Identifying Common Features in the Activation of Melanocortin-2 Receptors: Studies on the Xenopus tropicalis Melanocortin-2 Receptor.

The interaction between the pituitary hormone, adrenocorticotropin (ACTH), and melanocortin-2 receptor (MC2R) orthologs involves the H6 F7 R8 W9 and R/K15 K16 R17 R18 motifs in ACTH making contact with corresponding contact sites on MC2R. Earlier studies have localized the common HFRW binding site of all melanocortin receptors to residues in TM2, TM3, and TM6 that are located close to the extracellular space. The current study has identified residues in Xenopus tropicalis (xt) MC2R in TM4 (I158, F161), in EC2 (M166), and in TM5 (V172) that also are involved in activation of xtMC2R, and may be in the R/KKRR contact site of xtMC2R. These results are compared to earlier studies on the corresponding domains of human MC2R and rainbow trout MC2R in an effort to identify common features in the activation of teleost and tetrapod MC2R orthologs following stimulation with ACTH.

Molecular characterization and functional regulation of melanocortin 2 receptor (MC2R) in the sea bass. A putative role in the adaptation to stress.

The activation of melanocortin 2 receptor (MC2R) by ACTH mediates the signaling cascade leading to steroid synthesis in the interrenal tissue (analogous to the adrenal cortex in mammals) of fish. However, little is known about the functional regulation of this receptor in fish. In this work described, we cloned sea bass MC2R from a liver cDNA. SbMC2R requires the melanocortin 2 receptor accessory protein (MRAP) for its functional expression. Dietary cortisol but not long-term stress protocols downregulated interrenal sbMC2R expression. Data suggest the existence of a negative feedback on interrenal sbMC2R expression imposed by local or systemic glucocorticoids. This feedback could be involved in long-term stress adaptation by regulating interrenal sensitivity to ACTH. ACTH-induced MC2R activation stimulates hepatic lipolysis, suggesting that ACTH may mediate stress-induced effects upstream of cortisol release.

Characterization of murine melanocortin receptors mediating adipocyte lipolysis and examination of signalling pathways involved.

The melanocortin receptors (MCRs) belong to the G-protein coupled receptors (family A). So far, 5 different subtypes have been described (MC1R-MC5R) and of these MC2R and MC5R have been proposed to act directly in adipocytes and regulate lipolysis in rodents. Using ACTH and α-melanocyte stimulating hormone (α-MSH) generated from proopiomelanocortin (POMC), as well as synthetic MSH analogues to stimulate lipolysis in murine 3T3-L1 adipocytes it is shown that MC2R and MC5R are lipolytic mediators in differentiated 3T3-L1 adipocytes. Involvement of cAMP, phosphorylated extracellular signal-regulated kinase (ERK) 1/2, protein kinase B (PKB), adenosine 5' monophosphate activated protein kinase (AMPK) and Jun-amino-terminal kinase (JNK) in MCR mediated lipolysis were studied. Interestingly, results obtained in 3T3-L1 cells suggest that lipolysis stimulated by α-MSH, NDP-α-MSH, MT-II, SHU9119 and PG-901 is mediated through MC5R in a cAMP independent manner. Finally, we identify essential differences in MCR mediated lipolysis when using 3T3-L1 cells compared to primary adipocytes.

Observations on the ligand selectivity of the melanocortin 2 receptor.

The melanocortin 2 receptor (MC2R) is unique in terms of ligand selectivity and in vitro expression in mammalian cell lines as compared to the other four mammalian MCRs. It is well established that ACTH is the only melanocortin ligand that can activate the ACTH receptor (i.e., melanocortin 2 receptor). Recent studies have provided new insights into the presence of a common binding site for the HFRW motif common to all melanocortin ligands. However, the activation of the melanocortin 2 receptor requires an additional amino acid motif that is only found in the sequence of ACTH. This mini-review will focus on these two topics and provide a phylogenetic perspective on the evolution of MC2R ligand selectivity.

Molecular characterization, tissue-specific expression, and regulation of melanocortin 2 receptor in rainbow trout.

ACTH, the primary secretagogue for corticosteroid biosynthesis, binds to melanocortin 2 receptor (MC2R) and activates the signaling cascade leading to steroid biosynthesis in the adrenal cortex. Whereas MC2R regulation has been studied using mammalian models, little is known about the molecular mechanisms involved in ACTH signaling in nonmammalian vertebrates. A full-length cDNA encoding MC2R was sequenced from rainbow trout (Oncorhynchus mykiss) interrenal tissue (analogous to the adrenal cortex in mammals) and showed about 60 and about 44% amino acid sequence similarity to teleosts and humans, respectively. Phylogenetic analysis confirmed that MC2R from all species clustered together and was distant from other MCRs. Quantitative real-time PCR revealed a marked tissue-specific difference in MC2R mRNA abundance, with the highest levels observed in the interrenal tissue, ovary, and testis. Acute ACTH, but not alpha-MSH or [Nle4, d-Phe7]-MSH, stimulation resulted in a time- and dose-related elevation in MC2R mRNA abundance in the interrenal tissue. This corresponded with higher steroidogenic acute regulatory protein and cytochrome P450 side-chain cleavage enzyme gene expression as well as elevated cortisol production. An acute stressor transiently elevated plasma ACTH and cortisol levels at 1 h, and this was followed by a significant increase in MC2R mRNA abundance at 4 h after stressor exposure. Taken together, our results demonstrate that ACTH regulation of MC2R is highly conserved in vertebrates, whereas the tissue-specific distribution of this receptor transcript level leads us to propose a role for ACTH signaling in the stressor-mediated suppression of sex steroid levels in fish.

The melanocortin pathway: effects of voluntary exercise on the melanocortin-4 receptor knockout mice and ACTH(1-24) ligand structure activity relationships at the melanocortin-2 receptor.

The melanocortin pathway consists of endogenous agonists, antagonists, G-protein coupled receptors, and ancillary proteins that mediate the function of the endogenous antagonists. The melanocortin-4 receptor (MC4R) is involved in the regulation of obesity and the melanocortin-2 receptor (MC2R) is involved in the regulation of steroidogenesis. Herein, we present the effects of voluntary exercise on the MC4R knockout mice in terms of bypassing the morbid obesity and hyperphagia phenotypes associated with this genetic obesity model. Additionally, a systematic truncation study of the adrenocorticotropin hormone (ACTH 1-24) has been performed and characterized at the cloned MC2R.