The Asp298Asn polymorphism of melanocortin-4 receptor (MC4R) in pigs: evidence for its potential effects on MC4R constitutive activity and cell surface expression

In humans and mice, melanocortin receptor 4 (MC4R) and melanocortin receptor accessory protein 2 (MRAP2) can form a complex and control energy balance, thus regulating body weight and obesity. In pigs, a missense variant (p.Asp298Asn) of MC4R has been suggested to be associated with growth and fatness; however, the effect of Asp298Asn substitution on MC4R function is controversial, limiting its application in animal breeding. Here we examined the effect of this polymorphism on MC4R constitutive activity, cell surface expression and signaling, and its interaction with MRAP2 in pigs. We found that: (i) both pig MC4R(Asp)and MC4R(Asn)can be activated by its ligands (alpha-MSH and ACTH) and stimulate cAMP/PKA signaling pathway, as detected by pGL3-CRE-luciferase reporter assay, indicating that, like pMC4R(Asp), pMC4R(Asn)is coupled to the cAMP/PKA signaling pathway; (ii) compared with pMC4R(Asp), pMC4R(Asn)loses the basal constitutive activity and shows a decreased surface expression, as detected by Deual-luciferase reporter assay and Nano-HiBiT system; (iii) as in other vertebrates, both pMC4R(Asp)and pMC4R(Asn)can interact with pMRAP2, thus decreasing receptor surface expression and enhancing ligand sensitivity, although, in contrast to pMC4R(Asp), the basal constitutive activity of pMC4R(Asn)cannot be affected by pMRAP2; and (iv) RNA-seq data analysis revealed a co-expression ofMC4RandMRAP2in pig hypothalamus. Taken together, our data provide convincing evidence that Asp298Asn substitution decreases the constitutive activity and cell surface expression of MC4R or MC4R-MRAP2 complex, which may affect energy balance and be a valuable selection marker for breeding programs in pigs.