MRAP and MRAP2 are bidirectional regulators of the melanocortin receptor family.
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ABSTRACT:
SUBMITTER: Chan LF
PROVIDER: S-EPMC2661846 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:The melanocortin-2 (MC2) receptor accessory protein (MRAP) is required for trafficking of the G protein-coupled MC2 receptor to the plasma membrane. The mechanism of action and structure of MRAP, which has a single transmembrane domain, are unknown. Here, we show that MRAP displays a previously uncharacterized topology. Epitopes on both the N- and C-terminal ends of MRAP were localized on the external face of CHO cells at comparable levels. Using antibodies raised against N- and C-terminal MRAP peptides, we demonstrated that both ends of endogenous MRAP face the outside in adrenal cells. Nearly half of MRAP was glycosylated at the single endogenous N-terminal glycosylation site, and over half was glycosylated when the natural glycosylation site was replaced by one in the C-terminal domain. A mutant MRAP with potential glycosylation sites on both sides of the membrane was singly but not doubly glycosylated, suggesting that MRAP is not monotopic. Coimmunoprecipitation of differentially tagged MRAPs established that MRAP is a dimer. By selectively immunoprecipitating cell surface MRAP in one or the other orientation, we showed that MRAP homodimers are antiparallel and form a stable complex with MC2 receptor. In the absence of MRAP, MC2 receptor was trapped in the endoplasmic reticulum, but with MRAP, the MC2 receptor was glycosylated and localized on the plasma membrane, where it signaled in response to ACTH. MRAP acted specifically, because it did not increase surface expression of other melanocortin, beta2-adrenergic, or TSH-releasing hormone receptors. MRAP is the first eukaryotic membrane protein identified with an antiparallel homodimeric structure.
| S-EPMC2154416 | biostudies-literature
Project description: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.
| S-EPMC8004712 | biostudies-literature
Project description:The melanocortin-4 receptor (MC4R) is essential for control of energy homeostasis in vertebrates. MC4R interacts with melanocortin receptor accessory protein 2 (MRAP2) in vitro, but its functions in vivo are unknown. We found that MRAP2a, a larval form, stimulates growth of zebrafish by specifically blocking the action of MC4R. In cell culture, this protein binds MC4R and reduces the ability of the receptor to bind its ligand, α-melanocyte-stimulating hormone (α-MSH). A paralog, MRAP2b, expressed later in development, also binds MC4R but increases ligand sensitivity. Thus, MRAP2 proteins allow for developmental control of MC4R activity, with MRAP2a blocking its function and stimulating growth during larval development, whereas MRAP2b enhances responsiveness to α-MSH once the zebrafish begins feeding, thus increasing the capacity for regulated feeding and growth.
| S-EPMC4255277 | biostudies-literature
Project description:Phylogenetic analyses indicate that the genome of the cartilaginous fish, Callorhynchus milii (elephant shark), encodes a melanocortin-2 receptor (MC2R) ortholog. Expression of the elephant shark mc2r cDNA in Chinese hamster ovary (CHO) cells revealed that trafficking to the plasma membrane and functional activation of the receptor do not require coexpression with an exogenous melanocortin receptor-2 accessory protein (mrap) cDNA. Ligand selectivity studies indicated that elephant shark MC2R-transfected CHO cells produced cAMP in a dose-dependent manner when stimulated with either human ACTH (1-24) or [Nle(4), d-Phe(7)]-MSH. Furthermore, the order of ligand selectivity when elephant shark MC2R-transfected CHO cells were stimulated with cartilaginous fish melanocortins was as follows: ACTH (1-25) = γ-MSH = δ-MSH > αMSH = β-MSH. Elephant shark MC2R is the first vertebrate MC2R ortholog to be analyzed that does not require melanocortin receptor-2 accessory protein 1 for functional activation. In addition, elephant MC2R is currently the only MC2R ortholog that can be activated by either ACTH- or MSH-sized ligands. Hence, it would appear that MC2R dependence on melanocortin receptor-2 accessory protein 1 for functional activation and the exclusive selectivity of this melanocortin receptor for ACTH are features that emerged after the divergence of the ancestral cartilaginous fishes and the ancestral bony fishes more than 400 million years ago.
| S-EPMC3512025 | biostudies-literature
Project description: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.
| S-EPMC9251544 | biostudies-literature
Project description:Melanocortin 2 receptor accessory protein (MRAP) is highly expressed in adrenal gland and adipose tissue. In adrenal cells, MRAP is essential for adrenocorticotropic hormone (ACTH)-induced activation of the cAMP/protein kinase A (PKA) pathway by melanocortin 2 receptor (MC2R), leading to glucocorticoid production and secretion. Although ACTH was known to stimulate PKA-dependent lipolysis, the functional involvement of MRAP in adipocyte metabolism remains incompletely defined. Herein, we found that knockdown or overexpression of MRAP in 3T3-L1 adipocytes reduced or increased ACTH-induced lipolysis, respectively. Moreover, an unbiased proteomics screen and coimmunoprecipitation analysis identified Gαs as a novel interacting partner of MRAP. An MRAP mutant disabled in Gαs association failed to augment the activation of PKA and lipolytic response to ACTH. Furthermore, compared with wild-type mice, transgenic mice (aP2-MRAP) overexpressing MRAP fat specifically exhibited increased lipolytic response to ACTH. When fed a high-fat diet (HFD), the transgenic mice displayed a significant decrease in the gain of adiposity and body weight as well as an improvement in glucose and insulin tolerance. These phenotypes were accompanied by increased adipose expression of genes for mitochondrial fatty acid oxidation and thermogenesis, and overall energy expenditure. Collectively, our data strongly suggest that MRAP plays a critical role in the regulation of ACTH-induced adipose lipolysis and whole-body energy balance.
| S-EPMC5780064 | biostudies-literature
Project description:Melanocortin receptors (MCRs) and their accessory proteins (MRAPs) evolutionarily first appear in the genome of sea lamprey. The most ancient melanocortin system consists of only two melanocortin receptors (slMCa and slMCb) and one MRAP2 (slMRAP2) protein, but the physiological roles have not been fully explored in this primitive species. Here, we synthesize and characterize the pharmacological features of slMRAP2 protein on two slMCRs. Our results show that the slMRAP2 protein lacks the long carboxyl terminus; it directly interacts and decreases the surface expression but enhances the α-MSH-induced agonism of slMCa and slMCb. In comparison with higher organisms such as elephant shark and zebrafish, we also demonstrate the constantly evolving regulatory function of the carboxyl terminus of MRAP2 protein, the unique antiparallel topology of slMRAP2 dimer and the homo- and hetero-dimerization of two slMCRs. This study elucidates the presence and modulation of melanocortin receptor by the accessory protein of the agnathans for the first time, which provides a better insight of the melanocortin system in ancient species of chordates.
| S-EPMC6454295 | biostudies-literature
Project description:The melanocortin receptors (MCRs) are a gene family in the rhodopsin class of G protein-coupled receptors. Based on the analysis of several metazoan genome databases it appears that the MCRs are only found in chordates. The presence of five genes in the family (i.e., mc1r, mc2r, mc3r, mc4r, mc5r) in representatives of the tetrapods indicates that the gene family is the result of two genome duplication events and one local gene duplication event during the evolution of the chordates. The MCRs are activated by melanocortin ligands (i.e., ACTH, α-MSH, β-MSH, γ-MSH, δ-MSH) which are all derived from the polypeptide hormone/neuropeptide precursor, POMC, and as a result the functional evolution of the MCRs is intimately associated with the co-evolution of POMC endocrine and neuronal circuits. This review will consider the origin of the MCRs, and discuss the evolutionary relationship between MC2R, MC5R, and MC4R. In addition, this review will analyze the functional evolution of the mc2r gene in light of the co-evolution of the MRAP (Melanocortin-2 Receptor Accessory Protein) gene family.
| S-EPMC3622036 | biostudies-literature
Project description:RT-PCR analysis of gar pituitary and brain indicated that different combinations of gar melanocortin receptor mRNAs are present in the same tissues with mRNAs for gar mrap1 and gar mrap2. Against this background, an objective of this study was to determine whether the ligand sensitivity for either ACTH or α-MSH was affected when gar (g) melanocortin receptors (Mcrs) were co-expressed with either of the accessory proteins gMrap1 or gMrap2 in Chinese Hamster Ovary cells. The results indicated that gMc2r has an obligatory requirement for co-expression with gMrap1 in order for the receptor to be activated by hACTH(1-24). In addition, activation of gMc2r did not occur when the receptor was expressed alone or co-expressed with gMrap2. Furthermore, co-expression of gMc2r with gMrap1 followed by stimulation with NDP-MSH resulted in a low level of activation (only at 10-7 M and 10-6 M). However, gMc1r, gMc3r, gMc4r, and gMc5r responded to stimulation by NDP-MSH in a more robust manner. Co-expression of gMc1r, gMc3r, gMc4r, and gMc5r with gMRAP1 had no effect on sensitivity to stimulation by NDP-MSH or hACTH(1-24). Co-expression with gMRAP2 had no negative or positive effect on ligand sensitivity for gMc1r, gMc3r, and gMc5r, however this treatment did increase the activation of CHO cells transfected with gMc4r following stimulation with both hACTH(1-24) (p < 0.001), and NDP-MSH (p < 0.001). Co-expression of gMC5R with either gMRAP1 or gMRAP2 increased trafficking of gMC5R to the plasma membrane. These pharmacological observations are compared to the response of melanocortin receptors from other neopterygian fishes, cartilaginous fishes, and tetrapods to stimulation by ACTH(1-24) and forms of α-MSH.
| S-EPMC7263024 | biostudies-literature
Project description:BackgroundThe 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.MethodsHere, 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.Results36 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.ConclusionsCollectively, 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.
| S-EPMC9619224 | biostudies-literature