The interaction of melanocortin-4 (MC4R) and melanocortin-3 (MC3R) receptors with proopiomelanocortin (POMC)-derived peptides (e.g. α-MSH), agouti-related protein (AgRP) and melanocortin-2 receptor accessory protein 2 (MRAP2) is suggested to play critical roles in energy balance of vertebrates. However, evidence on their interaction in birds remains scarce. Our study aims to reveal their interaction in chickens and the results showed that (1) chicken (c-)MC3R and cMC4R expressed in Chinese hamster ovary (CHO) cells can be activated by α-MSH and ACTH_1–39equipotently, monitored by a pGL3-CRE-luciferase reporter system; (2) cMC3R and cMC4R, when co-expressed with cMRAP2 (or cMRAP, a cMRAP2 homolog), show increased sensitivity to ACTH treatment and thus likely act as ACTH-preferring receptors, and the interaction between cMC3R/cMC4R and cMRAP2 was demonstrated by co-immunoprecipitation assay; (3) both cMC3R and cMC4R display constitutive activity when expressed in CHO cells, as monitored by dual-luciferase reporter assay, and cMRAP2 (and cMRAP) can modulate their constitutive activity; (4) AgRP inhibits the constitutive activity of cMC3R/cMC4R, and it also antagonizes ACTH/α-MSH action on cMC4R/cMC3R, indicating that AgRP functions as the inverse agonist and antagonist for both receptors. These findings, together with the co-expression ofcMC4R,cMC3R,cMRAP2,cAgRPandcPOMCin chicken hypothalamus detected by quantitative real-time PCR, suggest that within the hypothalamus, α-MSH/ACTH, AgRP and MRAP2 may interact at the MC4R(/MC3R) interface to control energy balance. Furthermore, our data provide novel proof for the involvement of MRAP2 (and MRAP) in fine-tuning the constitutive activity and ligand sensitivity and selectivity of both MC3R and MC4R in vertebrates.

Apelin receptor(s) (APLNR) are suggested to mediate the actions of apelin and Elabela (ELA) peptides in many physiological processes, including cardiovascular development and food intake in vertebrates. However, the functionality of APLNR has not been examined in most vertebrate groups. Here, we characterized two APLNRs APLNR1, APLNR2) in chickens and red-eared sliders, and three APLNRs in zebrafish (APLNR2a, APLNR2b, APLNR3a), which are homologous to human APLNR. Using luciferase-reporter assays or Western blot, we demonstrated that in chickens, APLNR1 (not APLNR2) expressed in HEK293 cells was potently activated by chicken apelin-36 and ELA-32 and coupled to Gi-cAMP and MAPK/ERK signaling pathways, indicating a crucial role of APLNR1 in mediating apelin/ELA actions; in red-eared sliders, APLNR2 (not APLNR1) was potently activated by apelin-36/ELA-32, suggesting that APLNR2 may mediate apelin/ELA actions; in zebrafish, both APLNR2a and APLNR2b were potently activated by apelin-36/ELA-32 and coupled to Gi-cAMP signaling pathway, as previously proposed, whereas the novel APLNR3a was specifically and potently activated by apelin. Similarly, an apelin-specific receptor (APLNR3b) sharing 57% sequence identity with zebrafish APLNR3a was identified in Nile tilapia. Collectively, our data facilitates the uncovering of the roles of APLNR signaling in different vertebrate groups and suggests a key functional switch between APLNR1 and APLNR2/3 in mediating the actions of ELA and apelin during vertebrate evolution.