| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
The Impact of the Human Genome on Endocrinology: Original Articles |
Pediatric and Reproductive Endocrinology Branch (A.V., T.K., G.P.C.), National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892; and Endocrine Unit, Medicine B (H.C., P.L.), Centre Hospitalier Universitaire de Tours, 37044 Tours, France
Address all correspondence and requests for reprints to: Tomoshige Kino, M.D., Pediatric and Reproductive Endocrinology Branch, National Institute of Child Health and Human Development, National Institutes of Health, Building 10, Room 9D42, 10 Center Drive, MSC 1583, Bethesda, Maryland 20892-1583. E-mail: . kinot{at}mail.nih.gov
Abstract
Primary cortisol resistance is a rare, inherited or sporadic form of generalized end-organ insensitivity to glucocorticoids. Here, we report a kindred in which affected members had a heterozygous T to G base substitution at nucleotide 2373 of exon 9
of the GR gene, causing substitution of Ile by Met at position 747. This mutation was located close to helix 12, at the C terminus of the ligand-binding domain, which has a pivotal role in the formation of activation function (AF)-2, a subdomain that interacts with p160 coactivators. The affinity of the mutant GR for dexamethasone was decreased by about 2-fold, and its transcriptional activity on the glucocorticoid-responsive mouse mammary tumor virus promoter was compromised by 20- to 30-fold. In addition, the mutant GR functioned as a dominant negative inhibitor of wild-type receptor-induced transactivation. The mutant GR through its intact AF-1 domain bound to a p160 coactivator, but failed to do so through its AF-2 domain. Overexpression of a p160 coactivator restored the transcriptional activity and reversed the negative transdominant activity of the mutant GR. Interestingly, green fluorescent protein (GFP)-fused GRI747M had a slight delay in its translocation from the cytoplasm into the nucleus and formed coarser nuclear speckles than GFP-fused wild-type GR. Similarly, a GFP-fused p160 coactivator had a distinctly different distribution in the nucleus in the presence of mutant vs. wild-type receptor, presenting also as coarser speckling. We conclude that the mutation at amino acid 747 of the GR causes familial, autosomal dominant glucocorticoid resistance by decreasing ligand binding affinity and transcriptional activity, and by exerting a negative transdominant effect on the wild-type receptor. The mutant receptor has an ineffective AF-2 domain, which leads to an abnormal interaction with p160 coactivators and a distinct nuclear distribution of both.
GLUCOCORTICOID RESISTANCE IS a rare, familial or sporadic condition that is characterized by generalized end-organ inability to respond to normal glucocorticoid levels (1). The syndrome has been associated with qualitative and/or quantitative abnormalities of the GR, primarily low affinity for glucocorticoids, low concentrations, or abnormal stability (2). Inactivating mutations within the ligand-binding domain (LBD) of the GR, as well as a microdeletion of an exon-intron boundary of the GR gene, have been described in three kindreds and a sporadic case (3, 4, 5, 6). Three of these mutations influence both splice products of the human GR gene, namely the classic GR isoform and its nonligand-binding isoform ß (3, 5, 6). The latter exerts a specific dominant-negative effect on the function of the former; however, its role in the physiological modulation of glucocorticoid actions remains uncertain (7, 8, 9, 10, 11). Recently, we described a genetically determined, imbalanced expression of GR and GRß in cultured lymphocytes from a patient with generalized glucocorticoid resistance complicated by chronic leukemia and whose GR gene-coding regions were normal (12).
Binding of glucocorticoids to GR causes it to dissociate from a cytoplasmic hetero-oligomer with heat shock proteins and induces its translocation into the nucleus (13). The ligand-bound GR is imported into the nucleus with the assistance of two nuclear localization signals (NLS), NLS1 and NLS2 (14). After entering the nucleus, GR rapidly binds as a homo- or heterodimer with GRß to specific DNA enhancer elements, the glucocorticoid-response elements (GREs), to change the transcriptional activity of glucocorticoid-dependent genes (15). GR also influences the activities of other transcription factors, such as activator protein 1, nuclear factor 
B, and several signal transducers and activators of transcription, through protein-protein interactions, possibly as a monomer (13).
The ability of the ligand-bound GR to transactivate a steroid-responsive gene depends on the presence of activation function (AF)-1 and AF-2 interacting, bridging nucleoproteins, the coactivators, which have chromatin-remodeling and other enzymatic activities (16). The known coactivators of steroid receptors belong to several families. These include the p160 family, the recently described riboprotein coactivator steroid receptor RNA activator (SRA), the cAMP-responsive element-binding protein (CREB)-binding protein (CBP) and p300 family, and the CBP/p300-associated factor, PCAF. Both the p160 and CBP/p300 coactivators have multiple amphipathic LXXLL signal sequences (the coactivator motifs or nuclear receptor boxes), which, upon binding the ligand, serve as interfaces between these coactivators and a hydrophobic cleft formed by helixes 3, 4, and 12 of the nuclear receptors (17, 18, 19). p160 proteins may also interact directly and/or via the riboprotein SRA with the AF-1 domain (20). The p160, CBP/p300, and PCAF proteins all have histone acetylase activity, which loosens the DNA strands from the nucleosomes and allows the transcription initiation complex of RNA-polymerase II and its ancillary components, the TATA Box binding protein-associated factors, to initiate and promote transcription (21).
Glucocorticoid resistance is characterized by compensatory elevations of circulating ACTH and cortisol, which, although set at levels higher than normal, maintain a circadian rhythm and responsiveness to stressors, and no overt clinical evidence of hypo- or hypercortisolism (1). Of interest, the molecular mechanisms via which glucocorticoids exert their negative feedback effects on central nervous system genes regulating the hypothalamic-pituitary-adrenal axis, such as those of CRH and ACTH, involve both interaction with GREs and protein-protein interactions with other transcription factors controlling the activity of some of these genes (13, 15). The spectrum of clinical manifestations of glucocorticoid resistance is quite broad, varying from completely silent to mildly or severely symptomatic, including manifestations of androgen and/or mineralocorticoid excess (1, 2). The former may manifest as precocious puberty in children and as acne, hirsutism, male-pattern baldness, menstrual irregularities, and oligo-anovulation and infertility in women; the latter may appear as hypertension and/or hypokalemic alkalosis in either gender. In two kindreds, the presenting symptom was chronic fatigue (1, 2).
In this study, we assessed the molecular mechanisms of familial glucocorticoid resistance in a French family. Study of the structure and function of the GR gene, mRNA, and/or protein suggested that glucocorticoid resistance in this family was due to a novel missense mutation of the GR gene exon 9, which affected the sequence of only this isoform of the receptor. This mutation led to abnormal receptor interactions with p160 nuclear receptor coactivators and aberrant nuclear distribution of both the receptor and its coactivators.
Patients and Methods
Patients
The proband was an 18-yr-old woman (height, 155 cm; weight, 41 kg; blood pressure, 110/60 mm Hg) with hyperandrogenism, elevated plasma cortisol concentrations (1700 mmol/liter; normal < 700), and increased 24-h urinary free cortisol (UFC) excretion (1200 nmoles/d; normal < 220), but no stigmata of Cushing’s syndrome (Fig. 1A
). She had her menarche at age 13 yr, developed cystic acne and hirsutism at puberty, and had persistent oligo-amenorrhea. Her two brothers and her father were phenotypically silent, but her brothers had elevated serum cortisol (840 and 960 nmoles/liter) and UFCs (696 and 770 nmoles/d), whereas her mother had normal morning serum cortisol (<700 nmoles/liter) and UFC (<220 nmoles/d) values.
|
Epstein-Barr virus-transformed lymphoblast lines were established from peripheral blood lymphocytes from all members of the family. Cells were grown in RPMI 1640 medium, supplemented with 10% FBS in the presence of 100 U/ml penicillin and 100 µg/ml streptomycin (Life Technologies, Inc., Gaithersburg, MD).
Amplification of genomic DNA by PCR and sequencing of amplified DNA
Genomic DNA was extracted from Epstein-Barr virus-transformed lymphocytes using a rapid extraction protocol (QIAamp Blood, QIAGEN, Chatsworth, CA), according to the instructions provided by the manufacturer. PCR amplification of the GR gene was carried out using previously reported primer sequences (5). For the amplification of exons 3 through 9, intronic primer pairs complementary to sequences in the 5' and 3' exon-flanking regions were used. Exon 2, which encodes the entire N-terminal part of the GR, was amplified in three overlapping segments. PCR was performed in a GeneAmp PCR System 9700 (Perkin-Elmer Corp., Emeryville, CA) using Ampli-Taq polymerase (Perkin-Elmer Corp.).
The samples were analyzed for their sequence by using ABI PRIZM dRhodamine Terminator Cycle Sequencing Ready Reaction Kit (PE Applied Biosystems, Foster City, CA) and the ABI PRIZM Genetic Analyzer (PE Applied Biosystems).
Transformed and tumor cell lines
COS-7, CV-1, and HeLa cells, purchased from American Type Culture Collection (Rockville, MD), were cultured in DMEM that was enriched as described above for RPMI 1640.
Plasmids
Expression vectors pRShGRI747M, pBK/CMV-GRI747M, and pF25-hGRI747M, which carry the identified mutant GR cDNA, were constructed by PCR-assisted site-directed mutagenesis (Stratagene, La Jolla, CA), using pRShGR, pBK/CMV-hGR, and pF25-hGR as templates, respectively. pRShGR and pRSVerbA-1 were kindly provided by Dr. R. M. Evans (Salk Institute, La Jolla, CA). pGEX4T3-Glucocorticoid receptor-interacting protein 1 (GRIP1) (1–1462), and the -GRIP1 (596–774) and -GRIP1 (740–1217) expressing plasmids, which respectively contain an AF-2-directed GR binding site nuclear receptor interacting domain (NID) and an AF-1-directed GR binding site with NIDaux (22, 23, 24), were constructed by subcloning corresponding GRIP1 fragment cDNAs into pGEX-4T3 plasmid (Amersham Pharmacia Biotech, Piscataway, NJ). pSG5-GRIP1 full-length (fl) was a kind gift from Dr. M. Stallcup (University of Southern California, Los Angeles, CA). pM-GRIP1 fl, pM-GRIP1 (1101–1462), and pM-GRIP1 (1–740) were constructed by subcloning the corresponding fragment cDNAs into pM (CLONTECH Laboratories, Inc., Palo Alto, CA) (25). pEGFP-SRC1e was a kind gift from Dr. M. A. Mancini (Baylor College of Medicine, Houston, TX). pMMTV-luc, which codes for the luciferase cDNA under the control of the murine mammary tumor virus (MMTV) long terminal repeat promoter (which contains four GRE sites), was a kind gift from Dr. G. Hager (National Institutes of Health, Bethesda, MD). pSG5 and pSV40-ß-Gal were purchased from Stratagene and Promega Corp. (Madison, WI), respectively.
Transient transfection assay
COS-7 and CV-1 cells were transfected using lipofectin (Life Technologies, Inc.), and HeLa cells were transfected with lipofectin or the CaPO4 method, as previously described (26). COS-7 cells were transfected with 1.5 µg/well of either pRShGR or pRShGRI747M for whole cell ligand binding studies. For reporter assays, CV-1 cells were transfected with 0.05 µg/well of pRShGR or pRShGRI747M together with 0.3 µg/well of pMMTV-luc and 0.1 µg/well of pSV40-ß-Gal. Twenty-four hours after transfection, the cells were incubated with graded concentrations of dexamethasone (0, 10-12 to 10-5 M) over 24 h. Cell lysates were collected, and luciferase and ß-galactosidase activities were measured, as previously described (26). To examine the potential transdominant activity of GRI747M, CV-1 cells were transfected with 0.05 µg/well of pRShGR with increasing concentrations of pRShGRI747M (ratio, 1:0 to 1:10) together with 0.3 µg/well of pMMTV-luc and 0.1 g/well of pSV40-ß-Gal. To further address the effect of GRIP1 on the transdominant activity of GRI747M, 0.3 µg/well of plasmids expressing GRIP1 proteins were added. To keep the same amount of DNA in transfection, pRS-erbA-1 for pRShGR or pRShGRI747M, pSG5 for pSG5-GRIP1 fl and pM for pM-GRIP1s were used. The cells were similarly treated with appropriate doses of dexamethasone, and cell lysates were collected for luciferase and ß-galactosidase activity (26). All measurements were conducted in triplicate, and luciferase values were corrected by the ß-galactosidase values.
Whole-cell dexamethasone binding assay
COS-7 cells transfected with wild-type or mutant GR-expressing vectors were incubated in RPMI 1640 with six concentrations of [3H] dexamethasone (1.56–50 nM) at 37 C in the presence or absence of a 500-fold molar excess of unlabeled dexamethasone for 1 h. After incubation, the cells were washed three times with cold PBS to remove free steroid. Specific binding was calculated by subtracting nonspecific from total binding, and these data were analyzed as proposed by Scatchard. Binding capacity was expressed as femtomoles per 106 cells, and the apparent dissociation constant (Kd) was expressed in nanomoles.
Gel mobility shift assay
COS-7 cells were transfected with 2.0 µg/ml pRShGR or pRShGRI747M as described. Twenty-four hours after transfection, the medium was replaced with either normal medium or medium containing dexamethasone 10-7 M. Forty-eight hours after transfection, cells were washed twice with PBS, and nuclear extracts were prepared as previously described (27). Four picomoles of the 22-bp double-stranded GRE probe (5'-GATCAGAACACAGTGTTCTCTA- 3', purchased from Stratagene, was labeled using 4 U T4 polynucleotide kinase (Roche Molecular Biochemicals, Indianapolis, IN) and 25 µCi 
[32P]ATP (6,000 Ci/mmol; Amerham-Pharmacia Biotech). Fifteen micrograms of undiluted nuclear extract protein were co-incubated with 100,000 cpm of 32P-labeled GRE probe for 20 min on ice, and then 15 min at room temperature. Supershift was tested using a specific anti-GR polyclonal antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). PAGE was performed in a 0.5 Tris/Borate/EDTA buffer at constant voltage (130 V) for 3 h. The gel was dried, and radioactivity was visualized by autoradiography.
In vitro translation of GRs and production of glutathione-S-transferase (GST)-fused GRIP1s
We produced in vitro translated and 35S-labeled GR, GRI747M using pBK/CMV-hGR and pBK/CMV-hGRI747M as templates, respectively, as well as bacterially expressed GST-fused GRIP1, GRIP1 (596–774) or GRIP1 (740–1217), as previously described (26).
GST pull-down assay
In vitro interaction of GRs and GRIP fragments was tested using above in vitro translated and labeled GR or GRI747M, and bacterially produced GST-fused GRIP1, GRIP1 (596–774) or GRIP1 (740–1217), which contain binding sites for AF-2 or AF-1 of nuclear receptors, respectively. In vitro binding assays were performed as described previously (26). Samples were loaded and electrophoresed on an 8% SDS-PAGE gel, and the gel was fixed and dried as previously described (26). Radioactivity was detected by exposing a film on the gel.
Detection and localization of green fluorescent protein (GFP)-fused receptors and SRC1e
COS-7 or HeLa cells were plated on coated 25-mm glass-bottom dishes (MatTek Corporation, Ashland, MA) in phenol red-free DMEM containing 10% charcoal-treated FBS (HyClone Laboratories, Inc., Logan, UT). The cells were transfected with the indicated wild-type- or mutant receptor-expressing plasmids 24 h later. For the experiments examining trafficking of wild-type or mutant receptor, 10-6 M dexamethasone was added after 48 h of transfection, and fluorescence was detected sequentially by an inverted fluorescence microscope (Zeiss Axiovert 135; Carl Zeiss, Thornwood, NY), as described previously (28). For experiments using EGFP-fused SRC1e, 10-6 M dexamethasone or vehicle was added to the dishes 24 h after transfection. Forty-eight hours after transfection, and after 24-h incubation with dexamethasone, the fluorescence was analyzed with the same microscope. Twelve-bit black-and-white images were captured using a digital charge-coupled device camera (Photometrix, Tucson, AZ). Image analysis and presentation were performed using the IPLab Spectrum software (Scanalytics, Vienna, VA).
Results
Sequencing
The deduced mRNA nucleotide sequence of the GR from sequencing the GR gene (exons 2–8, 9 and 9ß) was normal except for a heterozygous base change at cDNA nucleotide position 2373, corresponding to exon 9, where the nucleotide thymidine of the wild-type sequence was substituted by guanosine (codon ATT vs. ATG) (Fig. 1B
). This predicted an amino acid change from Ile to Met at position 747, close to the C terminus of the LBD but outside the ligand-binding pocket (Fig. 2). This novel point mutation was present in the heterozygous state in all of the affected members of the family, but not in the unaffected mother who had a normal sequence (Fig. 1). To confirm the presence of the mutation and, therefore, exclude any PCR artifacts, sequencing analysis was performed several times.
|
). Properties of transfected mutant GR
Below we describe the results of the studies undertaken to examine the impact of the mutation identified on the functions of the GR. Binding studies performed in COS-7 cells transfected with either the mutant or wild-type receptors demonstrated that the former had a 2-fold increased Kd for dexamethasone (Kd, 29 ± 7.7 vs. 13.9 ± 2.6 nM), but no difference in Bmax. Western blots demonstrated similar levels of expression of both wild-type and mutant receptors in the transfected cells (data not shown).
In the absence of dexamethasone, GFP-GRI747M, similarly to GFP-GR, was primarily located in the cytoplasm of HeLa cells (Fig. 3A). Addition of 10-6 M dexamethasone induced rapid translocation of the mutant and wild-type receptor into the nucleus. The former translocated at a visibly slower rate and formed coarser speckles than the wild-type receptor (Fig. 3B).
|
I747M and the wild-type receptor expressed in COS-7 cells in the presence of 10-6 M dexamethasone bound similarly to a radiolabeled double stranded consensus GRE-containing oligonucleotide in a gel shift mobility assay, suggesting that DNA binding activity of the mutant receptor was preserved (data not shown).
In CV-1 cells cotransfected with the pMMTV-luc, the mutant receptor had an effective concentration (EC50) of dexamethasone that was approximately 20- to 30-fold higher than that of the wild-type receptor, indicating an impaired ability to activate GRE-mediated gene expression (Fig. 4A). It was approximately 4- to 5-fold less potent than the wild-type receptor at 10-9 M dexamethasone and half as potent at 10-6 M dexamethasone. Increasing amounts of transfected pRShGRI747M cotransfected with a constant concentration of the wild-type receptor inhibited GR transactivation function, leading from a 25% suppression at 1:1 GR/GRI747M ratio to an 85% suppression at the 1:10 GR/GRI747M ratio (Fig. 4B).
|
). One site, located N-terminally between amino acids 542 and 745, contains three LXXLL sequences known as coactivator signature motifs or nuclear receptor boxes (17, 31) and interacts with the AF-2 domain of steroid receptors in a ligand-dependent fashion. The other site, located C-terminally between amino acids 1121 and 1250, binds to the AF-1 domain of steroid receptors in a ligand-independent fashion (23, 24) (Fig. 5A). In vitro translated and labeled GR or GRI747M and bacterially produced GST-fused GRIP1, GRIP1 (596–774) and GRIP1 (740–1217) had the following interactions in a GST pull-down assay: both mutant and wild-type receptors interacted with full-length GRIP1; the mutant receptor did not bind to GRIP1 (596–774), whereas the wild-type receptor did; the mutant receptor bound to GRIP1 (740–1217) similarly to the wild-type receptor (Fig. 5B).
|
). Overexpression of GRIP1 did not change mutant receptor-driven transactivation activity at 10-9 M dexamethasone but moderately corrected it at high concentrations of dexamethasone. GRIP1 completely abolished the transdominant negative effect of GRI747M to the wild-type receptor. Overexpression of GRIP1 (1–774), however, did not influence the transdominant negative effect of the mutant receptor.
|
). In the absence of dexamethasone, EGFP-SRC1e was homogeneously spread in the nucleus of both wild-type and mutant receptor-expressing cells. In the presence of 10-6 M dexamethasone, however, it showed fine granular distribution in the nucleus of cells containing the wild-type receptor, whereas it demonstrated coarse granular distribution in the nucleus of cells containing the mutant receptor.
|
The heterozygous mutation at amino acid residue 747 in the C terminus of the LBD of the GR identified in affected members of this family lay outside the ligand-binding pocket but decreased the affinity of the receptor for dexamethasone by about 2-fold. This affinity defect was associated with an 8-fold elevation of 24-h UFC excretion in affected members of this family (eight times the normal mean value). Previously reported patients with glucocorticoid resistance, in whom one GR allele was not expressed, had a 6-fold elevation in UFC (5), whereas in two other families with point mutations of the receptor, 3- and 2-fold decreases in the affinity of the GR were associated with 40-fold (3) and 6-fold (4, 32) elevations of UFC, respectively. The differences in the UFC elevation between members of this family (8-fold) and the family with the one allele knockout (6-fold) suggest that in this family the mutant receptor has a dominant negative effect on the wild-type receptor expressed biochemically by compensatory elevations of cortisol secretion. On the basis of UFC excretion, the dominant negative effect of this family’s mutant receptor was less than that of the single sporadic case reported by Karl et al. (6) in which the UFC was approximately 12-fold higher than normal.
The 747 mutation was a few amino acids upstream of helix 12 of the LBD (Fig. 2). This helix plays a crucial role in the protein-protein interactions between nuclear receptors and p160 coactivators, and, hence, in the ability of the receptors for active transcription modulation (18, 19). In vitro expression studies showed that GRI747M had a severely impaired ability to activate gene expression through glucocorticoid-responsive elements, an abnormality explained by the location of the mutation. Furthermore, the mutant receptor had a dominant negative effect on the wild-type receptor, suggesting a further decrement in the ability of the patients’ cells to respond to glucocorticoids. At a 1:1 ratio, the mutant receptor inhibited about 25% of the activity of the wild-type receptor, suggesting that the cells of affected family members sense less than 40% of the glucocorticoid activity of normal cortisol concentrations.
The location of the mutation predicted that interaction of the mutant receptor with p160 coactivators would be defective. In the GST pull-down experiments, both the mutant and wild-type GRs interacted with the full GRIP1 molecule, whereas the former did not interact with the GRIP1 fragment carrying only the AF-2 interacting site (Fig. 5B). This suggests that, possibly, the mutant GR forms a complex with GRIP1, which is partially or completely ineffective, because one important interaction site is either weak or nonexistent. This concept was supported by the reporter assay, in which negative transdominant activity of the mutant receptor was completely abolished by overexpression of the p160 coactivator GRIP1. This phenomenon may be the result of restoring the defect of AF-2 activity and/or of overstimulating AF-1 activity. Given the location of the mutation in the AF-2 domain, it is possible that the interaction with AF-2 domain-interacting proteins other than p160 coactivators could also be defective. These include the CBP/p300 cointegrators and the vitamin D receptor-interacting proteins/thyroid hormone receptor associated proteins complex (21).
This is the second mutation of the GR reported to have a dominant negative effect on the wild-type receptor, with about 25% inhibition at the 1:1 ratio of mutant to wild-type receptor (6). This mutation differs markedly from the first in its location (C terminus vs. hinge region of the LBD) and effect on ligand binding affinity (2-fold decrease vs. no binding at all), but it exerts a similar degree of dominant negative effect on the wild-type receptor. Indeed, in the first case of a dominant negative GR mutation, the translocation of the mutant molecule to the nucleus was severely impaired, whereas the same molecule significantly delayed the translocation of the wild-type receptor (33). We hypothesize that, in both instances, the mutant receptors not only have severely compromised inherent activity as homodimers, but also form heterodimers with the wild-type receptors, producing functional impairment in one or more of the post ligand-binding steps, i.e. translocation into the nucleus or interaction with coregulators and/or general transcription factors (13). In this kindred, mutant and wild-type GR gene alleles of patients were equally expressed, indicating that their cells would produce up to 50% of GR mutant/wild-type heterodimers, forming a moderately compromised coactivator complex with p160 coactivators, up to 25% of wild-type homodimers, forming a fully active complex, and up to 25% of mutant homodimers, forming a severely compromised complex (Fig. 8).
|
and p160 coactivators in the nucleus. In a very recent series of reports, nuclear receptors and their coactivators were found to accumulate in nuclear bodies (speckles or ND10 bodies) containing promyelocytic leukemia protein, as well as several components of the ubiquitin-proteasome pathway (34, 35, 36, 37). These accumulations represent important sites of nuclear receptor and coactivator pools that are involved in the regulation of transcription. Visible coarse accumulation of our patients’ mutant receptors and p160 coactivators in nuclear speckles suggests that aberrantly interacting nuclear receptors and p160 coactivators are retained in such nuclear bodies and unavailable for participating in the formation of the transcriptosome of responsive genes.
Although the clinically overt syndrome of glucocorticoid resistance due to changes in the GR gene is rare, we believe that mild GR or non-GR-related forms of the condition in both women and men may not have been recognized (1, 2). Recently, two sisters with multiple, partial steroid resistance, with manifestations of isolated, generalized, partial glucocorticoid resistance, were described (38). In these patients, the defect appeared to be in a coregulator of the GR rather the receptor itself, influencing several nuclear hormone signal systems (16). On the other hand, the ability of the GRI747M mutant to interact abnormally with and probably sequester p160 coactivator molecules in ND10 bodies suggests that the affected members of our family might have subclinical resistance to other nuclear hormones, a testable hypothesis. Indeed, it would be important to analyze not only GRs but also other functionally related molecules, such as several nuclear hormone coregulators in common disorders in which the glucocorticoid signal transduction pathway might be pathophysiologically involved. Glucocorticoid resistance and hypersensitivity could be respectively associated with hyperandrogenism and/or hypertension, and visceral obesity, hypertension, and/or osteoporosis (1, 2).
Acknowledgments
We thank Drs. M. A. Mancini, M. Stallcup, R. M. Evans, and G. L. Hager for kindly providing their plasmids to us. We also thank B. S. Warren for kindly supplying purified mouse GR protein and K. Zachman, M.A., for excellent technical assistance.
Footnotes
A.V. and T.K. contributed equally to this work.
Abbreviations: AF, Activation function; CBP, cAMP-responsive element-binding protein (CREB)-binding protein; fl, full-length; GFP, green fluorescent protein; GRE, glucocorticoid-response element; GRIP1, GR-interacting protein 1; GST, glutathione-S-transferase; Kd, dissociation constant; LBD, ligand-binding domain; MMTV, murine mammary tumor virus; NID, nuclear receptor-interacting domain; NLS, nuclear localization signal; SRA, steroid receptor RNA activator; UFC, urinary free cortisol.
Received August 15, 2001.
Accepted January 18, 2002.
References
and steroid receptor coactivator-1. Mol Endocrinol 14:518–534 and coactivator turnover and for efficient estrogen receptor- transactivation. Mol Cell 5:939–948[CrossRef][Medline]
This article has been cited by other articles:
 |
|
 |
|
  E. Charmandari, T. Kino, T. Ichijo, and G. P. Chrousos Generalized Glucocorticoid Resistance: Clinical Aspects, Molecular Mechanisms, and Implications of a Rare Genetic Disorder J. Clin. Endocrinol. Metab., May 1, 2008; 93(5): 1563 - 1572. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Raef, E. Y Baitei, M. Zou, and Y. Shi Genotype-phenotype correlation in a family with primary cortisol resistance: possible modulating effect of the ER22/23EK polymorphism. Eur. J. Endocrinol., April 1, 2008; 158(4): 577 - 582. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. Charmandari, T. Kino, T. Ichijo, W. Jubiz, L. Mejia, K. Zachman, and G. P. Chrousos A Novel Point Mutation in Helix 11 of the Ligand-Binding Domain of the Human Glucocorticoid Receptor Gene Causing Generalized Glucocorticoid Resistance J. Clin. Endocrinol. Metab., October 1, 2007; 92(10): 3986 - 3990. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. Lin, P. Philibert, B. Ferraz-de-Souza, D. Kelberman, T. Homfray, A. Albanese, V. Molini, N. J. Sebire, S. Einaudi, G. S. Conway, et al. Heterozygous Missense Mutations in Steroidogenic Factor 1 (SF1/Ad4BP, NR5A1) Are Associated with 46,XY Disorders of Sex Development with Normal Adrenal Function J. Clin. Endocrinol. Metab., March 1, 2007; 92(3): 991 - 999. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. Carlsson, S. Burendahl, and L. Nilsson Unbinding of Retinoic Acid from the Retinoic Acid Receptor by Random Expulsion Molecular Dynamics Biophys. J., November 1, 2006; 91(9): 3151 - 3161. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. Charmandari, T. Kino, T. Ichijo, K. Zachman, A. Alatsatianos, and G. P. Chrousos Functional Characterization of the Natural Human Glucocorticoid Receptor (hGR) Mutants hGR{alpha}R477H and hGR{alpha}G679S Associated with Generalized Glucocorticoid Resistance J. Clin. Endocrinol. Metab., April 1, 2006; 91(4): 1535 - 1543. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Russcher, P. Smit, E. F. C. van Rossum, E. L. T. van den Akker, A. O. Brinkmann, L. J. M. de Heide, F. H. de Jong, J. W. Koper, and S. W. J. Lamberts Strategies for the Characterization of Disorders in Cortisol Sensitivity J. Clin. Endocrinol. Metab., February 1, 2006; 91(2): 694 - 701. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. A.E. Irving, L. Minto, S. Bailey, and A. G. Hall Loss of Heterozygosity and Somatic Mutations of the Glucocorticoid Receptor Gene Are Rarely Found at Relapse in Pediatric Acute Lymphoblastic Leukemia but May Occur in a Subpopulation Early in the Disease Course Cancer Res., November 1, 2005; 65(21): 9712 - 9718. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. S. Chriguer, L. L. K. Elias, I. M. da Silva Jr., J. G. H. Vieira, A. C. Moreira, and M. de Castro Glucocorticoid Sensitivity in Young Healthy Individuals: in Vitro and in Vivo Studies J. Clin. Endocrinol. Metab., November 1, 2005; 90(11): 5978 - 5984. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. Carlsson, K. F. Koehler, and L. Nilsson Glucocorticoid Receptor Point Mutation V571M Facilitates Coactivator and Ligand Binding by Structural Rearrangement and Stabilization Mol. Endocrinol., August 1, 2005; 19(8): 1960 - 1977. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. Charmandari, A. Raji, T. Kino, T. Ichijo, A. Tiulpakov, K. Zachman, and G. P. Chrousos A Novel Point Mutation in the Ligand-Binding Domain (LBD) of the Human Glucocorticoid Receptor (hGR) Causing Generalized Glucocorticoid Resistance: The Importance of the C Terminus of hGR LBD in Conferring Transactivational Activity J. Clin. Endocrinol. Metab., June 1, 2005; 90(6): 3696 - 3705. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. Charmandari, G. P. Chrousos, T. Ichijo, N. Bhattacharyya, A. Vottero, E. Souvatzoglou, and T. Kino The Human Glucocorticoid Receptor (hGR) {beta} Isoform Suppresses the Transcriptional Activity of hGR{alpha} by Interfering with Formation of Active Coactivator Complexes Mol. Endocrinol., January 1, 2005; 19(1): 52 - 64. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. Charmandari, T. Kino, E. Souvatzoglou, A. Vottero, N. Bhattacharyya, and G. P. Chrousos Natural Glucocorticoid Receptor Mutants Causing Generalized Glucocorticoid Resistance: Molecular Genotype, Genetic Transmission, and Clinical Phenotype J. Clin. Endocrinol. Metab., April 1, 2004; 89(4): 1939 - 1949. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. U. De Martino, N. Bhattachryya, S. Alesci, T. Ichijo, G. P. Chrousos, and T. Kino The Glucocorticoid Receptor and the Orphan Nuclear Receptor Chicken Ovalbumin Upstream Promoter-Transcription Factor II Interact with and Mutually Affect Each Other's Transcriptional Activities: Implications for Intermediary Metabolism Mol. Endocrinol., April 1, 2004; 18(4): 820 - 833. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. F.C. van Rossum and S. W.J. Lamberts Polymorphisms in the Glucocorticoid Receptor Gene and Their Associations with Metabolic Parameters and Body Composition Recent Prog. Horm. Res., January 1, 2004; 59(1): 333 - 357. [Abstract] [Full Text]
|
|
|
|
|
|
S. Kunz, R. Sandoval, P. Carlsson, J. Carlstedt-Duke, J. W. Bloom, and R. L. Miesfeld Identification of a Novel Glucocorticoid Receptor Mutation in Budesonide-Resistant Human Bronchial Epithelial Cells Mol. Endocrinol., December 1, 2003; 17(12): 2566 - 2582. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Koyano, Y. Saito, M. Nagano, K. Maekawa, Y. Kikuchi, N. Murayama, T. Fujino, S. Ozawa, T. Nakajima, K. Matsumoto, et al. Functional Analysis of Three Genetic Polymorphisms in the Glucocorticoid Receptor Gene J. Pharmacol. Exp. Ther., October 1, 2003; 307(1): 110 - 116. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Kino and G. P. Chrousos Tumor Necrosis Factor alpha Receptor- and Fas-associated FLASH Inhibit Transcriptional Activity of the Glucocorticoid Receptor by Binding to and Interfering with Its Interaction with p160 Type Nuclear Receptor Coactivators J. Biol. Chem., January 24, 2003; 278(5): 3023 - 3029. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||
