Helix 8 of the ligand binding domain of the glucocorticoid receptor (GR) is essential for ligand binding

Abstract

Membrane association of estrogen receptors (ER) depends on cysteine palmitoylation and two leucines in the ligand binding domain (LBD), conserved in most steroid receptors. The role of this region, corresponding to helix 8 of the glucocorticoid receptor (GR) LBD, on membrane association of GR was studied in 4B cells, expressing endogenous GR, and Cos-7 cells transfected EGFP-GR constructs. 4B cells preloaded with radiolabeled palmitic acid showed no radioactivity incorporation into immunoprecipitated GR. Moreover, mutation C683A (corresponding to ER palmitoylation site) did not affect corticosterone-induced membrane association of GR. Mutations L687-690A, L682A, E680G and K685G, prevented membrane and also nuclear localization through reduced ligand binding. L687-690A mutation decreased association of GR with heat shock protein 90 and transcriptional activity, without overt effects on receptor protein stability. The data demonstrate that palmitoylation does not mediate membrane association of GR, but that the region 680-690 (helix 8), is critical for ligand binding and receptor function.

Rapid Glucocorticoid Feedback Inhibition of ACTH Secretion Involves Ligand-Dependent Membrane Association of Glucocorticoid Receptors

“…In the case of the ER, these nongenomic actions are mediated by membrane association of the receptor. In agreement with previous immunohistochemical (14 -17), and western blot studies (24), the present experiments show association of the classical GR to membrane fractions in AtT-20 and 4B cell lines. This ligand-dependent increase in membrane GR was not due to contamination with nuclear proteins as shown by the absence of nuclear markers in membrane proteins.…”

“…The lower corticosterone concentration used, 10 nM increased nuclear immunoreactive GR by 2.8 Ϯ 0.4-fold (P Ͻ .01) and much larger increases of 6.5 Ϯ 0.3 and 7.5 Ϯ 0.1 were observed with 100 nm and 1 M, corticosterone respectively ( Figure 6). As previously described in 4B cells (24), a band corresponding to irGR was also observed in membrane protein fractions colocalized with pancadherin. One way ANOVA also showed a significant effect of corticosterone on GR levels in membrane proteins (F ϭ 11.1, P Ͻ .002).…”

“…Eighteen hours later medium was changed to serum-free medium containing 0.1% BSA and incubated for 2h before exposure to corticosterone as indicated in Results and Figures. Then cells were harvested and cytoplasmic, membrane and nuclear protein fractions were separated using the Subcellular Protein Fractionation Kit for Cultured Cells (Pierce, Life Technologies) as described (24). Protein concentrations were determined using the Bicinchoninic Acid (BCA) Protein Assay.…”

Novel insights into the molecular mechanisms underlying generalized glucocorticoid resistance and hypersensitivity syndromes

“…52 However, the nature of this membrane-anchoring GR has not yet been clarified. Although most of the steroid receptors were shown to incorporate palmitic acid, a post-translational modification termed "S-palmitoylation" that enables membrane binding of proteins and increases protein hydrophobicity, 53,54 we and others have demonstrated that GRα does not undergo S-palmitoylation, implying that other mechanisms are responsible for GRα membrane localization and mediate the nongenomic glucocorticoid actions [55][56][57] (Figure 2). …”

Molecular Regulation of Corticotropin-Releasing Hormone Gene Expression in Parvocellular Neurons of the Hypothalamic Paraventricular Nucleus

“…However, the lack of rapid effects of dexamethasone in hypothalamic slices of mice bearing targeted deletion of GR in the PVN, strongly suggests that the classical GR mediates non-genomic effects [43]. Supporting the involvement of the classical GR in mediating the rapid effects of glucocorticoids, immunohistochemical and biochemical studies have shown membrane localization of GR in a number of systems [26,[44][45][46][47].…”