The hypothalamic neuropeptide 26RFa is the most recently identified member of the RFamide peptide family, and this 26RFa signaling system has been shown to be implicated in regulating a variety of physiological processes. In zebrafish，26RFa and two putative receptors, DrGPR103A and DrGPR103B, have been in silico identified, and in vivo data derived from overexpression and loss of function mutation experiments suggest the 26RFa signaling system plays an important role in the hypothalamic regulation of sleep. However, the biochemical and pharmacological information on DrGPR103A/B receptors is still unknown. Here, after cloning of cDNAs of two putative 26RFa receptor genes, DrGPR103A and B, from the total RNA of zebrafish whole body, functional assays demonstrated that both receptors were activated by synthetic zebrafish 26RFa neuropeptide, leading to a significant increase in CRE-driven luciferase activity and intracellular Ca²⁺ mobilization in a G_αq inhibitor- and G_αi/o inhibitor-sensitive manner. Upon activation by 26RFa, DrGPR103A and B evoked ERK1/2 phosphorylation and underwent internalization. Further functional determination also revealed that zebrafish kisspeptin-1 exhibited a slight potency for activating both DrGPR103A and B, and vice versa, zebrafish 26RFa also showed some activity at zebrafish GPR54A and B. Our findings provided evidence that zebrafish GPR103A and B are two functional G_αq- and G_αi/o-dually coupled receptors for 26RFa, enabling the further elucidation of the endocrinological roles of zebrafish 26RFa signaling system in the regulation of physiological activities.

下丘脑神经肽 26RFa 是最近发现的 RFamide 肽家族成员，并且该 26RFa 信号系统已被证明与调节各种生理过程有关。在斑马鱼中，26RFa 和两种推定的受体 DrGPR103A 和 DrGPR103B 已被计算机识别，来自过度表达和功能丧失突变实验的体内数据表明 26RFa 信号系统在下丘脑调节睡眠中起着重要作用。然而，关于 DrGPR103A/B 受体的生化和药理信息仍然未知。在这里，在从斑马鱼全身的总 RNA 中克隆了两个假定的 26RFa 受体基因 DrGPR103A 和 B 的 cDNA 后，功能分析表明这两种受体都被合成斑马鱼 26RFa 神经肽激活，以 G _αq抑制剂和 G _αi/o抑制剂敏感的方式进行²⁺动员。在被 26RFa 激活后，DrGPR103A 和 B 引起 ERK1/2 磷酸化并进行内化。进一步的功能测定还表明，斑马鱼 Kisspeptin-1 对激活 DrGPR103A 和 B 表现出轻微的效力，反之亦然，斑马鱼 26RFa 也对斑马鱼 GPR54A 和 B 显示出一些活性。我们的研究结果提供了证据表明斑马鱼 GPR103A 和 B 是两种功能性 G _αq - 和 G _{αi/o -} 26RFa 双重偶联受体，可以进一步阐明斑马鱼 26RFa 信号系统在调节生理活动中的内分泌作用。