ESPE Abstracts

Background: In humans, glucocorticoids (GCs) regulate a broad spectrum of physiologic functions, exerting both genomic and nongenomic actions through their ubiquitously expressed glucocorticoid receptor (hGR). The rapid nongenomic 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 localization and occurs through a highly conserved nine amino acids motif in the ligand-binding domain (LBD) of steroid receptors. A highly homologous sequence (663YLCMKTLLL671) 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 the motif 663YLCMKTLLL671 in mediating rapid non-genomic glucocorticoid signaling following translocation and binding to the plasma membrane.

Methods and Results: Immunofluorescence experiments showed that both the hGRαWT and the mutant receptors hGRαY663A, hGRαC665A, and hGRαLL670/671AA demonstrated similar distribution under the plasma membrane. These results were confirmed by subcellular fractionation experiments. Addition of a palmitoylation inhibitor, 2-bromopalmitate, did not prevent membrane localization of the receptor or colocalization with caveolin-1. In kinase signaling assays, neither the mutant receptors nor the addition of 2-bromopalmitate prevented the diphasic activation of MAPK signaling pathway in the early time points. Moreover, there was no essential difference in the activation of PI3K pathway, both in the absence or presence of the 2-bromopalmitate. Finally, palmitoylation assays surprisingly showed that the hGRα did not undergo palmitoylation.

Conclusions: The motif 663YLCMKTLLL671 does not play a role in the membrane localization of hGRα and it is not involved in mediating rapid non-genomic glucocorticoid effects. Moreover, the hGRα is not a palmitoylated protein.