Poster Presentations: pp. 47–150

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.