Peptides (v.26, #10)

Contents List (v-vii).

Melanocortins (MCs) are multifunctional peptide hormones that regulate a diversity of physiological functions. MCs have been implicated in sexual function in animals. We document here that a MC analog, Melanotan II (MTII), can enhance sexual function in human males (erectile activity) and females (increased levels of sexual desire and genital arousal). Unlike other sexual-enhancement drugs, MTII works at the level of the brain, thus eliciting a rather natural sexual response with minimal or no undesirable side effects. The actions of the peptide were discovered accidentally while studying the effects of the peptide and related analogs on human skin pigmentation (tanning).
Keywords: Melanocortins; MTI and MTII; MSH;

a-MSH enhances activity-based anorexia by Jacquelien J.G. Hillebrand; Martien J.H. Kas; Roger A.H. Adan (1690-1696).
Activity-based anorexia (ABA) is considered an animal model of anorexia nervosa (AN). In ABA, scheduled feeding in combination with voluntary access to running wheels, results in hyperactivity, hypophagia, body weight loss and activation of the HPA axis. Since stimulation of the melanocortin (MC) system has similar effects, this system is a candidate system involved in ABA. Here it is shown that chronic a-MSH treatment enhances ABA by increasing running wheel activity (RWA), decreasing food intake and increasing HPA axis activation.
Keywords: Hyperactivity; Food restriction; Melanocortins; Anorexia nervosa;

Isogenic and congenic offspring from matings of inbred black a/a dams by sibling (or non-sibling from another inbred strain) yellow agouti A vy /a sires provide an animal model of obese yellow agouti A vy /a and isogenic lean pseudoagouti A vy /a mice exhibiting two different in vivo concentrations (high, very low) of ectopic agouti protein (ASP) with congenic lean black a/a mice as null controls. This makes it possible to differentiate between the high and very low dose levels of ectopic ASP with respect to interactions with diverse physiological and molecular pathways. Assay of differential responses to 12 or 24 months of carbonyl iron overload assessed the possible suitability of this animal model for the study of hemochromatosis. Agouti A/a B6C3F1 mice were used as non-congenic null controls. The age-related waxing and waning of body weight, food consumption, and caloric efficiency, as well as associated changes in pancreatic islets and islet cells, and formation of liver tumors were assayed. While the hypothesis that these mice might serve as a tool for investigating hemochromatosis was not confirmed, the data did provide evidence that even the very low levels of ASP in pseudoagouti A vy /a mice affect the network of molecular/metabolic/physiological response pathways that comprises the yellow agouti obese phenome. We suggest that the combination of yellow agouti A vy /a, pseudoagouti A vy /a, and black a/a congenic mice provides a practical tool for applying a dose–response systems biology approach to understanding the dysregulatory influence of ectopic ASP on the molecular-physiological matrix of the organism.
Keywords: Agouti protein; Caloric efficiency; Hepatocellular tumors; Hyperplasia; Iron overload; Melanocortin receptor 4; Obesity; Pancreatic islet cells; Pseudoagouti mice; Systems biology; Age-associated weight loss;

Melanocortin-4 receptor-null mice display normal affective licking responses to prototypical taste stimuli in a brief-access test by Shachar Eylam; Marcus Moore; Carrie Haskell-Luevano; Alan C. Spector (1712-1719).
We tested whether MC4R null mice display altered gustatory function relative to wild-type controls that may contribute to the characteristic hyperphagia and obesity associated with this gene deletion. Mice were tested for their licking responses to prototypical taste solutions (sucrose, NaCl, quinine, citric acid) in series of daily 30-min sessions in which a range of concentrations of each tastant was available in randomized blocks of 5-s trials. Notwithstanding some minor deviations, the concentration-response functions of the MC4R null and wild-type mice were basically the same for all of the prototypical compounds tested here. Thus, taste-based appetitive and avoidance behavior is expressed in the absence of the MC4 receptor, demonstrating that this critical component in the melanocortin system is not required for normal affective gustatory function to be maintained.
Keywords: Gustatory; Hedonics; Obesity; Hyperphagia; Neuropeptides; Sucrose; Quinine; NaCl; Citric acid; Knockout mice;

Ghrelin-induced food intake and growth hormone secretion are altered in melanocortin 3 and 4 receptor knockout mice by Amanda M. Shaw; Boman G. Irani; Marcus C. Moore; Carrie Haskell-Luevano; William J. Millard (1720-1727).
Ghrelin stimulates food intake in part by activating hypothalamic neuropeptide Y (NPY) neurons/agouti related peptide (AGRP) neurons. We investigated the role of AGRP/melanocortin signaling in ghrelin-induced food intake by studying melanocortin 3 and 4 receptor knockout (MC3R KO and MC4R KO) mice. We also determined whether reduced ghrelin levels and/or an altered sensitivity to the GH-stimulating effects of ghrelin accompany the obesity syndromes of MC3R KO and MC4R KO mice. Compared to wild-type (WT) mice, the effects of ghrelin on food intake were reduced in MC3R KO and MC4R KO mice and circulating ghrelin levels were reduced in female MC4R KO mice. Female MC3R KO and MC4R KO mice exhibited a diminished responsiveness to the GH-releasing effects of ghrelin. Thus, deletion of the MC3R or MC4R results in a decreased sensitivity to ghrelin and verifies the involvement in the melanocortin system in ghrelin-induced food intake.
Keywords: Ghrelin; Growth hormone; Melanocortin; Agouti related protein; Orexigenic;

Serotonergic pathways converge upon central melanocortin systems to regulate energy balance by Ligang Zhou; Todd Williams; Jennifer L. Lachey; Toshiro Kishi; Michael A. Cowley; Lora K. Heisler (1728-1732).
Multiple lines of research provide compelling support for an important role for central serotonergic (5-hydroxytryptamine, 5-HT) and melanocortin pathways in the regulation of food intake and body weight. In this brief review, we outline data supporting a model in which serotonergic pathways affect energy balance, in part, by converging upon central melanocortin systems to stimulate the release of the endogenous melanocortin agonist, alpha-melanocyte stimulating hormone (α-MSH). Further, we review the neuroanatomical mapping of a downstream target of α-MSH, the melanocortin 4 receptor (MC4R), in the rodent brain. We propose that downstream activation of MC4R-expressing neurons substantially contributes to serotonin's effects on energy homeostasis.
Keywords: Hypothalamus; Obesity; Melanocortin 4 receptor (MC4R); Serotonin2C receptor (5-HT2CR);

The mammalian melanocortin system has been established as a crucial regulatory component in an extraordinarily diverse number of physiological functions. In contrast, comparatively little is known about the avian melanocortin system: interest in the physiological role of α-MSH in birds has been limited by the fact that birds lack the intermediate lobe of the pituitary, the main source of circulating α-MSH in most vertebrates. Recently, however, the main avian melanocortin system genes, including POMC, AGRP, and all the melanocortin receptors, have been cloned and their physiological roles are the beginning to be elucidated. This review outlines our improved understanding of the avian melanocortin system, particularly in relation to two of the most widely studied physiological functions of the melanocortin system in mammals, the regulation of pigmentation and energy homeostasis. The data reviewed here indicate that the melanocortin system has been strongly conserved during vertebrate evolution and that α-MSH is produced locally in birds to act as an autocrine/paracrine hormone.
Keywords: α-MSH; ACTH; POMC; Melanocortin; Melanocortin receptor; Agouti-related protein; Neuropeptide-Y; Pigmentation; Energy homeostasis; Birds;

Peripheral influences on central melanocortin neurons by K.G. Murphy; S.R. Bloom (1744-1752).
The melanocortins are peptide products of post-translational processing of the pro-opiomelanocortin precursor protein. Melanocortin-expressing neurons are found in the arcuate nucleus of the hypothalamus and the nucleus of the solitary tract in the brain stem. The central melanocortin system is involved in a number of biological functions, including regulation of energy homeostasis. Hypothalamic and brain stem circuits interpret and integrate a number of peripheral inputs to provide a coordinated central response. This review examines the effect of these peripheral signals on central melanocortin signaling.
Keywords: Melanocortin; Pro-opiomelanocortin; Hypothalamus; Brain stem; Peripheral;

Sensing the fat: Fatty acid metabolism in the hypothalamus and the melanocortin system by Miguel López; Sulay Tovar; María Jesús Vázquez; Rubén Nogueiras; Rosa Señarís; Carlos Diéguez (1753-1758).
Recent evidence has demonstrated that circulating long chain fatty acids act as nutrient abundance signals in the hypothalamus. Moreover, pharmacological inhibition of fatty acid synthase (FAS) results in profound decrease in food intake and body weight in rodents. These anorectic actions are mediated by the modulation of hypothalamic neuropeptide systems, such as melanocortins. In this review, we summarize what is known about lipid sensing and fatty acid metabolism in the hypothalamus. Understanding these molecular mechanisms could provide new pharmacological targets for the treatment of obesity and appetite disorders, as well as novel concepts in the nutritional design.
Keywords: AGRP; Fatty acids; Fatty acid synthase; Feeding; Malonyl-CoA; Obesity; POMC;

Agouti-related protein: More than a melanocortin-4 receptor antagonist? by Lynn E. Pritchard; Anne White (1759-1770).
It is well established that agouti-related protein (AGRP) can act as a competitive antagonist to proopiomelanocortin (POMC)-derived peptides at the melanocortin-4 receptor (MC4R), and that this homeostatic mechanism is important as a means of coordinating appetite with perceived metabolic requirement. However, there are clearly additional facets to the physiological role of AGRP, given that it is active in MC4R knockout mice and it has strikingly long-lasting effects on food intake, compared with MC4R agonists. In this review we focus on: (i) evidence that AGRP is more sensitive to perturbations in energy balance than POMC and is therefore the primary basis of melanocortinergic regulation. (ii) Evidence that the bioactive peptide AGRP83–132, acts by alternate mechanism(s) to elicit its long-term effects on food intake. (iii) Evidence that AGRP is post-translationally cleaved to generate AGRP83–132 and one or more N terminal peptides, which may have an important physiological role(s) that are independent of the melanocortin system. A clear understanding of how proAGRP processing is regulated, and the role of resultant peptides, may define additional therapeutic targets in the treatment of obesity.
Keywords: Agouti-related protein; Melanocortin-3 receptor; Melanocortin-4 receptor; Post-translational processing;

The Agouti-related protein and its role in energy homeostasis by Adrian M. Stütz; Christopher D. Morrison; George Argyropoulos (1771-1781).
The melanocortin system plays an important role in the regulation of energy homeostasis. The Agouti-related protein (AGRP) is a natural antagonist of the action of alpha-melanocyte stimulating hormone (α-MSH) at the melanocortin receptors (MCR). AGRP is upregulated by fasting while intracerebroventricular injections of synthetic AGRP lead to increased appetite and food intake. Transgenic mice overexpressing AGRP are also hyperphagic and eventually become obese. AGRP is, therefore, a significant regulator of energy balance and a candidate gene for human fatness. Indeed, humans with common single nucleotide polymorphisms (SNPs) in the promoter or the coding region are leaner and resistant to late-onset obesity than wild-type individuals. AGRP is also expressed in the periphery. Recent studies show that AGRP in the adrenal gland is upregulated by fasting as much as it is in the hypothalamus. These data open up the possibility for a wider role by AGRP not only in food intake but also in the regulation of energy balance through its actions on peripheral tissues. This review summarizes recent advances in the biochemical and physiological properties of AGRP in an effort to enhance our understanding of the role this powerful neuropeptide plays in mammalian energy homeostasis.
Keywords: Obesity; Adrenal; Hypothalamus; Leptin; Polymorphism;

Roles for melanocortins and leptin in adipose tissue apoptosis and fat deposition by Mary Anne Della-Fera; Clifton A. Baile (1782-1787).
Leptin has a wide range of effects on physiological functions related to the regulation of body energy balance. Many of leptin's effects are mediated through neuropeptide-containing neurons and neuropeptide receptors in the hypothalamus. The melanocortin system includes both agonist (α-melanocyte stimulating hormone, αMSH) and antagonist peptides (agouti related peptide, AGRP). Increased melanocortin receptor stimulation following leptin administration plays an important role in leptin-induced hypophagia and increased sympathetic nervous system activity and is partly responsible for leptin-induced weight loss. However, melanocortins do not appear to mediate some of the more striking centrally-mediated effects of leptin on adipose tissue, including adipose tissue apoptosis, that lead to the extensive depletion of fat.
Keywords: αMSH; Leptin; NPY; Food intake; Body weight; Adipose apoptosis; Energy metabolism;

Feeding effects of melanocortin ligands—A historical perspective by Boman G. Irani; Carrie Haskell-Luevano (1788-1799).
The process of energy homeostasis is a highly regulated process involving interacting signals between a variety of anorexigenic and orexigenic peptides, proteins and signaling molecules. The melanocortin system is an important component of this complex regulatory network. Involvement of the melanocortin pathway in the control of food intake and body weight regulation has been studied extensively in the past two decades. Previous studies that involve central administration of melanocortin molecules and examination of molecules that effect food intake in melanocortin knockout (KO) mice (MC3R, MC4R, POMC, AGRP and NPY) have been examined. In this review, we have summarized feeding studies that have resulted in the recognition of the melanocortin system as a major contributor to the complex neuroendocrine system regulating energy homeostasis.
Keywords: Melanocortin; Obesity; Knockout; Food intake;

Regulation of thermogenesis by the central melanocortin system by Wei Fan; Adriana Voss-Andreae; Wei-Hua Cao; Shaun F. Morrison (1800-1813).
Adaptive thermogenesis represents one of the important homeostatic mechanisms by which the body maintains appropriate levels of stored energy and its core temperature. Dysregulation of adaptive thermogenesis promotes obesity. The central melanocortin system, in particular the melanocortin 4 receptor (MC4R) signaling pathway, influences the regulation of every aspect of energy balance, including thermogenesis, and plays a critical role in energy homeostasis in both rodent and man. This review will outline our current understanding of adaptive thermogenesis, focusing on the role of the central melanocortin pathway in the regulation of thermogenesis.
Keywords: Obesity; Melanocortin; POMC; AGRP; Thermogenesis; Sympathetic nervous system; BAT; UCP; Thyroid; Central circuits; Diet-induced thermogenesis; Pathogenic stimuli;

Two cysteine substitutions in the MC1R generate the blue variant of the arctic fox (Alopex lagopus) and prevent expression of the white winter coat by Dag Inge Våge; Eva Fuglei; Kristin Snipstad; Janne Beheim; Veslemøy Malm Landsem; Helge Klungland (1814-1817).
We have characterized two mutations in the MC1R gene of the blue variant of the arctic fox (Alopex lagopus) that both incorporate a novel cysteine residue into the receptor. A family study in farmed arctic foxes verified that the dominant expression of the blue color phenotype cosegregates completely with the allele harboring these two mutations. Additionally to the altered pigment synthesis, the blue fox allele suppresses the seasonal change in coat color found in the native arctic fox. Consequently, these findings suggest that the MC1R/agouti regulatory system is involved in the seasonal changes of coat color found in arctic fox.
Keywords: Pigmentation; MC1R; Blue fox; Winter coat; Arctic fox; Polar fox; Alopex lagopus;

Activation of the cAMP pathway by variant human MC1R alleles expressed in HEK and in melanoma cells by Richard A. Newton; Sonia E. Smit; Christopher C. Barnes; Julie Pedley; Peter G. Parsons; Richard A. Sturm (1818-1824).
α-Melanocyte-stimulating hormone (α-MSH) activates the melanocortin-1 receptor (MC1R) on melanocytes to promote a switch from red/yellow pheomelanin synthesis to darker eumelanins via positive coupling to adenylate cyclase. The human MC1R locus is highly polymorphic with the specific variants associated with red hair and fair skin (RHC phenotype) postulated to be loss-of-function receptors. We have examined the ability of MC1R variants to activate the cAMP pathway in stably transfected HEK293 cells. The RHC associated variants, Arg151Cys, Arg160Trp and Asp294His, demonstrated agonist-mediated increases in cAMP and phosphorylation of cAMP-responsive element-binding protein (CREB). Whereas the Asp294His variant showed severely impaired functional responses, the Arg151Cys and Arg160Trp variants retained considerable signaling capacity. Melanoma cells homozygous for either the Arg151Cys variant or consensus sequence both elicited CREB phosphorylation in response to α-MSH in the presence of IBMX. The common RHC alleles, Arg151Cys, Arg160Trp and Asp294His, are neither complete loss-of-function receptors nor are they functionally equivalent.
Keywords: α-Melanocyte-stimulating hormone; MC1R; Polymorphism; cAMP; CREB; Melanoma; Red hair; RHC;

Peptide–lipid interaction monitored by spin labeled biologically active melanocortin peptides by Roberto M. Fernandez; Renata F.F. Vieira; Clóvis R. Nakaie; Amando S. Ito; M. Teresa Lamy (1825-1834).
The present work comparatively analyzes the interaction of α-MSH and its more potent and long-acting analog [Nle4, D-Phe7]α-MSH (NDP-MSH) with lipid bilayers. The peptides were spin labeled with Toac (2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid) at the N-terminal, as those derivatives had been previously shown to keep their full biological activity. Due to the special rigidity of the Toac covalent binding to the peptide molecule, this spin label is highly sensitive to the peptide backbone conformation and dynamics. The peptides were investigated both by the electron spin resonance (ESR) of Toac0 and the time resolved fluorescence of Trp9 present in the peptides. The Toac0 ESR of the membrane-bound peptides indicates that the two peptides are inserted into the bilayer, close to the bilayer surface, in rather similar environments. A residue titration around pK a 7.5, possibly that of His6, can be clearly monitored by peptide–lipid partition. Trp9 time resolved fluorescence indicates that the peptides, and their Toac-labeled derivatives, present rather similar conformations when membrane bound, though Trp9 in NDP-MSH, and in its Toac-labeled derivative, goes somewhat further down into the bilayer. Yet, Toac0 ESR signal shows that the Toac-labeled N-terminal of NDP-MSH is in a shallower position in the bilayer, as compared to the hormone.
Keywords: Melanocortins; Toac; ESR; α-MSH; NDP-MSH; Bilayer-partition; pH titration; Trp; Time resolved fluorescence;

Rattus norvegicus melanocortin 3 receptor: A corrected sequence by Derek Daniels; Aae Suzuki; Edan Shapiro; Laiyi Luo; Daniel K. Yee; Steven J. Fluharty (1835-1841).
Keywords: Melanocortin receptor; Melanocyte stimulating hormone; MTII; cAMP;

Expression of the human melanocortin-2 receptor in different eukaryotic cells by Mohamed Rached; Haquima El Mourabit; Anna Buronfosse; Antonine Blondet; Danielle Naville; Martine Begeot; Armelle Penhoat (1842-1847).
The human melanocortin-2 receptor (hMC2R) is mainly present in the adrenal cortex and has been difficult to express in heterologous cells. The hMC2R fused to the EGFP at its C-terminus has been stably transfected in the murine M3 melanoma and HEK293 cells. In the M3 cells, the hMC2R-EGFP was well-addressed to the cell membrane and functional whereas in the HEK293 cells, the hMC2R-EGFP was retained intracellularly. These results suggest that some specific factors, missing in cells, which do not express any melanocortin receptor, are involved in the correct addressing of the hMC2R to the cell membrane.
Keywords: ACTH; Human melanocortin-2 receptor; Stable expression; Enhanced green fluorescent protein;

The melanocortin-1 receptor carboxyl terminal pentapeptide is essential for MC1R function and expression on the cell surface by J. Sánchez-Más; B.L. Sánchez-Laorden; L.A. Guillo; C. Jiménez-Cervantes; J.C. García-Borrón (1848-1857).
The pigmentary actions of the melanocortins are mediated by the melanocortin-1 receptor (MC1R), a seven transmembrane domains receptor positively coupled to Gs and the cAMP cascade. In order to define the structure–function relationships of potentially relevant domains in MC1R, particularly its C-terminal cytosolic tail, we generated and analyzed several variants with C-terminal deletions, as well as point mutants in selected residues of the human MC1R. We show that the MC1R C-terminal pentapeptide is essential for proper receptor expression on the plasma membrane, and that residues Thr314, Cys315 and Trp317 are at least partially responsible for this effect.
Keywords: G-protein coupled receptor; Melanocortin-1 receptor; α-Melanocyte-stimulating hormone; Receptor expression; Intracellular traffic;

Regulatory elements of the melanocortin 1 receptor by Francois Rouzaud; Vincent J. Hearing (1858-1870).
Among more than 120 genes that are now known to regulate mammalian pigmentation, one of the key genes is MC1R, which encodes the melanocortin 1 receptor, a seven transmembrane G protein-coupled receptor expressed on the surface of melanocytes. Since the monoexonic sequence of the gene was cloned and characterized more than a decade ago, tremendous efforts have been dedicated to the extensive genotyping of mostly red-haired populations all around the world, thus providing allelic variants that may or may not account for melanoma susceptibility in the presence or absence of ultraviolet (UV) exposure. Soluble factors, such as proopiomelanocortin (POMC) derivatives, agouti signal protein (ASP) and others, regulate MC1R expression, leading to improved photoprotection via increased eumelanin synthesis or in contrast, inducing the switch to pheomelanin. However, there is an obvious lack of knowledge regarding the numerous and complex regulatory mechanisms that govern the expression of MC1R at the intra-cellular level, from gene transcription in response to an external stimulus to the expression of the mature receptor on the melanocyte surface.
Keywords: Melanocortin 1 receptor (MC1R); Promoter; α-MSH; Pigmentation; Melanocyte;

Gene polymorphisms and their effects in the melanocortin system by Levi Carroll; Joanne Voisey; Angela van Daal (1871-1885).
In addition to its role in human pigmentation, components of the melanocortin system regulate appetite, energy homeostasis and hormone production. Recent studies have suggested possible roles of this system in immunity, transmission of pain signals, and reproductive potential. A number of polymorphisms have been identified in genes of the melanocortin system and are associated with pigmentation in humans, as well as being causative of disorders of adrenal hormone production and obesity. This review gives an outline of these polymorphisms, their functional significance and possible application to or impact on diagnosis and pharmacotherapy based on melanocortin pathways.
Keywords: Polymorphism; SNP; Agouti; Melanocortin;

Evolutionary conservation of the structural, pharmacological, and genomic characteristics of the melanocortin receptor subtypes by Helgi B. Schiöth; Tatjana Haitina; Maria K. Ling; Aneta Ringholm; Robert Fredriksson; José Miguel Cerdá-Reverter; Janis Klovins (1886-1900).
We have cloned melanocortin receptors (MCRs) from several species of fish. The MC4R and MC5R subtypes arose early in vertebrate evolution and their primary structure is remarkably conserved. Expression and pharmacological characterization of the MCRs in fish has revealed that they bind and respond to melanocortin peptides with high potency. Detailed characterization of the binding properties of the different subtypes suggests that MCRs in early vertebrates had preference for adrenocorticotropic hormone (ACTH) peptides, while the high sensitivity for the shorter proopiomelanocortin (POMC) products, such as the α-, β-, and γ-melanocyte-stimulating hormone (MSH), has appeared later, perhaps as the MCR subtypes gained more specialized functions. The MCR repertoire shows in general high similarities in their primary structures, while they are however not similar in terms of functional roles. The MCRs serve therefore as an interesting model family to understand the molecular mechanisms of how functions of the genes can diverge during evolution. In this review, we provide an overview of our recent studies on the cloning, expression, pharmacology, 3D modeling, and genomic studies of the MCRs in non-mammalian species.
Keywords: Melanocortin receptor; MSH; ACTH; GPCR; Evolution;

While there have been many advances in our understanding of the genetics of pathological skin pigmentation in humans, our knowledge about what determines variation in normal skin color is still incomplete. Variation in one gene, melanocortin 1 receptor (MC1R), has been associated with red hair and fair skin in Europeans. However, this gene might also play an important role in shaping pigmentation of other human populations, where it experiences different selective pressures. Below we review what is currently known about polymorphism and selection at the MC1R coding and promoter regions in human populations, the pattern of MC1R evolution in nonhuman primates, and the interaction of MC1R with other genes.
Keywords: MC1R; Pigmentation; Natural selection; Human evolution;

Obesity-associated mutations in the melanocortin 4 receptor provide novel insights into its function by Cedric Govaerts; Supriya Srinivasan; Astrid Shapiro; Sumei Zhang; Franck Picard; Karine Clement; Cecile Lubrano-Berthelier; Christian Vaisse (1909-1919).
Mutations in the Melanocortin 4 receptor are implicated in 1–6% of early onset or severe adult obesity cases. Most of the patients carry heterozygous missense mutations. Arguments for the pathogenicity of these mutations are based on the frequency of rare functionally relevant non-synonymous mutations in severely obese children and adults versus non-obese controls, the segregation of mutations with obesity in the family of the probands (although with incomplete penetrance) and the relevant functional defects described for these mutations. We have developed new assays to study the functional characteristics of these obesity-associated MC4R mutations. Systematic and comparative functional study of over 50 different obesity-associated mutations suggests that multiple functional alterations contribute to their pathogenicity. These studies also lead to new insights into the structure–function relationship of MC4R, provide novel hypotheses for the genetic predisposition to common obesity in humans and allow the development of new molecular tools for studying the physiological role of GPCRs.
Keywords: Obesity; Genetics; Melanocortin receptors;

Analyzing the radiation of the melanocortins in amphibians: Cloning of POMC cDNAs from the pituitary of the urodele amphibians, Amphiuma means and Necturus maculosus by Katarzyna Kozak; David Costantino; Stephanie Lecaude; Cristina Sollars; Phillip Danielson; Robert M. Dores (1920-1928).
Proopiomelanocortin (POMC) cDNAs were cloned and sequenced from brain extracts of two species of urodele amphibians: Amphiuma means and Necturus maculosus. Although the two species of urodele amphibians belong to separate families, and do not share a direct common ancestor, the level of primary sequence identity for the open reading of the POMC cDNAs was 90% at the amino acid level and 79% at the nucleotide level. It appears that the POMC gene in these urodele amphibians has been accumulating mutations at the amino acid level at a slower rate than the POMC gene in other sarcopterygian orders.
Keywords: POMC; Urodele amphibians; α-MSH; β-MSH; γ-MSH; ACTH;

Evidence for direct actions of melanocortin peptides on bone metabolism by Laurence M. Dumont; C.-S. Jenny Wu; Michele A. Tatnell; Jillian Cornish; Kathleen G. Mountjoy (1929-1935).
Expression of melanocortin-4 receptor (MC4R) mRNA in developing rat limb buds, teeth, and skull bone first indicated a possible role for MC4R in bone metabolism. We therefore investigated whether MC4R mRNA was expressed in the rat osteosarcoma UMR106.06 cell line and in primary rat osteoblast cells. Reverse transcriptase-polymerase chain reaction (RT-PCR), Northern blot analysis, and ribonuclease protection assay (RPA) were used to demonstrate MC4R mRNA expression in UMR106.06 and primary osteoblast cells. MC4R mRNA was found to be localized to the periosteum of mouse bone using in situ hybridization. We also used RT-PCR and rat specific MC2R and MC5R oligonucleotides to amplify the correct size DNA fragments for these melanocortin receptors from rat primary osteoblasts. In conclusion, melanocortin receptor expression in mouse periosteum and rat osteoblasts suggests a direct role for POMC derived peptides in bone development and bone metabolism.
Keywords: POMC; α-MSH; Melanocortin receptor; Agouti protein;

The regulation of α-MSH release by GABA is mediated by a chloride-dependent [Ca2+] c increase in frog melanotrope cells by Laurence Desrues; Hélène Castel; Maria M. Malagon; Hubert Vaudry; Marie-Christine Tonon (1936-1943).
In frog melanotrope cells, γ-aminobutyric acid (GABA) induces a biphasic effect, i.e. a transient stimulation followed by a more sustained inhibition of α-MSH release, and both phases of the GABA effect are mediated by GABAA receptors. We have previously shown that the stimulatory phase evoked by GABAA receptor agonists can be accounted for by calcium entry. In the present study, we have investigated the involvement of the chloride flux on GABA-induced [Ca2+] c increase and α-MSH release. We show that GABA evokes a concentration-dependent [Ca2+] c rise through specific activation of the GABAA receptor. The GABA-induced [Ca2+] c increase results from opening of voltage-activated L- and N-type calcium channels, and sodium channels. Variations of the extracellular Cl concentration revealed that GABA-induced [Ca2+] c rise and α-MSH release both depend on the Cl flux direction and driving force. These observations suggest for the first time that GABA-gated Cl efflux provokes an increase in [Ca2+] c increase that is responsible for hormone secretion.
Keywords: α-MSH; [Ca2+] c ; Chloride gradient; GABAA receptor; Melanotrope cell;

Role of gamma-MSH peptides in the regulation of adrenal steroidogenesis by Stephen C. Harmer; Andrew B. Bicknell (1944-1951).
α-, β- and γ-melanocyte stimulating hormones (MSHs) are peptides derived from the ACTH precursor, pro-opiomelanocortin. All three peptides have been highly conserved throughout evolution but their exact biological function in mammals is still largely obscure. In recent years, there has been a surge of interest in alpha-MSH and its role in the regulation of feeding. Gamma-MSH by contrast has been shown to be involved in the regulation of adrenal steroidogenesis and also has effects on the cardiovascular and renal systems. This review will provide an overview of the role that γ-MSH peptides play in the regulation of adrenal steroidogenesis.
Keywords: Adrenal; POMC; Gamma-MSH; Steroidogenesis; Melanocortin receptor;

Involvement of melanocortin-4 receptor in anxiety and depression by Shigeyuki Chaki; Shigeru Okuyama (1952-1964).
The melanocortins, which are derived from proopiomelanocortin, have a variety of physiological functions mediated membrane surface receptors. To date, five subtypes have been cloned. With the cloning of melanocortin receptors, studies with genetic models, and development of selective compounds, the physiological roles of the five melanocortin receptors have begun to be understood. The melanocortin-4 receptor (MC4R), which is predominantly expressed in the central nervous system, has in particular become the focus of much attention in recent years because of the critical roles it plays in a wide range of functions, including feeding, sexual behavior, and stress. Recent development of selective antagonists for the MC4R has provided pharmacological evidence that blockade of MC4R could be a useful way of alleviating numerous conditions such as anxiety/depression, pain, and addiction to drugs of abuse.
Keywords: Melanocortin-4 receptor; Stress; Anxiety; Depression;

The melanocortin 1 receptor (MC1R) is known to play an important role in determining physiological variation in human pigmentation, and consequently human susceptibility to ultraviolet radiation. A reason for wider interest is that the considerable phenotypic diversity has been in part generated by the effects of gene dosage, and the presence of a large number of mutations at this G-protein coupled receptor that are not functionally equivalent. Thus, a range of mutations at a single receptor locus can lead to a complex range of graded phenotypes.
Keywords: Melanocortin 1 receptor; Melanocyte stimulating hormone; G-protein coupled receptor; Ultraviolet radiation; Phenotype; Genetics; Erythema; Skin; Pigment;

Melanocortinergic control of penile erection by H. Wessells; J.E. Blevins; T.W. Vanderah (1972-1977).
Melanocortin receptors in the forebrain and spinal cord can be activated by endogenous or synthetic ligands to induce penile erection in rats and human subjects. To better understand how melanocortin circuits play a role in sex behavior, we review the contribution of melanocortin receptors and/or neurons in the hypothalamus, hindbrain, spinal cord and peripheral nerves to erectile function. New information regarding neuropeptides that mediate penile erection has extended our understanding of the central control of sex behavior, and melanocortin agonists may provide alternatives to existing treatment for highly prevalent problems including erectile dysfunction.
Keywords: MSH; Proopiomelanocortin; Hindbrain; Sex behavior;

Chimeras of the agouti-related protein: Insights into agonist and antagonist selectivity of melanocortin receptors by Pilgrim J. Jackson; Bin Yu; Benjamin Hunrichs; Darren A. Thompson; Biaoxin Chai; Ira Gantz; Glenn L. Millhauser (1978-1987).
The specific melanocortin receptors, MC3R and MC4R, are directly linked to metabolism and body weight control. These receptors are activated by the peptide hormone α-MSH and antagonized by the agouti-related protein (AGRP). Whereas α-MSH acts broadly on most members of the MCR family (with the exception of MC2R), AGRP is highly specific for only MC3R and MC4R. AGRP is a complex ligand of approximately 100 amino acids. Within AGRP, MCR recognition and antagonism is localized to a 34 residue, cysteine-rich domain that adopts an inhibitor cystine knot (ICK) fold. An oxidatively folded peptide corresponding to this domain, referred to as mini-AGRP, exhibits full antagonist function and selectivity for MC3R and MC4R. Here we investigate a series of chimera proteins based on the mini-AGRP scaffold. Amino acid sequences derived from peptide agonists are grafted into the mini-AGRP active loop, implicated in receptor recognition, with the goal of producing ICK based agonists specific for MC3R and MC4R. Several constructs indeed exhibited potent agonist activity; however, with all chimeras, receptor selectivity is significantly altered. Pharmacologic data indicate that the chimeras do not interact with MC receptors through native AGRP like contacts. A model to explain the data suggest that there is only partial overlap of the agonist versus antagonist binding surfaces within MC receptors. Moreover, accessibility to the binding pocket is highly receptor specific with MC3R being the least tolerant of ligand alterations.
Keywords: Agouti-related protein; Agouti signaling protein; Melanocortin receptors; Protein chimeras; Obesity; Diabetes;

Potent peptide agonists for human melanocortin 3 and 4 receptors derived from enzymatic cleavages of human β-MSH(5–22) by dipeptidyl peptidase I and dipeptidyl peptidase IV by Hansen M. Hsiung; David L. Smiley; Xing-yue Zhang; Lianshan Zhang; Liang Zeng Yan; Libbey Craft; Mark L. Heiman; Dennis P. Smith (1988-1996).
Human β-MSH(1–22) was first isolated from human pituitary as a 22-amino acid (aa) peptide derived from a precursor protein, pro-opiomelanocortin (POMC). However, Bertagna et al. demonstrated that a shorter human β-MSH(5–22), (DEGPYRMEHFRWGSPPKD), is a true endogenous peptide produced in human hypothalamus. In this report, we demonstrated that in vitro enzymatic cleavage of native human β-MSH(5–22) with two ubiquitous dipeptidyl peptidases (DPP), DPP-I and DPP-IV, generated two potent MC3/4R peptide analogues, β-MSH(7–22) (GPYRMEHFRWGSPPKD) and β-MSH(9–22) (YRMEHFRWGSPPKD). In fact, the MC4R binding affinity and functional potency of β-MSH(7–22) (K i  = 4.6 nM, EC50 = 0.6 nM) and β-MSH(9–22) (K i  = 5.7 nM, EC50 = 0.6 nM) are almost an order of magnitude greater than those of their parent peptide, β-MSH(5–22) (MC4R, K i  = 23 nM, EC50 = 3 nM). Furthermore, the DPP-I/DPP-IV cleaved peptide, β-MSH(9–22), when administered intracerebroventricularly (ICV) at a dose of 3 nmol/rat, potently induced an acute negative energy balance in a diet-induced obese rat model, while its parent molecule, β-MSH(5–22), administered at the same dose did not have any effect. These data suggest that DPP-I and DPP-IV may play a role in converting the endogenous β-MSH(5–22) to more potent peptides that regulate energy homeostasis in the hypothalamus.
Keywords: Melanocortin 3 and 4 receptors; β-Melanin stimulating hormone (β-MSH);

N-alkylated dipeptide amides and related structures as imitations of the melanocortins’ active core by Felikss Mutulis; Ilze Mutule; Edvards Liepinsh; Aleh Yahorau; Maris Lapinsh; Sergei Kopantshuk; Santa Veiksina; Ago Rinken; Jarl E.S. Wikberg (1997-2016).
Thirty-three low molecular mass structures combining both peptide and peptoid features were prepared and tested on human melanocortin receptors MC1,3–5R. Most of them displayed low micromolar activity with preference for diamines, guanidino and 2-naphthyl derivatives compared to monoacetylated, amino and 3-indolyl counterparts. Some contained l- or d-histidine residues, but the change did not influence affinity. QSAR modelling yielded excellent models for the MC3–5 receptors explaining R 2 Y  = 0.89–0.91 and predicting Q 2  = 0.77–0.80 of the affinity variations. One compound (12c) displayed MC1R selectivity (13-fold and more). An NMR study of 12c showed that it exists as a mixture of four rotamers at its tertiary amide bonds. Comparisons with earlier data for melanocortin core tetrapeptide analogues indicate that the novel peptide–peptoids interact with the melanocortin receptors in a different way.
Keywords: Melanocortins; Peptidomimetics; Peptoid–peptide hybrids; Solid phase organic synthesis; Structure–activity; QSAR; NMR;

Structure–activity relationship of linear tetrapeptides Tic-DPhe-Arg-Trp-NH2 at the human melanocortin-4 receptor and effects on feeding behaviors in rat by Zhixiong Ye; Tanya MacNeil; David H. Weinberg; Rubana N. Kalyani; Rui Tang; Alison M. Strack; Beth A. Murphy; Ralph T. Mosley; D. Euan MacIntyre; Lex H.T. Van der Ploeg; Arthur A. Patchett; Matthew J. Wyvratt; Ravi P. Nargund (2017-2025).
The melanocortin subtype-4 receptor (MC4R) has been implicated in the control of feeding behavior and body weight regulation. A series of tetrapeptides, based on Tic-DPhe-Arg-Trp-NH2—a mimic of the putative message sequence “His-Phe-Arg-Trp” and modified at the DPhe position, were prepared and pharmacologically characterized for potency and selectivity. Substitution of His with Tic gave peptides with significant increases in selectivity. The effects of the substitution pattern of DPhe were investigated and it has significant influences on potency and the level of the maximum cAMP accumulation. Intracerebroventricular administration of peptide 10 induced significant inhibition of cumulative overnight food intake and feeding duration in rats.
Keywords: α-MSH; Melanocortin; MC4R; Agonist; Tetrapeptide; Food intake;

A review of melanocortin receptor small molecule ligands by Aleksandar Todorovic; Carrie Haskell-Luevano (2026-2036).
The melanocortin system (MC) is implicated in the regulation of a variety of physiological pathways including pigmentation, steroid function, energy homeostasis, food intake, obesity, cardiovascular, sexual function, and normal gland regulation. The melanocortin system consists of five receptors identified to date (MC1–5R), melanocortin agonists derived from the pro-opiomelanocortin prohormone (POMC) and two naturally existing antagonists. Melanocortin receptor ligand structure–activity studies have been performed since the 1960s, primarily focused on the pigmentation aspect of physiology. During the 1990s, the melanocortin-4 receptor was identified to play a significant physiological role in the regulation of both food intake and obesity. Subsequently, a concerted drug design effort has focused on the design and discovery of melanocortin receptor small molecules. Herein, we present an overview of melanocortin receptor heterocyclic small molecules.
Keywords: Melanocortin; Peptidomimetic; Synthesis; Piperidine; Piperazine; Heterocyclic;