Functional characterization of two melanocortin (MC) receptors in lamprey showing orthology to the MC1 and MC4 receptor subtypes.

BACKGROUND: The melanocortin (MC) receptors have a key role in regulating body weight and pigmentation. They belong to the rhodopsin family of G protein-coupled receptors (GPCRs). The purpose of this study was to identify ancestral MC receptors in agnathan, river lamprey. RESULTS: We report cloning of two MC receptors from river lamprey. The lamprey receptors, designated MCa and MCb, showed orthology to the MC1 and MC4 receptor subtypes, respectively. The molecular clock analysis suggested that lamprey MC receptor genes were not duplicated recently and diverged from each other more than 400 MYR ago. Expression and pharmacological characterization showed that the lamprey MCa receptor was able to bind and be activated by both lamprey and human MSH peptides. The lamprey MCa receptor had relatively high affinity for ACTH derived peptides similarly to the fish MC receptors. We found that both of the lamprey MC receptors were expressed in skin, while the MCb receptor was also found in liver, heart and skeletal muscle. CONCLUSION: This study shows presence of MC receptors in agnathans indicating early signs of specific functions of melanocortin receptor subtypes.

Evolutionary conservation of the structural, pharmacological, and genomic characteristics of the melanocortin receptor subtypes.

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 alpha-, beta-, and gamma-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.

Pharmacological characterization of loss of function mutations of the human melanocortin 1 receptor that are associated with red hair.

Variation in skin color is the major host risk factor for melanoma and other forms of skin cancer. Individuals with red hair show an increased ratio of phaeomelanin to eumelanin in both hair and skin. This ratio is regulated by the melanocortin (MC) 1 receptor. There are several common point mutations in the human MC1 receptor that are overrepresented in North European red-heads, and in individuals with pale skin. In order to determine the functional significance of these mutations, we expressed the Asp84Glu, Val92Met, Arg163Gln, and Asp294His variants of the human MC1 receptors in eukaryotic cells and determined their ability to bind alpha-melanocyte stimulating hormone (MSH) peptides and increase intracellular cAMP. The mutants Asp84Glu and Asp294His showed a much lower response to alpha-MSH in cAMP and a slightly impaired ability to bind alpha-MSH, and the Val92Met mutant bound alpha-MSH with 100-fold lower affinity as compared with the wild-type. The Arg163Gln variant, widely found in some Asian populations, reached normal level of cAMP response but had just slightly lower potency for alpha-MSH in binding and second messenger studies. The results provide important pharmacological characterization of common MC1 receptor variants in various world populations.

Molecular cloning, pharmacological characterization, and brain mapping of the melanocortin 4 receptor in the goldfish: involvement in the control of food intake.

We report cloning, pharmacological characterization, tissue distribution, detailed brain mapping, and role in control of food intake of melanocortin 4 receptor in goldfish (gMC4R). The gMC4R protein has 68% identity with the human ortholog and is conserved in important functional domains. Pharmacological profiling showed similar affinities and potency order to hMC4R for MSH peptides, whereas MTII and HS024 were identified as high-affinity agonist and antagonist analogs, respectively. The gMC4R-mRNA was found in brain and some peripheral tissues including the ovary, gill, and spleen. Detailed MC4R-mRNA mapping showed expression in main neuroendocrine and food intake-controlling areas. High expression levels were found in the telencephalon, preoptic area, ventral thalamus, tuberal hypothalamus, and hypothalamic inferior lobe. By RT-PCR, low levels were also detected in the cerebellum, medulla, and spinal cord. Intracerebroventricular MTII administration inhibited food intake in 24-h fasted animals in a dose-dependent manner, whereas HS024 stimulated food intake in fed animals, suggesting that melanocortins exert a tonic inhibitory effect on food intake, which is mediated through central MC4R signaling. The conserved central expression pattern and physiological role in regulation of food intake for the MC4R suggests that neuronal pathways of the melanocortin system may be important for regulation of energy homeostasis in most vertebrates.

The melanocortin receptor subtypes in chicken have high preference to ACTH-derived peptides.

1 Melanocortin (MC) receptors are widely distributed throughout the body of chicken, like in mammals, and participate in a wide range of physiological functions. 2 To clarify the pharmacological impact of ligands acting in the MC system, we expressed the chicken MC1, MC2, MC3, MC4 and MC5 (cMC1-5) receptors in eukaryotic cells and performed comprehensive pharmacological characterization of the potency of endogenous and synthetic melanocortin peptides. 3 Remarkably, the cMC receptors displayed high affinity for ACTH-derived peptides and in general low affinity for alpha-MSH. It is evident that not only the cMC2 receptor but also the other cMC receptors interact with ACTH-derived peptide through an epitope beyond the sequence of alpha-MSH. 4 The synthetic ligand MTII was found to be a potent agonist whereas HS024 was a potent antagonist at the cMC4 receptor, indicating that these ligands are suitable for physiological studies in chicken. 5 We also show the presence of prohormone convertase 1 (PC1) and PC2 genes in chicken, and that these peptides are coexpressed with proopiomelanocortin (POMC) in various tissues.

Functional role, structure, and evolution of the melanocortin-4 receptor.

The melanocortin (MC)-4 receptor participates in regulating body weight homeostasis. We demonstrated early that acute blockage of the MC-4 receptor increases food intake and relieves anorexic conditions in rats. Our recent studies show that 4-week chronic blockage of the MC-4 receptor leads to robust increases in food intake and development of obesity, whereas stimulation of the receptor leads to anorexia. Interestingly, the food conversion ratio was clearly increased by MC-4 receptor blockage, whereas it was decreased in agonist-treated rats in a transient manner. Chronic infusion of an agonist caused a transient increase in oxygen consumption. Our studies also show that the MC-4 receptor plays a role in luteinizing hormone and prolactin surges in female rats. The MC-4 receptor has a role in mediating the effects of leptin on these surges. The phylogenetic relation of the MC-4 receptor to other GPCRs in the human genome was determined. The three-dimensional structure of the protein was studied by construction of a high-affinity zinc binding site between the helices, using two histidine residues facing each other. We also cloned the MC-4 receptor from evolutionary important species and showed by chromosomal mapping a conserved synteny between humans and zebrafish. The MC-4 receptor has been remarkably conserved in structure and pharmacology for more than 400 million years, implying that the receptor participated in vital physiological functions early in vertebrate evolution.

High diversity in functional properties of melanocortin 1 receptor (MC1R) in divergent primate species is more strongly associated with phylogeny than coat color.

We have characterized the biochemical function of the melanocortin 1 receptor (MC1R), a critical regulator of melanin synthesis, from 9 phylogenetically diverse primate species with varying coat colors. There is substantial diversity in melanocyte-stimulating hormone (MSH) binding affinity and basal levels of activity in the cloned MC1Rs. MSH binding was lost independently in lemur and New World monkey lineages, whereas high basal levels of MC1R activity occur in lemurs and some New World monkeys and Old World monkeys. Highest levels of basal activity were found in the MC1R of ruffed lemurs, which have the E94K mutation that leads to constitutive activation in other species. In 3 species (2 lemurs and the howler monkey), we report the novel finding that binding and inhibition of MC1R by agouti signaling protein (ASIP) can occur when MSH binding has been lost, thus enabling continuing regulation of the melanin type via ASIP expression. Together, these findings can explain the previous paradox of a predominantly pheomelanic coat in the red ruffed lemur (Varecia rubra). The presence of a functional, MSH-responsive MC1R in orangutan demonstrates that the mechanism of red hair generation in this ape is different from the prevalent mechanism in European human populations. Overall, we have found unexpected diversity in MC1R function among primates and show that the evolution of the regulatory control of MC1R activity occurs by independent variation of 3 distinct mechanisms: basal MC1R activity, MSH binding and activation, and ASIP binding and inhibition. This diversity of function is broadly associated with primate phylogeny and does not have a simple relation to coat color phenotype within primate clades.

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.

Cloning of two melanocortin (MC) receptors in spiny dogfish: MC3 receptor in cartilaginous fish shows high affinity to ACTH-derived peptides while it has lower preference to gamma-MSH.

We report the cloning and characterization of two melanocortin receptors (MCRs) from the spiny dogfish (Squalus acanthias) (Sac). Phylogenetic analysis shows that these shark receptors are orthologues of the MC3R and MC5R subtypes, sharing 65% and 70% overall amino acid identity with the human counterparts, respectively. The SacMC3R was expressed and pharmacologically characterized in HEK293 cells. The radioligand binding results show that this receptor has high affinity for adrenocorticotropic hormone (ACTH)-derived peptides while it has comparable affinity for alpha- and beta-melanocyte stimulating hormone (MSH), and slightly lower affinity for gamma-MSH when compared with the human orthologue. ACTH(1-24) has high potency in a second-messenger cAMP assay while alpha- and gamma-MSH had slightly lower potency in cells expressing the SacMC3R. We used receptor-enhanced green fluorescence protein (EGFP) fusion to show the presence of SacMC3R in plasma membrane of Chinese hamster ovary and HEK293 cells but the SacMC5R was retained in intracellular compartments of these cells hindering pharmacological characterization. The anatomical distribution of the receptors were determined using reverse transcription PCR. The results showed that the SacMC3R is expressed in the hypothalamus, brain stem and telencephalon, optic tectum and olfactory bulbs, but not in the cerebellum of the spiny dogfish while the SacMC5R was found only in the same central regions. This report describes the first molecular characterization of a MC3R in fish. The study indicates that many of the important elements of the MC system existed before radiation of gnathostomes, early in vertebrate evolution, at least 450 million years ago.

One melanocortin 4 and two melanocortin 5 receptors from zebrafish show remarkable conservation in structure and pharmacology.

We report the cloning, genome mapping, functional expression, pharmacology and anatomical distribution of three melanocortin (MC) receptors from zebrafish (z). Phylogenetic analysis showed with high bootstrap support that these genes represent one MC4 receptor and two MC5 receptors. Chromosomal mapping showed conserved synteny between regions containing zMC4 and human (h) MC4 receptors, whereas the two zMC5 receptor genes map on chromosome segments in which the zebrafish has several genes with two orthologues of a single mammalian gene. It is likely that the two copies of zMC5 receptors arose through a separate duplication in the teleost lineage. The zMC4, zMC5a, and zMC5b receptors share 70-71% overall amino acid identity with the respective human orthologues and over 90% in three TM regions believed to be most important for ligand binding. All three zebrafish receptors also show pharmacological properties remarkably similar to their human orthologues, with similar affinities and the same potency order, when expressed and characterized in radioligand binding assay for the natural MSH) peptides alpha-, beta-, and gamma-MSH. Stimulation of transfected mammalian cells with alpha-MSH caused a dose-dependent increase in intracellular cAMP levels for all three zebrafish receptors. All three genes were expressed in the brain, eye, ovaries and gastrointestinal tract, whereas the zMC5b receptor was also found in the heart, as determined by RT-PCR. Our studies, which represent the first characterization of MC receptors in a nonamniote species, indicate that the MC receptor subtypes arose very early in vertebrate evolution. Important pharmacological and functional properties, as well as gene structure and syntenic relationships have been highly conserved over a period of more than 400 million years implying that these receptors participate in vital physiological functions.

Presence of melanocortin (MC4) receptor in spiny dogfish suggests an ancient vertebrate origin of central melanocortin system.

We report the cloning, expression, pharmacological characterization and tissue distribution of a melanocortin (MC) receptor gene in a shark, the spiny dogfish (Squalus acanthias) (Sac). Phylogenetic analysis showed that this receptor is an ortholog of the MC4 subtype, sharing 71% overall amino acid identity with the human (Hsa) MC4 receptor. When expressed and characterized by radioligand binding assay for the natural MSH (melanocyte-stimulating hormone) peptides alpha-, beta-, and gamma-MSH, the SacMC4 receptor showed pharmacological properties very similar to the HsaMC4 receptor. Stimulation of SacMC4 receptor transfected cells with alpha-MSH caused a dose-dependent increase in intracellular cAMP levels. The SacMC4 receptor has Ala in position 59 where all other cloned MC receptors have Glu. We confirmed that this was not due to individual polymorphism and subsequently mutated the residue 'back' to Glu but the mutation did not affect the pharmacological properties of the receptor. SacMC4 receptor mRNA was detected by RT-PCR in the optic tectum, hypothalamus, brain stem, telencephalon and olfactory bulb but not in cerebellum or in peripheral tissues. This study describes the first characterization of an MC receptor in a cartilaginous fish, the most distant MC receptor gene cloned to date. Conservation of gene structure, pharmacological properties and tissue distribution suggests that this receptor may have similar roles in sharks as in mammals and that these were established more than 450 million years ago.