In conclusion, we cloned the full-length sequence of chicken MC5R gene and characterized its promoter activity in DF1 chicken embryonic fibroblasts. The functional assay demonstrated that MC5R exhibited higher sensitivity to chicken ACTH/α-MSH compared to β-MSH/γ-MSH. It also showed that both MRAP1 and MRAP2 inhibited the trafficking of chicken MC5R to the plasma membrane, and that only MRAP2 significantly reduced the sensitivity of MC5R to stimulation by α-MSH. MC5R and MRAP1 mRNA were co-expressed in the liver of post-hatch chickens. We found that ACTH may increase glucose production, decrease triglyceride content, and dose-dependently downregulate the expression levels of ELOVL6 and THRSPA genes in hepatocytes, suggesting that ACTH has a unique endocrine role in regulating hepatic glucolipid metabolism. However, further studies are needed to characterize and confirm the critical role of the ACTH-MC5R/MRAP1 axis in regulating glucose and lipid metabolism in chicken liver.

In summary, our data sets a critical reference point to reveal the novel aspects of pituitary biology across vertebrates, and helps to figure out how the anterior pituitary, as a pivotal signal converging and output center for the brain and peripheral tissues, can orchestrate so many physiological processes (e.g., growth, reproduction, metabolism, and stress) to meet the changing physiological demands or pathological status observed in vertebrates, including birds and humans.

In addition, we found that pig MRAP2 can decrease surface expression, increase the ligand sensitivity and selectivity of pMC4RAsp/pMC4RAsn, and enhance the basal constitutive activity of pMC4RAsp.