGR in mediating rapid glucocorticoid signaling following translocation and binding to the PM.Methods: In vitro studies were performed to determine the specific residues within the 9 amino acid motif of the LBD of hGRα that are crucial for rapid glucocorticoid signaling.Specifically, we determined whether mutation of the amino acids at position -2, 0 and +5/6, relative to cysteine in the 9 amino acid motif, significantly reduces localization of the receptor to the PM, Spalmitoylation, association with caveolin-1, and MAPK and PI3K activation.Results: Both the wild-type and the mutant receptors hGRαY663A, hGRαC665A and hGRáLL670/671AA showed similar distribution to the PM.Addition of 2-bromopalmitate, an S-palmitoylation inhibitor, did not prevent PM localization of the receptor or colocalization with caveolin-1.Compared with the wild-type hGR, all hGR mutant receptors resulted in decreased activation of MAPK signaling from 60 min onwards.A similar reduction in wildtype GR-induced MAPK signaling at 60 min was observed after treatment with 2-bromopalmitate.Conclusions: S-palmitoylation facilitates sustained activation of the MAPK pathway.Further studies are required to confirm that the hGR protein mediates this effect through the 9 amino acid motif in the LBD.
Nicolas C. Nicolaides, Tomoshige Kino, Michael L. Roberts, Eleni Katsantoni, Amalia Sertedaki, Paraskevi Moutsatsou, Anna‐Maria G. Psarra, George Chrousos, Evangelia Charmandari
Michael L. Roberts, Tomoshige Kino, Nicolas C. Nicolaides, Darrell E. Hurt, Eleni Katsantoni, Amalia Sertedaki, Filadelfia Komianou, K. Kassiou, George Chrousos, Evangelia Charmandari
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