Aneta Ringholm

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.

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 melanocyte stimulating hormone (MSH) peptides α‐, β‐, and γ‐MSH. Stimulation of transfected mammalian cells with α‐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.

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

Melanocortin (MC) receptors are widely distributed throughout the body of chicken, like in mammals, and participate in a wide range of physiological functions. 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. Remarkably, the cMC receptors displayed high affinity for ACTH‐derived peptides and in general low affinity for α‐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 α‐MSH. 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. 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 characterization of two melanocortin (MC) receptors in lamprey showing orthology to the MC1 and MC4 receptor subtypes

BackgroundThe 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.ResultsWe 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.ConclusionThis study shows presence of MC receptors in agnathans indicating early signs of specific functions of melanocortin receptor subtypes.

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.

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

Abstract: 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.