Melanocortin 2 Receptor Accessory Protein Research Articles

Abstract 003: Mrap2 Dissociate Mc4r-Mediated Sympathetic Control of Metabolism and Blood Pressure

The brain melanocortin-4 receptor (MC4R) plays an important in the control of food intake and energy expenditure by increasing thermogenic sympathetic nerve activity (SNA). In addition, brain MC4R-mediated activation of SNA subserving cardiovascular organs such as the kidneys increase blood pressure. The melanocortin receptor accessory protein 2 (MRAP2) regulates the activity of the MC4R and other G-protein coupled receptors, but its specific role in MC4R-containing neurons is not clear . We hypothesized that MRAP2 in MC4R neurons is essential for the regulation of SNA, energy homeostasis and blood pressure. We generated mice (MRAP2 MC4R ) that lack MRAP2 in MC4R neurons by crossing MRAP2 fl/fl mice with MC4R Cre mice and confirmed Cre recombinase activity in MC4R neurons using td-Tomato reporter mice. Interestingly, the MRAP2 MC4R mice displayed obesity as indicated by the increased (p<0.05) body weight (males: 40.1±2.2 vs 32.9±0.9g in littermate controls, females: 28.6±2.8 vs 24.2±1.3g in controls at 16 weeks of age) and fat mass, but not lean mass. Weights of fat pads and liver were also significantly elevated in the MRAP2 MC4R relative to littermate controls. Food intake was increased in the MRAP2 MC4R mice relative to littermate controls (males: 4.7±0.3 vs 3.9±0.3g and females: 3.5±0.2 vs 2.8±0.2g). Glucose and insulin tolerance tests revealed impaired glucose tolerance and insulin-induced glucose clearance in MRAP2 MC4R mice compared to littermate controls. Baseline brown adipose tissue (BAT) SNA tended to be lower in MRAP2 MC4R mice relative to controls. Moreover, intracerebroventricular (icv) injection of MTII (MC4R agonist, 1 μg) increased BAT SNA in control mice (107±31%) but not in MRAP2 MC4R mice (18±24%, p=0.02). On the other hand, icv the MTII (1 and 2 μg)-induced increase in renal SNA (p=0.2) and blood pressure (p=0.4) was not different in MRAP2 MC4R (100±32% and 15±8mmHg, respectively) relative to controls (74±24% and 12±6mmHg). These findings support a critical role for MRAP2 in uncoupling neuronal MC4R-mediated control of regional sympathetic traffic impacting energy metabolism versus blood pressure.

Pathological cyclic strain promotes proliferation of vascular smooth muscle cells via the ACTH/ERK/STAT3 pathway.

Abnormal proliferation of vascular smooth muscle cells (VSMCs) is closely related to hyperplasia in hypertension. Our previous study suggested that adrenocorticotropic hormone (ACTH) is mechano-responsive and may regulate VSMC proliferation. However, the molecular mechanism of VSMC abnormal proliferation induced by conditions of high cyclic strain, especially the role of ACTH in this process, is unclear. Our results revealed that ACTH and its specific receptor melanocortin receptor type 2 (MC2R) were highly expressed in hypertensive rat models. Furthermore, it was demonstrated that the expression of ACTH and MC2R was up-regulated when exposed to high cyclic strain in vitro, accompanied by abnormal proliferation of VSMCs. Next, it was proved that ACTH-dependent cell proliferation was related to the phosphorylation of extracellular regulated protein kinases (ERK) and signal transducer and activator of transcription 3 (STAT3). The study also found that ACTH could promote dimerization and glycosylation of melanocortin 2 receptor accessory protein (MRAP), which had a significant effect on MC2R membrane localization and signal activation. When VSMCs were treated with PD98059, a mitogen-activated protein kinase (MAP kinase) cascade antagonist, it was determined that phosphorylation of STAT3 at Ser727 was dependent on ERK phosphorylation. In summary, these data demonstrated that the abnormal proliferation of VSMCs induced by conditions of high cyclic strain is in part attributed to ACTH and its receptor MC2R. Identifying the mechanism of ACTH-dependent proliferation of VSMCs may help to provide new therapeutic targets for hypertension.

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/β-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.

Pharmacological effect of human melanocortin-2 receptor accessory protein 2 variants on hypothalamic melanocortin receptors.

Melanocortin-3 receptor (MC3R), melanocortin-4 receptor (MC4R), and a recently identified melanocortin-2 receptor accessory protein 2 (MRAP2), are highly expressed in hypothalamus and coordinately regulate energy homeostasis, but the single cellular transcriptome of melanocortin system remains unknown. Several infrequent MRAP2 variants are reported from severe obese human patients but the mechanisms on how they affect melanocortin signaling are unclear. First, we performed in silico analysis of mouse hypothalamus RNA sequencing datasets at single-cell resolution from two independent studies. Next, we inspected the three-dimensional conformational alteration of three mutations on MRAP2 protein. Finally, the influence of MRAP2 variants on MC3R and MC4R signaling was analyzed in vitro. (1) We confirmed the actual co-expression of Mrap2 with Mc3r and Mc4r, and demonstrated more broad distribution of Mrap2-positive neuronal populations than Mc3r or Mc4r in mouse hypothalamus. (2) Compared with wild-type MRAP2, MRAP2N88Y, and MRAP2R125C showed impaired α-MSH-induced MC4R or MC3R stimulation. (3) MRAP2N88Yexhibited enhanced interaction with MC4R protein and its own. This is the first dedicated description of single-cell transcriptome signature of Mrap2, Mc3r, and Mc4r in the central nerve system and the first evidence describing the unique dimer formation, conformational change, and pharmacological effect of MRAP2 mutations on MC3R signaling.

Molecular characterization of feline melanocortin 4 receptor and melanocortin 2 receptor accessory protein 2

Melanocortin 4 receptor (MC4R), which is a member of the G protein-coupled receptor (GPCR) family, mediates regulation of energy homeostasis upon the binding of α-melanocyte-stimulating hormone (α-MSH) in the central nervous system (CNS). Melanocortin 2 receptor accessory protein 2 (MRAP2) modulates the function of MC4R. We performed cDNA cloning of cat MC4R and MRAP2 and characterized their amino acid sequences, mRNA expression patterns in cat tissues, protein–protein interactions, and functions. We found high sequence homology (>88%) with other mammalian MC4R and MRAP2 encoding 332 and 206 amino acid residues, respectively. Reverse transcription-polymerase chain reaction analysis revealed that cat MC4R and MRAP2 mRNA were expressed highly in the CNS. In CHO-K1 cells transfected with cat MC4R, stimulation with α-MSH increased intracellular cyclic adenosine monophosphate (cAMP) concentration in a dose-dependent manner. Furthermore, the presence of MRAP2 enhanced the cat MC4R-mediated cAMP production. These results suggested that cat MC4R acts as a neuronal mediator in the CNS and that its function is modulated by MRAP2. In addition, our NanoBiT study showed the dynamics of their interactions in living cells; stimulation with α-MSH slightly affected the interaction between MC4R and MRAP2, and did not affect MC4R homodimerization, suggesting that they interact in the basal state and that structural change of MC4R by activation may affect the interaction between MC4R and MRAP2.

MRAP2 regulates ghrelin receptor signaling and hunger sensing

Ghrelin is the only known circulating orexigenic hormone. It is primarily secreted by the stomach and acts at its receptor, the growth hormone secretagogue receptor 1a (GHSR1a), in the hypothalamus to signal hunger and promote food intake. The melanocortin receptor accessory protein 2 (MRAP2) was previously shown to regulate energy homeostasis through the modulation of the activity of the melanocortin-4 receptor and prokineticin receptors. In this study we identify MRAP2 as a partner of ghrelin-GHSR1a signaling. We show that MRAP2 interacts with GHSR1a and potentiates ghrelin-stimulated signaling both in vitro and in vivo. We demonstrate that in the absence of MRAP2, fasting fails to activate agouti-related protein neurons. In addition, we show that the orexigenic effect of ghrelin is lost in mice lacking MRAP2. Our results suggest that MRAP2 is an important modulator of the energy homeostasis machinery that operates through the regulation of multiple GPCRs throughout the hypothalamus.

Regions of MRAP2 required for the inhibition of orexin and prokineticin receptor signaling

The Melanocortin Receptor Accessory Protein 2 (MRAP2) regulates the activity of several GPCRs involved in the control of food intake and energy expenditure. While MRAP2 was originally thought to exclusively interact with melanocortin receptors we have recently shown that it interacts with and inhibits the trafficking and signaling of the prokineticin receptor 1 (PKR1). In this study we demonstrate a new role of MRAP2 in the regulation of the orexin receptor 1 (OX1R) and identify the specific regions of MRAP2 required for the regulation of OX1R and PKR1. Importantly, like MC4R and PKRs, OX1R is predominately expressed in the brain where it regulates food intake. By demonstrating that MRAP2 modulates the activity of OX1R we further establish the critical role of MRAP2 in the control of energy homeostasis.

Genomewide association studies of suicide attempts in US soldiers.

Suicide is a global public health problem with particular resonance for the US military. Genetic risk factors for suicidality are of interest as indicators of susceptibility and potential targets for intervention. We utilized population-based nonclinical cohorts of US military personnel (discovery: N = 473 cases and N = 9778 control subjects; replication: N = 135 cases and N = 6879 control subjects) and a clinical case-control sample of recent suicide attempters (N = 51 cases and N = 112 control subjects) to conduct GWAS of suicide attempts (SA). Genomewide association was evaluated within each ancestral group (European-, African-, Latino-American) and study using logistic regression models. Meta-analysis of the European ancestry discovery samples revealed a genomewide significant locus in association with SA near MRAP2 (melanocortin 2 receptor accessory protein 2) and CEP162 (centrosomal protein 162); 12 genomewide significant SNPs in the region; peak SNP rs12524136-T, OR = 2.88, p = 5.24E-10. These findings were not replicated in the European ancestry subsamples of the replication or suicide attempters samples. However, the association of the peak SNP remained significant in a meta-analysis of all studies and ancestral subgroups (OR = 2.18, 95%CI 1.70, 2.80). Polygenic risk score (PRS) analyses showed some association of SA with bipolar disorder. The association with SNPs encompassing MRAP2, a gene expressed in brain and adrenal cortex and involved in neural control of energy homeostasis, points to this locus as a plausible susceptibility gene for suicidality that should be further studied. Larger sample sizes will be needed to confirm and extend these findings.

Analyzing the effects of co-expression of chick (Gallus gallus) melanocortin receptors with either chick MRAP1 or MRAP2 in CHO cells on sensitivity to ACTH(1–24) or ACTH(1–13)NH2: Implications for the avian HPA axis and avian melanocortin circuits in the hypothalamus

In order to better understand the roles that melanocortin receptors (cMCRs) and melanocortin-2 receptor accessory proteins (cMRAP1 and cMRAP2) play in the HPA axis and hypothalamus, adrenal gland and hypothalamus mRNA from 1day-old white leghorn chicks (Gallus gallus), were analyzed by real-time PCR. mRNA was also made for kidney, ovary, and liver. Mrap1 mRNA could be detected in adrenal tissue, but not in any of the other tissues, and mrap2 mRNA was also detected in the adrenal gland. Finally, all five melanocortin receptors mRNAs could be detected in the adrenal gland; mc2r and mc5r mRNAs were the most abundant. To evaluate any potential interactions between MRAP1 and the MCRs that may occur in adrenal cells, individual chick mcr cDNA constructs were transiently expressed in CHO cells either in the presence or absence of a chick mrap1 cDNA, and the transfected cells were stimulated with hACTH(1–24) at concentrations ranging from 10−13M to 10−6M. As expected, MC2R required co-expression with MRAP1 for functional expression; whereas, co-expression of cMC3R with cMRAP1 had no statistically significant effect on sensitivity to hACTH(1–24). However, co-expression of MC4R and MC5R with MRAP1, increased sensitivity for ACTH(1–24) by approximately 35 fold and 365 fold, respectively. However, co-expressing of cMRAP2 with these melanocortin receptors had no effect on sensitivity to hACTH(1–24). Since the real-time PCR analysis detected mrap2 mRNA and mc4r mRNA in the hypothalamus, the interaction between cMC4R and cMRAP2 with respect to sensitivity to ACTH(1–13)NH2 stimulation was also evaluated. However, no effect, either positive or negative, was observed. Finally, the highest levels of mc5r mRNA were detected in liver cells. This observation raises the possibility that in one-day old chicks, activation of the HPA axis may also involve a physiological response from liver cells.

The interaction of MC3R and MC4R with MRAP2, ACTH, α-MSH and AgRP in chickens.

The interaction of melanocortin-4 (MC4R) and melanocortin-3 (MC3R) receptors with proopiomelanocortin (POMC)-derived peptides (e.g. α-MSH), agouti-related protein (AgRP) and melanocortin-2 receptor accessory protein 2 (MRAP2) is suggested to play critical roles in energy balance of vertebrates. However, evidence on their interaction in birds remains scarce. Our study aims to reveal their interaction in chickens and the results showed that (1) chicken (c-)MC3R and cMC4R expressed in Chinese hamster ovary (CHO) cells can be activated by α-MSH and ACTH1-39 equipotently, monitored by a pGL3-CRE-luciferase reporter system; (2) cMC3R and cMC4R, when co-expressed with cMRAP2 (or cMRAP, a cMRAP2 homolog), show increased sensitivity to ACTH treatment and thus likely act as ACTH-preferring receptors, and the interaction between cMC3R/cMC4R and cMRAP2 was demonstrated by co-immunoprecipitation assay; (3) both cMC3R and cMC4R display constitutive activity when expressed in CHO cells, as monitored by dual-luciferase reporter assay, and cMRAP2 (and cMRAP) can modulate their constitutive activity; (4) AgRP inhibits the constitutive activity of cMC3R/cMC4R, and it also antagonizes ACTH/α-MSH action on cMC4R/cMC3R, indicating that AgRP functions as the inverse agonist and antagonist for both receptors. These findings, together with the co-expression of cMC4R, cMC3R, cMRAP2, cAgRP and cPOMC in chicken hypothalamus detected by quantitative real-time PCR, suggest that within the hypothalamus, α-MSH/ACTH, AgRP and MRAP2 may interact at the MC4R(/MC3R) interface to control energy balance. Furthermore, our data provide novel proof for the involvement of MRAP2 (and MRAP) in fine-tuning the constitutive activity and ligand sensitivity and selectivity of both MC3R and MC4R in vertebrates.

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.

ACTH Receptor (MC2R) Specificity: What Do We Know About Underlying Molecular Mechanisms?

Coincidentally, the release of this Research Topic in Frontiers in Endocrinology takes place 25 years after the discovery of the adrenocorticotropic hormone receptor (ACTHR) by Mountjoy and colleagues. In subsequent years, following the discovery of other types of mammalian melanocortin receptors (MCRs), ACTHR also became known as melanocortin type 2 receptor (MC2R). At present, five types of MCRs have been reported, all of which share significant sequence similarity at the amino acid level, and all of which specifically bind melanocortins (MCs)—a group of biologically active peptides generated by proteolysis of the proopiomelanocortin precursor. All MCs share an identical –H–F–R–W– pharmacophore sequence. α-Melanocyte-stimulating hormone (α-MSH) and adrenocorticotropic hormone (ACTH) are the most extensively studied MCs and are derived from the same region. Essentially, α-MSH is formed from the first 13 amino acid residues of ACTH. ACTHR is unique among MCRs because it binds one sole ligand—ACTH, which makes it a very attractive research object for molecular pharmacologists. However, much research has failed, and functional studies of this receptor are lagging behind other MCRs. The reason for these difficulties has already been outlined by Mountjoy and colleagues in their publication on ACTHR coding sequence discovery where the Cloudman S91 melanoma cell line was used for receptor expression because it was a “more sensitive assay system.” Subsequent work showed that ACTHR could be successfully expressed only in endogenous MCR-expressing cell lines, since in other cell lines it is retained within the endoplasmic reticulum. The resolution of this methodological problem came in 2005 with the discovery of melanocortin receptor accessory protein, which is required for the formation of functionally active ACTHR. The decade that followed this discovery was filled with exciting research that provided insight into the molecular mechanisms underlying the action of ACTHR. The purpose of this review is to summarize the advances in this fascinating research field.

Association of estradiol on expression of melanocortin receptors and their accessory proteins in the liver of chicken (Gallus gallus)

The melanocortin receptor accessory proteins (MRAP and MRAP2) are small single-pass transmembrane proteins that regulate the biological functions of the melanocortin receptor (MCR) family. MCRs comprise five receptors (MC1R–MC5R) with diverse physiological roles in mammals. Five MCR members and two MRAPs were also predicted in the chicken (Gallus gallus) genome. However, little is known about their expression, regulation and biological functions. In this study, we cloned the MRAP and MRAP2 genes. Sequencing analysis revealed that the functional domains of MRAP and MRAP2 were conserved among species, suggesting that the physiological roles of chicken MRAP and MRAP2 could be similar to their mammalian counterparts. Tissue expression analysis demonstrated that MRAP was expressed in the adrenal gland, liver, spleen, glandular stomach and lungs, while MRAP2 is predominantly expressed in the adrenal gland. All five MCRs were present in the adrenal gland, but showed different expression patterns in other tissues. The MC5R was the only MCR member that was expressed in the chicken liver. The expression levels of MRAP in chicken liver were significantly increased at sexual maturity stage, and were significantly up-regulated (P<0.05) when chickens and chicken primary hepatocytes were treated with 17β-estradiol in vivo and in vitro, respectively; however, expression levels of PPARγ were down-regulated, and no effect on MC5R was observed. Our results suggested that estrogen could stimulate the expression of MRAP in the liver of chicken through inhibiting the expression of transcription regulation factor PPARγ, and MRAP might play its biological role in a different way rather than forming an MRAP/MC2R complex in chicken liver during the egg-laying period.

ACTH Antagonists.

Adrenocorticotropin (ACTH) acts via a highly selective receptor that is a member of the melanocortin receptor subfamily of type 1 G protein-coupled receptors. The ACTH receptor, also known as the melanocortin 2 receptor (MC2R), is unusual in that it is absolutely dependent on a small accessory protein, melanocortin receptor accessory protein (MRAP) for cell surface expression and function. ACTH is the only known naturally occurring agonist for this receptor. This lack of redundancy and high degree of ligand specificity suggests that antagonism of this receptor could provide a useful therapeutic aid and a potential investigational tool. Clinical situations in which this could be useful include (1) Cushing’s disease and ectopic ACTH syndrome – especially while preparing for definitive treatment of a causative tumor, or in refractory cases, or (2) congenital adrenal hyperplasia – as an adjunct to glucocorticoid replacement. A case for antagonism in other clinical situations in which there is ACTH excess can also be made. In this article, we will explore the scientific and clinical case for an ACTH antagonist, and will review the evidence for existing and recently described peptides and modified peptides in this role.

Decreased melanocortin-4 receptor function conferred by an infrequent variant at the human melanocortin receptor accessory protein 2 gene.

The melanocortin receptor accessory protein 2 (MRAP2) is relevant for weight regulation in mice and humans. This function is likely mediated by regulation of the melanocortin-4 receptor (MC4R). Functional implications of human MRAP2 mutations have not been described yet. A mutation screen was conducted in MRAP2 in 184 children and adolescents with (extreme) obesity and in 184 lean controls. Detected nonsynonymous variants were genotyped in larger independent study groups (300 people with obesity and 436 individuals with normal weight). The influence of mutant MRAP2 on MC4R signaling was analyzed in vitro. (1) Three (two novel) nonsynonymous MRAP2 variants were detected: p.Ala137Thr, p.Gln174Arg, p.Arg125His (rs115655382), two synonymous variants, and three intronic variants. (2) The impact of MRAP2 on MC4R function was dependent on the ratio between the two co-expressed proteins. Increased MC4R signaling was detected at MRAP2/MC4R ratios of 2 + 1 and above. (3) The function of MC4R was reduced with the infrequent allele at the MRAP2 p.Gln174Arg variant. (4) The three nonsynonymous mutations were each only detected once among the 484 people with obesity and not among 620 individuals with normal weight. This was the first study describing an effect of a MRAP2 mutation on MC4R function.

Dual Topology of the Melanocortin-2 Receptor Accessory Protein Is Stable.

Melanocortin 2 receptor accessory protein (MRAP) facilitates trafficking of melanocortin 2 (MC2) receptors and is essential for ACTH binding and signaling. MRAP is a single transmembrane domain protein that forms antiparallel homodimers. These studies ask when MRAP first acquires this dual topology, whether MRAP architecture is static or stable, and whether the accessory protein undergoes rapid turnover. To answer these questions, we developed an approach that capitalizes on the specificity of bacterial biotin ligase, which adds biotin to lysine in a short acceptor peptide sequence; the distinct mobility of MRAP protomers of opposite orientations based on their N-linked glycosylation; and the ease of identifying biotin-labeled proteins. We inserted biotin ligase acceptor peptides at the N- or C-terminal ends of MRAP and expressed the modified proteins in mammalian cells together with either cytoplasmic or endoplasmic reticulum-targeted biotin ligase. MRAP assumed dual topology early in biosynthesis in both CHO and OS3 adrenal cells. Once established, MRAP orientation was stable. Despite its conformational stability, MRAP displayed a half-life of under 2 h in CHO cells. The amount of MRAP was increased by the proteasome inhibitor MG132 and MRAP underwent ubiquitylation on lysine and other amino acids. Nonetheless, when protein synthesis was blocked with cycloheximide, MRAP was rapidly degraded even when MG132 was included and all lysines were replaced by arginines, implicating non-proteasomal degradation pathways. The results show that although MRAP does not change orientations during trafficking, its synthesis and degradation are dynamically regulated.

Loss of Mrap2 is associated with Sim1 deficiency and increased circulating cholesterol.

Melanocortin receptor accessory protein 2 (MRAP2) is a transmembrane accessory protein predominantly expressed in the brain. Both global and brain-specific deletion of Mrap2 in mice results in severe obesity. Loss-of-function MRAP2 mutations have also been associated with obesity in humans. Although MRAP2 has been shown to interact with MC4R, a G protein-coupled receptor with an established role in energy homeostasis, appetite regulation and lipid metabolism, the mechanisms through which loss of MRAP2 causes obesity remains uncertain. In this study, we used two independently derived lines of Mrap2 deficient mice (Mrap2tm1a/tm1a) to further study the role of Mrap2 in the regulation of energy balance and peripheral lipid metabolism. Mrap2tm1a/tm1a mice have a significant increase in body weight, with increased fat and lean mass, but without detectable changes in food intake or energy expenditure. Transcriptomic analysis showed significantly decreased expression of Sim1, Trh, Oxt and Crh within the hypothalamic paraventricular nucleus of Mrap2tm1a/tm1a mice. Circulating levels of both high-density lipoprotein and low-density lipoprotein were significantly increased in Mrap2 deficient mice. Taken together, these data corroborate the role of MRAP2 in metabolic regulation and indicate that, at least in part, this may be due to defective central melanocortin signalling.

A novel role for pigment genes in the stress response in rainbow trout (Oncorhynchus mykiss).

In many vertebrate species visible melanin-based pigmentation patterns correlate with high stress- and disease-resistance, but proximate mechanisms for this trait association remain enigmatic. Here we show that a missense mutation in a classical pigmentation gene, melanocyte stimulating hormone receptor (MC1R), is strongly associated with distinct differences in steroidogenic melanocortin 2 receptor (MC2R) mRNA expression between high- (HR) and low-responsive (LR) rainbow trout (Oncorhynchus mykiss). We also show experimentally that cortisol implants increase the expression of agouti signaling protein (ASIP) mRNA in skin, likely explaining the association between HR-traits and reduced skin melanin patterning. Molecular dynamics simulations predict that melanocortin 2 receptor accessory protein (MRAP), needed for MC2R function, binds differently to the two MC1R variants. Considering that mRNA for MC2R and the MC1R variants are present in head kidney cells, we hypothesized that MC2R activity is modulated in part by different binding affinities of the MC1R variants for MRAP. Experiments in mammalian cells confirmed that trout MRAP interacts with the two trout MC1R variants and MC2R, but failed to detect regulation of MC2R signaling, possibly due to high constitutive MC1R activity.

Hypothesis and Theory: Revisiting Views on the Co-evolution of the Melanocortin Receptors and the Accessory Proteins, MRAP1 and MRAP2.

The evolution of the melanocortin receptors (MCRs) is closely associated with the evolution of the melanocortin-2 receptor accessory proteins (MRAPs). Recent annotation of the elephant shark genome project revealed the sequence of a putative MRAP1 ortholog. The presence of this sequence in the genome of a cartilaginous fish raises the possibility that the mrap1 and mrap2 genes in the genomes of gnathostome vertebrates were the result of the chordate 2R genome duplication event. The presence of a putative MRAP1 ortholog in a cartilaginous fish genome is perplexing. Recent studies on melanocortin-2 receptor (MC2R) in the genomes of the elephant shark and the Japanese stingray indicate that these MC2R orthologs can be functionally expressed in CHO cells without co-expression of an exogenous mrap1 cDNA. The novel ligand selectivity of these cartilaginous fish MC2R orthologs is discussed. Finally, the origin of the mc2r and mc5r genes is reevaluated. The distinctive primary sequence conservation of MC2R and MC5R is discussed in light of the physiological roles of these two MCR paralogs.

Identifying the activation motif in the N-terminal of rainbow trout and zebrafish melanocortin-2 receptor accessory protein 1 (MRAP1) orthologs

The activation of mammalian melanocortin-2 receptor (MC2R) orthologs is dependent on a four-amino acid activation motif (LDYL/I) located in the N-terminal of mammalian MRAP1 (melanocortin-2 receptor accessory protein). Previous alanine substitution analysis had shown that the Y residue in this motif appears to be the most important for mediating the activation of mammalian MC2R orthologs. Similar, but not identical amino acid motifs were detected in rainbow trout MRAP1 (YDYL) and zebrafish MRAP1 (YDYV). To determine the importance of these residues in the putative activation motifs, rainbow trout and zebrafish MRAP1 orthologs were individually co-expressed in CHO cells with rainbow trout MC2R, and the activation of this receptor with either the wild-type MRAP1 ortholog or alanine-substituted analogs of the two teleost MRAP1s was analyzed. Alanine substitutions at all four amino acid positions in rainbow trout MRAP1 blocked activation of the rainbow trout MC2R. Single alanine substitutions of the D and Y residues in rainbow trout and zebrafish MRAP1 indicate that these two residues play a significant role in the activation of rainbow trout MC2R. These observations indicate that there are subtle differences in the way that teleost and mammalian MRAPs are involved in the activation of their corresponding MC2R orthologs.