The melanocortin-3 receptor (MC3R) is a G protein–coupled receptor and potentially important in production traits. Three naturally occurring mutations (M54L, G104S, and L151R) in chicken MC3R (cMC3R) were reported previously to be associated with production traits. Here, we inserted the full-length cMC3R coding sequence into pcDNA3.1(+) and generated the 3 mutations by site-directed mutagenesis. The total and cell surface expression of the receptors was measured by flow cytometry. We analyzed the pharmacological characteristics, including binding and cyclic adenosine monophosphate (cAMP) and mitogen-activated protein kinase (MAPK) signaling, using 6 ligands ([Nle⁴, D-Phe⁷]-α-melanocyte stimulating hormone (MSH), α-, β-, γ-, and D-Trp⁸-γ-MSHs, and agouti-related peptide). All mutants had similar total and cell surface expression as the wild-type (WT) cMC3R. M54L had similar pharmacological properties as the WT cMC3R. G104S did not exhibit any specific binding but had minimal response to α-, β-, γ-, and D-Trp⁸-γ-MSH, although it generated 24% WT response when stimulated by NDP-MSH. Although L151R had normal binding, the responses to agonists were reduced to approximately 25% of that of the WT. In MAPK signaling, all 3 mutants showed significantly increased agonist-stimulated phosphorylation of extracellular signal-regulated protein kinases 1/2, indicating the existence of biased signaling at G104S and L151R. In summary, our studies demonstrated that although all 3 mutations are significantly associated with production traits, only G104S and L151R had severe defects in receptor pharmacology. How M54L might cause production trait differences remains to be investigated.

黑皮质素 3 受体 (MC3R) 是一种 G 蛋白偶联受体，在生产性状中可能很重要。先前报道了鸡 MC3R (cMC3R) 中三个自然发生的突变（M54L、G104S 和 L151R）与生产性状相关。在这里，我们将全长 cMC3R 编码序列插入 pcDNA3.1(+) 并通过定点诱变产生了 3 个突变。通过流式细胞术测量受体的总表达和细胞表面表达。我们使用 6 种配体（[Nle ⁴ , D-Phe ⁷ ]-α-促黑素细胞刺激素 (MSH)、α -、β-、γ-和 D-Trp ⁸-γ-MSH 和刺鼠相关肽）。所有突变体具有与野生型 (WT) cMC3R 相似的总表达和细胞表面表达。M54L 具有与 WT cMC3R 相似的药理学特性。G104S 没有表现出任何特异性结合，但对 α-、β-、γ- 和 D-Trp ^{8 的}反应很小-γ-MSH，虽然它在受 NDP-MSH 刺激时产生了 24% 的 WT 响应。尽管 L151R 具有正常结合，但对激动剂的反应降低到 WT 的约 25%。在 MAPK 信号传导中，所有 3 个突变体都显示出显着增加的激动剂刺激的细胞外信号调节蛋白激酶 1/2 的磷酸化，表明 G104S 和 L151R 处存在偏向性信号传导。总之，我们的研究表明，尽管所有 3 个突变都与生产性状显着相关，但只有 G104S 和 L151R 在受体药理学方面存在严重缺陷。M54L 如何导致生产性状差异仍有待研究。