The Function Analyses on p.R236H and p.D298N of MC4R Gene in the Pig

The melanocortin-4 receptor (MC4R) is an important member of G protein-coupled receptor family involved in the regulation of energy balance and food intake in mammals. Two SNPs in the coding region of porcine MC4R gene, p.R236H and p.D298N respectively were identified in our previous study, and the occurrence of p.D298N is widespread in breeds, but p.R236H is rare. The association analysis indicated that both SNPs were significantly associated with fatness and growth traits. To better understand the biological significance of these two SNPs, the function of two single mutants and double mutant were detected respectively by ligand affinity and signal detection. The results showed that, compared with wild-type, two single mutants had normal ligand binding to a-MSH and b-MSH. When D298N and double mutant were stimulated with a-MSH and b-MSH, they were defective in cAMP signaling compared with wild-type; Similarly, there was a significant decrease in basal cAMP level. In ERK1/2 pathway, stimulated with a-MSH, all of mutants had normal pERK1/2 level. While being stimulated with b-MSH, D298N had significantly decreased pERK1/2 level. p.R236H had the similar cAMP and ERK1/2 signaling to wild-type. The results demonstrated that p.D298N may affect lipid metabolism by both Gs-cAMP-PKA and MAPK signaling pathways, while p.R236H may interact with p.D298N and affect lipid metabolism by Gs-cAMP-PKA pathway. Both SNPs were useful molecular markers for selection of fat deposits traits in pigs.