A direct correlation between the concentration of GR in a cell and sensitivity to glucocorticoids has been demonstrated in many studies (21, 22, 49-53). Genetic abnormalities of the glucocorticoid receptor - primarily inactivating mutations of the ligand-binding domain or mutations leading to functional knock-out of one of the two GR gene alleles - have been described (54).

Until now, over 10 families and a few sporadic cases have been described (47, 55-58), but the molecular mechanism has been elucidated only in 5 families (59-63)  and 3 sporadic cases (48, 64) (Table 1; Fig. 5).

The first two patients, a father and a son, with long term "hypercortisolism" not associated with clinical manisfestations of Cushing's syndrome, were described in 1976 (2, 3, 15, 54). The propositus of this family was a homozygote for a single nonconservative point mutation, replacing aspartate at amino acid position 641 with valine in the hormone binding domain of the GR; this mutation caused a three-fold reduction in glucocorticoid receptor binding affinity for dexamethasone and caused loss of transactivation activity on the MMTV promoter (59). In the absence of ligand, the mutant receptor was primarily localized in the cytoplasm of cells. Exposure to dexamethasone (10-6M) induced a slow translocation into the nucleus, which took 22 min as opposed to 12 min required for nuclear translocation of the wild type receptor (65). Finally, the mutant receptor interacted with the amino-terminal but not with the carboxyl-terminal fragment or full-length GRIP1 in vitro (65). 

The second family was described by Karl et al. in 1993; the proposita of this family was a young woman with manifestations of hyperandrogenism. Molecular analysis showed a 4-base deletion at 3'-boundary of exon 6, removing a donor splice site. This resulted in complete ablation of expression of one of the GR alleles associated with a decrease of GR protein by 50% in the affected members of the family (60).

The propositus of the third kindred had a single homozygotic point mutation at amino acid 729 (valine to isoleucine) in the hormone binding domain, which reduced both the affinity and transactivation activity of the GR (61). The mutant receptor was localized primarily in the nucleus of cells in the absence of ligand, while further translocation from the cytoplasm into the nucleus required longer (120 min) exposure to dexamethasone (10-6M), and demonstrated a weak, ligand-dependent interaction with the full-length and carboxyl-terminal fragment but not with the amino-terminal fragment of GRIP1 in vitro (65).

The first sporadic case of a man with a history of infertility and hypertension with 5-10 fold elevation of his urinary free cortisol levels was described in 1996 (48). This patient had a de novo, germ-line heterozygotic mutation at amino acid 559 (isoleucine to asparagine) in the hormone-binding domain, at the hinge region of the GR, which abolished ligand binding but exerted dominant negative activity on the wild type receptor. This receptor had a 2-3-fold more potent dominant negative activity than GRβ in the same MMTV-luciferase assay, and was expressed in a 1:1 ratio with the normal GRα in the patient's cells. Interestingly, this mutant receptor had a markedly delayed nuclear translocation (180 min), an effect that would be overcome at very high dexamethasone concentrations. There was no interaction between the mutant receptor and GRIP1 (65). Later this patient developed severe Cushing disease due to an ACTH-secreting pituitary adenoma (48).

Our group studied a fifth case/kindred with glucocorticoid resistance, whose proband was a young woman with signs of hyperandrogenism but no other complaint (62). Molecular analysis detected a heterozygotic T to G substitution at nucleotide 2373 of exon 9α of GR, in the ligand-binding domain at amino acid position 747, replacing isoleucine with methionine. 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 mutant receptor had reduced affinity for dexamethasone about two-fold and its transcriptional activity on the glucocorticoid-responsive mouse mammary tumor virus (MMTV) promoter was compromized by 20-30-fold; interestingly, it also had dominant negative activity upon the wild-type. 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, GFP-fused GRαI747M had a slight delay in its translocation from the cytoplasm into the nucleus and formed coarser nuclear speckles than GFP-fused wild type GRα. Similarly, a GFP-fused p160 coactivator had a distinctly different distribution in the nucleus in the presence of the mutant vs. wild type receptor, presenting also a coarser speckling, suggesting that defective interaction of the mutant receptor with p160 coactivator may explain its dominant negative activity on the wild type receptor (62, 65).

An artificial mutation placed in position 747 of the ligand-binding domain of GR, leading to a different amino acid substitution, was described (66). Interestingly, this mutation induced a conformational change in GRα-ligand binding. Thus, they suggested that mutations in the loop between helix 11 and 12 could specifically alter the conformational state induced by agonist binding, probably by altering the position of helix 12, and this positioning is affected differently depending on the amino acids.

The sixth and seventh sporadic cases were due to heterozygous mutations, replacing arginine by histidine at amino acid 477 and glycine by serine at amino acid 679, respectively (64). The former was located in the second zinc finger in the DBD. This mutant receptor had no transactivation activity due to impaired binding to GREs. The latter mutation was found in the LBD, outside of the ligand-binding pocket. This mutation caused 50% reduction of ligand-binding affinity with comparable reduction of the transactivation activity. Since these two mutant receptors were found in the heterozygous condition, they might behave as dominant negative mutants, suppressing the biological activity of the wild type receptor.

The proposita of the eighth familial case had a homozygotic point mutation replacing valine by alanine at amino acid 571 of the LBD (63). The mutant receptor had six-fold reduction in its binding affinity to dexamethasone and 10-50-fold less transactivation activity than the wild type receptor. The nuclear translocation of the mutant receptor was delayed (25 min), while its interaction with the GRIP1 coactivator occurred mostly via its AF-1 domain (65). Interestingly, this baby born with ambiguous genitalia also suffered from 21-hydroxylase deficiency, suggesting that this congenital disease exacerbated the hyperandrogenism and virilization potential of the glucocorticoid resistance syndrome.

We have also studied 2 patients with glucocorticoid resistance who had no abnormalities of the coding sequences and intron-exon boundaries of their GR genes; one of these patients was published earlier (67). Also, in a recent article 5 Dutch patients with clinical and biochemical evidence of glucocorticoid resistance were studied, but no abnormalities in their GR gene were found (68).

The natural glucocorticoid receptor mutants causing familial glucocorticoid resistance has been studied by our group (65). In particular, each of the mutations described up to now exerted different functional defects upon the GR signal transduction pathway, which explain the autosomal recessive or dominant transmission of the disorder and may in part explain its variable clinical phenotype. However, one must not underestimate the importance of background genetic and constitutional factors with epistatic actions on the expression of the disorder (54). Thus, factors that define the activity of the hypothalamic-pituitary-adrenal axis, renin-angiotensin-aldosterone system, and gonadal axis as well as the target tissue sensitivity to glucocorticoids, and androgens, are bound to play important roles in the clinical manifestation of this condition (65).

Recently, the molecular analyses of these five families and three sporadic cases were reviewed, while the possible contribution of newly identified molecules, such as the HIV-1 accessory proteins Vpr and Tat, FLICE-associated huge protein (FLASH) and chicken ovalbumin upstream promoter-transcription factor II (COUP-TFII)  to  the molecular regulation of GR activity, as well as their possible contribution to changes in tissue sensitivity to glucocorticoids in pathologic conditions were discussed (52). Vpr, a 96-amino acid virion-associated protein with multiple functions (69, 70), enhanced GR transactivation by functioning as a coactivator in vitro (71). Indeed, Vpr contains a nuclear receptor signature motif LXXLL at amino acids 64-68, through which host nuclear receptor coactivators bind to nuclear receptors (72). Vpr directly bound to the GR through this motif and cooperatively enhanced its activity on its responsive promoters along with host p160 nuclear receptor coactivators and p300/CBP (71). Vpr also directly bound p300 at its C- terminal amino acids 2045-2191, at which host coactivator p160 proteins also bind, and functioned as an adaptor linking GR and the coactivator complex. Another HIV-1 accessory protein, Tat, which functions as a major transactivator of the HIV-1 long term repeat promoter (73) also moderately potentiated GR activity, by promoting the accumulation of the positive transcription elongation factor b (P-TEFb) (74-79).

Although FLASH was originally found as a component of a cytoplasmic complex located under the plasma membrane, it contains two putative nuclear localization signals (NLS) and one nuclear export signal (NES), findings that have led to the speculation that it might translocate into the nucleus in response to certain stimuli (80). Kino et al. found that this molecule inhibited both GR transactivation and its enhancement by GRIP1 on a glucocorticoid-responsive promoter by interfering with GR binding to GRIP1 (81). FLASH is a mediator of Fas ligand and TNFalpha effects in the cell and its glucocorticoid signaling system-neutralizing activity  may increase the apoptosis-promoting activities of these  cytokines.

To explore the presence of transcription factors which bind to the GR and influence the GR metabolic activity, Kino et al. performed yeast two-hybrid screening using the GR LBD as bait and found that the orphan nuclear receptor COUP-TFII specifically interacted with the GR (82, 83). In particular, the ligand-activated GR formed a complex with COUP-TFII and enhanced its transcriptional activity on a COUP-TFII-responsive promoter in several cell lines (82, 83). GR also enhanced the transcriptional activity of COUP-TFII on the promoter of the PEPCK, a key regulatory enzyme in gluconeogenesis, suggesting that their interaction might also be important in glucose metabolism.

The importance of GRβ in physiologic conditions is controversial, but it has been proposed that in pathologic situations, such as glucocorticoid resistance, it might play a role. Taking into consideration that increasing amount of GRβ determines a dose-dependent decrease of the wild-type GRα transcriptional activity (84), an imbalance in the expression of these two isoforms might determine an altered sensitivity to glucocorticoids. Supporting the possible role of GRβ on glucocorticoid sensitivity, a genetically-determined imbalanced expression of the glucocorticoid receptor isoforms was reported in cultured lymphocytes from a patient with congenital generalized glucocorticoid resistance and chronic leukemia (67); in this patient a low GRα to GRβ ratio was found compared to a group of normal controls. This alteration in the expression of the two isoforms could explain the glucocorticoid resistance of this patient since no abnormalities in the sequence of the entire cDNA and individual exons of this patient’s gene were found (67).

Glucocorticoids are the mainstay of the treatment of many chronic autoimmune/inflammatory and allergic diseases, such as rheumatoid arthritis, inflammatory bowel disease and asthma, as well as lymphoproliferative neoplasms, such as lymphoma or leukemia, and other conditions. Their use in some patients, however, is hampered by systemic side effects, while in others it fails to produce a therapeutic response even at very high doses. A good example of this is glucocorticoid-resistant asthma. In peripheral blood from glucocorticoid resistant asthma type 1 patients, a significantly higher number of GRβ immunoreactive cells was present than in glucocorticoid sensitive asthmatic patients and normal controls (85). This finding was confirmed and was more pronounced in bronchial lavage cells. The explanation was that the GRβ expression was cytokine- inducible and, therefore, the enhanced expression of GRβ might be directly involved in the development of glucocorticoid insensitivity in certain patients with chronic asthma. In patients with the rarer glucocorticoid resistant asthma type 2, a genetically determined nonreversible decrease of the GRα/GRβ ratio could be responsible for glucocorticoid resistance of the immune cells.

During glucocorticoid treatment of an immune-related disease, several mechanisms may seriously diminish the efficacy of the therapy. Pro-inflammatory signals activate NF-kB and increase the expression level of isoform GRβ, which will result in decreased sensitivity to glucocorticoid treatment of inflamed tissue. Glucocorticoid treatment itself might also result in down-regulation of the glucocorticoid receptor levels in most cell types, thereby decreasing the efficacy of treatment. Further insight into glucocorticoid resistance might help determine  an individual’s glucocorticoid responsiveness before treatment and to improve glucocorticoid therapy (53).

There have been 17 polymorphisms detected in the glucocorticoid receptor gene, including two restriction fragment length polymorphisms (86). In contrast to the mutation cluster in the LBD, the majority of polymorphisms were localized in the amino-terminal transactivation domain. The most common polymorphism studied in a variety of diseases is N363S. One polymorphism was reported in the promoter region of the GR gene and was associated with decreased transcription of the gene (87). Two silent polymorphisms (D678D and N766N) were found in the LBD. Two intronic point mutations were reported as well as one point mutation in the 3’UTR of GRβ.

Animal models of systemic glucocorticoid resistance, such as New World Primates, including squirrel, marmoset, and owl monkeys, have been described. These animals have total plasma cortisol levels which are 7-20 times higher than in humans or other Old World primates (88), while the concentration, affinity and predicted amino acid sequence of their GR are similar to those of the human receptor (89). Interestingly, these animals exhibit resistance to a variety of other steroid/sterol hormones, including estrogens, progesterone, androgens, aldosterone, and vitamin D (15, 90, 91). Immunoreactivity of both isoforms of the GR has been found in Epstein-Barr virus-transformed B-lymphocytes from marmosets, with the β isoform being ~ 10 times overexpressed compared to the corresponding human cells (44). An altered splicing pattern of the GR pre-mRNA or differential mRNA translation or degradation and/or GR protein degradation rates might contribute to the steroid resistance of these animals. Alternatively, these animals may have decreases in the activity of coactivators and/or increases in the activity of corepressors leading to their 'pansteroid' resistance (31).

Guinea pigs, like New World primates, are also resistant to glucocorticoids. This is likely due to a natural lower affinity of the GR for the ligand. The guinea pig GR differs from the human GR by 24 amino acids in the LBD (92). The molecular basis for glucocorticoid resistance is currently unknown. However, the decreased sensitivity of the GR is due to sequences in the first third of the LBD (93).

Prairie voles are resistant to glucocorticoids but, similarly to the guinea pig, the alteration in the GR responsible for this  is unknown. The prairie vole and  human GRs differ in amino acids of the amino-terminus and the  LBD, which may influence the  function of the GR (94).

In addition to mutations in the GR gene, steroid receptor coactivator defects may also account for generalized glucocorticoid resistance and/or resistance to other steroid hormones. Two very interesting sisters with manifestations of glucocorticoid resistance were described by New et al. (31, 95). Their evaluation revealed resistance not limited to glucocorticoids, but also to mineralocorticoids and androgens, but no resistance to vitamin D or thyroid hormones. The diagnosis of these patients was multiple, partial steroid resistance (95). The New World primate physiologic biochemical syndrome and the two pathologic human multiple steroid resistance syndrome cases are the first conditions in which a defective steroid receptor coregulator was suggested to be responsible for an altered clinical and/or biochemical picture (31, 95).