Genome-wide identification and expression analysis of neuropeptide G protein-coupled receptors in Urechis unicinctus (Annelida, Echiura).

Neuropeptide G protein-coupled receptors (GPCRs) are membrane protein receptors with 7 transmembrane helices, regulating physiological processes like reproduction, development, and behavior. However, little is known about neuropeptide GPCRs in marine benthic invertebrates. To explore the GPCR composition and potential neuropeptide GPCRs involved in larval development, we performed a genome-wide search of neuropeptide GPCR family genes in the echiuran worm Urechis unicinctus, a marine benthic burrowing Annelida with typical larval settlement and metamorphosis processes during the larval development. A total of 71 putative neuropeptide GPCR genes were identified including 60 family-A GPCRs and 11 family-B GPCRs. Both families contained the conserved 7-transmembrane domain, with family-B GPCRs evolving more conservatively. The clustering of family-A GPCRs formed a large central cluster, where 12 GPCR genes were likely ancestral genes in the evolution of this family. About one-third of neuropeptide GPCR genes were highly expressed in embryos, larvae and adult organs of U. unicinctus. However, the specific GPCRs involved in larval development and adult organ function may differ. In addition, the binding of several neuropeptide GPCRs to larval settlement-related mature peptides was predicted by AlphaFold3 docking. The dual luciferase reporter gene assay showed that the affinity of neuropeptide GPCR Uu-A25 to MIP2 was higher than that to MIP1 in U. unicinctus. Inhibitor (H89 and calmidazolium) and activator (forskolin and Bay K 8644) experiments proved that the cAMP and Ca pathways, mediated commonly by neuropeptide GPCRs, play important roles in the regulation of the larval settlement of U. unicinctus. This study provides a better understanding of neuropeptide GPCR evolution in marine benthic invertebrates and provides fundamental data for future functional exploration of the specific neuropeptide GPCRs in U. unicinctus larval development.