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. 2017;6(1):3-12.
Epub 2017 Apr 15.

The Role of S-Palmitoylation of the Human Glucocorticoid Receptor (hGR) in Mediating the Nongenomic Glucocorticoid Actions

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Free PMC article

The Role of S-Palmitoylation of the Human Glucocorticoid Receptor (hGR) in Mediating the Nongenomic Glucocorticoid Actions

Nicolas C Nicolaides et al. J Mol Biochem. .
Free PMC article

Abstract

Background: Many rapid nongenomic glucocorticoid actions are mediated by membrane-bound glucocorticoid receptors (GRs). S-palmitoylation is a lipid post-translational modification that mediates the membrane localization of some steroid receptors. A highly homologous amino acid sequence (663YLCM KTLLL671) is present in the ligand-binding domain of hGRα, suggesting that hGRα might also undergo S-palmitoylation.

Aim: To investigate the role of the motif 663YLCMKTLLL671 in membrane localization of the hGRα and in mediating rapid nongenomic glucocorticoid signaling.

Methods and results: We showed that the mutant receptors hGRαY663A, hGRαC665A and hGRαLL670/671AA, and the addition of the palmitoylation inhibitor 2-bromopalmitate did not prevent membrane localization of hGRα and co-localization with caveolin-1, and did not influence the biphasic activation of mitogen-activated protein kinase (MAPK) signaling pathway in the early time points. Finally, the hGRα was not shown to undergo S-palmitoylation.

Conclusions: The motif 663YLCMKTLLL671 does not play a role in membrane localization of hGRα and does not mediate the nongenomic glucocorticoid actions.

Conflict of interest statement

Conflicts of interest None.

Figures

Figure 1
Figure 1
Stereotactic conformation of the agonist form of the LBD of hGRα. The yellow arrow indicates the 9-amino acid sequence, 663YLCMKTLL670, which is located on helix 8. H: Helix
Figure 2
Figure 2
(A–D) Localization of the wild-type hGRα (hGRαWT) or its mutant receptors expressed in COS-7 cells in the absence or presence of dexamethasone. (E) Localization of hGRαWT in the presence of the palmitoylation inhibitor. The localization of hGRα-related proteins was examined with immunofluorescent staining. The yellow arrows point to membrane localization of hGRα. Dex: dexamethasone; i: palmitoylation inhibitor 2-Br.
Figure 3
Figure 3
Cytoplasmic, membrane and nuclear localization of the hGRα in the COS-7 cells expressing the wild-type hGRα (hGRαWT) or the mutant receptors. Amounts of hGRα-related proteins were examined in subcellular fractionated samples with Western blotting.
Figure 4
Figure 4
Co-localization of hGRα and caveolin-1 in COS-7 cells expressing (4A) the wild-type hGRα (hGRαWT) or (4B) its mutant receptors or the hGRαWT in the presence of 2-Br. (4C) Addition of dexamethasone resulted in nuclear translocation of both the hGRαWT and the mutant receptors.
Figure 5
Figure 5
(A) Time-course effects of dexamethasone on the MAPK activity in COS-7 cells expressing the wild-type hGRα (hGRαWT) or its mutant receptors. (B) The effect of 2-Br on the MAPK activity in COS7 cells expressing the wild-type hGRα (hGRαWT) or its mutant receptors. Expression levels of total ERK and p-ERK were examined by Western blotting.
Figure 6
Figure 6
(A) Time-course effect of dexamethasone on the AKT activity in COS-7 cells expressing the wild-type hGRα (hGRαWT) or its mutant receptors. (B) The effect of 2-Br on the AKT activity in COS-7 cells expressing the wild-type hGRα (hGRαWT) or its mutant receptors. Expression levels of the total AKT and p-Akt were examined with Western blotting.
Figure 7
Figure 7
Palmitoylation of wild-type hGRα and caveolin-1. Caveolin-1 incorporated 9,10-3H(N) palmitic acid, whereas the wild-type hGRα (hGRαWT) did not incorporate the tritiated substrate. Bars represent mean ± SEM of at least three independent experiments. **: P<0.01, n.s.: not significant, compared to control.

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