Redundancy of a functional melanocortin 1 receptor in the anti-inflammatory actions of melanocortin peptides: studies in the recessive yellow (e/e) mouse suggest an important role for melanocortin 3 receptor

The issue of which melanocortin receptor (MC-R) is responsible for the anti-inflammatory effects of melanocortin peptides is still a matter of debate. Here we have addressed this aspect using a dual pharmacological and genetic approach, taking advantage of the recent characterization of more selective agonists/antagonists at MC1 and MC3-R as well as of the existence of a naturally defective MC1-R mouse strain, the recessive yellow (e/e) mouse. RT-PCR and ultrastructural analyses showed the presence of MC3-R mRNA and protein in peritoneal macrophages (M phi) collected from recessive yellow (e/e) mice and wild-type mice. This receptor was functional as Mphi incubation (30 min) with melanocortin peptides led to accumulation of cAMP, an effect abrogated by the MC3/4-R antagonist SHU9119, but not by the selective MC4-R antagonist HS024. In vitro M phi activation, determined as release of the CXC chemokine KC and IL-1 beta, was inhibited by the more selective MC3-R agonist gamma(2)-melanocyte stimulating hormone but not by the selective MC1-R agonist MS05. Systemic treatment of mice with a panel of melanocortin peptides inhibited IL-1 beta release and PMN accumulation elicited by urate crystals in the murine peritoneal cavity. MS05 failed to inhibit any of the inflammatory parameters either in wild-type or recessive yellow (e/e) mice. SHU9119 prevented the inhibitory actions of gamma(2)-melanocyte stimulating hormone both in vitro and in vivo while HS024 was inactive in vivo. In conclusion, agonism at MC3-R expressed on peritoneal M phi leads to inhibition of experimental nonimmune peritonitis in both wild-type and recessive yellow (e/e) mice.     

The Yellow Mouse Obesity Syndrome and Mechanisms of Agouti-Induced Obesity

The yellow mouse obesity syndrome is due to dominant mutations at the Agouti locus, which is characterized by obesity, hyperinsulinemia, insulin resistance, hyperglycemia, hyperleptinemia, increased linear growth, and yellow coat color. This syndrome is caused by ectopic expression of Agouti in multiple tissues. Mechanisms of Agouti action in obesity seem to involve, at least in part, competitive melanocortin antagonism. Both central and peripheral effects have been implicated in Agouti-induced obesity. An Agouti-Related Protein (AGRP) has been described recently. It has been shown to be expressed in mice hypothalamus and to act similarly to agouti as a potent antagonist to central melanocortin receptor MC4-R, suggesting that AGRP is an endogenous MC4-R ligand. Mice lacking MC4-R become hyperphagic and develop obesity, implying that agouti may lead to obesity by interfering with MC4-R signaling in the brain and consequently regulating food intake. Furthermore, food intake is inhibited by intracerebro-ventricular injection of a potent melanocortin agonist and was reversed by administration of an MC4-R antagonist. The direct cellular actions of Agouti include stimulation of fatty acid and triglyceride synthesis via a Ca²⁺-dependent mechanism. Agouti and insulin act in an additive manner to increase lipogenesis. This additive effect of agouti and insulin is demonstrated by the necessity of insulin in eliciting weight gain in transgenic mice expressing agouti specifically in adipose tissue. This suggests that agouti expression in adipose tissue combined with hyperinsulinemia may lead to increased adiposity. The roles of melanocortin receptors or agouti-specific receptor(s) in agouti regulation of adipocyte metabolism and other peripheral effects remain to be determined. In conclusion, both central and peripheral actions of agouti contribute to the yellow mouse obesity syndrome and this action is mediated at least in part by antagonism with melanocortin receptors and/or regulation of intracellular calcium.     

Conformation of the core sequence in melanocortin peptides directs selectivity for the melanocortin MC3 and MC4 receptors.

Melanocortin peptides regulate a variety of physiological processes. Five melanocortin receptors (MC-R) have been cloned and the MC3R and MC4R are the main brain MC receptors. The aim of this study was to identify structural requirements in both ligand and receptor that determine gamma-melanocyte-stimulating hormone (MSH) selectivity for the MC3R versus the MC4R. Substitution of Asp10 in [Nle4]Lys-gamma2-MSH for Gly10 from [Nle4]alpha-MSH, increased both activity and affinity for the MC4R while the MC3R remained unaffected. Analysis of chimeric MC3R/MC4Rs and mutant MC4Rs showed that Tyr268 of the MC4R mainly determined the low affinity for [Nle4]Lys-gamma2-MSH. The data demonstrate that Asp10 determines selectivity for the MC3R, however, not through direct side chain interactions, but probably by influencing how the melanocortin core sequence is presented to the receptor-binding pocket. This is supported by mutagenesis of Tyr268 to Ile in the MC4R which increased affinity and activity for [Nle4]Lys-gamma2-MSH, but decreased affinity for two peptides with constrained cyclic structure of the melanocortin core sequence, MT-II and [D-Tyr4]MT-II, that also displayed lower affinity for the MC3R. This study provides a general concept for peptide receptor selectivity, in which the major determinant for a selective receptor interaction is the conformational presentation of the core sequence in related peptides to the receptor-binding pocket.     

Characterization of cell lines stably expressing human normal or mutated EGFP-tagged MC4R

The melanocortin receptor type 4 (MC4-R) is involved in food intake and represents a potential target for the treatment of some forms of obesity. The fluorescent protein EGFP was fused to the wild-type or mutated coding sequence of the human MC4-R. After transfection in HEK 293, clones stably expressing hMC4-R-EGFP were selected. Wild-type chimeric hMC4-R was well addressed to the cell membrane as demonstrated using confocal microscopy and displayed the same pharmacological characteristics as native hMC4R. NDP-alpha MSH induced a time-dependent internalization of MC4-R that was partially prevented by AgRP. The two mutated chimeric receptors studied here (CTCT-deleted and C271A) showed a high alteration of their response to ligand and were retained inside the cells. In conclusion, we have developed a model of clones stably expressing EGFP-tagged-hMC4-R. This is the only such model available to date and it provides a useful tool to follow the trafficking of MC4-R inside living cells.     

Anxiety-like behavior induced by IL-1beta is modulated by alpha-MSH through central melanocortin-4 receptors

The proinflammatory cytokine interleukin-1beta (IL-1beta) influences neuroendocrine activity and produces other effects, including fever and behavioral changes such as anxiety. The melanocortin neuropeptides, such as alpha-melanocyte-stimulating hormone (alpha-MSH), antagonize many actions of IL-1, including fever, anorexia and hypothalamic-pituitary-adrenal (HPA) axis activation through specific melanocortin receptors (MC-R) in the central nervous system. The objective of the present study was to establish the effect of MSH peptides on IL-1beta-induced anxiety-like behavior and the melanocortin receptors involved. We evaluated the effects of intracerebroventricular (i.c.v.) administration of IL-1beta (30 ng) and melanocortin receptor agonists: alpha-MSH, an MC3/MC4-R agonist (0.2 microg) or gamma-MSH, an MC3-R agonist (2 microg) or HS014, an MC4-R antagonist (2 microg), on an elevated plus-maze (EPM) test. Injection of IL-1beta induced an anxiogenic-like response, as indicated by reduced open arms entries and time spent on open arms. The administration of alpha-MSH reversed IL-1beta-induced anxiety with co-administration of HS014 inhibiting the effect of alpha-MSH. However, the associated treatment with gamma-MSH did not affect the anxiety response to IL-1beta. These data suggest that alpha-MSH, through central MC4-R can modulate the anxiety-like behavior induced by IL-1beta.     

Opposite Effects of the Melanocortin-2 (MC2) Receptor Accessory Protein MRAP on MC2 and MC5 Receptor Dimerization and Trafficking

MC2 (ACTH) receptors require MC2 receptor accessory protein (MRAP) to reach the cell surface. In this study, we show that MRAP has the opposite effect on the closely related MC5 receptor. In enzyme-linked immunosorbent assay and microscopy experiments, MC2 receptor was retained in the endoplasmic reticulum in the absence of MRAP and targeted to the plasma membrane with MRAP. MC5 receptor was at the plasma membrane in the absence of MRAP, but trapped intracellularly when expressed with MRAP. Using bimolecular fluorescence complementation, where one fragment of yellow fluorescent protein (YFP) was fused to receptors and another to MRAP, we showed that MC2 receptor-MRAP dimers were present at the plasma membrane, whereas MC5 receptor-MRAP dimers were intracellular. Both MC2 and MC5 receptors co-precipitated with MRAP. MRAP did not alter expression of β2-adrenergic receptors or co-precipitate with them. To determine if MRAP affects formation of receptor oligomers, we co-expressed MC2 receptors fused to YFP fragments in the presence or absence of MRAP. YFP fluorescence, reporting MC2 receptor homodimers, was readily detectable with or without MRAP. In contrast, MC5 receptor homodimers were visible in the absence of MRAP, but little fluorescence was observed by microscopic analysis when MRAP was co-expressed. Co-precipitation of differentially tagged receptors confirmed that MRAP blocks MC5 receptor dimerization. The regions of MRAP required for its effects on MC2 and MC5 receptors differed. These results establish that MRAP forms stable complexes with two different melanocortin receptors, facilitating surface expression of MC2 receptor but disrupting dimerization and surface localization of MC5 receptor.In mammals, the five members of the melanocortin (MC²) receptor family play diverse physiological roles. MC1 receptors (melanocyte-stimulating hormone receptors) control pigmentation in many animals, MC2 receptors (ACTH receptors) regulate adrenal corticosteroid synthesis, MC3 and MC4 receptors in brain influence food intake and energy expenditure, and MC5 receptors control exocrine gland secretion (1). Melanocortin receptors (MC1 through MC5) are structurally related G protein-coupled receptors that respond to agonists with an increase in cAMP. The receptors differ in their affinity for physiological agonists (α-, β-, and γ-melanocyte-stimulating hormone and ACTH) and antagonists (agouti and agouti-related protein).Unlike other melanocortin receptors, MC2 receptors are selectively regulated by ACTH. The MC2 receptor is also unusual in its requirement for an accessory protein, the MC2 receptor accessory protein (MRAP) (2). MRAP must be expressed with the MC2 receptor in order for the receptor to undergo glycosylation, traffic to the plasma membrane, bind ACTH, and stimulate adenylyl cyclase (2–4). Individuals with inactivating mutations of either the MC2 receptor or MRAP suffer from ACTH resistance and severe glucocorticoid deficiency (2).MRAP is a small protein with a conserved amino terminus, single membrane-spanning domain, and non-conserved carboxyl terminus that can also differ among splice variants. MRAP forms antiparallel homodimers, which are exceedingly rare structures, and it is these dimers that co-precipitate with the MC2 receptor (3). Mutagenic analysis has revealed that the carboxyl-terminal region of MRAP is not essential, but the amino-terminal and transmembrane regions are necessary for function (3–5). Deletion or alanine substitution of a critical four-amino acid segment, LDYI, at residues 18–21 of the mouse sequence, results in an MRAP molecule that facilitates expression of the MC2 receptor on the plasma membrane but does not allow the receptor to bind agonist or signal (4). MRAP is not required for cell surface expression of other melanocortin receptors and can inhibit expression or signaling in some cases (3, 6).The MC5 receptor was identified on the basis of its homology to other melanocortin receptors and is thought to be the ancestral ACTH (MC2) receptor (7). Analysis of MC5 receptor knock-out mice revealed that the MC5 receptor is important in controlling exocrine gland secretion (8) and behavioral responses depending on pheromones secreted by the preputial gland (9). MC2 and MC5 receptors are closely related, with 46% identity and 67% homology at the amino acid level, respectively. MC2 and MC5 receptors are both found on 3T3-L1 adipocytes and in some adipose tissues in animals (10, 11). Furthermore, MC2 and MC5 receptors are both expressed in adrenal cortex during embryonic development, when the MC5 receptor appears before the MC2 receptor (12). Mammalian MC2 and MC5 receptors are activated by different pro-opiomelanocortin peptides, responding physiologically to ACTH and melanocyte-stimulating hormone, respectively. Here we demonstrate that MC2 and MC5 receptors are also differentially regulated by MRAP, which exerts opposite effects on surface expression and dimerization of the two receptors.     

Activation of MAP kinase by MC4-R through PI3 kinase

The melanocortin 4 receptor (MC4-R) is a G_s-coupled receptor known to increase cAMP production following agonist stimulation. We demonstrate that the mitogen-activated protein kinases p42 (ERK2) and p44 (ERK1) are also activated by MC4-R following treatment with the MC4-R agonist NDP--MSH in stably transfected CHO-K1 cells. This time- and dose-dependent response is abolished by the MC4-R antagonist SHU-9119. p42/p44 MAPK activation is blocked by the phosphatidylinositol 3-kinase (PI3K) inhibitors wortmannin and LY294002 but not by the protein kinase A (PKA) inhibitor Rp-cAMPS, indicating that that signal activating the p42/p44 MAPK pathway is conveyed through inositol triphosphate.     

Metal ion-mediated agonism and agonist enhancement in melanocortin MC1 and MC4 receptors

An endogenous metal-ion site in the melanocortin MC1 and MC4 receptors was characterized mainly in transiently transfected COS-7 cells. ZnCl(2) alone stimulated signaling through the Gs pathway with a potency of 11 and 13 microm and an efficacy of 50 and 20% of that of alpha-melanocortin stimulating hormone (alpha-MSH) in the MC1 and MC4 receptors, respectively. In the presence of peptide agonist, Zn(II) acted as an enhancer on both receptors, because it shifted the dose-response curves to the left: most pronounced was a 6-fold increase in alpha-MSH potency on the MC1 receptor. The effect of the metal ion appeared to be additive, because the maximal cAMP response for alpha-MSH in the presence of Zn(II) was 60% above the maximal response for the peptide alone. The affinity of Zn(II) could be increased through binding of the metal ion in complex with small hydrophobic chelators. The binding affinities and profiles were similar for a number of the 2,2'-bipyridine and 1,10-phenanthroline analogs in complex with Zn(II) in the MC1 and MC4 receptors. However, the potencies and efficacies of the metal-ion complexes were very different in the two receptors, and close to full agonism was obtained in the MC1 receptor. Metal ion-chelator complexes having antagonistic properties were also found. An initial attempt to map the metal-ion binding site in the MC1 receptor indicated that Cys(271) in extracellular loop 3 and possibly Asp(119) at the extracellular end of TM-III, which are both conserved among all MC receptors, are parts of the site. It is concluded that the function of the MC1 and MC4 receptors can be positively modulated by metal ions acting both as partial agonists and as potentiators for other agonists, including the endogenous peptide ligand alpha-MSH at Zn(II) concentrations that could be physiological. Furthermore, the metal ion-chelator complexes may serve as leads in the development of novel melanocortin receptor modulators.     

Synthesis and biological evaluation of imidazole-based small molecule antagonists of the melanocortin 4 receptor (MC4-R)

A novel series of imidazole-based small molecule antagonists of the melanocortin 4 receptor (MC4-R) is reported. Members of this series have been identified, which exhibit sub-micromolar binding affinity for the MC4-R, functional potency <100nM, and good oral exposure in rat. Antagonists of the MC4-R are potentially useful in the therapeutic treatment of involuntary weight loss due to advanced age or disease (e.g. cancer or AIDS), an area of large, unmet medical need.     

Modeling the interactions between MC2R and ACTH models from human

Melanocortin system is composed of four peptide hormones namely α-, β-, -γ, and adrenocorticotropic hormone (ACTH), derived from post-translational cleavage of a polypeptide precursor ‘proopiomelanocortin (POMC).’ Among these hormones, ACTH, a 38 amino acid residue peptide fragment is an important hormone as it is involved in steroid secretion. In addition to this, to cite a few, this hormone is also known to induce variety of other effects, such as alterations in motor/sexual behavior, improvement in memory, and anti-inflammatory effects. To date, five melanocortin receptors (MC1R–MC5R) have been characterized with tissue-specific expression patterns and different binding affinities for each of the melanocortin hormones to regulate various biological functions. In the present work, three-dimensional (3D) models of MC2R and ACTH from human have been predicted, followed by docking and molecular dynamics simulation. While the 3D model of MC2R receptor has been predicted through threading approach, structure of ACTH was built based on ab initio technique. The MC2R model was later successfully docked onto the ACTH structure. Molecular dynamics (MD) simulation for 20?ns was used to compute the binding free energy of MC2R with ACTH model under implicit solvent conditions.     

Further evidence for ancient role of ACTH peptides at melanocortin (MC) receptors; pharmacology of dogfish and lamprey peptides at dogfish MC receptors

The cloning of melanocortin (MC) receptors in distant species has provided us tools to get insight in how the ligand-receptors interactions in the MC system have evolved. We have however lacked studies on pharmacology of native ancient melanocortin peptides at the ancient MC receptors. In this paper we synthesized melanocortin peptides from both the sea lamprey (Petromyzon marinus) and spiny dogfish (Squalus acanthias) and tested them on the MC3 and MC4 receptors from spiny dogfish. The results show that both the dogfish and lamprey ACTH peptides have similar or higher affinity than the dogfish alpha-, beta- and gamma-MSH peptides to the dogfish MC3 and MC4 receptors. Moreover, both the dogfish and lamprey ACTH peptides have more than 10-fold higher affinity than alpha-MSH to the dogfish MC4 receptor. We also show that dogfish delta-MSH is able to bind to MC receptors and its potency is higher than of dogfish beta-MSH, which is considered to be its precursor. Our results provide the first evidence that native ACTH ligands from dogfish and lamprey have a preference above native MSH peptides to ancient version of the MC3 and MC4 receptors. This further strengthens the hypotheses that the ligand contributing to the first version of the melanocortin ligand-receptor system resembled ACTH.     

Monogenic disorders of obesity and body fat distribution.

Recently, great progress has been made towards understanding the molecular basis of body fat regulation. Identification of mutations in several genes in spontaneous monogenic animal models of obesity and development of transgenic models have indicated the physiological roles of many genes in the regulation of body fat distribution. In humans, mutations in leptin, leptin receptor, prohormone convertase 1 (PC1), pro-opiomelanocortin (POMC), melanocortin 4-receptor (MC4-R), and peroxisome proliferator-activated receptor (PPAR) gamma2 genes have been described in patients with severe obesity. Most of these obesity disorders exhibit a distinct phenotype with varying degrees of hypothalamic and pituitary dysfunction and a recessive inheritance, whereas MC4-R mutation has a nonsyndromic phenotype with dominant inheritance. These mutations suggest the critical role of central signaling systems composed of leptin/leptin receptor and alpha-melanocyte stimulating hormone/MC4-R in human energy homeostasis. Although the genetic basis of monogenic disorders of body fat distribution, such as congenital generalized lipodystrophy and familial partial lipodystrophy, Dunnigan variety, is still unknown, the genes for these have recently been localized to chromosomes 9q34 and 1q21-22, respectively. The advances in our knowledge of the phenotypic manifestations and underlying molecular mechanisms of genetic body fat disorders may lead to better treatment and prevention of obesity and other disorders of adipose tissue in the future.     

Remarkable synteny conservation of melanocortin receptors in chicken,human, and other vertebrates

The melanocortin receptors (MCR) belong to the superfamily of G-protein-coupled receptors that participate in both peripheral and central functions, including regulation of energy balance. Genomic clones of the five chicken (GGA) MCRs were isolated and used to find the chromosomal location of each of the loci. The genes encoding MC2R and MC5R mapped to the middle part of the long arm of chromosome 2 (GGA2q22-q26) and MC4R proximally on the same chromosome arm, close to the centromere (2q12). This arrangement seems to be conserved on chromosome 18 in the human (HSA18). The MC1R and MC3R genes mapped to different microchromosomes that also appear to share homology with the respective human localization. The conserved synteny of the MC2R, MC5R, and MC4R cluster in chicken (GGA2), human (HSA18), and other mammals suggests that this cluster is ancient and was formed by local gene duplications that most likely occurred early in vertebrate evolution. Analysis of conserved synteny with mammalian genomes and paralogon segments prompted us to predict an ancestral gene organization that may explain how this family was formed through both local duplication and tetraploidization processes.