Functional characterization of neuropeptide 26RFa receptors GPR103A and GPR103B in zebrafish, Danio rerio

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.