Distinct binding and signaling activity of Acthar Gel compared to other melanocortin receptor agonists.

PURPOSE: To compare the binding and agonistic activity of Acthar® Gel and synthetic melanocortin receptor (MCR) agonists and examine how the activity of select agonists affects the in vivo production of corticosterone. MATERIALS AND METHODS: In vitro binding was determined using concentration-dependent displacement of the ligand [125I]Nle4, D-Phe7-α-melanocyte-stimulating hormone (α-MSH) on cells expressing MC1R, MC3R, MC4R, or MC5R. Functional activity was determined using a time-resolved fluorescence cyclic adenosine monophosphate (cAMP) assay in cells expressing MC1R, MC2R, MC3R, MC4R, or MC5R. In vivo corticosterone analyses were performed by measuring plasma corticosterone levels in Sprague Dawley rats. RESULTS: Acthar Gel and synthetic MCR agonists exhibited the highest binding at MC1R, lowest binding at MC5R, and moderate binding at MC3R and MC4R. Acthar Gel stimulated the production of cAMP in all 5 MCR-expressing cell lines, with MC2R displaying the lowest level of full agonist activity, 3-, 6.6-, and 10-fold lower than MC1R, MC3R, and MC4R, respectively. Acthar Gel was a partial agonist at MC5R. The synthetic MCR agonists induced full activity at all 5 MCRs, with the exception of α-MSH having no activity at MC2R. Acthar Gel treatment had less of an impact on in vivo production of corticosterone compared with synthetic ACTH1-24 depot. CONCLUSIONS: Acthar Gel bound to and activated each MCR tested in this study, with partial agonist activity at MC5R and the lowest level of full agonist activity at MC2R, which distinguished it from synthetic MCR agonists. The minimal activity of Acthar Gel at MC2R corresponded to lower endogenous corticosteroid production.

Molecular cloning and expression analysis of mc5r like genes (mc5rl) in Ruditapes philippinarum (Manila clam) after aerial exposure and low-temperature stress.

The melanocortin-5 receptor (mc5r) plays an important role in exocrine function, lipid metabolism, obesity, and stress response in the vertebrate. However, the functions of the mc5r in mollusks have been rarely investigated. We cloned the full length of Ruditapes philippinarum mc5r like gene (mc5rl) and the sequence structure and phylogenetic relationship of mc5rl were analyzed. Besides, we detected the tissue distribution and the expression pattern of R. philippinarum mc5r like (mc5rl) genes after aerial exposure and low-temperature stress. The full-length cDNA of the mc5rl-1 was 2143 bp, consisting of a 1224 bp open reading frame encoding (ORF) 408 amino acids. Sequence and phylogenetic analyses revealed that the nucleotide and amino acid sequences of Manila clam mc5rl were highly homologous with mc5r of Crassostrea virginica, Crassostrea gigas, Mizuhopecten yessoensis, and Pecten maximus (32%-36%) and low homologous with vertebrates. The results of the distribution of mc5rl genes showed that mc5rl genes were dominant in the mantle, gonad, and hepatopancreas in R. philippinarum. The expression of mc5rl genes was significantly increased after aerial exposure and low-temperature stress in R. philippinarum in hepatopancreas. Aerial exposure and low-temperature stress could induce mc5rl expressed. Mc5rl might serve as a sensor and promote stress response in R. philippinarum. The cloning and expression characteristics of mc5rl will facilitate the investigation of its function in stress response and other physiological processes in R. philippinarum.

Evolution of the MC5R gene in placental mammals with evidence for its inactivation in multiple lineages that lack sebaceous glands.

MC5R is one of five melanocortin receptor genes found in placental mammals. MC5R plays an important role in energy homeostasis and is also expressed in the terminal differentiation of sebaceous glands. Among placental mammals there are multiple lineages that either lack or have degenerative sebaceous glands including Cetacea (whales, dolphins, and porpoises), Hippopotamidae (hippopotamuses), Sirenia (manatees and dugongs), Proboscidea (elephants), Rhinocerotidae (rhinos), and Heterocephalus glaber (naked mole rat). Given the loss or diminution of sebaceous glands in these taxa, we procured MC5R sequences from publicly available genomes and transcriptomes, supplemented by a newly generated sequence for Choeropsis liberiensis (pygmy hippopotamus), to determine if this gene remains intact or is inactivated in association with loss/reduction of sebaceous glands. Our data set includes complete MC5R sequences for 114 placental mammal species including two individuals of Mammuthus primigenius (woolly mammoth) from Oimyakon and Wrangel Island. Complete loss or inactivation of the MC5R gene occurs in multiple placental lineages that have lost sebaceous glands (Cetacea, West Indian manatee, African elephant, white rhinoceros) or are characterized by unusual skin (pangolins, aardvarks). Both M. primigenius individuals share inactivating mutations with the African elephant even though sebaceous glands have been reported in the former. MC5R remains intact in hippopotamuses and the naked mole rat, although slightly elevated dN/dS ratios in these lineages allow for the possibility that the accumulation of inactivating mutations in MC5R may lag behind the relaxation of purifying selection. For Cetacea and Hippopotamidae, the absence of shared inactivating mutations in two different skin genes (MC5R, PSORS1C2) is consistent with the hypothesis that semi-aquatic lifestyles were acquired independently in these clades following divergence from a common ancestor.

Spliceosomal intron insertions in genome compacted ray-finned fishes as evident from phylogeny of MC receptors, also supported by a few other GPCRs.

BACKGROUND: Insertions of spliceosomal introns are very rare events during evolution of vertebrates and the mechanisms governing creation of novel intron(s) remain obscure. Largely, gene structures of melanocortin (MC) receptors are characterized by intron-less architecture. However, recently a few exceptions have been reported in some fishes. This warrants a systematic survey of MC receptors for understanding intron insertion events during vertebrate evolution. METHODOLOGY/PRINCIPAL FINDINGS: We have compiled an extended list of MC receptors from different vertebrate genomes with variations in fishes. Notably, the closely linked MC2Rs and MC5Rs from a group of ray-finned fishes have three and one intron insertion(s), respectively, with conserved positions and intron phase. In both genes, one novel insertion was in the highly conserved DRY motif at the end of helix TM3. Further, the proto-splice site MAG↑R is maintained at intron insertion sites in these two genes. However, the orthologs of these receptors from zebrafish and tetrapods are intron-less, suggesting these introns are simultaneously created in selected fishes. Surprisingly, these novel introns are traceable only in four fish genomes. We found that these fish genomes are severely compacted after the separation from zebrafish. Furthermore, we also report novel intron insertions in P2Y receptors and in CHRM3. Finally, we report ultrasmall introns in MC2R genes from selected fishes. CONCLUSIONS/SIGNIFICANCE: The current repository of MC receptors illustrates that fishes have no MC3R ortholog. MC2R, MC5R, P2Y receptors and CHRM3 have novel intron insertions only in ray-finned fishes that underwent genome compaction. These receptors share one intron at an identical position suggestive of being inserted contemporaneously. In addition to repetitive elements, genome compaction is now believed to be a new hallmark that promotes intron insertions, as it requires rapid DNA breakage and subsequent repair processes to gain back normal functionality.

Differential expressions of melanocortin receptor subtypes in melanophores and xanthophores of barfin flounder.

alpha-Melanocyte-stimulating hormone (alpha-MSH) is a member of the melanocortin (MC) family, and the MC receptor (MCR) is a member of the G protein-coupled receptor (GPCR) superfamily. We previously found that in barfin flounder, a flatfish, alpha-MSH with an acetyl group at the N-terminus stimulated pigment dispersion in xanthophores; however, this effect was not observed in melanophores. Therefore, the present study was undertaken to find which MCR subtypes are expressed in these pigment cells in order to elucidate how acetylation regulates activities of alpha-MSH-related peptides. Here, we also report the cloning of Mc1r and Mc5r from barfin flounder. Three types of cells-melanophores, xanthophores, and nonchromatophoric dermal cells-were isolated from the skin samples collected from the dorsal fin. These cells were then tested for the expression of Mc1r and Mc5r as well as Mc2r and Mc4r that we had previously cloned. Mc1r and Mc5r transcripts were detected in melanophores, and a sole Mc5r transcript was detected in xanthophores. We had previously found that the efficiency of alpha-MSH was higher than that of desacetyl-alpha-MSH for pigment dispersion in xanthophores. Acetylated MSH peptide may have increased binding affinity to MC5R, whereas alpha-MSH lacks melanin-dispersing activity. Increasing evidences indicate that many GPCRs form heterodimers, and this may affect the affinity of the ligand for the corresponding GPCR. Taken together, the expression of two different Mcr subtypes in melanophores may suggest that a heterodimer consisting of MC1R and MC5R may have a low binding affinity toward alpha-MSH. The present results clarify the types of MCRs that are expressed in melanophores and xanthophores of barfin flounder; furthermore, the results provide important clues about the functional regulation of alpha-MSH-related peptides.

Subtype-specific pharmacological properties of the melanocortin receptors in chicken.

The melanocortin (MC) receptors belong to the family of G-protein-coupled receptors and have five subtypes (MC1-5) in mammals and chicken. We have expressed the chicken MC1-5 (cMC1-5) receptors and seven allelic variants of the cMC1 receptor and performed pharmacological characterization of these receptors. Three variants of the cMC1 receptor, all sharing a Glu to Lys mutation in position 92, are associated with dark feather coloration, and these were pharmacologically described as constitutively active. The pharmacological characterization of the cMC1-5 receptors provides insight of how this receptor family has evolved and useful information for the use of chicken in physiological experiments.