IDENTIFICATION of NLP-5 and NLP-6 as potential ligands for the NPR-9 receptor in Caenorhabditis elegans

Neuropeptides in Caenorhabditis elegans regulate physiological and behavioural responses to environmental cues, influencing locomotion, feeding, and fat storage via interactions with G-protein coupled receptors (GPCRs). C. elegans expresses a diverse repertoire of neuropeptides, including FMRFamide-related peptides, neuropeptide-like peptides (NLPs), and insulin-like peptides (INSs). Among these, the galanin/allatostatin-like GPCR, NPR-9, localized in the AIB interneurons, regulates locomotory behaviours (roaming and dwelling) and fat accumulation by inhibiting AIB activity. Recent studies identified NLP-1 as a ligand for NPR-9, modulating behaviour through direct receptor interaction. However, our research explored whether other neuropeptides, specifically NLP-5 and NLP-6 (allatostatin A-type/galanin-like neuropeptides), could also function as NPR-9 ligands, despite evidence suggesting NLP-1 as the primary ligand. In this study, we characterized phenotypes associated with NPR-9 receptor function, including Omega turns, roaming, and fat accumulation. Loss-of-function mutations in nlp-5, nlp-6, and nlp-1 exhibited behavioural phenotypes similar to npr-9 mutants, suggesting that NLP-5 and NLP-6 may act as additional ligands for NPR-9 or affect NPR-9 signalling. Furthermore, double-mutant analyses with candidate ligand genes suppressed phenotypes associated with NPR-9 overexpression, reinforcing the hypothesis that these neuropeptides may regulate NPR-9-mediated signalling.