Li F. Chan

MRAP and MRAP2 are bidirectional regulators of the melanocortin receptor family

The melanocortin receptor (MCR) family consists of 5 G protein-coupled receptors (MC1R–MC5R) with diverse physiologic roles. MC2R is a critical component of the hypothalamic–pituitary–adrenal axis, whereas MC3R and MC4R have an essential role in energy homeostasis. Mutations in MC4R are the single most common cause of monogenic obesity. Investigating the way in which these receptors signal and traffic to the cell membrane is vital in understanding disease processes related to MCR dysfunction. MRAP is an MC2R accessory protein, responsible for adrenal MC2R trafficking and function. Here we identify MRAP2 as a unique homologue of MRAP, expressed in brain and the adrenal gland. We report that MRAP and MRAP2 can interact with all 5 MCRs. This interaction results in MC2R surface expression and signaling. In contrast, MRAP and MRAP2 can reduce MC1R, MC3R, MC4R, and MC5R responsiveness to [Nle4,D-Phe7]alpha-melanocyte-stimulating hormone (NDP-MSH). Collectively, our data identify MRAP and MRAP2 as unique bidirectional regulators of the MCR family.

Loss of function of the melanocortin 2 receptor accessory protein 2 is associated with mammalian obesity.

Accessory to Obesity? Melanocortin receptors are a family of cell membrane receptors that control diverse physiological functions. Mutations in the gene encoding melanocortin 4 receptor (MC4R) are a cause of familial early-onset obesity. Asai et al. (p. 275) studied the function of an accessory protein for MC4R signaling, MRAP2, and found that mice genetically deficient in MRAP2 develop severe obesity. Sequencing of MRAP2 in unrelated, severely obese humans revealed one individual with a clearly disruptive genetic variant, suggesting that MRAP2 mutations might also be a rare cause of human obesity. In a zebrafish model, Sebag et al. (p. 278) studied two paralogs of the MRAP2 accessory protein, one of which enhanced MC4R responsiveness to α–melanocyte-stimulating hormone, which regulates feeding and growth. Disruption of a protein required for effective signaling by a melanocortin receptor causes severe obesity in mice. Melanocortin receptor accessory proteins (MRAPs) modulate signaling of melanocortin receptors in vitro. To investigate the physiological role of brain-expressed melanocortin 2 receptor accessory protein 2 (MRAP2), we characterized mice with whole-body and brain-specific targeted deletion of Mrap2, both of which develop severe obesity at a young age. Mrap2 interacts directly with melanocortin 4 receptor (Mc4r), a protein previously implicated in mammalian obesity, and it enhances Mc4r-mediated generation of the second messenger cyclic adenosine monophosphate, suggesting that alterations in Mc4r signaling may be one mechanism underlying the association between Mrap2 disruption and obesity. In a study of humans with severe, early-onset obesity, we found four rare, potentially pathogenic genetic variants in MRAP2, suggesting that the gene may also contribute to body weight regulation in humans.

Paediatric Cushing's syndrome: epidemiology, investigation and therapeutic advances

Cushings syndrome (CS), which is caused by excessive circulating glucocorticoid concentrations, is rare in the paediatric age range but presents a diagnostic and therapeutic challenge. Most paediatric endocrinologists have limited experience of managing children or adolescents with CS and thus benefit from close consultation with colleagues who treat adult patients. A protocol for investigation is required that broadly follows the model for adult patients. Here, the epidemiology and diagnosis of different causes of CS are discussed according to typical age of presentation. Treatment strategies for adrenocorticotrophic hormone (ACTH)-independent and ACTH-dependent CS are described and critically appraised. The management of paediatric CS patients after cure also presents challenges for optimizing growth, bone health, reproduction and body composition from childhood into and during adult life.

Distinct Melanocortin 2 Receptor Accessory Protein Domains Are Required for Melanocortin 2 Receptor Interaction and Promotion of Receptor Trafficking

Melanocortin 2 receptor (MC2R) is the receptor for the pituitary hormone ACTH. When activated, MC2R stimulates cAMP production and adrenal steroidogenesis. The functional expression of the receptor requires melanocortin 2 receptor accessory protein (MRAP), a single-transmembrane domain protein involved in the trafficking of MC2R from the endoplasmic reticulum to the cell surface. Mutations in both MC2R and MRAP cause the inherited disease familial glucocorticoid deficiency. At present, little is known regarding the mechanism of MRAP in MC2R functional expression. Here we report the characterization of MRAP in the trafficking of MC2R to the cell surface and the formation of a functional receptor. We identify the transmembrane domain of MRAP as the MC2R interaction domain and a conserved N-terminal tyrosine-rich domain of MRAP that is required for trafficking MC2R to the cell surface.

The Majority of Adrenocorticotropin Receptor (Melanocortin 2 Receptor) Mutations Found in Familial Glucocorticoid Deficiency Type 1 Lead to Defective Trafficking of the Receptor to the Cell Surface

CONTEXT There are at least 24 missense, nonconservative mutations found in the ACTH receptor [melanocortin 2 receptor (MC2R)] that have been associated with the autosomal recessive disease familial glucocorticoid deficiency (FGD) type 1. The characterization of these mutations has been hindered by difficulties in establishing a functional heterologous cell transfection system for MC2R. Recently, the melanocortin 2 receptor accessory protein (MRAP) was identified as essential for the trafficking of MC2R to the cell surface; therefore, a functional characterization of MC2R mutations is now possible. OBJECTIVE Our objective was to elucidate the molecular mechanisms responsible for defective MC2R function in FGD. METHODS Stable cell lines expressing human MRAPalpha were established and transiently transfected with wild-type or mutant MC2R. Functional characterization of mutant MC2R was performed using a cell surface expression assay, a cAMP reporter assay, confocal microscopy, and coimmunoprecipitation of MRAPalpha. RESULTS Two thirds of all MC2R mutations had a significant reduction in cell surface trafficking, even though MRAPalpha interacted with all mutants. Analysis of those mutant receptors that reached the cell surface indicated that four of six failed to signal, after stimulation with ACTH. CONCLUSION The majority of MC2R mutations found in FGD fail to function because they fail to traffic to the cell surface.

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.

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.

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.

Phenotypic characteristics of familial glucocorticoid deficiency (FGD) type 1 and 2

Context  Familial glucocorticoid deficiency (FGD) is a rare autosomal recessive disorder as a result of mutation in genes encoding either the ACTH receptor [melanocortin 2 receptor (MC2R)] or its accessory protein [melanocortin 2 receptor accessory protein (MRAP)]. The disorder is known as FGD type 1 and 2, respectively.

Familial Glucocorticoid Deficiency: Advances in the Molecular Understanding of ACTH Action

Familial glucocorticoid deficiency (FGD), otherwise known as hereditary unresponsiveness to ACTH, is a rare autosomal recessive disease characterized by glucocorticoid deficiency in the absence of mineralocorticoid deficiency. Mutations of the ACTH receptor, also known as the melanocortin-2 receptor (MC2R), account for approximately 25% of FGD cases. More recently a second gene, MRAP (melanocortin-2 receptor accessory protein), was identified and found to account for a further 15–20%. MRAP encodes a small single transmembrane domain protein, which is essential in the trafficking of the MC2R to the cell surface. In this review, we will firstly summarize the clinical presentation and genetic aetiology of this condition. Secondly, we will discuss how the discovery of MRAP has enhanced our understanding of the mechanisms of ACTH/MC2R action. Finally, we will explore future developments in this field.