Establishment of human induced pluripotent stem cell line SDQLCHi029-A from one Type 1 familial glucocorticoid deficiency patient carrying compound heterozygote mutations in MC2R gene.

Type 1 familial glucocorticoid deficiency (FGD) is a rare autosomal recessive disorder due to variation of the melanocortin-2-receptor (MC2R) gene. Induced pluripotent stem cell (iPSC) line SDQLCHi029-A was successfully generated from peripheral blood mononuclear cells obtained from a 5-day-old girl with MC2R mutations (c.428C > T and c.409C > T). The iPSC line showed genetically stable and matched the donor's PBMCs. displayed a normal karyotype, expressed high pluripotent markers, and exhibited differentiation potential of three germ layers in vitro. The iPSC line could be a good model to study the pathogenesis of FGD type 1 and screen new drugs for the disease.

[Clinical characteristics and genetic analysis of two children with Familial glucocorticoid deficiency type 1 due to variants of MC2R gene].

OBJECTIVE: To improve the recognition of Familial glucocorticoid deficiency type 1 (FGD1) due to variants of melanocortin 2 receptor (MC2R) gene. METHODS: Two children with FGD1 diagnosed at the Henan Children's Hospital respectively in 2019 and 2021 were selected as the study subjects. Clinical data, treatment, follow-up and results of genetic testing were collected and retrospectively analyzed. RESULTS: Whole exome sequencing revealed that both children had harbored compound heterozygous variants of the MC2R gene, including c.433C>T (p.R145C) and c.710T>C (p.L237P) in child 1, and c.145delG (p.V49Cfs*35) and c.307G>A (p.D103N) in child 2, among which c.710T>C (p.L237P) and c.145delG (p.V49Cfs*35) were unreported previously. CONCLUSION: FGD1 is clinically rare, and genetic sequencing is crucial for the definite diagnosis. Discovery of the and novel variants has enriched the mutational spectrum of the FGD1 gene.

Functional characterization of melanocortin 2 receptor (Mc2r) from a lobe-finned fish (Protopterus annectens) and insights into the molecular evolution of melanocortin receptors.

Recent studies from our group on melanocortin 2 receptors (Mc2r) from basal families of actinopterygians have served to resolve that Mrap1 dependence and ACTH selectivity are features of even the most basal ray-finned fishes. However, there have been no studies on Mc2r function of the basal sarcopterygians, the lobe-finned fishes, represented by the extant members coelacanths and lungfishes. Here, we offer the first molecular and functional characterization of an Mc2r from a lobe-finned fish, the West African lungfish (Protopterus annectens). Plasmids containing cDNA constructs of lungfish (lf) Mc2r and Mrap1 were expressed in mammalian and zebrafish cell lines. Cells were then stimulated by human ACTH(1-24) and melanocyte stimulating hormone (α-MSH), as well as alanine-substituted analogs of hACTH(1-24) targeting residues within the H6F7R8W9 and K15K16R17R18P19 motifs. Activation of lfMc2r was assessed using a cAMP-responsive luciferase reporter gene assay. In these assays, lfMc2r required co-expression with lfMrap1, was selective for ACTH over α-MSH at physiological concentrations of the ligands, and was completely inhibited by multiple-alanine substitutions of the HFRW (A6-9) and KKRRP (A15-19) motifs. Single- and partial-alanine substitutions of the HFRW and KKRRP motifs varied in their impacts on receptor-ligand affinity from having no effect to completely inhibiting lfMc2r activation. This characterization of the Mc2r of a lobe-finned fish fulfills the last major extant vertebrate group for which Mc2r function had yet to be characterized. In doing so, we resolve that all basal bony vertebrate groups exhibit Mc2r function that substantially differs from that of the cartilaginous fishes, indicating that rapid and dramatic shift in Mc2r function occurred between the radiation of cartilaginous fishes and the emergence of bony fishes. We support this interpretation with a molecular clock analysis of the melanocortin receptors, which demonstrates the uniquely high rate of sequence divergence in Mc2r. Much remains to be understood regarding the molecular evolution of Mc2r during the early radiation of vertebrates that resulted in the derived functional characteristics of Mrap1 dependence and exclusive selectivity for ACTH.

Molecular and pharmacological analysis of the melanocortin-2 receptor and its accessory proteins Mrap1 and Mrap2 in a Squalomorph shark, the Pacific spiny dogfish.

The hypothalamus-pituitary-adrenal/interrenal (HPA/I) axis is a conserved vertebrate neuroendocrine mechanism regulating the stress response. The penultimate step of the HPA/I axis is the exclusive activation of the melanocortin-2 receptor (Mc2r) by adrenocorticotropic hormone (ACTH), requiring an accessory protein, Mrap1 or Mrap2. Limited data for only three cartilaginous fishes support the hypothesis that Mc2r/Mrap1 function in bony vertebrates is a derived trait. Further, Mc2r/Mrap1 functional properties appear to contrast among cartilaginous fishes (i.e., the holocephalans and elasmobranchs). This study sought to determine whether functional properties of Mc2r/Mrap1 are conserved across elasmobranchs and in contrast to holocephalans. The deduced amino acid sequences of Pacific spiny dogfish (Squalus suckleyi; pd) pdMc2r, pdMrap1, and pdMrap2 were obtained from a de novo transcriptome of the interrenal gland and validated against the S. suckleyi genome. pdMc2r showed high primary sequence similarity with elasmobranch and holocephalan Mc2r except at extracellular domains 1 and 2, and transmembrane domain 5. pdMraps showed similarly high sequence similarity with holocephalan and other elasmobranch Mraps, with all cartilaginous fish Mrap1 orthologs lacking an activation motif. cAMP reporter gene assays demonstrated that pdMc2r requires an Mrap for activation, and can be activated by stingray (sr) ACTH(1-24), srACTH(1-13)NH2 (i.e., α-MSH), and γ-melanocyte-stimulating hormone at physiological concentrations. However, pdMc2r was three orders of magnitude more sensitive to srACTH(1-24) than srACTH(1-13)NH2. Further, pdMc2r was two orders of magnitude more sensitive to srACTH(1-24) when expressed with pdMrap1 than with pdMrap2. These data suggest that functional properties of pdMc2r/pdMrap1 reflect other elasmobranchs and contrast what is seen in holocephalans.

Hypothalamus-pituitary-interrenal (HPI) axis signaling in Atlantic sturgeon (Acipenser oxyrinchus) and sterlet (Acipenser ruthenus).

In vertebrates, the hypothalamic-pituitaryadrenal/interrenal (HPA/HPI) axis is a highly conserved endocrine axis that regulates glucocorticoid production via signaling by corticotropin releasing hormone (CRH) and adrenocorticotropic hormone (ACTH). Once activated by ACTH, Gs protein-coupled melanocortin 2 receptors (Mc2r) present in corticosteroidogenic cells stimulate expression of steroidogenic acute regulatory protein (Star), which initiates steroid biosynthesis. In the present study, we examined the tissue distribution of genes involved in HPI axis signaling and steroidogenesis in the Atlantic sturgeon (Acipenser oxyrinchus) and provided the first functional characterization of Mc2r in sturgeon. Mc2r of A. oxyrinchus and the sterlet sturgeon (Acipenser ruthenus) are co-dependent on interaction with the melanocortin receptor accessory protein 1 (Mrap1) and highly selective for human (h) ACTH over other melanocortin ligands. A. oxyrinchus expresses key genes involved in HPI axis signaling in a tissue-specific manner that is indicative of the presence of a complete HPI axis in sturgeon. Importantly, we co-localized mc2r, mrap1, and star mRNA expression to the head kidney, indicating that this is possibly a site of ACTH-mediated corticosteroidogenesis in sturgeon. Our results are discussed in the context of other studies on the HPI axis of basal bony vertebrates, which, when taken together, demonstrate a need to better resolve the evolution of HPI axis signaling in vertebrates.

A rare homozygous variant of MC2R gene identified in a Chinese family with familial glucocorticoid deficiency type 1: A case report.

Background: Melanocortin-2 receptor (MC2R), a member of the G protein-coupled receptor family, is selectively activated by adrenocorticotropic hormone (ACTH). variants in MC2R are associated with family glucocorticoid deficiency 1 (FGD1). Case presentation: We first reported a Chinese family with two affected siblings with a homozygotic variant of c.712C>T/p.H238Y in MC2R, presenting with skin hyperpigmentation, hyperbilirubinemia, and tall stature. These individuals showed novel clinical features, including congenital heart defects, not been found in other FGD1 patients. Conclusions: We reported a Chinese family with affected siblings having a homozygotic variant of c.712C>T/p.H238Y in MC2R.Our report may expand the genetic and clinical spectrum of FGD1.

Trends in the evolution of the elasmobranch melanocortin-2 receptor: Insights from structure/function studies on the activation of whale shark Mc2r.

To understand the mechanism for activation of the melanocortin-2 receptor (Mc2r) of the elasmobranch, Rhincodon typus (whale shark; ws), wsmc2r was co-expressed with wsmrap1 in CHO cells, and the transfected cells were stimulated with alanine-substituted analogs of ACTH(1-24) at the "message" motif (H6F7R8W9) and the "address" motif (K15K16R17R18P19). Complete alanine substitution of the H6F7R8W9 motif blocked activation, whereas single alanine substitution at this motif indicated the following hierarchy of position importance for activation: W9 > R8, and substitution at F7 and H6 had no effect on activation. The same analysis was done on a representative bony vertebrate Mc2r ortholog (Amia calva; bowfin; bf) and the order of position importance for activation was W9 > R8 = F7, (alanine substitution at H6 was negligible). Complete alanine substitution at the K15K16R17R18P19 motif resulted in distinct outcomes for wsMc2r and bfMc2r. For bfMc2r, this analog blocked activation-an outcome typical for bony vertebrate Mc2r orthologs. For wsMc2r, this analog resulted in a shift in sensitivity to stimulation of the analog as compared to ACTH(1-24) by two orders of magnitude, but the dose response curve did reach saturation. To evaluate whether the EC2 domain of wsMc2r plays a role in activation, a chimeric wsMc2r was made in which the EC2 domain was replaced with the EC2 domain from a melanocortin receptor that does not interact with Mrap1 (i.e., Xenopus tropicalis Mc1r). This substitution did not negatively impact the activation of the chimeric receptor. In addition, alanine substitution at a putative activation motif in the N-terminal of wsMrap1 did not affect the sensitivity of wsMc2r to stimulation by ACTH(1-24). Collectively, these observations suggest that wsMc2r may only have a HFRW binding site for melanocortin-related ligand which would explain how wsMc2r could be activated by either ACTH or MSH-sized ligands.

MRAP2 regulates energy homeostasis by promoting primary cilia localization of MC4R.

The G protein-coupled receptor melanocortin-4 receptor (MC4R) and its associated protein melanocortin receptor-associated protein 2 (MRAP2) are essential for the regulation of food intake and body weight in humans. MC4R localizes and functions at the neuronal primary cilium, a microtubule-based organelle that senses and relays extracellular signals. Here, we demonstrate that MRAP2 is critical for the weight-regulating function of MC4R neurons and the ciliary localization of MC4R. More generally, our study also reveals that GPCR localization to primary cilia can require specific accessory proteins that may not be present in heterologous cell culture systems. Our findings further demonstrate that targeting of MC4R to neuronal primary cilia is essential for the control of long-term energy homeostasis and suggest that genetic disruption of MC4R ciliary localization may frequently underlie inherited forms of obesity.

Structural basis of signaling regulation of the human melanocortin-2 receptor by MRAP1.

G protein-coupled receptors (GPCRs) are regulated by various downstream proteins, of which the melanocortin receptor accessory protein 1 (MRAP1) is closely involved in the regulation of melanocortin receptor 2 (MC2R). Assisted by MRAP1, MC2R responds to adrenocorticotropic hormone (ACTH) and stimulates glucocorticoid biogenesis and cortisol secretion. MC2R activation plays an essential role in the hypothalamic-pituitary-adrenal (HPA) axis that regulates stress response, while its dysfunction causes glucocorticoid insufficiency- or cortisol excess-associated disorders. Here, we present a cryo-electron microscopy (cryo-EM) structure of the ACTH-bound MC2R-Gs-MRAP1 complex. Our structure, together with mutagenesis analysis, reveals a unique sharp kink at the extracellular region of MRAP1 and the 'seat-belt' effect of MRAP1 on stabilizing ACTH binding and MC2R activation. Mechanisms of ACTH recognition by MC2R and receptor activation are also demonstrated. These findings deepen our understanding of GPCR regulation by accessory proteins and provide valuable insights into the ab initio design of therapeutic agents targeting MC2R.

Clinical characteristics and genetic analysis of two children with Familial glucocorticoid deficiency type 1 due to variants of MC2R gene / 中华医学遗传学杂志

OBJECTIVE@#To improve the recognition of Familial glucocorticoid deficiency type 1 (FGD1) due to variants of melanocortin 2 receptor (MC2R) gene.@*METHODS@#Two children with FGD1 diagnosed at the Henan Children's Hospital respectively in 2019 and 2021 were selected as the study subjects. Clinical data, treatment, follow-up and results of genetic testing were collected and retrospectively analyzed.@*RESULTS@#Whole exome sequencing revealed that both children had harbored compound heterozygous variants of the MC2R gene, including c.433C>T (p.R145C) and c.710T>C (p.L237P) in child 1, and c.145delG (p.V49Cfs*35) and c.307G>A (p.D103N) in child 2, among which c.710T>C (p.L237P) and c.145delG (p.V49Cfs*35) were unreported previously.@*CONCLUSION@#FGD1 is clinically rare, and genetic sequencing is crucial for the definite diagnosis. Discovery of the and novel variants has enriched the mutational spectrum of the FGD1 gene.

Structure/Function Studies on the Activation Motif of Two Non-Mammalian Mrap1 Orthologs, and Observations on the Phylogeny of Mrap1, Including a Novel Characterization of an Mrap1 from the Chondrostean Fish, Polyodon spathula.

In derived bony vertebrates, activation of the melanocortin-2 receptor (Mc2r) by its ACTH ligand requires chaperoning by the Mc2r accessory protein (Mrap1). The N-terminal domain of the non-mammalian tetrapod MRAP1 from chicken (c; Gallus gallus) has the putative activation motif, W18D19Y20I21, and the N-terminal domain in the neopterygian ray-finned fish Mrap1 from bowfin (bf; Amia calva) has the putative activation motif, Y18D19Y20I21. The current study used an alanine-substitution paradigm to test the hypothesis that only the Y20 position in the Mrap1 ortholog of these non-mammalian vertebrates is required for activation of the respective Mc2r ortholog. Instead, we found that for cMRAP1, single alanine-substitution resulted in a gradient of inhibition of activation (Y20 >> D19 = W18 > I21). For bfMrap1, single alanine-substitution also resulted in a gradient of inhibition of activation (Y20 >> D19 > I21 > Y18). This study also included an analysis of Mc2r activation in an older lineage of ray-finned fish, the paddlefish (p), Polyodon spathula (subclass Chondronstei). Currently no mrap1 gene has been found in the paddlefish genome. When pmc2r was expressed alone in our CHO cell/cAMP reporter gene assay, no activation was observed following stimulation with ACTH. However, when pmc2r was co-expressed with either cmrap1 or bfmrap1 robust dose response curves were generated. These results indicate that the formation of an Mc2r/Mrap1 heterodimer emerged early in the radiation of the bony vertebrates.

Modulation of Canine Melanocortin-3 and -4 Receptors by Melanocortin-2 Receptor Accessory Protein 1 and 2.

The neural melanocortin receptors (MCRs), melanocortin-3 and -4 receptors (MC3R and MC4R), have crucial roles in regulating energy homeostasis. The melanocortin-2 receptor accessory proteins (MRAPs, MRAP1 and MRAP2) have been shown to regulate neural MCRs in a species-specific manner. The potential effects of MRAP1 and MRAP2 on canine neural MCRs have not been investigated before. Herein, we cloned canine (c) MC3R and identified one canine MRAP2 splice variant, MRAP2b, with N-terminal extension of cMRAP2a. Canine MC3R showed higher maximal responses to five agonists than those of human MC3R. We further investigated the modulation of cMRAP1, cMRAP2a, and cMRAP2b, on cMC3R and cMC4R pharmacology. For the cMC3R, all MRAPs had no effect on trafficking; cMRAP1 significantly decreased Bmax whereas cMRAP2a and cMRAP2b significantly increased Bmax. Both MRAP1 and MRAP2a decreased Rmaxs in response to α-MSH and ACTH; MRAP2b only decreased α-MSH-stimulated cAMP generation. For the MC4R, MRAP1 and MRAP2a increased cell surface expression, and MRAP1 and MRAP2a increased Bmaxs. All MRAPs had increased affinities to α-MSH and ACTH. MRAP2a increased ACTH-induced cAMP levels, whereas MRAP2b decreased α-MSH- and ACTH-stimulated cAMP production. These findings may lead to a better understanding of the regulation of neural MCRs by MRAP1 and MRAP2s.

Human Melanocortin-2 Receptor: Identifying a Role for Residues in the TM4, EC2, and TM5 Domains in Activation and Trafficking as a Result of Co-Expression with the Accessory Protein, Mrap1 in Chinese Hamster Ovary Cells.

Human melanocortin-2 receptor (hMC2R) co-expressed with the accessory protein mouse (m)MRAP1 in Chinese Hamster Ovary (CHO) cells has been used as a model system to investigate the activation and trafficking of hMC2R. A previous study had shown that the N-terminal domain of mMRAP1 makes contact with one of the extracellular domains of hMC2R to facilitate activation of hMC2R. A chimeric receptor paradigm was used in which the extracellular domains of hMC2R were replaced with the corresponding domains from Xenopus tropicalis MC1R, a receptor that does not interact with MRAP1, to reveal that EC2 (Extracellular domain 2) is the most likely contact site for hMC2R and mMRAP1 to facilitate activation of the receptor following an ACTH binding event. Prior to activation, mMRAP1 facilitates the trafficking of hMC2R from the ER to the plasma membrane. This process is dependent on the transmembrane domain (TM) of mMRAP1 making contact with one or more TMs of hMC2R. A single alanine substitution paradigm was used to identify residues in TM4 (i.e., I163, M165), EC2 (F167), and TM5 (F178) that play a role in the trafficking of hMC2R to the plasma membrane. These results provide further clarification of the activation mechanism for hMC2R.

A basal actinopterygian melanocortin receptor: Molecular and functional characterization of an Mc2r ortholog from the Senegal bichir (Polypterus senegalus).

In bony vertebrates, melanocortin 2 receptor (Mc2r) specifically binds adrenocorticotropic hormone (ACTH) and is responsible for mediating anterior pituitary signaling that stimulates corticosteroid production in the adrenal gland/interrenal cells. In bony fishes Mc2r requires the chaperoning of an accessory protein (Mrap1) to traffic to the membrane surface and bind ACTH. Here, we evaluated the structure and pharmacological properties of Mc2r from the Senegal bichir (Polypterus senegalus), which represents the most basal bony fish from which an Mc2r has been pharmacologically studied to date. In our experiments, cDNA constructs of the Mc2r from the Senegal bichir (sbMc2r) and various vertebrate Mrap1s were heterologously co-expressed in Chinese hamster ovary (CHO) cells, stimulated by ACTH or melanocyte-stimulating hormone (α-MSH) ligands, and assessed using a luciferase reporter gene assay. When expressed without an Mrap1, sbMc2r was not activated by ACTH. When co-expressed with Mrap1 from either chicken (Gallus gallus) or bowfin (Amia calva), sbMc2r could be activated in a dose-dependent manner by ACTH, but not α-MSH. Co-expression of sbMrap2 with sbMc2r resulted in no detectable activation of the receptor. Collectively, these results demonstrate that sbMc2r has pharmacological properties similar to those of Mc2rs of later-evolved bony fishes, such as Mrap1 dependence and ACTH selectivity, indicating that these qualities of Mc2r function are ancestral to all bony fish Mc2rs.

Identification of genes associated with human-canine communication in canine evolution.

The dog (Canis familiaris) was the first domesticated animal and hundreds of breeds exist today. During domestication, dogs experienced strong selection for temperament, behaviour, and cognitive ability. However, the genetic basis of these abilities is not well-understood. We focused on ancient dog breeds to investigate breed-related differences in social cognitive abilities. In a problem-solving task, ancient breeds showed a lower tendency to look back at humans than other European breeds. In a two-way object choice task, they showed no differences in correct response rate or ability to read human communicative gestures. We examined gene polymorphisms in oxytocin, oxytocin receptor, melanocortin 2 receptor, and a Williams-Beuren syndrome-related gene (WBSCR17), as candidate genes of dog domestication. The single-nucleotide polymorphisms on melanocortin 2 receptor were related to both tasks, while other polymorphisms were associated with the unsolvable task. This indicates that glucocorticoid functions are involved in the cognitive skills acquired during dog domestication.

Pharmacological Evaluation of Melanocortin 2 Receptor Accessory Protein 2 on Axolotl Neural Melanocortin Signaling.

The Melanocortin-3 receptor (MC3R) and Melanocortin-4 receptor (MC4R), two members of the key hypothalamic neuropeptide signaling, function as complex mediators to control the central appetitive and energy homeostasis. The melanocortin 2 receptor accessory protein 2 (MRAP2) is well-known for its modulation on the trafficking and signaling of MC3R and MC4R in mammals. In this study, we cloned and elucidated the pharmacological profiles of MRAP2 on the regulation of central melanocortin signaling in a relatively primitive poikilotherm amphibian species, the Mexican axolotl (Ambystoma mexicanum). Our results showed the higher conservation of axolotl mc3r and mc4r across species than mrap2, especially the transmembrane regions in these proteins. Phylogenetic analysis indicated that the axolotl MC3R/MC4R clustered closer to their counterparts in the clawed frog, whereas MRAP2 fell in between the reptile and amphibian clade. We also identified a clear co-expression of mc3r, mc4r, and mrap2 along with pomc and agrp in the axolotl brain tissue. In the presence of MRAP2, the pharmacological stimulation of MC3R by α-MSH or ACTH significantly decreased. MRAP2 significantly decreased the cell surface expression of MC4R in a dose dependent manner. The co-localization and formation of the functional complex of axolotl MC3R/MC4R and MRAP2 on the plasma membrane were further confirmed in vitro. Dramatic changes of the expression levels of mc3r, mrap2, pomc, and agrp in the fasting axolotl hypothalamus indicated their critical roles in the metabolic regulation of feeding behavior and energy homeostasis in the poikilotherm aquatic amphibian.

Silver nanoparticles modify the hypothalamic-pituitary-interrenal axis and block cortisol response to an acute stress in zebrafish, Danio rerio.

The present study aimed at assessing the effects of exposure to silver nanoparticle (AgNP) and a subsequent acute stress on the expression of various genes involved in the hypothalamus-pituitary-interrenal (HPI) axis in zebrafish, Danio rerio. The fish were exposed to 0 (Control), 0.1 (LC), 0.4 (MC), and 1.2 (HC) mg Ag/L (as AgNP) over a 2-week period, followed by an acute air exposure stress. The whole body cortisol and the expression of selected genes in the fish brain and kidney were analyzed, before and after the acute stress. The results showed that AgNP increased basal cortisol levels and the expression of corticotropin releasing factor, prohormone convertase 1, pro-opiomelanocortin, and melanocortin 2 receptor; however, it suppressed/inhibited whole body cortisol, brain corticotropin releasing factor responses, pro-opiomelanocortin, and the kidney melanocortin 2 receptor responses to the acute stress. AgNP down-regulated the expression of the steroidogenic acute regulatory protein, but it intensified the gene expression in response to the acute stress. Before the acute stress, LC treatment exhibited an up-regulation in Cytochrome P450-11A-1 expression, but MC and HC treatments induced down-regulation. After the acute stress, the AgNP-exposed fish exhibited decreased Cytochrome P450-11A-1 expressions, compared with the Control. Exposure to AgNP significantly increased Cytochrome P450-11B expression. However, after the acute stress, LC treatment exhibited an up-regulation, but MC and HC treatments exhibited down-regulation in the Cytochrome P450-11B gene expression. In conclusion, AgNP suppressed cortisol response to stress, which appears to be a consequence of alterations in the HPI axis at the transcriptomic levels.

Regulation of Melanocortin-3 and -4 Receptors by Isoforms of Melanocortin-2 Receptor Accessory Protein 1 and 2.

The neural melanocortin receptors (MCRs), melanocortin-3 and -4 receptors (MC3R and MC4R), play essential non-redundant roles in the regulation of energy homeostasis. Interaction of neural MCRs and melanocortin-2 receptor accessory proteins (MRAPs, MRAP1 and MRAP2) is suggested to play pivotal roles in MC3R and MC4R signaling. In the present study, we identified two new human (h) MRAP2 splice variants, MRAP2b (465 bp open reading frame) and MRAP2c (381 bp open reading frame). Human MRAP2s are different in C-termini. We investigated the effects of five isoforms of MRAPs, hMRAP1a, hMRAP1b, hMRAP2a, hMRAP2b, and hMRAP2c, on MC3R and MC4R pharmacology. At the hMC3R, hMRAP1a and hMRAP2c increased and hMRAP1b decreased the cell surface expression. hMRAP1a increased affinity to ACTH. Four MRAPs (hMRAP1a, hMRAP1b, hMRAP2a, and hMRAP2c) decreased the maximal responses in response to α-MSH and ACTH. For hMC4R, hMRAP1a, hMRAP2a, and hMRAP2c increased the cell surface expression of hMC4R. Human MRAP1b significantly increased affinity to ACTH while MRAP2a decreased affinity to ACTH. Human MRAP1a increased ACTH potency. MRAPs also affected hMC4R basal activities, with hMRAP1s increasing and hMRAP2s decreasing the basal activities. In summary, the newly identified splicing variants, hMRAP2b and hMRAP2c, could regulate MC3R and MC4R pharmacology. The two MRAP1s and three MRAP2s had differential effects on MC3R and MC4R trafficking, binding, and signaling. These findings led to a better understanding of the regulation of neural MCRs by MRAP1s and MRAP2s.

Hypothesis and Theory: Evaluating the Co-Evolution of the Melanocortin-2 Receptor and the Accessory Protein MRAP1.

The melanocortin receptors (MCRs) and the MRAP accessory proteins belong to distinct gene families that are unique to the chordates. During the radiation of the chordates, the melancortin-2 receptor paralog (MC2R) and the MRAP1 paralog (melanocortin-2 receptor accessory protein 1) have co-evolved to form a heterodimer interaction that can influence the ligand selectivity and trafficking properties of MC2R. This apparently spontaneous interaction may have begun with the ancestral gnathostomes and has persisted in both the cartilaginous fishes and the bony vertebrates. The ramifications of this interaction had profound effects on the hypothalamus/anterior pituitary/adrenal-interrenal axis of bony vertebrates resulting in MC2R orthologs that are exclusively selective for the anterior pituitary hormone, ACTH, and that are dependent on MRAP1 for trafficking to the plasma membrane. The functional motifs within the MRAP1 sequence and their potential contact sites with MC2R are discussed. The ramifications of the MC2R/MRAP1 interaction for cartilaginous fishes are also discussed, but currently the effects of this interaction on the hypothalamus/pituitary/interrenal axis is less clear. The cartilaginous fish MC2R orthologs have apparently retained the ability to be activated by either ACTH or MSH-sized ligands, and the effect of MRAP1 on trafficking varies by species. In this regard, the possible origin of the dichotomy between cartilaginous fish and bony vertebrate MC2R orthologs with respect to ligand selectivity and trafficking properties is discussed in light of the evolution of functional amino acid motifs within MRAP1.

MC2R/MRAP2 activation could affect bovine ovarian steroidogenesis potential after ACTH treatment.

Stressors activate the hypothalamic-pituitary-adrenal (HPA) axis, reducing fertility by interfering with the mechanisms that regulate the timing of events within the follicular phase of the estrous cycle. In the HPA axis, melanocortin 2 receptor (MC2R) mediates responses to adrenocorticotropic hormone (ACTH) in concert with melanocortin receptor accessory protein 2 (MRAP2). The aims of the present study were: (1) to evaluate the effects of ACTH administered in cows in the preovulatory period on the expression of the MC2R/MRAP2 complex in the dominant follicle; and (2) to analyze the involvement of Extracellular signal Regulated Kinase 1 (ERK1) signaling in the activation of MC2R and the expression of key enzymes involved in the biosynthesis of glucocorticoids (GCs) in the dominant follicle. To this end, 100 IU ACTH was administered to Holstein cows from a local dairy farm during pro-estrus every 12 h for four days until ovariectomy, which was performed before ovulation. Protein immunostaining of MC2R was higher in the dominant follicles of ACTH-treated cows (p < 0.05). Also, Western blot analysis showed higher activation of the ERK1 signaling pathway in ACTH-treated cows (p < 0.05). Finally, immunohistochemistry performed in the dominant follicles of ACTH-treated cows detected higher expression of CYP17A1 and CYP21A2 (p < 0.05). These results suggest that the bovine ovary is able to respond locally to ACTH as a consequence of stress altering the expression of relevant steroidogenic enzymes. The results also confirm that the complete GC biosynthesis pathway is present in bovine dominant follicle and therefore GCs could be produced locally.