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

•Helix 8 in LBD of GR matches conserved region mediating membrane association of ER.•Unlike the ER, cysteine 683 in helix 8 of rat GR does not undergo palmitoylation.•The leucine repeat and selected amino acids in helix 8 of the LBD are critical for ligand binding.

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.