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 (α‐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 dual‐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 of MC4R and MRAP2 in 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.

中文翻译：

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猪黑素皮质素4受体（MC4R）的Asp298Asn多态性：证明其对MC4R组成型活性和细胞表面表达的潜在影响。

在人类和小鼠中，黑皮质素受体4（MC4R）和黑皮质素受体辅助蛋白2（MRAP2）可以形成复合物并控制能量平衡，从而调节体重和肥胖症。在猪中，MC4R的一个错义变异体（p.Asp298Asn）被认为与生长和肥胖有关。然而，Asp298Asn取代对MC4R功能的影响是有争议的，限制了其在动物育种中的应用。在这里，我们研究了这种多态性对猪MC4R组成型活性，细胞表面表达和信号传导及其与MRAP2相互作用的影响。我们发现：（i）猪MC4R ^Asp和MC4R ^Asn均可被其配体激活（α‐MSH和ACTH），并通过pGL3-CRE-萤光素酶报告基因检测法检测到刺激cAMP / PKA信号通路，表明pMC4R ^Asn像pMC4R ^Asp一样与cAMP / PKA信号通路偶联；（ii）与pMC4R ^Asp相比，pMC4R ^Asn失去了基本的组成活性，并通过双荧光素酶报告基因检测和Nano-HiBiT系统检测到了表面表达降低；（ⅲ）如在其它脊椎动物中，既pMC4R^天冬氨酸和pMC4R^的Asn可以与pMRAP2相互作用，从而降低受体的表面表达和增强配体灵敏度，尽管在对比pMC4R^天冬氨酸，pMC4R的基组成性活性^的Asn不受pMRAP2的影响；（iv）RNA-seq数据分析显示，猪下丘脑中MC4R和MRAP2共表达。综上所述，我们的数据提供了令人信服的证据，表明Asp298Asn取代降低了MC4R或MC4R–MRAP2复合物的本构活性和细胞表面表达，这可能会影响能量平衡，并成为猪育种程序的有价值的选择标记。