The melanocortin-4 receptor (MC4R) plays a critical role in the regulation of energy homeostasis in both mammals and fish. Several fish MC4Rs recently characterized have high constitutive activities, potentially associated with food intake and growth rate. In the present study, we systematically investigated the effects of four human MC4R (hMC4R) antagonists, including agouti-related peptide (AgRP), Ipsen 5i, ML00253764, and MCL0020, on the cAMP and ERK1/2 signaling of two fish MC4Rs: spotted scat (Scatophagus argus) MC4R (saMC4R) and grass carp (Ctenopharyngodon idella) MC4R (ciMC4R), with hMC4R as a control. We showed that both saMC4R and ciMC4R were constitutively active with significantly increased basal cAMP levels. AgRP acted as an inverse agonist in cAMP signaling pathway in both fish MC4Rs whereas MCL0020 functioned as an inverse agonist for ciMC4R but a weak neutral antagonist for saMC4R. Ipsen 5i and MCL0020 behaved as neutral allosteric modulators in the cAMP signaling of fish MC4Rs. The saMC4R and ciMC4R had similar basal pERK1/2 levels as hMC4R and the pERK1/2 levels of the two fish MC4Rs were significantly increased upon stimulation with all four ligands. In summary, our studies demonstrated the existence of biased signaling in fish MC4R. We also showed dramatic pharmacological differences of human and fish MC4Rs with synthetic ligands. Our data provided novel insights and led to a better understanding of fish MC4R pharmacology.

melanocortin-4受体（MC4R）在调节哺乳动物和鱼类的能量稳态中起着至关重要的作用。最近鉴定出的几种鱼类MC4R具有很高的本构活动，可能与食物摄入和生长速度有关。在本研究中，我们系统地研究了四种人类MC4R（hMC4R）拮抗剂，包括刺古豚相关肽（AgRP），Ipsen 5i，ML00253764和MCL0020，对两种鱼类MC4R的cAMP和ERK1 / 2信号的影响：粪便（Scatophagus argus）MC4R（saMC4R）和草鱼（Ctenopharyngodon idella）MC4R（ciMC4R），并以hMC4R作为对照。我们显示，saMC4R和ciMC4R都具有组成型活性，且基础cAMP水平显着增加。AgRP在两条鱼MC4R中均充当cAMP信号通路的反向激动剂，而MCL0020对ciMC4R起到反向激动剂的作用，但对saMC4R呈弱中性拮抗剂。益普生5i和MCL0020在鱼类MC4Rs的cAMP信号传导中充当中性变构调节剂。saMC4R和ciMC4R的基础pERK1 / 2水平与hMC4R相似，两种鱼MC4R的pERK1 / 2水平在全部四个配体刺激下均显着增加。总而言之，我们的研究证明了鱼类MC4R中存在偏向信号。我们还显示了人类和鱼类带有合成配体的MC4R的巨大药理差异。