title: 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.
authors: Section on Endocrine Physiology, PDEGEN, NICHD, NIH, Bethesda, Maryland, USA; College of Animal Sciences, Jilin University, China.
authors: Qiong
authors: Q
authors: Deng
authors: Section on Organelle Biology, CBMP, NICHD, NIH, Bethesda, Maryland, USA.
authors: Bennett
authors: B
authors: Waxse
authors: Section on Endocrine Physiology, PDEGEN, NICHD, NIH, Bethesda, Maryland, USA.
authors: Denise
authors: D
authors: Riquelme
authors: College of Animal Sciences, Jilin University, China.
authors: Jiabao
authors: J
authors: Zhang
authors: Section on Endocrine Physiology, PDEGEN, NICHD, NIH, Bethesda, Maryland, USA. Electronic address: Greti_Aguilera@nih.gov.
authors: Greti
authors: G
authors: Aguilera
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D017124:Conserved Sequence; D003345:Corticosterone / Q000494:pharmacology; D003545:Cysteine / Q000378:metabolism; D003907:Dexamethasone / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D018841:HSP90 Heat-Shock Proteins / Q000378:metabolism; D006801:Humans; D008024:Ligands; D054878:Lipoylation / Q000187:drug effects; D008156:Luciferases / Q000378:metabolism; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000737:chemistry; D009154:Mutation / Q000235:genetics; D019308:Palmitic Acid / Q000378:metabolism; D011485:Protein Binding / Q000187:drug effects; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D021381:Protein Transport / Q000187:drug effects; D051381:Rats; D011965:Receptors, Glucocorticoid / Q000737:chemistry* / Q000378:metabolism*; D020449:Repetitive Sequences, Amino Acid; D016415:Sequence Alignment; D013329:Structure-Activity Relationship; D014158:Transcription, Genetic / Q000187:drug effects; D014316:Tritium / Q000378:metabolism
chemical_list: D018841:HSP90 Heat-Shock Proteins; D008024:Ligands; D050505:Mutant Proteins; D011965:Receptors, Glucocorticoid; D014316:Tritium; D019308:Palmitic Acid; D003907:Dexamethasone; D008156:Luciferases; D003545:Cysteine; D003345:Corticosterone
keywords: Dexamethasone binding; Glucocorticoid receptor; HSP90; Helix 8; Ligand binding domain; Palmitoylation