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A Novel Point Mutation in Helix 11 of the Ligand-Binding Domain of the Human Glucocorticoid Receptor Gene Causing Generalized Glucocorticoid Resistance

Section on Pediatric Endocrinology (E.C., T.K., T.I., K.Z., G.P.C.), Reproductive Biology and Medicine Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892; First Department of Pediatrics (G.P.C.), Athens University Medical School, Athens 11527, Greece; and Centro de Endocrinologia (W.J., L.M.), Metabolismo y Diabetes and Clinica, Fundacion Valle del Lili, Cali, Colombia

Address all correspondence and requests for reprints to: Evangelia Charmandari, M.D., Section on Endocrinology and Metabolism, Foundation for Biomedical Research of the Academy of Athens, 4 Soranou Efessiou, Athens 11527, Greece. E-mail: evangelia.charmandari{at}googlemail.com.

	Abstract

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Background: Generalized glucocorticoid resistance is a rare condition characterized by partial, end-organ insensitivity to glucocorticoids, compensatory elevations in adrenocorticotropic hormone and cortisol secretion, and increased production of adrenal steroids with androgenic and/or mineralocorticoid activity. We have identified a new case of glucocorticoid resistance caused by a novel mutation of the human glucocorticoid receptor (hGR) gene and studied the molecular mechanisms through which the mutant receptor impairs glucocorticoid signal transduction.

Methods and Results: We identified a novel, single, heterozygous nucleotide (T C) substitution at position 2209 (exon 9) of the hGR gene, which resulted in phenylalanine (F) to leucine (L) substitution at amino acid position 737 within helix 11 of the ligand-binding domain of the protein. Compared with the wild-type receptor, the mutant receptor hGRF737L demonstrated a significant ligand-exposure time-dependent decrease in its ability to transactivate the glucocorticoid-inducible mouse mammary tumor virus promoter in response to dexamethasone and displayed a 2-fold reduction in the affinity for ligand, a 12-fold delay in nuclear translocation, and an abnormal interaction with the glucocorticoid receptor-interacting protein 1 coactivator. The mutant receptor preserved its ability to bind to DNA and exerted a dominant-negative effect on the wild-type hGR only after a short duration of exposure to the ligand.

Conclusions: The mutant receptor hGRF737L causes generalized glucocorticoid resistance because of decreased affinity for the ligand, marked delay in nuclear translocation, and/or abnormal interaction with the glucocorticoid receptor-interacting protein 1 coactivator. These findings confirm the importance of the C terminus of the ligand-binding domain of the receptor in conferring transactivational activity.

	Introduction

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GENERALIZED GLUCOCORTICOID resistance is a rare condition characterized by partial, end-organ insensitivity to glucocorticoids, compensatory elevations in circulating cortisol and ACTH concentrations and resistance of the hypothalamic-pituitary-adrenal axis to dexamethasone suppression (1, 2, 3, 4). The excess ACTH secretion results in increased production of adrenal steroids with mineralocorticoid and/or androgenic activity and the corresponding clinical phenotype(s). The molecular basis of the condition has been ascribed to mutations in the human glucocorticoid receptor (hGR) gene, which impair glucocorticoid signal transduction, thereby altering tissue sensitivity to glucocorticoids. Inactivating mutations within the ligand-binding (LBD) and DNA-binding domains of the receptor and a 4-bp deletion at the 3'-boundary of exon 6 of the gene have been described in five kindreds and four sporadic cases (5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15).

In the present study, we report a new case of generalized glucocorticoid resistance caused by a novel, heterozygous, point mutation of the hGR gene, and we present the molecular mechanisms through which the mutant receptor impairs glucocorticoid signal transduction.

	Subject and Methods

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Case report

A 7-yr-old boy presented with severe hypertension and hypokalemia. He had been treated with ß-blockers, calcium channel blockers, and large doses of potassium supplements with no improvement. The past medical history and family history were unremarkable. On examination, he had elevated blood pressure (BP) [systolic BP 180 mm Hg (+11 SD score [SDS]), diastolic BP 120 mm Hg (+8.62 SDS)] but no evidence of hyperandrogenism and no signs suggestive of Cushing’s syndrome. His weight was 31.0 kg (+2.58 SDS), his height 133.8 cm (+2.56 SDS), and his body mass index 17.3 kg/m² (+0.9 SDS). Biochemical and endocrinologic evaluation at presentation revealed hypokalemia (2.6 mmol/liter); elevated serum cortisol concentrations, which maintained circadian rhythmicity (0800 h cortisol: 160 µg/dl, 1700 h cortisol: 50 µg/dl; normal range 8–19 µg/dl); and elevated 0800 h plasma ACTH (425 pg/ml; normal range 10–60 pg/ml) concentrations. A low (1 mg) and high (8 mg) dose overnight dexamethasone suppression test revealed resistance of the hypothalamic-pituitary-adrenal axis to dexamethasone suppression (0800 h serum cortisol: 29 and 15 µg/dl, respectively). Abdominal computed tomography scan confirmed bilateral adrenal hyperplasia. Chest x-rays and magnetic resonance scans of the pituitary and hypothalamic regions did not reveal any pathology. Written informed consent was obtained from the parents of the patient for further molecular studies.

Sequencing of the hGR gene

Genomic DNA was extracted from peripheral lymphocytes, and the entire coding region of the hGR gene was amplified by the PCR and sequenced as previously described (14).

Plasmids

The plasmids used in this study included pRShGR, pF25GFP-hGR, pBK/CMV-hGR, pMMTV-luc, pSV40-ß-gal, pRSV-erbA^–1, pGEX4T3-GRIP1(1–1462), pGEX4T3-GRIP1 (596–774), and pGEX4T3-GRIP1(740–1217) (12, 13, 14, 15). The plasmids pRShGRF737L, pF25GFP-hGRF737L, and pBK/CMV-hGRF737L were constructed by introducing the F737L mutation into pRShGR, pF25GFP-hGR, and pBK/CMV-hGR, respectively, using PCR-assisted site-directed mutagenesis (12, 13, 14, 15).

Transactivation assays

CV-1 cells were cotransfected with pRShGR, pRShGRF737L, or pRSV-erbA^–1 (0.05 µg/well), pMMTV-luc (0.5 µg/well), and pSV40-ß-gal (0.1 µg/well) using lipofectin. In further experiments, cells were cotransfected with pMMTV-luc, pSV40-ß-gal, a constant amount of pRShGR (0.05 µg/well), and progressively increasing concentrations of pRShGRF737L. Forty-eight hours after transfection, cells were exposed to dexamethasone for 3–24 h. Luciferase and ß-galactosidase activities were determined in the cell lysates as previously described (12, 13, 14, 15).

Western blot analyses

CV-1 and COS-7 cells were transfected with pRShGR or pRShGRF737L (15 µg/flask) using lipofectin. Western blot analyses were performed as previously described (13, 14, 15).

Dexamethasone-binding assays

COS-7 cells were transfected with pRShGR or pRShGRF737L (1.5 µg/well) using lipofectin. Confluent cells were 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 h. Dexamethasone-binding assays were performed as previously described (13, 14, 15).

Nuclear translocation studies

HeLa cells were transfected with pF25GFP-hGR or pF25GFP-hGRF737L (2 µg/dish) using FuGENE 6 according to the instructions of the manufacturer (Roche Diagnostics Corp., Indianapolis, IN). In further experiments, cells were transfected with pF25GFP-hGR and pRShGRF737L (1.5 µg/dish). Nuclear translocation studies were performed as previously described (13, 14, 15).

Chromatin immunoprecipitation assays

HCT-116 cells, in which the mouse mammary tumor virus (MMTV) promoter was stably integrated within chromatin, were transiently transfected with pRShGR or pRShGRF737L (10 µg/dish). Chromatin immunoprecipitation assays were performed as previously described (14, 15, 16).

Glutathione-S-transferase pull-down assay

In vitro transcription/translation reactions were used to produce ³⁵S-labeled hGR and hGRF737L in rabbit reticulocyte lysate by using pBK/CMV-hGR and pBK/CMV-hGRF737L, respectively, as templates. The in vitro interaction between hGR-related plasmids and glutathione-S-transferase-fused glucocorticoid receptor-interacting protein 1 (GRIP1) proteins was tested as previously described (13, 14, 15).

	Results

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Sequencing of the hGR gene

A single, heterozygous thymine to cytosine (T C) substitution was identified at nucleotide position 2209 in exon 9 of the gene, which resulted in phenylalanine (F) to leucine (L) substitution at amino acid position 737 in the LBD of the receptor. Using the three-dimensional crystal structure of the LBD of hGR, we determined that the F737L mutation is located in helix 11 of this domain (17) (Fig. 1A).

View larger version (24K): [in this window] [in a new window]   FIG. 1. A, Crystal structure of the agonist-bound (left panel) and antagonist-bound (right panel) ligand-binding domain of hGR. The upper arrows indicate the position of helix 11 of the receptor. The lower arrows indicate the position of the F737L mutation (in yellow) identified in our patient. B, Transcriptional activity of the wild-type hGR and mutant receptor hGRF737L. Compared with the wild-type receptor, the mutant receptor demonstrated a 2-fold reduction in its ability to transactivate the MMTV promoter in response to dexamethasone. C, Absence of a dominant-negative effect of the mutant receptor hGRF737L on the wild-type hGR after exposure to the ligand for 24 h. D, Dominant-negative effect of the mutant receptor hGRF737L on the wild-type hGR after exposure to the ligand for 3 and 6 h. Cotransfection with a constant amount of hGR and progressively increasing concentrations of hGRF737L revealed a dose-dependent inhibition of the hGR-mediated transactivation of the MMTV promoter only after a shorter duration (up to 6 h) of exposure to dexamethasone. Bars represent mean ± SE of at least five independent experiments.

The mutant receptor hGRF737L displays decreased transcriptional activity and exerts a dominant negative effect upon the wild-type hGR only after short-duration exposure to the ligand

Compared with hGR, hGRF737L displayed a 2-fold reduction in its ability to transactivate glucocorticoid-responsive genes (Fig. 1B). The decreased transcriptional activity of hGRF737L was ligand-exposure time-dependent and progressively more pronounced at shorter exposure times. Cotransfection with a constant amount of hGR and increasing concentrations of hGRF737L did not result in a dose-dependent inhibition of hGR-mediated transactivation of the MMTV promoter, suggesting that hGRF737L does not exert a dominant-negative effect on the wild-type hGR (Fig. 1C). The latter does not preclude a partial dominant-negative effect in more complex transcriptional systems or in different cell lines. Indeed, exposure to dexamethasone for shorter periods of time (3 and 6 h) revealed a dominant-negative effect of hGRF737L on hGR (Fig. 1D).

The mutant receptor hGRF737L demonstrates decreased affinity for the ligand

The apparent dissociation constant of hGRF737L was significantly higher than that of hGR (13.9 ± 1.7 vs. 7.8 ± 0.3 nM, P = 0.006), indicating that hGRF737L had a 2-fold lower affinity for the ligand than hGR. No difference in the number of dexamethasone-binding sites was noted between hGR and hGRF737L. Western blot analyses demonstrated no differences in the expression of hGR and hGRF737L proteins in CV-1 or COS-7 cells, indicating that the above-described findings did not reflect differences at the protein expression level.

The mutant receptor hGRF737L demonstrates marked delay in nuclear translocation

In the absence of ligand, hGR was primarily localized in the cytoplasm of cells. Exposure to dexamethasone resulted in nuclear translocation of the receptor within 15 min (14.75 ± 0.25 min) (Fig. 2A). The mutant receptor hGRF737L was observed in both the cytoplasm and nucleus of cells in the absence of ligand, whereas exposure to the same concentration of dexamethasone induced a 12-fold delay in nuclear translocation (175.00 ± 5.00 min) (Fig. 2B). Coexpression of hGR and hGRF737L at a 1:1 ratio had no apparent effect on the nuclear translocation of hGR (Fig. 2C).

View larger version (24K): [in this window] [in a new window]   FIG. 2. Nuclear translocation of GFP-hGR (A), GFP-hGRF737L (B), and GFP-hGR in the presence of pRShGRF737L (C) before and after exposure to dexamethasone. HeLa cells transiently expressing GFP-hGR or GFP-hGRF737L were exposed to the same concentration of dexamethasone (10–6 M). Images of the same cells were obtained at the indicated time points.

The mutant receptor hGRF737L preserves its ability to bind to DNA

Both hGR and hGRF737L coprecipitated with MMTV glucocorticoid-response elements similarly, in a ligand-dependent fashion, suggesting that hGRF737L preserves its ability to bind to DNA.

The mutant receptor hGRF737L interacts with the GRIP1 coactivator in vitro only through its activation function (AF)-1

Both hGR and hGRF737L bound to full-length GRIP1; however, hGRF737L did not demonstrate a ligand-dependent increase in its interaction with GRIP1. Also, although hGR interacted with the amino-terminal fragment of GRIP1 in a ligand-dependent fashion, there was no interaction between hGRF737L and this fragment of GRIP1. Both hGR and hGRF737L bound to the carboxyl-terminal fragment of GRIP1 in a ligand-independent fashion. These results suggest that hGRF737L interacts with the GRIP1 coactivator in vitro only through its AF-1.

	Discussion

Top Abstract Introduction Subject and Methods Results Discussion References

 
We have identified a novel, heterozygous point mutation in exon 9 of the hGR gene and studied the molecular mechanisms through which the mutant receptor impairs glucocorticoid signal transduction. We showed that hGRF737L demonstrated ligand-exposure time-dependent decreased transcriptional activity, reduced affinity for the ligand, and a marked delay in nuclear translocation. The mutant receptor preserved its ability to bind to glucocorticoid-response elements, interacted with the GRIP1 coactivator in vitro only through its AF-1 domain, and exerted a dominant-negative effect on the transcriptional activity of hGR only after up to 6 h of exposure to dexamethasone. These findings suggest that hGRF737L causes generalized glucocorticoid resistance by affecting multiple steps in the cascade of hGR action, and further underscore the importance of the C terminus of hGR LBD in conferring transactivational activity.

The decreased affinity of the mutant receptor hGRF737L for the ligand most likely reflects the location of the F737L mutation in helix (H) 11 of the LBD of hGR. The structure of the hGR LBD contains 12 -helices and four small ß-strands that fold into a three-layer helical domain (17) (Fig. 1). The ligand-binding pocket of hGR has a similar architecture to that of other nuclear receptors and can be described as a cavity closed by a lid, which involves residues from H11 and H12. Divergent residues contributing to the ligand-binding pocket may determine ligand specificity. For example, the C742G mutation of the mouse glucocorticoid receptor, which corresponds to the C736G mutation of hGR and is located in the lid region of the hGR ligand-binding pocket in H11, has been associated with dexamethasone-resistant lymphoma (18). Therefore, the presence of the F737L mutation in the LBD of the receptor may affect the affinity of the receptor for the ligand directly.

Upon ligand binding, the receptor undergoes major conformational changes, which alter the position of H11 and H12 and generate an interaction surface that allows coactivators to bind to the LBD through their LXXLL motifs. That hGRF737L interacted with the GRIP1 coactivator in vitro only through its AF-1 highlights the importance of H11 of the LBD of the receptor in facilitating the formation of the AF-2 surface that interacts with coactivators (17, 18).

The mutant receptor hGRF737L was localized in both the cytoplasm and nucleus of cells in the absence of ligand, whereas exposure to dexamethasone induced a markedly delayed nuclear translocation, which required up to 3 h. These findings suggest that the F737L mutation affects the nucleocytoplasmic shuttling of hGR, probably through impairment of nuclear localization signal (NL) 1 and/or NL2 function (19). Given that the hGRß isoform, which has a defective LBD, as well as hGR mutants lacking their LBD, constitutively localize primarily in the nucleus (10), it is likely that the LBD of hGR plays an important role in the cytoplasmic retention of the receptor in the absence of ligand. Therefore, a single-point mutation resulting in amino acid substitution within the LBD of hGR might alter this activity of the LBD, resulting in nuclear retention of the receptor. Alternatively, defective mechanisms that relate to delayed nuclear export might account for the nuclear localization of the unliganded hGRF737L (20), an effect that might be similar to the nuclear retention of hGRß (10).

We conclude that hGRF737L causes generalized glucocorticoid resistance by affecting multiple steps in the cascade of the glucocorticoid receptor signaling pathway. These findings confirm the importance of the C terminus of the LBD of hGR in conferring transactivational activity.

	Footnotes

 
This work was supported in part by the intramural program of the National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland.

Disclosure Statement: The authors have nothing to disclose.

First Published Online July 17, 2007

Abbreviations: AF, Activation function; BP, blood pressure; F, phenylalanine; GRIP1, glucocorticoid receptor-interacting protein 1; H, helix; hGR, human glucocorticoid receptor; L, leucine; LBD, ligand-binding domain; MMTV, mouse mammary tumor virus; SDS, SD score.

Received December 20, 2006.

Accepted July 10, 2007.

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This Article Abstract Full Text (PDF) Submit a related Letter to the Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Copyright Permission Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Charmandari, E. Articles by Chrousos, G. P. Search for Related Content PubMed PubMed Citation Articles by Charmandari, E. Articles by Chrousos, G. P. Related Collections Adrenal and Hypertension