MRAP2a Binds and Modulates Activity and Localisation of Prokineticin Receptor 1 in Zebrafish.

The prokineticin system plays a role in hypothalamic neurons in the control of energy homeostasis. Prokineticin receptors (PKR1 and PKR2), like other G-protein-coupled receptors (GPCRs) are involved in the regulation of energy intake and expenditure and are modulated by the accessory membrane protein 2 of the melanocortin receptor (MRAP2). The aim of this work is to characterise the interaction and regulation of the non-melanocortin receptor PKR1 by MRAP2a in zebrafish (zMRAP2a) in order to use zebrafish as a model for the development of drugs targeting accessory proteins that can alter the localisation and activity of GPCRs. To this end, we first showed that zebrafish PKR1 (zPKR1) is able to interact with both zMRAP2a and human MRAP2 (hMRAP2). This interaction occurs between the N-terminal region of zPKR1 and the C-terminal domain of zMRAP2a, which shows high sequence identity with hMRAP2 and a similar propensity for dimer formation. Moreover, we demonstrated that in Chinese hamster ovary (CHO) cells, zMRAP2a or hMRAP2 are able to modulate zPKR1 activation induced by zebrafish PK2 (zPK2) resulting in an impaired ERK and STAT3 activation.

A novel pathogenic variant in MRAP2 in an obese patient with successful outcome of bariatric surgery.

Mutations in genes encoding proteins located in the leptin/melanocortin pathway have been identified in the rare cases of genetic obesities. Heterozygous variants of MRAP2, encoding a G coupled-protein receptor accessory protein implicated in energy control notably via the melanocortin-4 receptor, have been recently identified. A 24-year-old patient with early-onset severe obesity (body mass index [BMI]: 64 kg/m2) associated with hypertension, respiratory complications, nonalcoholic fatty liver disease, and type 2 diabetes was referred to our department. Sleeve gastrectomy was successful. A new heterozygous variant in MRAP2 (NM_138409.4: c.154G>C/p.G52R) variant was identified in the patient DNA. Functional assessment confirmed that this new variant was pathogenic. We report a new pathogenic loss-of-function mutation in MRAP2 in a patient suffering from a severe multicomplicated obesity. This confirms the metabolic phenotype in patients with this monogenic form of obesity. Longer follow-up will be necessary. Our finding will allow a personalized medicine.

Pharmacology of orange-spotted grouper (Epinephelus coioides) melanocortin-5 receptor and its modulation by Mrap2.

The mammalian melanocortin-5 receptors (MC5Rs) are involved in various functions, including exocrine gland secretion, glucose uptake, adipocyte lipolysis, and immunity. However, the physiological role of fish Mc5r is rarely studied. Melanocortin-2 receptor accessory protein 2 (MRAP2) modulates pharmacological properties of melanocortin receptors. Herein, to lay the foundation for future physiological studies, we cloned the orange-spotted grouper (Epinephelus coioides) mc5r, with a 1008 bp open reading frame and a predicted protein of 334 amino acids. Grouper mc5r had abundant expression in the brain, skin, and kidney. Four ligands could bind to grouper Mc5r and dose-dependently increase intracellular cAMP levels. Grouper Mrap2 did not affect binding affinity or potency of Mc5r; however, grouper Mrap2 decreased cell surface expression and maximal binding of Mc5r. Mrap2 also significantly decreased the maximal response to a superpotent agonist but not the endogenous agonist. This study provided new data on fish Mc5r pharmacology and its regulation by Mrap2.

Activation of amygdala prokineticin receptor 2 neurons drives the anorexigenic activity of the neuropeptide PK2.

Energy homeostasis is a complex system involving multiple hormones, neuropeptides, and receptors. Prokineticins (PK1 and PK2) are agonists to two G protein-coupled receptors, prokineticin receptor 1 and 2 (PKR1 and PKR2), which decrease food intake when injected in rodents. The relative contribution of PKR1 and PKR2 to the anorexigenic effect of PK2 and their site of action in the brain have not yet been elucidated. While PKR1 and PKR2 are both expressed in the hypothalamus, a central region involved in the control of energy homeostasis, PKR2 is also present in the amygdala, which has recently been shown to regulate food intake in response to several anorexigenic signals. PKR trafficking and signaling are inhibited by the melanocortin receptor accessory protein 2 (MRAP2), thus suggesting that MRAP2 has the potential to alter the anorexigenic activity of PK2 in vivo. In this study, we investigated the importance of PKR1 and PKR2 for PK2-mediated inhibition of food intake, the brain region involved in this function, and the effect of MRAP2 on PK2 action in vivo. Using targeted silencing of PKR2 and chemogenetic manipulation of PKR2 neurons, we show that the anorexigenic effect of PK2 is mediated by PKR2 in the amygdala and that altering MRAP2 expression in PKR2 neurons modulates the activity of PK2. Collectively, our results provide evidence that inhibition of food intake by PKs is not mediated through activation of hypothalamic neurons but rather amygdala PKR2 neurons and further establishes the importance of MRAP2 in the regulation of energy homeostasis.

The interaction of MC3R and MC4R with MRAP2a in rainbow trout (Oncorhynchus mykiss).

Melanocortin 3 and 4 receptors are two important neural G protein-coupled receptors that regulate energy homeostasis in vertebrates. Melanocortin receptor accessory protein 2 (MRAP2) is also involved in the regulation of food intake and body weight as a variable regulator of melanocortin receptors. Rainbow trout (Oncorhynchus mykiss) is a valuable cold-water fish cultured worldwide. In the rainbow trout model, we cloned and identified mrap2a, a paralog of mrap2. Rainbow trout mrap2a consisted of a 690 bp ORF and was expected to encode a putative protein of 229 amino acids. The qPCR results showed that rainbow trout mrap2a was expressed at high levels in brain tissue similar to mc3r and mc4r. In addition, co-immunoprecipitation verified that MRAP2a interacts with MC3R and MC4R in vitro and that MRAP2a is involved in and regulates the constitutive activity and signaling of MC3R and MC4R. MRAP2a reduced constitutive and agonist-stimulated cAMP levels of MC3R; furthermore, MRAP2a increased constitutive ERK1/2 activation but reduced ligand-induced stimulation at high levels of expression. For MC4R, MRAP2a showed decreased cAMP basal activity but increased agonist-stimulated cAMP signaling and increased ACTH ligand sensitivity. However, MRAP2a failed to affect MC4R constitutive activity and agonist-induced ERK1/2 signaling. Undoubtedly, our study will have great significance for revealing the conserved role of MC4R and MC3R signaling in teleost fish, especially in cold-water fish growth and energy homeostasis.

Identification of MRAP protein family as broad-spectrum GPCR modulators.

BACKGROUND: The melanocortin receptor accessory proteins (MRAP1 and MRAP2) are well-known endocrine regulators for the trafficking and signalling of all five melanocortin receptors (MC1R-MC5R). The observation of MRAP2 on regulating several non-melanocortin G protein-coupled receptors (GPCRs) has been sporadically reported, whereas other endogenous GPCR partners of the MRAP protein family are largely unknown. METHODS: Here, we performed single-cell transcriptome analysis and drew a fine GPCR blueprint and MRAPs-associated network of two major endocrine organs, the hypothalamus and adrenal gland at single-cell resolution. We also integrated multiple bulk RNA-seq profiles and single-cell datasets of human and mouse tissues, and narrowed down a list of 48 GPCRs with strong endogenous co-expression correlation with MRAPs. RESULTS: 36 and 46 metabolic-related GPCRs were consequently identified as novel interacting partners of MRAP1 or MRAP2, respectively. MRAPs exhibited protein-protein interactions and varying pharmacological properties on the surface translocation, constitutive activities and ligand-stimulated downstream signalling of these GPCRs. Knockdown of MRAP2 expression by hypothalamic administration of adeno-associated virus (AAV) packed shRNA stimulated body weight gain in mouse model. Co-injection of corticotropinreleasing factor (CRF), the agonist of corticotropin releasing hormone receptor 1 (CRHR1), suppressed feeding behaviour in a MRAP2-dependent manner. CONCLUSIONS: Collectively, our study has comprehensively elucidated the complex GPCR networks in two major endocrine organs and redefined the MRAP protein family as broad-spectrum GPCR modulators. MRAP proteins not only serve as a vital endocrine pivot on the regulation of global GPCR activities in vivo that could explain the composite physiological phenotypes of the MRAP2 null murine model but also provide us with new insights of the phenotyping investigation of GPCR-MRAP functional complexes.

Arginine 125 Is an Essential Residue for the Function of MRAP2.

MRAP2 is a small simple transmembrane protein arranged in a double antiparallel topology on the plasma membrane. It is expressed in the paraventricular nucleus of the hypothalamus, where it interacts with various G protein-coupled receptors, such as the prokineticin receptors, and regulates energy expenditure and appetite. The aim of this work was to analyze the functional role of the specific arginine residue at position 125 of MRAP2, which affects protein conformation, dimer formation, and PKR2 binding. Results obtained with the MRAP2 mutants R125H and R125C, which are found in human patients with extreme obesity, and mouse MRAP2, in which arginine 125 is normally replaced by histidine, were compared with those obtained with human MRAP2. Understanding the mechanism by which MRAP2 regulates G protein-coupled receptors helps in elucidating the metabolic pathways involved in metabolic dysfunction and in developing new drugs as specific targets of the MRAP2-PKR2 complex.

Pharmacological Modulation of Melanocortin 1 Receptor Signaling by Mrap Proteins in Xenopus tropicalis.

The melanocortin system consists of five G protein-coupled receptors (MC1R-MC5R), the bidirectional endogenous ligands (MSH and Agouti families), and accessory proteins (MRAP1 and MRAP2). Accumulative studies of vertebrate species find high expression level of melanocortin 1 receptor (MC1R) in the dermal melanocyte and elucidate the essential roles in the skin and fur pigmentation, morphological background adaptation, and stress response. The diploid amphibian Xenopus tropicalis (xt) has been utilized as a fantastic animal model for embryonic development and studies of physiological cryptic colouring and environmental adaptiveness. However, the interaction of xtMc1r signaling with xtMrap proteins has not been assessed yet. In this study, we carried out in silico evolutionary analysis of protein alignment and genetic phylogenetic and genomic synteny of mc1r among various vertebrates. Ubiquitous expression of mrap1 and mrap2 and the co-expression with mc1r transcripts in the skin were clearly observed. Co-immunoprecipitation (ip) and fluorescent complementary approach validated the direct functional interaction of xtMc1r with xtMrap1 or xtMrap2 proteins on the plasma membrane. Pharmacological assay showed the improvement of the constitutive activity and alpha melanocyte-stimulating hormone (α-MSH) stimulated plateau without dramatic alteration of the cell surface translocation of xtMc1r in the presence of xtMrap proteins. Overall, the pharmacological modulation of xtMc1r by dual xtMrap2 proteins elucidated the potential role of this protein complex in the regulation of proper dermal function in amphibian species.

The Multifaceted Melanocortin Receptors.

The 5 known melanocortin receptors (MCs) have established physiological roles. With the exception of MC2, these receptors can behave unpredictably, and since they are more widely expressed than their established roles would suggest, it is likely that they have other poorly characterized functions. The aim of this review is to discuss some of the less well-explored aspects of the 4 enigmatic members of this receptor family (MC1,3-5) and describe how these are multifaceted G protein-coupled receptors (GPCRs). These receptors appear to be promiscuous in that they bind several endogenous agonists (products of the proopiomelanocortin [POMC] gene) and antagonists but with inconsistent relative affinities and effects. We propose that this is a result of posttranslational modifications that determine receptor localization within nanodomains. Within each nanodomain there will be a variety of proteins, including ion channels, modifying proteins, and other GPCRs, that can interact with the MCs to alter the availability of receptor at the cell surface as well as the intracellular signaling resulting from receptor activation. Different combinations of interacting proteins and MCs may therefore give rise to the complex and inconsistent functional profiles reported for the MCs. For further progress in understanding this family, improved characterization of tissue-specific functions is required. Current evidence for interactions of these receptors with a range of partners, resulting in modulation of cell signaling, suggests that each should be studied within the full context of their interacting partners. The role of physiological status in determining this context also remains to be characterized.

Reversion of MRAP2 Protein Sequence Generates a Functional Novel Pharmacological Modulator for MC4R Signaling.

As a member of the melanocortin receptor family, melanocortin 4 receptor (MC4R) plays a critical role in regulating energy homeostasis and feeding behavior, and has been proven as a promising therapeutic target for treating severe obesity syndrome. Numerous studies have demonstrated that central MC4R signaling is significantly affected by melanocortin receptor accessory protein 2 (MRAP2) in humans, mice and zebrafish. MRAP2 proteins exist as parallel or antiparallel dimers on the plasma membrane, but the structural insight of dual orientations with the pharmacological profiles has not yet been fully studied. Investigation and optimization of the conformational topology of MRAP2 are critical for the development of transmembrane allosteric modulators to treat MC4R-associated disorders. In this study, we synthesized a brand new single transmembrane protein by reversing wild-type mouse and zebrafish MRAP2 sequences and examined their dimerization, interaction and pharmacological activities on mouse and zebrafish MC4R signaling. We showed that the reversed zebrafish MRAPa exhibited an opposite function on modulating zMC4R signaling and the reversed mouse MRAP2 lost the capability for regulating MC4R trafficking but exhibited a novel function for cAMP cascades, despite proper expression and folding. Taken together, our results provided new biochemical insights on the oligomeric states and membrane orientations of MRAP2 proteins, as well as its pharmacological assistance for modulating MC4R signaling.

Analyzing the Hypothalamus/Pituitary/Interrenal axis of the neopterygian fish, Lepisosteus oculatus: Co-localization of MC2R, MC5R, MRAP1, and MRAP2 in interrenal cells.

RT-PCR analysis indicated that steroidogenic tissues are located along the length of the kidney of the neopterygian fish, Lepisosteus oculatus (spotted gar; g). However, RT-PCR analysis of the distribution of mc2r mRNA and mrap1 mRNA, critical components of the gar hypothalamus/pituitary/interrenal (HPI) axis, was only associated with the anterior and medial regions of the kidney. Steroidogenic cells were designated as interrenal cells that possess star mRNA (in situ hybridization) and lipid vesicles (histological analysis) within the kidney. RT-PCR also detected mc5r mRNA along the length of the tissues associated with the kidney. In situ hybridization analysis of the putative interrenal cells revealed co-expression of mc2r, and mc5r mRNAs in the same steroidogenic cells. Co-expression of gar Mc2r (gMc2r) and Mrap1 (gMrap1) in Chinese Hamster Ovary (CHO) cells stimulated with ACTH(1-24) resulted in activation with an EC50 value of 1.0 × 10-11M +/- 4.6 × 10-11); whereas stimulation of CHO cells co-expressed with gar Mc5r (gMc5r) and gMrap1 and stimulated with ACTH(1-24) resulted in an EC50 value that was 3 orders of magnitude lower (2.1 × 10-8 M +/- 3.5 × 10-9). Interesting, when CHO cells were co-transfected with gMc2r, gMc5r, and gMrap1 there was a decline in activation as measured by the Vmax values for CHO cells stimulated with either ACTH(1-24) or α-MSH. These results suggest that some interaction may occur between gMc2r and gMc5r when both receptors are expressed in the same cells. Phylogenetic and selection pressure analyses of vertebrate mc2r and mc5r genes concluded that the two genes are evolving at different rates after duplication from a proposed common ancestral gene.

Determination of the Interaction and Pharmacological Modulation of MCHR1 Signaling by the C-Terminus of MRAP2 Protein.

Melanin concentrating hormone (MCH), an orexigenic neuropeptide, is primarily secreted by the hypothalamus and acts on its receptor, the melanin-concentrating hormone receptor 1 (MCHR1), to regulate appetite and energy homeostasis. The Melanocortin Receptor Accessory Protein 2 (MRAP2), a small single transmembrane protein broadly expressed in multiple tissues, has been defined as a vital endocrine modulator of five melanocortin receptors (MC1R-MC5R) and several other GPCRs in the regulation of central neuronal activities and peripheral energy balance. Here, we demonstrated the interaction between MRAP2 and MCHR1 by immunoprecipitation and bimolecular fluorescent assay and found that MRAP2 could inhibit MCHR1 signaling in vitro. A series of functional truncations of different regions further identified that the C-terminal domains of MRAP2 protein were required for the pharmacological modulation of intracellular Ca2+ coupled cascades and membrane transport. These findings elucidated the broad regulatory profile of MRAP2 protein in the central nervous system and may provide implications for the modulation of central MCHR1 function in vivo.

Identification of Regions Involved in the Physical Interaction between Melanocortin Receptor Accessory Protein 2 and Prokineticin Receptor 2.

Melanocortin Receptor Accessory Protein 2 (MRAP2) modulates the trafficking and signal transduction of several G-protein-coupled receptors (GPCRs) involved in the control of energy homeostasis, such as Prokineticin receptors (PKRs). They bind the endogenous ligand prokineticin 2 (PK2), a novel adipokine that has an anorexic effect and modulates thermoregulation and energy homeostasis. In the present work, we used biochemical techniques to analyze the mechanism of interaction of MRAP2 with PKR2 and we identified the specific amino acid regions involved in the complex formation. Our results indicate that MRAP2 likely binds to the N-terminal region of PKR2, preventing glycosylation and consequently the correct receptor localization. We also identified a C-terminal region of MRAP2 that is critical for the interaction with PKR2. Consequently, we analyzed the role of the prokineticin transduction system in the regulation of MRAP2 expression in tissues involved in the control of food intake: at the central level, in hypothalamic explants, and at the peripheral level, in adipocytes. We demonstrated the modulation of MRAP2 expression by the prokineticin transduction system.

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.

Pharmacological properties of whale shark (Rhincodon typus) melanocortin-2 receptor and melancortin-5 receptor: Interaction with MRAP1 and MRAP2.

In the current study, the whale shark (ws; Rhincodon typus) melanocortin-2 receptor (MC2R) co-expressed with wsMRAP1 in Chinese Hamster Ovary (CHO) Cells could be stimulated in a dose dependent manner by ACTH(1-24) with an EC50 of 2.6 × 10-10 M ± 9.7 × 10-11. When the receptor was expressed alone, stimulation was only observed at [10-6 M]. A comparable increase in sensitivity to stimulation by srDes-Ac-αMSH was also observed when the receptor was co-expressed with wsMRAP1. Furthermore, co-expression with wsMRAP1 significantly increased the trafficking of wsMC2R to the plasma membrane of CHO cells. Surprisingly, co-expression with wsMRAP2 also increased sensitivity to stimulation by ACTH(1-24) and srDes-Ac-αMSH, and increased trafficking of the receptor to the plasma membrane. These observations are in sharp contrast to the response of MC2R orthologs of bony vertebrates which have an obligate requirement for co-expression with MRAP1 for both trafficking to the plasma membrane and activation, whereas, co-expression with MRAP2 increases trafficking, but has minimal effects on activation. In addition, when comparing the activation features of wsMC2R with those of the elephant shark MC2R and red stingray MC2R orthologs, both similarities and differences are observed. The spectrum of features for cartilaginous fish MC2R orthologs will be discussed. A second objective of this study was to determine whether wsMC5R has features in common with wsMC2R in terms of ligand selectivity and interaction with wsMRAP paralogs. While wsMC5R can be activated by either srACTH(1-24) or srDes-Ac-αMSH, and co-expression with wsMRAP1 enhances this activation, wsMRAP1 had no effect on the trafficking of wsMC5R. In addition, co-expression with wsMRAP2 had no positive or negative effect on either ligand sensitivity or trafficking of wsMC5R.

Functional Characterization of the Internal Symmetry of MRAP2 Antiparallel Homodimer.

The melanocortin receptors are defined as a series of vital pharmaceutical targets to regulate neuronal appetite and maintain controllable body weight for mammals and teleosts. Melanocortin receptor accessory protein 2 (MRAP2) functions as an essential accessory player that modulates the surface translocation and binding to a variety of endogenous or synthetic hormones of central melanocortin-4 receptor (MC4R) signaling. MRAP2 is a single-transmembrane protein and could form a functional symmetric antiparallel homodimer topology. Here, we inverted the N-terminal, transmembrane, and C-terminal domains and generated six distinct conformational variants of the mouse MRAP2 to explore the functional orientations and the internal symmetry of MRAP2 dimers. These remolded MRAP2 mutants showed proper assembly of the antiparallel homodimer and binding to the MC4R, but slightly altered the regulatory profile on the surface expression and the ligand-stimulated cAMP cascades of MC4R. This study elucidated the importance of the orientation of each domain of the single-transmembrane protein and revealed the pharmacological properties of the internal symmetry of the antiparallel homodimer for MRAP2.

The emerging role of heterodimerisation and interacting proteins in ghrelin receptor function.

Ghrelin is a peptide hormone secreted primarily by the stomach that acts upon the growth hormone secretagogue receptor (GHSR1), a G protein-coupled receptor whose functions include growth hormone secretion, appetite regulation, energy expenditure, regulation of adiposity, and insulin release. Following the discovery that GHSR1a stimulates food intake, receptor antagonists were developed as potential therapies to regulate appetite. However, despite reductions in signalling, the desired effects on appetite were absent. Studies in the past 15 years have demonstrated GHSR1a can interact with other transmembrane proteins, either by direct binding (i.e. heteromerisation) or via signalling cross-talk. These interactions have various effects on GHSR1a signalling including preferential coupling to one pathway (i.e. biased signalling), coupling to a unique G protein (G protein switching), suppression of GHSR1a signalling, and enhancement of signalling by both receptors. While many of these interactions have been shown in cells overexpressing the proteins of interest and remain to be verified in tissues, substantial evidence exists showing that GHSR1a and the dopamine receptor D1 (DRD1) form heteromers, which promote synaptic plasticity and formation of hippocampal memory. Additionally, a reduction in GHSR1a-DRD1 complexes in favour of establishment of GHSR1a-Aß complexes correlates with Alzheimer's disease, indicating that GHSR1a heteromers may have pathological functions. Herein, we summarise the evidence published to date describing interactions between GHSR1a and transmembrane proteins, discuss the experimental strengths and limitations of these studies, describe the physiological evidence for each interaction, and address their potential as novel drug targets for appetite regulation, Alzheimer's disease, insulin secretion, and inflammation.

Regulation of melanocortin-5 receptor pharmacology by two isoforms of MRAP2 in ricefield eel (Monopterus albus).

The melanocortin-5 receptor (MC5R) has been implicated in the regulation of exocrine gland secretion, immune regulation, and muscle fatty acid oxidation in mammals. Melanocortin-2 receptor accessory protein 2 (MRAP2) can modulate trafficking, ligand binding, and signaling of melanocortin receptors. To explore potential interaction between ricefield eel (Monopterus albus) MC5R and MRAP2s (maMC5R, maMRAP2X1, and maMRAP2X2), herein we studied the pharmacological characteristics of maMC5R and its modulation by maMRAP2s expressed in the human embryonic kidney cells. Three agonists, α-melanocyte-stimulating hormone (α-MSH), ACTH (1-24), and [Nle4, D-Phe7]-α-MSH, could bind to maMC5R and induce intracellular cAMP production dose-dependently. Compared with human MC5R (hMC5R), maMC5R displayed decreased maximal binding but higher binding affinity to α-MSH or ACTH (1-24). When stimulated with α-MSH or ACTH (1-24), maMC5R showed significantly lower EC50 and maximal response than hMC5R. Two maMRAP2s had no effect on cell surface expression of maMC5R, whereas they significantly increased maximal binding. Only maMRAP2X2 significantly decreased the binding affinity of ACTH (1-24). Both maMRAP2X1 and maMRAP2X2 significantly reduced maMC5R efficacy but did not affect ligand sensitivity. The availability of maMC5R pharmacological characteristics and modulation by maMRAP2s will assist the investigation of its roles in regulating diverse physiological processes in ricefield eel.

Role of N-Linked Glycosylation in PKR2 Trafficking and Signaling.

Prokineticin receptors are GPCRs involved in several physiological processes including the regulation of energy homeostasis, nociception, and reproductive function. PKRs are inhibited by the endogenous accessory protein MRAP2 which prevents them from trafficking to the plasma membrane. Very little is known about the importance of post-translational modification of PKRs and their role in receptor trafficking and signaling. Here we identify 2 N-linked glycosylation sites within the N-terminal region of PKR2 and demonstrate that glycosylation of PKR2 at position 27 is important for its plasma membrane localization and signaling. Additionally, we show that glycosylation at position 7 results in a decrease in PKR2 signaling through Gαs without impairing Gαq/ 11 signaling.

MRAP2 Interaction with Melanocortin-4 Receptor in SnakeHead (Channa argus).

The melanocortin-4 receptor (MC4R) plays an important role in the regulation of food intake and energy expenditure. Melanocortin-2 receptor accessory protein 2 (MRAP2) modulates trafficking, ligand binding, and signaling of MC4R. The Northern snakehead (Channa argus) is an economically important freshwater fish native to East Asia. To explore potential interaction between snakehead MC4R and MRAP2, herein we cloned snakehead mc4r and mrap2. The snakehead mc4r consisted of a 984 bp open reading frame encoding a protein of 327 amino acids, while snakehead mrap2 contained a 693 bp open reading frame encoding a protein of 230 amino acids. Synteny analysis indicated that mc4r was highly conserved with similar gene arrangement, while mrap2 contained two isoforms in teleost with different gene orders. Snakehead mc4r was primarily expressed in the brain, whereas mrap2 was expressed in the brain and intestine. Snakehead mc4r and mrap2 expression was modulated by fasting and refeeding. Further pharmacological experiments showed that the cloned snakehead MC4R was functional, capable of binding to peptide agonists and increasing intracellular cAMP production in a dose-dependent manner. Snakehead MC4R exhibited high constitutive activity. MRAP2 significantly decreased basal and agonist-stimulated cAMP signaling. These findings suggest that snakehead MC4R might be involved in energy balance regulation by interacting with MRAP2. Further studies are needed to elucidate MC4R in regulating diverse physiological processes in snakehead.