Natural Glucocorticoid Receptor Mutants Causing Generalized Glucocorticoid Resistance: Molecular Genotype, Genetic Transmission and Clinical Phenotype

E. Charmandari, T. Kino, G. P. Chrousos

Pediatric and Reproductive Endocrinology Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892, U.S.A.

Glucocorticoid resistance is a rare, familial or sporadic condition characterized by generalized, partial end-organ insensitivity to physiologic glucocorticoid concentrations. Patients have compensatory elevations in circulating cortisol and adrenocorticotropic hormone (ACTH) concentrations, and resistance of the hypothalamic-pituitary-adrenal (HPA) axis to dexamethasone suppression, but no clinical evidence of hypo- or hypercortisolism. The clinical spectrum of the condition is broad, ranging from completely asymptomatic to severe cases of hyperandrogenism, fatigue and/or mineralocorticoid excess. The molecular basis of glucocorticoid resistance in several families and sporadic cases has been ascribed to mutations in the human glucocorticoid receptor-α (hGRα) gene, which impair one or more of the molecular mechanisms of glucocorticoid receptor function, thus altering tissue sensitivity to glucocorticoids (Fig. [1], Table [1]).

Fig. 1 Location of the known mutations of the hGRα gene.

Table 1 Mutations of the human glucocorticoid receptor gene causing glucocorticoid resistance Mutation Position cDNA Amino acid Biochemical Phenotype Present Study Genotype Transmission Phenotype 2054 (A→T) 641 (D→V) Affinity for ligand ↓Transactivation ↓ Transcriptional activity of LBD ↓ Transdominance (-)Nuclear translocation ↓DNA binding (+) Abnormal interaction with GRIP1 Homozygous Autosomal Recessive HypertensionHypokalemic alkalosis 4 bp deletion in exon 6 hGRα number: 50 % of control Inactivation of the affected allele Heterozygous Autosomal Dominant Hirsutism Male-pattern hair-lossMenstrual irregularities 2317 (G→A) 729 (V→I) Affinity for ligand ↓Transactivation ↓ Transcriptional activity of LBD ↓ Transdominance (-)Nuclear translocation ↓ DNA binding (+)Abnormal interaction with GRIP1 Homozygous Autosomal Recessive Precocious puberty Hyperandrogenism 1808 (T→A) 559 (I→N) Affinity for ligand ↓Transactivation ↓Transdominance (+)Nuclear translocation ↓ Transcriptional activity of LBD ↓ DNA binding (+)Abnormal interaction with GRIP1 Heterozygous Sporadic HypertensionOligospermia, Infertility 1844 (C→T) 571 (V→A) Affinity for ligand ↓ Transactivation ↓ Transcriptional activity of LBD ↓ Transdominance (-)Nuclear translocation ↓DNA binding (+) Homozygous Autosomal Recessive Ambiguous genitaliaHypertension Hyperandrogenism Hypokalemia 2373 (T→G) 747 (I→M) Affinity for ligand ↓ Transactivation ↓Transdominance (+) Transcriptional activity of LBD ↓ DNA binding (+)Nuclear translocation ↓ Heterozygous Autosomal Dominant Cystic acneHirsutismOligoamenorrhea 1430 (G→A) 477 (R→H) Transactivation ↓ Heterozygous Sporadic Hirsutism, FatigueHypertension 2035 (G→A) 679 (G→S) Affinity for ligand ↓Transactivation ↓ Heterozygous Sporadic Hirsutism,Fatigue,Hypertension

We systematically investigated the molecular mechanisms through which natural, ligand-binding domain (LBD) hGRα mutants, including hGRαI559N, hGRαV571A, hGRαD641V, hGRαV729I and hGRαI747M, produce a defective signal, and determined whether their differential effects on hGRα function might account for the type of genetic transmission of the disorder and the variable clinical phenotype of the affected subjects.

Our findings suggest that all five mutant receptors studied have LBDs with decreased intrinsic transcriptional activity. Unlike hGRαI559N and hGRαI747M, which exert a dominant negative effect upon the transcriptional activity of hGRα, hGRαV571A, hGRαD641V and hGRαV729I do not have such an effect. All five mutants studied demonstrate varying degrees of decreased affinity for the ligand in a standard dexamethasone-binding assay, and preserve their ability to bind DNA. Fusion of the wild type and mutant receptors with GFP enabled the study of their subcellular localization in HeLa cells in the absence or presence of ligand. In the absence of dexamethasone, GFP-fused-hGRα was primarily localized in the cytoplasm. Addition of 10^{- 6} M dexamethasone resulted in translocation of the wild-type receptor into the nucleus within 12 min. The pathologic mutant receptors GFP-hGRαI559N, GFP-hGRαV571A, GFP-hGRαD641V and GFP-hGRαI747M were predominantly observed in the cytoplasm in the absence of ligand. Exposure to 10^{- 6} M dexamethasone induced a slow translocation of these mutant receptors into the nucleus, which took 180, 25, 22 and 30 min, respectively. The mutant receptor GFP-hGRαV729I was observed predominantly in the nucleus in the absence of ligand, while further translocation from the cytoplasm into the nucleus required longer exposure (120 min) to the same concentration of dexamethasone. Finally, all five mutants studied displayed an abnormal interaction with the GRIP1 coactivator in vitro.

We conclude that each of the above hGRα mutations imparts different functional defects upon the glucocorticoid signal transduction pathway, which explain the recessive or dominant transmission of the disorder, but might only in part explain its variable clinical phenotype.