Stability and Turnover of the ACTH Receptor Complex.

Glucocorticoid production in mammals is principally regulated by the action of the pituitary hormone adrenocorticotropin (ACTH) acting on its cognate membrane receptor on the zona fasciculata cells of the adrenal cortex. The receptor for ACTH consists of two essential components, a small seven transmembrane domain G protein-coupled receptor of the melanocortin receptor subgroup known as the melanocortin 2 receptor (MC2R) and a small single transmembrane domain protein that adopts a antiparallel homodimeric form and which is known as the melanocortin 2 receptor accessory protein (MRAP). MRAP is essential for the trafficking of the MC2R to the cell surface as well as being required for receptor responsiveness to ACTH at physiological concentrations-probably by facilitating ACTH binding, but possibly also by supporting G protein interaction with the MC2R. A number of studies have shown that ACTH stimulates the expression of functional receptor at the cell surface and the transcription of both MC2R and MRAP mRNA. However, the time course of these transcriptional effects differs such that MRAP is expressed relatively rapidly whereas MC2R transcription responds much more slowly. Furthermore, recent data suggests that MRAP protein is turned over with a short half-life whereas MC2R has a significantly longer half-life. These findings imply that these two ACTH receptor proteins have distinct trajectories and that it is likely that MRAP-independent MC2R is present at the cell surface. In such a situation newly transcribed and translated MRAP could enable the rapid recruitment of functional receptor at the plasma membrane without the need for new MC2R translation. This may be advantageous in circumstances of significant stress in that the potentially complex and perhaps inefficient process of de novo MC2R translation, folding, post-translational modification and trafficking can be avoided.

ACTH signalling and adrenal development: lessons from mouse models.

The melanocortin-2-receptor (MC2R), also known as the ACTH receptor, is a critical component of the hypothalamic-pituitary-adrenal axis. The importance of MC2R in adrenal physiology is exemplified by the condition familial glucocorticoid deficiency (FGD), a potentially fatal disease characterised by isolated cortisol deficiency. MC2R mutations cause ~25% of cases. The discovery of a MC2R accessory protein MRAP, mutations of which account for ~20% of FGD, has provided insight into MC2R trafficking and signalling. MRAP is a single transmembrane domain accessory protein highly expressed in the adrenal gland and essential for MC2R expression and function. Mouse models helped elucidate the action of ACTH. The Mc2r-knockout (Mc2r - / - ) mice was the first mouse model developed to have adrenal insufficiency with deficiencies in glucocorticoid, mineralocorticoid and catecholamines. We recently reported the generation of the Mrap - / - mice which better mimics the human FGD phenotype with isolated glucocorticoid deficiency alone. The adrenal glands of adult Mrap - / - mice were grossly dysmorphic with a thickened capsule, deranged zonation and deranged WNT4/beta-catenin and sonic hedgehog (SHH) pathway signalling. Collectively, these mouse models of FGD highlight the importance of ACTH and MRAP in adrenal progenitor cell regulation, cortex maintenance and zonation.

MRAP deficiency impairs adrenal progenitor cell differentiation and gland zonation.

Melanocortin 2 receptor accessory protein (MRAP) is a single transmembrane domain accessory protein and a critical component of the hypothamo-pituitary-adrenal axis. MRAP is highly expressed in the adrenal gland and is essential for adrenocorticotropin hormone (ACTH) receptor expression and function. Human loss-of-function mutations in MRAP cause familial glucocorticoid (GC) deficiency (FGD) type 2 (FGD2), whereby the adrenal gland fails to respond to ACTH and to produce cortisol. In this study, we generated Mrap-null mice to study the function of MRAP in vivo. We found that the vast majority of Mrap-/- mice died at birth but could be rescued by administration of corticosterone to pregnant dams. Surviving Mrap-/- mice developed isolated GC deficiency with normal mineralocorticoid and catecholamine production, recapitulating FGD2. The adrenal glands of adult Mrap-/- mice were small, with grossly impaired adrenal capsular morphology and cortex zonation. Progenitor cell differentiation was significantly impaired, with dysregulation of WNT4/ß-catenin and sonic hedgehog pathways. These data demonstrate the roles of MRAP in both steroidogenesis and the regulation of adrenal cortex zonation. This is the first mouse model of isolated GC deficiency and reveals the role of MRAP in adrenal progenitor cell regulation and cortex zonation.-Novoselova, T. V., Hussain, M., King, P. J., Guasti, L., Metherell, L. A., Charalambous, M., Clark, A. J. L., Chan, L. F. MRAP deficiency impairs adrenal progenitor cell differentiation and gland zonation.

Promiscuity among the MRAPs.

The melanocortin 2 receptor accessory protein (MRAP) was originally discovered to be an essential co-receptor for the ACTH receptor/melanocortin 2 receptor, and it physically interacts with this receptor and is required for receptor trafficking and ligand binding. A related molecule, MRAP2, is mainly expressed in the CNS and appears to have a role with the melanocortin 4 receptor. Consistent with this is the observation that a massively obese phenotype develops when the Mrap2 gene is deleted in mice. However, the characteristics of this phenotype differ from those of Mc4r-deleted mice and suggest that an additional role, possibly resulting from an interaction with other receptors is possible. In support of this, a functional interaction with the prokineticin receptors was recently reported. Evidence for other receptor interactions and aspects of the tissue distribution of MRAP and MRAP2 gene expression may indicate that these accessory proteins have a wider role than with the melanocortin receptors alone.

60 YEARS OF POMC: The proopiomelanocortin gene: discovery, deletion and disease.

The cloning of the bovine proopiomelanocortin (POMC) cDNA in 1978 by Nakanishi and colleagues was the result of a remarkable series of exacting and ingenious experiments. With this work, they instantly confirmed the single precursor hypothesis for adrenocorticotrophic hormone-ß-lipotropin, as it was then known, and in so doing revealed the existence of additional, largely unpredicted, N-terminal peptides that together formed the POMC precursor peptide. This work paved the way for a host of additional studies into the physiology of these peptides and their regulation. Furthermore, the cloning of the murine Pomc gene was essential for subsequent studies, in which Pomc was intentionally deleted in the mouse illuminating its substantial role in body weight regulation and adrenal function. Contemporaneously with this work, naturally occurring mutations in human POMC came to light underlining the vital role of this gene in appetite regulation. This article reviews each of these aspects of POMC with the benefit of several decades of hindsight and informed by more recent genomic and transcriptomic data.

Whole-Exome Sequencing in the Differential Diagnosis of Primary Adrenal Insufficiency in Children.

Adrenal insufficiency is a rare, but potentially fatal medical condition. In children, the cause is most commonly congenital and in recent years a growing number of causative gene mutations have been identified resulting in a myriad of syndromes that share adrenal insufficiency as one of the main characteristics. The evolution of adrenal insufficiency is dependent on the variant and the particular gene affected, meaning that rapid and accurate diagnosis is imperative for effective treatment of the patient. Common practice is for candidate genes to be sequenced individually, which is a time-consuming process and complicated by overlapping clinical phenotypes. However, with the availability, and increasing cost effectiveness of whole-exome sequencing, there is the potential for this to become a powerful diagnostic tool. Here, we report the results of whole-exome sequencing of 43 patients referred to us with a diagnosis of familial glucocorticoid deficiency (FGD) who were mutation negative for MC2R, MRAP, and STAR the most commonly mutated genes in FGD. WES provided a rapid genetic diagnosis in 17/43 sequenced patients, for the remaining 60% the gene defect may be within intronic/regulatory regions not covered by WES or may be in gene(s) representing novel etiologies. The diagnosis of isolated or familial glucocorticoid deficiency was only confirmed in 3 of the 17 patients, other genetic diagnoses were adrenal hypo- and hyperplasia, Triple A, and autoimmune polyendocrinopathy syndrome type I, emphasizing both the difficulty of phenotypically distinguishing between disorders of PAI and the utility of WES as a tool to achieve this.

Thioredoxin Reductase 2 (TXNRD2) mutation associated with familial glucocorticoid deficiency (FGD).

CONTEXT: Classic ACTH resistance, due to disruption of ACTH signaling, accounts for the majority of cases of familial glucocorticoid deficiency (FGD). Recently FGD cases caused by mutations in the mitochondrial antioxidant, nicotinamide nucleotide transhydrogenase, have highlighted the importance of redox regulation in steroidogenesis. OBJECTIVE: We hypothesized that other components of mitochondrial antioxidant systems would be good candidates in the etiology of FGD. DESIGN: Whole-exome sequencing was performed on three related patients, and segregation of putative causal variants confirmed by Sanger sequencing of all family members. A TXNRD2-knockdown H295R cell line was created to investigate redox homeostasis. SETTING: The study was conducted on patients from three pediatric centers in the United Kingdom. PATIENTS: Seven individuals from a consanguineous Kashmiri kindred, six of whom presented with FGD between 0.1 and 10.8 years, participated in the study. INTERVENTIONS: There were no interventions. MAIN OUTCOME MEASURE: Identification and functional interrogation of a novel homozygous mutation segregating with the disease trait were measured. RESULTS: A stop gain mutation, p.Y447X in TXNRD2, encoding the mitochondrial selenoprotein thioredoxin reductase 2 (TXNRD2) was identified and segregated with disease in this extended kindred. RT-PCR and Western blotting revealed complete absence of TXNRD2 in patients homozygous for the mutation. TXNRD2 deficiency leads to impaired redox homeostasis in a human adrenocortical cell line. CONCLUSION: In contrast to the Txnrd2-knockout mouse model, in which embryonic lethality as a consequence of hematopoietic and cardiac defects is described, absence of TXNRD2 in humans leads to glucocorticoid deficiency. This is the first report of a homozygous mutation in any component of the thioredoxin antioxidant system leading to inherited disease in humans.

Ligand-specific conformational change of the G-protein-coupled receptor ALX/FPR2 determines proresolving functional responses.

Formyl-peptide receptor type 2 (FPR2), also called ALX (the lipoxin A4 receptor), conveys the proresolving properties of lipoxin A4 and annexin A1 (AnxA1) and the proinflammatory signals elicited by serum amyloid protein A and cathelicidins, among others. We tested here the hypothesis that ALX might exist as homo- or heterodimer with FPR1 or FPR3 (the two other family members) and operate in a ligand-biased fashion. Coimmunoprecipitation and bioluminescence resonance energy transfer assays with transfected HEK293 cells revealed constitutive dimerization of the receptors; significantly, AnxA1, but not serum amyloid protein A, could activate ALX homodimers. A p38/MAPK-activated protein kinase/heat shock protein 27 signaling signature was unveiled after AnxA1 application, leading to generation of IL-10, as measured in vitro (in primary monocytes) and in vivo (after i.p. injection in the mouse). The latter response was absent in mice lacking the ALX ortholog. Using a similar approach, ALX/FPR1 heterodimerization evoked using the panagonist peptide Ac2-26, identified a JNK-mediated proapoptotic path that was confirmed in primary neutrophils. These findings provide a molecular mechanism that accounts for the dual nature of ALX and indicate that agonist binding and dimerization state contribute to the conformational landscape of FPRs.

Melanocortin 4 receptor becomes an ACTH receptor by coexpression of melanocortin receptor accessory protein 2.

Melanocortin 2 receptor (MC2R) is the only canonical ACTH receptor. Its functional expression requires the presence of an accessory protein, known as melanocortin receptor 2 accessory protein 1 (MRAP1). The vertebrate genome exhibits a paralogue gene called MRAP2, which is duplicated in zebrafish (MRAP2a and MRAP2b), although its function remains unknown. In this paper, we demonstrate that MRAP2a enables MC4R, a canonical MSH receptor, to be activated by ACTH with a similar sensitivity to that exhibited by MC2R. Both proteins physically interact and are coexpressed in the neurons of the preoptic area, a key region in the control of the energy balance and hypophyseal secretion in fish. ACTH injections inhibit food intake in wild-type zebrafish but not in fish lacking functional MC4R. Both MRAP1 and MRAP2a are hormonally regulated, suggesting that these proteins are substrates for feed-back regulatory pathways of melanocortin signaling. Fasting has no effect on the central expression of MRAP2a but stimulates MRAP2b expression. This protein interacts and is colocalized with MC4R in the tuberal hypothalamic neurons but has no effect on the pharmacologic profile of MC4R. However, MRPA2b is able to decrease basal reporter activity in cell lines expressing MC4R. It is plausible that MRAP2b decreases the constitutive activity of the MC4R during fasting periods, driving the animal toward a positive energy balance. Our data indicate that MRAP2s control the activity of MC4R, opening up new pathways for the regulation of melanocortin signaling and, by extension, for the regulation of the energy balance and obesity.

Familial glucocorticoid deficiency: a diagnostic challenge during acute illness.

UNLABELLED: Familial glucocorticoid deficiency (FGD) is a heterogeneous condition of isolated glucocorticoid deficiency due to adrenocorticotropic hormone (ACTH) resistance. Patients have adrenal failure with normal electrolytes. We report two Arab children with different forms of FGD, in whom the diagnosis was initially masked by their acute illness and discuss the reasons for the delay in the diagnosis of FGD in both patients. Patient 1 presented at 12 days with Serratia sepsis. She received hydrocortisone for septic shock and needed dexamethasone courses to wean her off ventilation. At 13 weeks, she had normal electrolytes, low cortisol and high ACTH in keeping with FGD. A homozygous missense mutation (T159) in MC2R confirmed the diagnosis of FGD type 1. Patient 2 was admitted at 4.5 years, with an acute exacerbation of chronic asthma. At presentation, he had hypotension, hypoglycaemia and normal electrolytes. He was given IV hydrocortisone to treat his severe asthma, and his lip hyperpigmentation was thought to be central cyanosis. Two weeks later, his lips remained dark, and cortisol was low, with markedly elevated ACTH. Family history revealed a sister aged 22 years with cerebral palsy and a healthy 15-year-old brother, who were both severely pigmented with high ACTH levels. The diagnosis of FGD type 2 was confirmed by identifying a homozygous missense mutation (p.Y59D) in MRAP in the three siblings. CONCLUSIONS: FGD can be easily overlooked during acute illness. In a sick child, paired measurement of serum cortisol with ACTH prior to starting steroid therapy would be useful in making the diagnosis of FGD.

The melanocortin receptors and their accessory proteins.

The five melanocortin receptors (MCRs) named MC1R-MC5R have diverse physiological roles encompassing pigmentation, steroidogenesis, energy homeostasis and feeding behavior as well as exocrine function. Since their identification almost 20 years ago much has been learnt about these receptors. As well as interacting with their endogenous ligands the melanocortin peptides, there is now a growing list of important peptides that can modulate the way these receptors signal, acting as agonists, antagonists, and inverse agonists. The discovery of melanocortin 2 receptor accessory proteins as a novel accessory factor to the MCRs provides further insight into the regulation of these important G protein-coupled receptor.

Familial glucocorticoid deficiency: New genes and mechanisms.

Familial Glucocorticoid deficiency (FGD), in which the adrenal cortex fails to produce glucocorticoids, was first shown to be caused by defects in the receptor for ACTH (MC2R) or its accessory protein (MRAP). Certain mutations in the steroidogenic acute regulatory protein (STAR) can also masquerade as FGD. Recently mutations in mini chromosome maintenance-deficient 4 homologue (MCM4) and nicotinamide nucleotide transhydrogenase (NNT), genes involved in DNA replication and antioxidant defence respectively, have been recognised in FGD cohorts. These latest findings expand the spectrum of pathogenetic mechanisms causing adrenal disease and imply that the adrenal may be hypersensitive to replicative and oxidative stresses. Over time patients with MCM4 or NNT mutations may develop other organ pathologies related to their impaired gene functions and will therefore need careful monitoring.

Genome-wide methylation and gene expression changes in newborn rats following maternal protein restriction and reversal by folic acid.

A large body of evidence from human and animal studies demonstrates that the maternal diet during pregnancy can programme physiological and metabolic functions in the developing fetus, effectively determining susceptibility to later disease. The mechanistic basis of such programming is unclear but may involve resetting of epigenetic marks and fetal gene expression. The aim of this study was to evaluate genome-wide DNA methylation and gene expression in the livers of newborn rats exposed to maternal protein restriction. On day one postnatally, there were 618 differentially expressed genes and 1183 differentially methylated regions (FDR 5%). The functional analysis of differentially expressed genes indicated a significant effect on DNA repair/cycle/maintenance functions and of lipid, amino acid metabolism and circadian functions. Enrichment for known biological functions was found to be associated with differentially methylated regions. Moreover, these epigenetically altered regions overlapped genetic loci associated with metabolic and cardiovascular diseases. Both expression changes and DNA methylation changes were largely reversed by supplementing the protein restricted diet with folic acid. Although the epigenetic and gene expression signatures appeared to underpin largely different biological processes, the gene expression profile of DNA methyl transferases was altered, providing a potential link between the two molecular signatures. The data showed that maternal protein restriction is associated with widespread differential gene expression and DNA methylation across the genome, and that folic acid is able to reset both molecular signatures.

Multiple segmental uniparental disomy associated with abnormal DNA methylation of imprinted Loci in silver-russell syndrome.

BACKGROUND: Silver-Russell syndrome (SRS; online inheritance in man 180860) is a low-birth-weight syndrome characterized by postnatal growth restriction and variable dysmorphic features. Although maternal uniparental disomy (UPD) of chromosome 7 and hypomethylation of H19 have been reported in up to 50% of all cases, no unifying mechanism is apparent. SUBJECTS AND METHODS: Ten patients and their parents were studied using the Illumina GoldenGate methylation array and the Illumina 370K HumHap single-nucleotide polymorphism array to identify aberrations in DNA methylation as well as genomic changes including copy number changes and uniparental disomy events. RESULTS: We found evidence of UPD events outside chromosome 7 in all patients. In up to 30% of patients with SRS, DNA methylation changes occur in imprinted gene loci outside 11p15.5 (PEG3, PLAGL1, and GRB10), not previously consistently linked with SRS. Furthermore, hypermethylation of GRB10 was associated with increased mRNA expression. In addition, 20% of patients appear to have DNA methylation abnormalities within multiple loci. Not all the imprinted loci with methylation defects were affected directly by UPD. CONCLUSIONS: The association of widespread UPD associated with abnormal methylation and mRNA expression in imprinted genes in SRS is consistent with the concept of UPD as an initial genomic abnormality leading to unstable DNA methylation within the regulatory network of imprinted genes. Furthermore, disruption of any one of these genes may contribute to the heterogeneous clinical spectrum of SRS.

Mutations in NNT encoding nicotinamide nucleotide transhydrogenase cause familial glucocorticoid deficiency.

Using targeted exome sequencing, we identified mutations in NNT, an antioxidant defense gene, in individuals with familial glucocorticoid deficiency. In mice with Nnt loss, higher levels of adrenocortical cell apoptosis and impaired glucocorticoid production were observed. NNT knockdown in a human adrenocortical cell line resulted in impaired redox potential and increased reactive oxygen species (ROS) levels. Our results suggest that NNT may have a role in ROS detoxification in human adrenal glands.

MCM4 mutation causes adrenal failure, short stature, and natural killer cell deficiency in humans.

An interesting variant of familial glucocorticoid deficiency (FGD), an autosomal recessive form of adrenal failure, exists in a genetically isolated Irish population. In addition to hypocortisolemia, affected children show signs of growth failure, increased chromosomal breakage, and NK cell deficiency. Targeted exome sequencing in 8 patients identified a variant (c.71-1insG) in minichromosome maintenance-deficient 4 (MCM4) that was predicted to result in a severely truncated protein (p.Pro24ArgfsX4). Western blotting of patient samples revealed that the major 96-kDa isoform present in unaffected human controls was absent, while the presence of the minor 85-kDa isoform was preserved. Interestingly, histological studies with Mcm4-depleted mice showed grossly abnormal adrenal morphology that was characterized by non-steroidogenic GATA4- and Gli1-positive cells within the steroidogenic cortex, which reduced the number of steroidogenic cells in the zona fasciculata of the adrenal cortex. Since MCM4 is one part of a MCM2-7 complex recently confirmed as the replicative helicase essential for normal DNA replication and genome stability in all eukaryotes, it is possible that our patients may have an increased risk of neoplastic change. In summary, we have identified what we believe to be the first human mutation in MCM4 and have shown that it is associated with adrenal insufficiency, short stature, and NK cell deficiency.