|The neural melanocortin receptors (MCRs), melanocortin-3 and -4 receptors (MC3R and MC4R), play essential non-redundant roles in the regulation of energy homeostasis. Melanocortin-2 receptor accessory proteins (MRAPs, MRAP1 and MRAP2) were shown to regulate neural MCRs. To gain a better understanding of the regulation of neural MCRs by MRAPs, we identified MRAP2 splice variants from humans and canine, and investigated effects of isoforms of MRAPs on human and canine MC3R/MC4R pharmacology. We also conducted experiments to explore modulation of fish Mc3r/Mc4r by two isoforms of Mrap2.
We identified two new human (h) MRAP2 splice variants, MRAP2b (465 bp open reading frame) and MRAP2c (381 bp open reading frame). Human MRAP2s are different in C-termini. We investigated effects of five isoforms of MRAPs, hMRAP1a, hMRAP1b, hMRAP2a, hMRAP2b, and hMRAP2c, on MC3R and MC4R pharmacology. At the hMC3R, hMRAP1a and hMRAP2c increased and hMRAP1b decreased the cell surface expression. hMRAP1a increased affinity to ACTH. Four MRAPs (hMRAP1a, hMRAP1b, hMRAP2a, and hMRAP2c) decreased the maximal responses in response to -MSH and ACTH. For hMC4R, hMRAP1a, hMRAP2a, and hMRAP2c increased the cell surface expression of hMC4R. Human MRAP1b significantly increased affinity to ACTH while MRAP2a decreased affinity to ACTH. Human MRAP1a increased ACTH potency. MRAPs also affected hMC4R basal activities, with hMRAP1s increasing and hMRAP2s decreasing the basal activities. In summary, the newly identified splicing variants, hMRAP2b and hMRAP2c, could regulate MC3R and MC4R pharmacology. The two MRAP1s and three MRAP2s had differential effects on MC3R and MC4R trafficking, binding, and signaling.
The effect of MRAP1 and MRAP2 on canine neural MCRs are not understood. Herein, we cloned canine (c) mc3r and identified an canine MRAP2 splice variant, MRAP2b, with extension at N-terminus of cMRAP2a. Canine MC3R showed higher Bmax and Rmaxs to five agonists than that of hMC3R. We further investigated modulation of cMRAP1, cMRAP2a, and cMRAP2b, on cMC3R and cMC4R pharmacology. All MRAPs had no effect on cMC3R trafficking. Canine MRAP1 significantly decreased the Bmax, whereas cMRAP2a and cMRAP2b increased Bmax of cMC3R. Both MRAP1 and MRAP2a decreased Rmaxs in response to -MSH and ACTH; MRAP2b only decreased -MSH-stimulated cAMP generation cAMP production of cMC3R. At cMC4R, MRAP1 and MRAP2a increased cell surface expression of cMC4R. MRAP1 and MRAP2a increased Bmaxs of cMC4R. All MRAPs increased affinities to -MSH and ACTH. MRAP2a increased ACTH-induced cAMP levels of cMC4R, whereas MRAP2b decreased -MSH- and ACTH-stimulated cAMP production of cMC4R. All MRAPs decreased the basal activities of cMC4R.
Considering the crucial importance of energy metabolism, understanding the endocrine modulation of energy homeostasis is important for economically important fishes and may potentially lead to novel approaches to manipulate fish growth, feed efficiency, and final product quality in cultured fish. Hence, it is not surprising that Mc3r/Mc4r has also attracted some attention in fish. Topmouth culter (Culter alburnus) is an economically important freshwater fish in China. In this study, we cloned culter mc3r, mrap2a, and mrap2b. All agonists could bind and stimulate caMc3r to increase dose-dependently intracellular cAMP accumulation. Compared to hMC3R, culter Mc3r showed higher constitutive activity, higher efficacies and Rmax to -MSH, des--MSH, and ACTH. Both caMrap2a and caMrap2b markedly decreased caMc3r basal cAMP production. However, only caMrap2a significantly decreased cell surface expression, Bmax and Rmax of caMc3r. These data indicated that the cloned caMc3r was a functional receptor. Mrap2a and Mrap2a had different effects on expression and signaling of caMc3r.
We also cloned culter mc4r, consisting of a 981 bp open reading frame encoding a protein of 326 amino acids. Culter Mc4r could bind to four peptide agonists and increase intracellular cAMP production dose dependently. Culter Mc4r could be constitutively active in both cAMP and Erk1/2 pathways, which differentially regulated by caMrap2a and caMrap2b. Culter Mrap2a significantly increased Bmax and decreased agonist-stimulated cAMP, while Mrap2b increased cell surface and total expression but did not affect Bmax and agonist-stimulated cAMP. These results will aid investigate the diverse physiological processes of Mc4r in topmouth culter.
In summary, we systemically investigated the modulation of MRAP1 and MRAP2 on MC3R/MC4R in humans, canine, and fish, and found that the effect of MRAPs on MC3R/MC4R is in species-dependent manner. We also studied functionality of Mc3r/Mc4r in an economically important fish, laying the foundation for further physiological studies of fish Mc3r or Mc4r that might provide new strategies for promoting growth and culture.