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The melanocortin-5 receptor (MC5R) has been implicated in the regulation of exocrine gland secretion, immune regulation, and muscle fatty acid oxidation. However, its function in fish is not well established. Melanocortin-2 receptor accessory protein 2 (MRAP2) can modulate trafficking, ligand binding, and signaling of melanocortin receptors. Ricefield eel (Monopterus albus) is an economically and evolutionarily important fish widely distributed in tropics and subtropics. To explore potential interaction between eel MC5R and MRAP2, herein we cloned ricefield eel mc5r, mrap2X1 and mrap2X2. Eel mc5r consisted of a 1056 bp open reading frame encoding a protein of 351 amino acids. Eel mrap2X1 consisted of a 708 bp open reading frame encoding a protein of 235 amino acids, while eel mrap2X2consisted of a 567 bp open reading frame encoding a protein of 188 amino acids. Multiple sequence alignment showed that maMRAP2X1 and maMRAP2X2 shared 90.43% identity. Interestingly, maMRAP2X2 lost the transmembrane domain. Phylogenetic analysis showed that maMC5R and maMRAP2s were closely related to piscine MC5Rs and MRAP2s, respectively. The maMC5R was further demonstrated to be a functional receptor and could be modulated by maMRAP2s in pharmacological studies. Three agonists, [Nle4, D-Phe7]-alpha-melanocyte stimulating hormone (NDP-MSH), alpha-MSH, and adrenocorticotropin (ACTH), could bind to maMC5R and induce intracellular cAMP production dose-dependently. Compared to human MC5R (hMC5R), maMC5R displayed a significantly decreased Bmax but higher binding affinity to alpha-MSH or ACTH. No significant difference in constitutive activity was observed between hMC5R and maMC5R. When stimulated with α-MSH and ACTH, maMC5R showed significantly lower EC50 and Rmax than that of hMC5R. Eel MRAP2s had no effect on cell surface and total expression of maMC5R, whereas they significantly increased Bmax. Only maMRAP2X2 significantly decreased the binding affinity of ACTH. Both maMRAP2X1 and maMRAP2X2 significantly reduced Rmax but did not affect EC50 in response to alpha-MSH or ACTH stimulation. The availability of maMC5R pharmacological characteristics and the modulation by maMRAP2s will facilitate the investigation of its function in regulating diverse physiological processes in ricefield eel.

Regulation of Melanocortin-5 Receptor Pharmacology by Two Isoforms of MRAP2 in Ricefield Eel (Monopterus albus)