A Novel Point Mutation of the Human Glucocorticoid Receptor Gene Causes Primary Generalized Glucocorticoid Resistance Through Impaired Interaction With the LXXLL Motif of the p160 Coactivators: Dissociation of the Transactivating and Transreppressive Activities

Abstract

Context:

Primary generalized glucocorticoid resistance is a rare genetic disorder characterized by generalized, partial, target-tissue insensitivity to glucocorticoids. The molecular basis of the condition has been ascribed to inactivating mutations in the human glucocorticoid receptor (hGR) gene.

Objective:

The objective of the study was to present three new cases caused by a novel mutation in the hGR gene and to delineate the molecular mechanisms through which the mutant receptor impairs glucocorticoid signal transduction.

Design and Results:

The index case (father) and his two daughters presented with increased urinary free cortisol excretion and resistance of the hypothalamic-pituitary-adrenal axis to dexamethasone suppression in the absence of clinical manifestations suggestive of Cushing syndrome. All subjects harbored a novel, heterozygous, point mutation (T→G) at nucleotide position 1724 of the hGR gene, which resulted in substitution of valine by glycine at amino acid 575 of the receptor. Compared with the wild-type receptor, the hGRαV575G demonstrated a significant (33%) reduction in its ability to transactivate the mouse mammary tumor virus promoter in response to dexamethasone, a 50% decrease in its affinity for the ligand, and a 2.5-fold delay in nuclear translocation. Although it did not exert a dominant negative effect on the wild-type receptor and preserved its ability to bind to DNA, hGRαV575G displayed significantly enhanced (∼80%) ability to transrepress the nuclear factor-κΒ signaling pathway. Finally, the mutant receptor hGRαV575G demonstrated impaired interaction with the LXXLL motif of the glucocorticoid receptor-interacting protein 1 coactivator in vitro and in computer-based structural simulation via its defective activation function-2 (AF-2) domain.

Conclusions:

The natural mutant receptor hGRαV575G causes primary generalized glucocorticoid resistance by affecting multiple steps in the glucocorticoid signaling cascade, including the affinity for the ligand, the time required for nuclear translocation, and the interaction with the glucocorticoid-interacting protein-1 coactivator.

Primary generalized glucocorticoid resistance is a rare, familial, or sporadic genetic condition characterized by generalized, partial, target-tissue insensitivity to glucocorticoids, compensatory elevations in circulating cortisol and ACTH concentrations, and resistance of the hypothalamic-pituitary-adrenal (HPA) axis to dexamethasone suppression. The molecular basis of the condition has been ascribed to mutations in the human glucocorticoid receptor (hGR) gene, which impair the molecular mechanisms of hGR action (1–7). In the present study, we describe three new cases caused by a novel heterozygous mutation of the hGR gene and present the molecular mechanisms through which the mutant receptor alters glucocorticoid signal transduction.

Case reports

A 70-year-old man presented with bilateral adrenal hyperplasia detected during melanoma surveillance. He was otherwise asymptomatic, normotensive (130/64 mm Hg) and had no clinical manifestations suggestive of Cushing syndrome. Endocrinological evaluation revealed elevated 8:00 am serum cortisol concentrations [661 nmol/L; normal range (nr) 220–525 nmol/L] and increased urinary free cortisol excretion (507–749 nmol/d; nr 100–380 nmol/d). A low-dose dexamethasone suppression test (0.5 mg dexamethasone every 6 h for 48 h) revealed resistance of the HPA axis to dexamethasone suppression (8:00 am serum cortisol, 98 nmol/L; nr < 50 nmol/L). A pituitary magnetic resonance imaging scan was normal.

His two daughters, aged 41 and 47 years, presented with mild hirsutism and increased 24-hour urinary free cortisol excretion (556 and 687 nmol/d, respectively). They had normal blood pressure of 130/60 mm Hg and 120/62 mm Hg, respectively. An overnight dexamethasone suppression test (1 mg) revealed resistance of the HPA axis to dexamethasone suppression (8:00 am serum cortisol of 351 and 175 nmol/L, respectively; nr < 50 nmol/L). A pituitary magnetic resonance imaging scan was normal and a computed tomography scan revealed no adrenal hyperplasia.

The above clinical findings suggested the diagnosis of primary generalized glucocorticoid resistance. Written informed consent was obtained and additional molecular studies were undertaken.

Materials and Methods

Amplification and sequencing of hGR gene

Genomic DNA was isolated from peripheral blood lymphocytes, and the coding sequences and intron-exon junctions of the hGR gene were PCR amplified and sequenced (8).

Plasmids

The plasmids used included pRShGRα, pF25GFP-hGRα, pBK/CMV-hGRα, pMMTV-luc, pGL4.73[hRluc/SV40], pRSV-erbA-1, pGEX4T3-GRIP1(1–1462), pGEX4T3-GRIP1 (596–774), and pGEX4T3-GRIP1(740–1217) (8). The plasmids pRShGRαV575G, pF25GFP-hGRαV575G, and pBK/CMV-hGRαV575G were constructed by introducing the V575G mutation into the pRShGRα, pF25GFP-hGRα, and pBK/CMV-hGRα, respectively. The plasmids pRSVC(p50)-NF-κB and pRSVC(p65)-RelA express, respectively, the p50 and the p65 subunits of nuclear factor-κB (NF-κB) under control of the Rous sarcoma virus promoter. The p(IκB)3-luc expresses luciferase under the control of a NF-κB-inducible promoter (9).

Transactivation and transrepression assays

CV-1 cells were cotransfected with pRShGRα or pRShGRαV575G (0.0125 μg/well) and pGL4.73[hRluc/SV40] (0.02 μg/well) together with pMMTV-luc (0.125 μg/well) (for transactivation assays) or pRSVC(p50)-NF-κB (0.0125 μg/well), pRSVC(p65)-RelA (0.0125 μg/well), and p(IκB)3-luc (0.125 μg/well) (for transrepression assays) using Lipofectin (Invitrogen). Forty-eight hours later, cells were exposed to dexamethasone or vehicle for 24 hours. Firefly and renilla luciferase activities were determined in the cell lysates (10).

Western blot analyses

CV-1 and COS-7 cells were transfected with pRShGRα or pRShGRαV575G (15 μg/flask) using Lipofectin (Invitrogen). Western blot analyses were performed as previously described (8).

Dexamethasone-binding assays

COS-7 cells were transfected with pRShGRα or hGRαV575G (1.5 μg/well) using Lipofectin (Invitrogen) and incubated with six different concentrations of [³H]-dexamethasone at 37°C in the presence or absence of a 500-fold molar excess of nonradioactive dexamethasone for 1 hour. Dexamethasone-binding assays were performed as previously described (8).

Nuclear translocation studies

HeLa cells were transfected with pF25GFP-hGRα or pF25GFP-hGRαV575G (2 μg/dish) using FuGENE 6. Nuclear translocation studies were performed as previously described (8).

Chromatin immunoprecipitation (ChIP) assays

HCT-116 cells were transfected with pRShGRα or pRShGRαV575G expressing plasmids using Lipofectamine 2000 (Invitrogen). ChIP assays were performed as previously described (10).

Glutathione-S-transferase (GST) pull-down assays

GST-fused glucocorticoid receptor-interacting protein 1 (GRIP1) (1–1462), GRIP1 (559–774), and GRIP1(740–1217) were bacterially produced, purified, and immobilized on GST beads. The in vitro interaction of pRShGRα and pRShGRαV575G with the GST-fused GRIP1 proteins was tested as previously described (10).

Structural biology studies

Molecular dynamics simulations for the hGR ligand-binding domain (LBD; PDB ID [Protein Data Bank ID]: 1M2Z) with and without the V575G mutation were performed in NAMD 2.7 using the CHARMM force field packaged within VMD 1.8.7 as previously described (10).

Results

Sequencing of the hGR gene

A single heterozygous T→G substitution was identified at nucleotide position 1724 in all three subjects. This mutation resulted in valine (Val) to glycine (Gly) substitution (GTG→GGG) at amino acid position 575 (exon 5) in the LBD of the receptor (Figure 1A).

Figure 1.

A, Sequencing of the entire coding region of the hGR gene revealed a novel heterozygous T→G substitution at nucleotide position 1724, resulting in replacement of Val by Gly (GTG→GGG) at amino acid position 575 (exon 5) in the LBD of the receptor. B, Transcriptional activity of the wild-type (WT) hGRα and the mutant receptor hGRαV575G. Compared with the wild-type receptor, the mutant receptor demonstrated an approximately 33% reduction in its ability to transactivate the glucocorticoid-inducible mouse mammary tumor virus promoter in response to increasing concentrations of dexamethasone. Bars represent mean ± SEM of at least five independent experiments. Asterisks indicate statistically significant differences. C and D, Subcellular localization and nuclear translocation studies of the wild-type and mutant receptors. GFP-hGRαV575G demonstrates delayed nuclear translocation compared with the GFP-hGRα after exposure to dexamethasone. HeLa cells transiently expressing GFP-hGRα (C) or GFP-hGRαV575G (D) were treated with 10⁻⁶ M of dexamethasone. Images of the same cells were obtained at the indicated time points.

hGRαV575G demonstrates decreased transactivational activity but does not exert dominant negative effect upon the wild-type hGRα

Compared with the wild-type receptor, the hGRαV575G demonstrated a significant reduction (33%) in its ability to transactivate the mouse mammary tumor virus promoter in response to dexamethasone (Figure 1B). Cotransfection with a constant amount of hGRα and increasing concentrations of hGRαV575G showed that hGRαV575G did not exert a dominant negative effect upon the hGRα. Western blot analyses demonstrated no differences in the expression of hGRα and hGRαV575G proteins.

hGRαV575G Displays Enhanced Ability to Transrepress the NF-κB Signaling Pathway Compared with the Wild-type hGRα

Compared with the hGRα, the hGRαV575G demonstrated significantly enhanced ability (∼80%) to transrepress the activity of NF-κB on the inhibitory-κB (IκB)-3 promoter in response to dexamethasone.

hGRαV575G demonstrates lower affinity for the ligand compared with the wild-type hGRα

The apparent dissociation constant of hGRαV575 was significantly higher than that of hGRα (11.6 ± 1.5 nM vs 6.0 ± 0.6 nM, P < .05), indicating that the affinity of the mutant receptor for the ligand was 50% lower than that of the wild-type receptor. No difference in the number of hGR binding sites was noted between hGRα and hGRαV575G.

hGRαV575G demonstrates delayed nuclear translocation compared with the wild-type hGRα

In the absence of dexamethasone, both the wild-type and the mutant receptors were localized in the cytoplasm of cells. Addition of dexamethasone (10⁻⁶ M) resulted in translocation of hGRα and hGRαV575G into the nucleus within 15 minutes (mean ± SE, 12.6 ± 0.4 min) and 36 minutes (mean ± SE, 34.8 ± 1.4 min; P < .001), respectively (Figure 1, C and D), suggesting a 2.6-fold delay in nuclear translocation of the hGRαV575G.

hGRαV575G preserves its ability to bind to DNA

ChIP assays showed no significant differences in the ability of hGRα and hGRαV575G to bind to glucocorticoid response elements of the endogenous G6Pase and GILZ genes.

hGRαV575G demonstrates impaired interaction with the GRIP1 coactivator due to defective activation function (AF)-2 domain

The hGRαV575G demonstrated decreased ability to interact with the nuclear receptor binding domain of GRIP1 and the full-length GRIP1, whereas its interaction to the carboxyl-terminal fragment of this coactivator was preserved. Therefore, the hGRαV575G displays an impaired interaction with the GRIP1 coactivator through its defective AF-2 surface.

hGRαV575G displays impaired interaction with the LXXLL motif in computer-based structural simulation

In the 3-dimensional crystallographic structural model of the hGRα LBD bound to dexamethasone, V575 is located in the C-terminal portion of helix 5. Val has a large, hydrophobic side chain, which protrudes into the AF-2 surface and creates a noncovalent interaction with the second and third leucine of the LXXLL motif (Figure 2Α). In the LBD of hGRαV575G, this Val residue is replaced by Gly. In our molecular simulation for hGRαV575G LBD, two noncovalent bonds observed between V575 and the leucines of the LXXLL motif were completely lost, suggesting that hGRαV575G displays impaired interaction with the LXXLL motif (Figure 2B). We further calculated the root mean square deviation for the helical residue containing the LXXLL motif (amino acids 743–750) of the human GRIP1 against the AF-2 surface. As expected, the hGRαV575G LBD demonstrated a 2-fold increase in the number of distance between LXXLL motif and AF-2 surface (Figure 2C); hence, this mutant LBD has reduced affinity for the LXXLL motif. We also demonstrated that the hGRαV575G LBD had a significantly wider AF-2 surface (Figure 2D). Taken together, these results provide a molecular explanation for the reduced transcriptional activity of hGRαV575G and its defective interaction with the NRB domain of the GRIP1 coactivator.

Figure 2.

Three-dimensional crystallographic structural model of the LBD of the wild-type (WT) hGRα bound to dexamethasone. In the wild-type receptor (A), the V575 creates a noncovalent interaction with the second and third leucine (L) of the LXXLL motif of the GRIP1 coactivator, thereby facilitating their interaction through the AF-2 domain of the hGRα. In the LBD of hGRαV575G (B), the replacement of Val by Gly resulted in a loss of the two noncovalent bonds and a reduced interaction of the mutant receptor with the LXXLL motif through an increase in the distance between the AF-2 domain and the LXXLL motif (C). Estimation of the area of the AF-2 surface of the WT and mutant receptors (D) indicated that the LBD of the mutant receptor had significantly wider AF-2 surface than that of the WT receptor. RMSD, root mean square deviation.

Discussion

In the present study, we identified a novel heterozygous point mutation in exon 5 of the hGR gene in three new cases of primary generalized glucocorticoid resistance, which resulted in substitution of Val to Gly at amino acid position 575 in the LBD of hGRα. We systematically investigated the molecular mechanisms through which hGRαV575G affects normal glucocorticoid signal transduction. The mutant receptor hGRαV575G demonstrated a significant reduction in its ability to transactivate glucocorticoid-responsive genes, had a 50% lower affinity for the ligand, and a 2.5-fold delay in nuclear translocation, it preserved its ability to bind to DNA and demonstrated an impaired interaction with the GRIP1 coactivator due to its defective AF-2 domain. Our findings suggest that hGRαV575G affects multiple steps in the glucocorticoid signaling pathway.

The reduced affinity of the mutant receptor for the ligand is most likely due to the location of the mutation within the LBD of the receptor, given that substitution of Val to Gly at amino acid position 575 in helix 5 might induce a conformational change of this domain. In addition, hGRαV575G showed a 2.5-fold delay in nuclear translocation, indicating that the V575G mutation alters the nucleocytoplasmic shuttling of hGRα. This defect might be caused by impairment of the nuclear localization sequence 1 and/or nuclear localization sequence 2 functions or occurs secondary to the reduced affinity for the ligand (8, 10–17).

In our molecular simulation for hGRαV575G LBD, we found that the V575 of hGRα is part of the AF-2 surface and that the hydrophobic side chain of this amino acid contributed directly to the attraction of the LXXLL motif by forming a noncovalent bond with its second and third leucine. The G575 of the mutant receptor lost these bonds to the LXXLL motif; therefore, it interacted with the NRB domain of GRIP1 less efficiently than the wild-type receptor. These results concur with our previous studies of other hGR mutations located in the LBD of the receptor; however, the hGRαV575G is the first mutant receptor that affects directly a residue within the AF-2 surface (8, 10–17).

Interestingly, the transrepression assays showed that hGRαV575G displayed enhanced ability to transrepress the NF-κΒ signaling pathway. Although the underlying mechanism is not known, a stronger interaction of the mutant receptor hGRαV575G with the p65 subunit of NF-κΒ heterodimer and/or reduced induction of glucocorticoid-responsive IκB by the activated mutant receptor might play a role.

The insensitivity to glucocorticoids in patients with pathological hGRα mutations accounts for the hyperactivity of HPA axis and consequent alterations in stress response (18, 19). The elevated CRH concentrations may be further accentuated by activation of the mineralocorticoid receptors in the hippocampus by glucocorticoids (20).

In summary, we identified and functionally characterized a novel point mutation in the hGR gene causing primary generalized glucocorticoid resistance. The hGRαV575G impairs multiple steps in the pathway of glucocorticoid signal transduction, including the affinity for the ligand, the time required for nuclear translocation, and the interaction with the GRIP1 coactivator, and expresses significant dissociation between its transactivating and transrepressive activities.