Melanocortin 2 Receptor Accessory Protein Research Articles

9293 Do Obesity-Associated MRAP2 Variants Modulate MC4R and GHSR Signaling?

Abstract Disclosure: A.V. Rodriguez Rondon: None. F. Volker: None. M.S. Welling: None. C. de Groot: None. M.M. van Haelst: None. E.L. van den Akker: None. E.F. van Rossum: None. P.J. Delhanty: None. J.A. Visser: None. Melanocortin 2 receptor accessory protein 2 (MRAP2) is a membrane-bound protein expressed on hypothalamic neurons that modulate the function of appetite-regulating receptors, including melanocortin-4 receptor (MC4R) and growth hormone secretagogue receptor (GHSR). While pathogenic variants in the MRAP2 gene are associated with obesity, the mechanism for this remains unclear. The aim of this study is to investigate the functional impact of wildtype (WT) MRAP2 and MRAP2 variants on MC4R and GHSR signaling. Four MRAP2 variants (S80F, R125C, P167A and Q174X) were identified in patients with obesity by obesity gene panel analysis. The functional effects of these variants were analyzed by co-transfecting HEK293 cells with expression plasmids encoding WT or variant MRAP2 and WT MC4R or WT GHSR. Endpoint analyses included cell surface expression, and ligand-induced second messenger responses (α-MSH-induced cAMP response for MC4R and ghrelin-induced Ca2+ mobilization for GHSR), β-arrestin-2 recruitment, and internalization. WT MRAP2 significantly decreased basal MC4R cell surface expression by 68.1±4.3% (p<0.001). In the absence of MRAP2, α-MSH induced MC4R internalization. However, in the presence of WT MRAP2, MC4R cell surface expression was increased by 89.4±6.9% upon α-MSH stimulation (p<0.001). GHSR cell surface expression and ghrelin-induced internalization were not affected by WT MRAP2. Furthermore, in the cells expressing MC4R with WT MRAP2, the maximal α-MSH-induced cAMP and β-arrestin-2 recruitment responses were increased by 29.8±5.2% and 81.3±4.2% respectively (p<0.001), compared with those lacking MRAP2. We also investigated GHSR ghrelin-induced Ca2+ mobilization and found that, in the presence of WT MRAP2, this increased significantly by 95.37±21.9%, (p<0.0003). However, ghrelin-induced β-arrestin-2 recruitment was suppressed by 69.8±7.6%, (p<0.001). Using these endpoints we found no effect of the variants on the ability of MRAP2 to modulate ligand-induced signaling by MC4R and GHSR. Our results indicate that WT MRAP2 enhances the cAMP response by increasing MC4R cell membrane expression upon α-MSH stimulation, and enhances GHSR Ca2+ mobilization by decreasing β-arrestin-2 recruitment upon ghrelin stimulation. The MRAP2 variants assessed in this study behaved as WT MRAP2 in α-MSH-induced MC4R and ghrelin-induced GHSR signaling, suggesting that these may be benign variants. However, since MRAP2 can modulate multiple receptors, we cannot rule out that these MRAP2 variants affect other appetite-regulating receptors and thereby influence body weight regulation via other pathways. Presentation: 6/3/2024

6789 Do Obesity-Associated MRAP2 Variants Modulate MC4R and GHSR Signaling?

Abstract Disclosure: A.V. Rodriguez Rondon: None. F. Volker: None. M.S. Welling: None. C. de Groot: None. M.M. van Haelst: None. E.L. van den Akker: None. E.F. van Rossum: None. P.J. Delhanty: None. J.A. Visser: None. Melanocortin 2 receptor accessory protein 2 (MRAP2) is a membrane-bound protein expressed on hypothalamic neurons that modulate the function of appetite-regulating receptors, including melanocortin-4 receptor (MC4R) and growth hormone secretagogue receptor (GHSR). While pathogenic variants in the MRAP2 gene are associated with obesity, the mechanism for this remains unclear. The aim of this study is to investigate the functional impact of wildtype (WT) MRAP2 and MRAP2 variants on MC4R and GHSR signaling. Four MRAP2 variants (S80F, R125C, P167A and Q174X) were identified in patients with obesity by obesity gene panel analysis. The functional effects of these variants were analyzed by co-transfecting HEK293 cells with expression plasmids encoding WT or variant MRAP2 and WT MC4R or WT GHSR. Endpoint analyses included cell surface expression, and ligand-induced second messenger responses (α-MSH-induced cAMP response for MC4R and ghrelin-induced Ca2+ mobilization for GHSR), β-arrestin-2 recruitment, and internalization. WT MRAP2 significantly decreased basal MC4R cell surface expression by 68.1±4.3% (p<0.001). In the absence of MRAP2, α-MSH induced MC4R internalization. However, in the presence of WT MRAP2, MC4R cell surface expression was increased by 89.4±6.9% upon α-MSH stimulation (p<0.001). GHSR cell surface expression and ghrelin-induced internalization were not affected by WT MRAP2. Furthermore, in the cells expressing MC4R with WT MRAP2, the maximal α-MSH-induced cAMP and β-arrestin-2 recruitment responses were increased by 29.8±5.2% and 81.3±4.2% respectively (p<0.001), compared with those lacking MRAP2. We also investigated GHSR ghrelin-induced Ca2+ mobilization and found that, in the presence of WT MRAP2, this increased significantly by 95.37±21.9%, (p<0.0003). However, ghrelin-induced β-arrestin-2 recruitment was suppressed by 69.8±7.6%, (p<0.001). Using these endpoints we found no effect of the variants on the ability of MRAP2 to modulate ligand-induced signaling by MC4R and GHSR. Our results indicate that WT MRAP2 enhances the cAMP response by increasing MC4R cell membrane expression upon α-MSH stimulation, and enhances GHSR Ca2+ mobilization by decreasing β-arrestin-2 recruitment upon ghrelin stimulation. The MRAP2 variants assessed in this study behaved as WT MRAP2 in α-MSH-induced MC4R and ghrelin-induced GHSR signaling, suggesting that these may be benign variants. However, since MRAP2 can modulate multiple receptors, we cannot rule out that these MRAP2 variants affect other appetite-regulating receptors and thereby influence body weight regulation via other pathways. Presentation: 6/3/2024

MRAP2 Inhibits β-Arrestin-2 Recruitment to the Prokineticin Receptor 2.

Melanocortin receptor accessory protein 2 (MRAP2) is a membrane protein that binds multiple G protein-coupled receptors (GPCRs) involved in the control of energy homeostasis, including prokineticin receptors. These GPCRs are expressed both centrally and peripherally, and their endogenous ligands are prokineticin 1 (PK1) and prokineticin 2 (PK2). PKRs couple all G-protein subtypes, such as Gαq/11, Gαs, and Gαi, and recruit β-arrestins upon PK2 stimulation, although the interaction between PKR2 and β-arrestins does not trigger receptor internalisation. MRAP2 inhibits the anorexigenic effect of PK2 by binding PKR1 and PKR2. The aim of this work was to elucidate the role of MRAP2 in modulating PKR2-induced β-arrestin-2 recruitment and β-arrestin-mediated signalling. This study could allow the identification of new specific targets for potential new drugs useful for the treatment of the various pathologies correlated with prokineticin, in particular, obesity.

SAT654 Regulation Of The Melanocortin-4 Receptor (mc4r) By Attractin Like Protein 1 (alp1)

Abstract Disclosure: P. Buscaglia: None. J. Sebag: None. The Melanocortin-4 Receptor (MC4R) plays a central role in the regulation of energy homeostasis. Mutations in MC4R are responsible for up to 6% of early onset obesity in humans and deletion of MC4R in mice results in severe obesity due to hyperphagia. MC4R is a GPCR expressed in different region of the brains including the paraventricular nucleus of the hypothalamus. Stimulation of MC4R by its agonist αMSH and inhibition by its endogenous inverse agonist AGRP result in inhibition or stimulation of food intake respectively. MC4R is known to interact with two single transmembrane proteins, the Melanocortin Receptor Accessory Protein 2 (MRAP2) and Attractin Like Protein 1 (ALP1). Whereas MRAP2 has been shown to promote MC4R signaling in vitro and in vivo, the pharmacological and physiological relevance of ALP1 for MC4R signaling and actions is not known. To determine if ALP1 alters MC4R signaling, we measured αMSH-stimulated cAMP production in cells transfected with MC4R alone or with either ALP1 or MRAP2. We found that ALP1 increases MC4R signaling to a larger extent than MRAP2. We also showed that ALP1 inhibits β-arrestin recruitment to MC4R, thus likely preventing desensitization of the receptor. To assess the physiological importance of ALP1, we generated an ALP1Flox mouse and bred it to MC4RCRE mice. Deletion of ALP1 in MC4R neurons resulted in increased body weight compared to MC4RCRE control on standard diet. This increase in body weight was further exacerbated when animals were fed a high fat diet. In conclusion our results suggest that ALP1 regulates MC4R signaling and that deletion of ALP1 from MC4R neurons causes weight gain that may be due to decreased MC4R activity. Presentation: Saturday, June 17, 2023

OR0302 Regulation Of Human Melanocortin-5 Receptor By Isoforms Of Melanocortin-2 Receptor Accessory Proteins

Abstract Disclosure: S. Jiang: None. Y. Tao: None. Melanocortin-5 receptor (MC5R) is ubiquitously expressed in central nervous system and peripheral tissues, has unique pharmacological properties, and physiological functions. Melanocortin-2 receptor accessory proteins (MRAPs) are regulators of melanocortin receptor family. To investigate the effects of MRAPs on trafficking, ligand binding and signaling properties of human (h) MC5R, HEK293T cells were transiently co-transfected with plasmids encoding MC5R and different isoforms of MRAPs (hMRAP1a, hMRAP1b, hMRAP2a, hMRAP2b, and hMRAP2c). The results showed that hMRAP1a and hMRAP2a increased the cell surface expression of hMC5R. All MRAPs have no effect on affinity to the superpotent analog of α-melanocyte stimulating hormone (α-MSH), NDP-MSH, while hMRAP2c significantly increased maximal binding. Additionally, α-MSH induced ERK1/2 activation. A higher basal level of ERK1/2 phosphorylation was observed when co-transfected with some MRAPs. All MRAPs had no effect on α-MSH-stimulated cAMP production (Gs-cAMP pathway). In summary, the two MRAP1s and three MRAP2s had differential effects on MC5R trafficking, binding, and signaling. These findings led to a better understanding of the regulation of MC5R by MRAP1s and MRAP2s. Presentation: Thursday, June 15, 2023

OR0303 Syndecans Enhance Ghrelin-Induced GHSR1a Signaling

Abstract Disclosure: K. Prins: None. M. Huisman: None. R. Mies: None. P.J. Delhanty: None. J.A. Visser: None. Ghrelin is a gut hormone that enhances food intake and growth hormone secretion through its receptor, the G-protein coupled receptor (GPCR) GHSR1a. Recently, we showed that ghrelin interacts with syndecans (SDCs), a family of four membrane proteins that have been linked to obesity in genome-wide association studies. Here, we investigated whether SDCs impact ghrelin signaling at GHSR1a. We used HEK293 cells that expressed GHSR1a and SDC1, SDC2, SDC3, or SDC4 through transient transfection. GHSR1a mainly signals through the Gαq pathway, so we first explored the effects of SDCs on ghrelin-induced intracellular Ca2+ influx. Using a mitochondrial-targeted aequorin, this Ca2+ response can be measured seconds after ghrelin stimulation. We found that all SDCs increased the maximum Ca2+ response at least 4.2-fold (P<0.001), without affecting the EC50. This enhanced Ca2+ response was not caused by increased receptor availability: SDCs reduced cell membrane receptor expression by 51-85% (P<0.001). This low GHSR1a membrane availability contrasted with the increased Ca2+ response, and raised the question whether SDCs could be an alternate receptor for ghrelin. To test this, we transfected cells with SDC1 alone, but this did not permit a ghrelin-stimulated Ca2+ response, indicating that the ghrelin-induced Ca2+ response was GHSR1a-dependent. Moreover, the SDC-potentiated response was also Gαq-dependent, since GNAQ knockout cells transfected with GHSR1a and SDC1 expression plasmids failed to generate a Ca2+ response. However, using a Gq biosensor, we found that the level of activation of Gαq by ghrelin was not affected by SDC1 transfection, suggesting an effect of SDC1 either downstream of Gαq or on the quenching of GHSR. Thus, we investigated the interaction between GHSR1a and β-arrestin 2. Β-arrestin 2 downregulates GHSR function following activation by sterically hindering G-protein coupling and mediating receptor internalization. Four minutes after ghrelin stimulation, SDCs reduced β-arrestin 2 recruitment by 69-87% (P<0.001). SDC-transfected cells also reached a maximum β-arrestin 2 response 8-14 minutes later than GHSR1a only-transfected cells. Notably, these differences in β-arrestin 2 recruitment occurred minutes after the Ca2+ response. These findings suggest that the reduction in β-arrestin 2 recruitment does not cause the increased Ca2+ response in the presence of SDCs, and that the effect of SDCs is probably downstream of Gαq. Interestingly, the effects of SDCs mimic those of melanocortin receptor accessory protein 2 (MRAP2). MRAP2 also enhances the ghrelin-stimulated activation of the Gαq signaling pathway, independently of β-arrestin recruitment. Altogether, we showed that SDCs enhanced Ca2+ influx and reduced β-arrestin 2 recruitment to GHSR1a in response to ghrelin. This could be a novel mechanism through which SDCs affect metabolism and obesity. Presentation: Thursday, June 15, 2023

Single transmembrane GPCR modulating proteins: neither single nor simple.

The discovery of G-protein coupled receptor (GPCR) accessory proteins has fundamentally redefined the pharmacological concept of GPCR signaling, demonstrating a more complex molecular basis for receptor specificity on the plasma membrane and impressionable downstream intracellular cascades. GPCR accessory proteins not only contribute to the proper folding and trafficking of receptors, but also exhibit selectable receptor preferences. The melanocortin receptor accessory proteins (MRAP1 and MRAP2) as well as receptor activity-modifying proteins (RAMPs) are two well-known single transmembrane partners for the regulation of the melanocortin receptors (MC1R-MC5R) and the glucagon receptor (GCGR), respectively. Especially, MRAP family participates in the pathological control of multiple endocrine disorders and RAMPs contribute to the endogenous regulation of glucose homeostasis. However, the precise mechanisms by which the MRAP and RAMP proteins regulate receptor signaling at the atomic resolution remain unknown. Recent progress made in the determination of RAMP2-bound GCGR complexes published on Cell (Krishna Kumar et al., 2023) unraveled the importance of RAMP2 for the promotion of extracellular receptor dynamics leading to cytoplasmic surface inactivation. Moreover, the new findings on Cell Research (Luo et al., 2023) of the adrenocorticotropic hormone (ACTH)-bound MC2R-Gs-MRAP1 complex disclosed the essential role of MRAP1 for MC2R activation and specificity of ligand recognition. In this article, we reviewed a series of key findings of MRAP proteins in the last decade, and the recent structural investigation of MRAP-MC2R and RAMP-GCGR functional complex and the expanded identification of new GPCR partners of MRAP proteins. In-depth understanding of GPCR modulation by single transmembrane accessory proteins will provide valuable insights for the therapeutic drug development to treat multiple GPCR associated human disorders.

RAMP and MRAP accessory proteins have selective effects on expression and signalling of the CB1, CB2, GPR18 and GPR55 cannabinoid receptors.

Receptor activity-modifying proteins (RAMPs) and melanocortin receptor accessory proteins (MRAPs) modulate expression and signalling of calcitonin and melanocortin GPCRs. Interactions with other GPCRs have also been reported. The cannabinoid receptors, CB1 and CB2, and two putative cannabinoid receptors, GPR18 and GPR55, exhibit substantial intracellular expression and there are discrepancies in ligand responsiveness between studies. We investigated whether interactions with RAMPs or MRAPs could explain these phenomena. Receptors and accessory proteins were co-expressed in HEK-293 cells. Selected receptors were studied at basal expression levels and also with enhanced expression produced by incorporation of a preprolactin signal sequence/peptide (pplss). Cell surface and total expression of receptors and accessory proteins were quantified using immunocytochemistry. Signalling was measured using cAMP (CAMYEL) and G protein dissociation (TRUPATH Gα13) biosensors. MRAP2 enhanced surface and total expression of GPR18. Pplss-GPR18 increased detection of cell surface MRAP2. MRAP1α and MRAP2 reduced GPR55 surface and total expression, correlating with reduced constitutive, but not agonist-induced, signalling. GPR55, pplss-CB1 and CB2 reduced detection of MRAP1α at the cell surface. Pplss-CB1 agonist potency was reduced by MRAP2 in Gα13 but not cAMP assays, consistent with MRAP2 reducing pplss-CB1 expression. Some cannabinoid receptors increased RAMP2 or RAMP3 total expression without influencing surface expression. Mutual influences on expression and/or function for specific accessory protein-receptor pairings raises the strong potential for physiological and disease-relevant consequences. Sequestration and/or hetero-oligomerisation of cannabinoid receptors with accessory proteins is a possible novel mechanism for receptor crosstalk. This article is part of a themed issue Therapeutic Targeting of G Protein-Coupled Receptors: hot topics from the Australasian Society of Clinical and Experimental Pharmacologists and Toxicologists 2021 Virtual Annual Scientific Meeting. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v181.14/issuetoc.

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.

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.

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 invivo. 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 invivo. 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 690bp 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.

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.

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.

PSUN97 Regulation of melanocortin-3 and -4 receptors by isoforms of melanocortin-2 receptor accessory protein 1 and 2

Abstract 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 MRAP2 splice variants, MRAP2b (465 bp open reading frame) and MRAP2c (381 bp open reading frame). Human (h) MRAP2s are different in C-termini. We investigated effects of hMRAP1a, hMRAP1b, hMRAP2a, hMRAP2b, and hMRAP2c on MC3R and MC4R pharmacology. At the hMC3R, hMRAP1a increased affinity to ACTH, and hMRAP1b decreased maximal binding (Bmax). All MRAPs decreased the maximal responses in response to ACTH. For hMC4R, hMRAP1a and hMRAP1b significantly decreased Bmax, and MRAP2a increased Bmax. 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 binding and signaling. These findings led to a better understanding of the regulation of neural MCRs by MRAP1s and MRAP2s. Presentation: Sunday, June 12, 2022 12:30 p.m. - 2:30 p.m.

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

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. 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. 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. 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.

Melanocortin-5 Receptor: Pharmacology and Its Regulation of Energy Metabolism.

As the most recent melanocortin receptor (MCR) identified, melanocortin-5 receptor (MC5R) has unique tissue expression patterns, pharmacological properties, and physiological functions. Different from the other four MCR subtypes, MC5R is widely distributed in both the central nervous system and peripheral tissues and is associated with multiple functions. MC5R in sebaceous and preputial glands regulates lipid production and sexual behavior, respectively. MC5R expressed in immune cells is involved in immunomodulation. Among the five MCRs, MC5R is the predominant subtype expressed in skeletal muscle and white adipose tissue, tissues critical for energy metabolism. Activated MC5R triggers lipid mobilization in adipocytes and glucose uptake in skeletal muscle. Therefore, MC5R is a potential target for treating patients with obesity and diabetes mellitus. Melanocortin-2 receptor accessory proteins can modulate the cell surface expression, dimerization, and pharmacology of MC5R. This minireview summarizes the molecular and pharmacological properties of MC5R and highlights the progress made on MC5R in energy metabolism. We poInt. out knowledge gaps that need to be explored in the future.

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

Simple SummaryReversion of the wild-type protein sequences of single transmembrane melanocortin accessory protein families (MRAP2) in mice and zebrafish created novel functional pharmacological modulators for regulating melanocortin 4 receptor (MC4R) signaling. All of the brand new reversed MRAP2 (rMRAP2) proteins could form proper dimeric topology on the plasma membrane and interact with and affect the ligand-stimulated pharmacological profiles of zebrafish and mouse MC4R signaling in vitro.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.

MRAP2 inhibits β-arrestin recruitment to the ghrelin receptor by preventing GHSR1a phosphorylation

The melanocortin receptor accessory protein 2 (MRAP2) is essential for several physiological functions of the ghrelin receptor growth hormone secretagogue receptor 1a (GHSR1a), including increasing appetite and suppressing insulin secretion. In the absence of MRAP2, GHSR1a displays high constitutive activity and a weak G-protein–mediated response to ghrelin and readily recruits β-arrestin. In the presence of MRAP2, however, G-protein–mediated signaling via GHSR1a is strongly dependent on ghrelin stimulation and the recruitment of β-arrestin is significantly diminished. To better understand how MRAP2 modifies GHSR1a signaling, here we investigated the role of several phosphorylation sites within the C-terminal tail and third intracellular loop of GHSR1a, as well as the mechanism behind MRAP2-mediated inhibition of β-arrestin recruitment. We show that Ser252 and Thr261 in the third intracellular loop of GHSR1a contribute to β-arrestin recruitment, whereas the C-terminal region is not essential for β-arrestin interaction. Additionally, we found that MRAP2 inhibits GHSR1a phosphorylation by blocking the interaction of GRK2 and PKC with the receptor. Taken together, these data suggest that MRAP2 alters GHSR1a signaling by directly impacting the phosphorylation state of the receptor and that the C-terminal tail of GHSR1a prevents rather than contribute to β-arrestin recruitment.