Identification of a G Protein-Coupled Receptor for Buccalin-Type Peptides in the Mollusk Aplysia: Evolutionary Insights into Neuropeptide Signaling.

Neuropeptides play a crucial role in modulating behavior, with certain signaling systems, such as those for Neuropeptide Y and Cholecystokinin (CCK), being clearly conserved across protostomes and deuterostomes. However, evolutionary relationships for other signaling systems are less evident. Buccalin (Buc) and allatostatin A (AstA) were independently discovered in the mollusk Aplysia californica and the arthropod Diploptera punctate and are now recognized as homologous. These peptides share a conserved C-terminal Y/F-X-F-G-L/I-amide motif. Bioinformatic evidence further suggests homology between this signaling system and the deuterostome galanin (GAL) and kisspeptin (KISS) signaling systems. Nonetheless, uncertainties persist, particularly due to the limited characterization of Buc/AstA receptors in lophotrochozoans, the superphylum that encompasses mollusks and annelids. To date, only one AstA receptor has been experimentally characterized in the annelid Platynereis dumerilii, while no receptors have been characterized in Aplysia. In this study, we combined bioinformatics, molecular biology, and a cell-based assay to identify an Aplysia buccalin receptor, designated as apBuc/AstA-R. The 19 mature buccalin peptides encoded by the buccalin precursor activated apBuc/AstA-R in a dose-dependent manner, with EC50 values ranging from 23 to 320 nM. Importantly, the potencies of buccalin A and B corresponded to their previously characterized physiological effects. To assess cross-activity, we tested whether Drosophila AstAs and human KISS/GAL can activate apBuc/AstA-R. While receptor activation was observed with Drosophila AstAs and human KISS, human GAL was not active. Phylogenetic and chromosomal gene analyses reinforced the idea that the protostome Buc/AstA signaling system is orthologous to the deuterostome GAL systems and paralogous with the KISS system. Additionally, bioinformatic data suggest the presence of a KISSR-like receptor in Aplysia, though it was not activated by either human KISS/GAL or by Aplysia and Drosophila Buc/AstAs. Overall, our study provides the first comprehensive characterization of the Buc/AstA signaling system in Aplysia and provides further evidence for its close evolutionary relationship with the deuterostome GAL and KISS signaling systems.