Functional analysis of the LXXLL motifs of the human glucocorticoid receptor: Association with altered ligand affinity

Dong, Diane; Jewell, Christine M.; Bienstock, Rachelle J.; Cidlowski, John A.

doi:10.1016/j.jsbmb.2006.06.010

Cited by 16 publications

(9 citation statements)

References 41 publications

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“…When digested with trypsin, WT-GR is cleaved at several positions, producing a number of fragments, including the production of a characteristic 16-kDa fragment. In addition, previous reports show that a conformational change within GR upon Dex binding prevents the formation of this fragment (15). In accordance with previous results, we, too, found that digestion of WT-GR with trypsin produced a 16-kDa fragment, whereas Dex treatment prevented it (Fig.…”

Section: Fig 3 Effects Of Ser404 Phosphorylation On Gr Cellular Locsupporting

confidence: 93%

Glycogen Synthase Kinase 3β-Mediated Serine Phosphorylation of the Human Glucocorticoid Receptor Redirects Gene Expression Profiles

Galliher-Beckley¹,

Williams²,

Collins

et al. 2008

Molecular and Cellular Biology

114

102

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Aberrant glycogen synthase kinase 3␤ (GSK-3␤) activity is associated with the progression of several pathological conditions such as diabetes, Alzheimer's, and cancer. GSK-3␤ regulates cellular processes by directly phosphorylating metabolic enzymes and transcription factors. Here, we discovered a new target for GSK-3␤ phosphorylation: the human glucocorticoid receptor (GR). Glucocorticoid signaling is essential for life and regulates diverse biological functions from cell growth to metabolism to apoptosis. Specifically, we found hormone-dependent GR phosphorylation on serine 404 by GSK-3␤. Cells expressing a GR that is incapable of GSK-3␤ phosphorylation had a redirection of the global transcriptional response to hormone, including the activation of additional signaling pathways, in part due to the altered ability of unphosphorylatable GR to recruit transcriptional cofactors CBP/p300 and the p65 (RelA) subunit of NF-B. Furthermore, GSK-3␤-mediated GR phosphorylation inhibited glucocorticoid-dependent NF-B transrepression and attenuated the glucocorticoid-dependent cell death of osteoblasts. Collectively, our results describe a novel convergence point of the GSK-3␤ and the GR pathways, resulting in altered hormone-regulated signaling. Our results also provide a mechanism by which GSK-3␤ activity can dictate how cells will ultimately respond to glucocorticoids.

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Section: Fig 3 Effects Of Ser404 Phosphorylation On Gr Cellular Locsupporting

confidence: 93%

Glycogen Synthase Kinase 3β-Mediated Serine Phosphorylation of the Human Glucocorticoid Receptor Redirects Gene Expression Profiles

Galliher-Beckley¹,

Williams²,

Collins

et al. 2008

Molecular and Cellular Biology

114

102

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“…Recent findings have indicated that both mechanisms contribute to local GC levels and a large body of evidence supports the hypothesis that human skin is capable of generating appreciable levels of GC through steroidogenesis (reviewed in Slominski et al 2012). However, here we demonstrate that the contribution made by steroidogenesis in mouse skin is negligible compared with GC activation by 11b-HSD1, which generates corticosterone at concentrations capable of GR activation (Dong et al 2006). Furthermore, while 11b-HSD1 expression and activity increase during wound healing, corticosteroidogenic enzymes display variable mRNA regulation but decreased activity.…”

Section: Discussionsupporting

confidence: 81%

Increased glucocorticoid activation during mouse skin wound healing

Tiganescu

Hupe

Uchida

et al. 2014

Journal of Endocrinology

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Glucocorticoid (GC) excess inhibits wound healing causing increased patient discomfort and infection risk. 11b-hydroxysteroid dehydrogenase type 1 (11b-HSD1) activates GCs (converting 11-dehydrocorticosterone to corticosterone in rodents) in many tissues including skin, where de novo steroidogenesis from cholesterol has also been reported. To examine the regulation of 11b-HSD1 and steroidogenic enzyme expression during wound healing, 5 mm wounds were generated in female SKH1 mice and compared at days 0, 2, 4, 8, 14, and 21 relative to unwounded skin. 11b-HSD1 expression (mRNA and protein) and enzyme activity were elevated at 2 and 4 days post-wounding, with 11b-HSD1 localizing to infiltrating inflammatory cells. 11b-HSD2 (GC-deactivating) mRNA expression and activity were undetectable. Although several steroidogenic enzymes displayed variable expression during healing, expression of the final enzyme required for the conversion of 11-deoxycorticosterone to corticosterone, 11b-hydroxylase (CYP11B1), was lacking in unwounded skin and post-wounding. Consequently, 11-deoxycorticosterone was the principal progesterone metabolite in mouse skin before and after wounding. Our findings demonstrate that 11b-HSD1 activates considerably more corticosterone than is generated de novo from progesterone in mouse skin and drives GC exposure during healing, demonstrating the basis for 11b-HSD1 inhibitors to accelerate wound repair.

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“…A number of studies have demonstrated that amino acid mutations within the carboxy terminus result in altered ligand binding. For example, mutation of the 532–536 LXXLL motif of the human GR to LXXAA suppresses dexamethasone induced transcription and ligand binding, while the 718–722 LXXAA mutant is fully active at high ligand concentrations (Dong et al 2006). A number of additional mutations in the ligand binding domain have been shown to decrease or suppress ligand binding affinity (Chakraborti et al 1991; Garabedian & Yamamoto 1992; Schmitt & Stunnenberg 1993; Lanz et al 1994), for review see (Simons 1994).…”

Section: Discussionmentioning

confidence: 99%

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

Deng

Waxse

Riquelme

et al. 2015

Molecular and Cellular Endocrinology

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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.

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Functional analysis of the LXXLL motifs of the human glucocorticoid receptor: Association with altered ligand affinity

Cited by 16 publications

References 41 publications

Glycogen Synthase Kinase 3β-Mediated Serine Phosphorylation of the Human Glucocorticoid Receptor Redirects Gene Expression Profiles

Glycogen Synthase Kinase 3β-Mediated Serine Phosphorylation of the Human Glucocorticoid Receptor Redirects Gene Expression Profiles

Increased glucocorticoid activation during mouse skin wound healing

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

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