Functional analysis of LFRFamide signaling in Pacific abalone, Haliotis discus hannai

The invertebrate LFRFamide (LFRFa) and short neuropeptide F (sNPF), consisting of 6 to 10 amino acids, are orthologs for bilaterian NPF/Y, which consist of 36 to 40 amino acids. Recently, a molluscan G protein-coupled receptor (GPCR) for NPF was characterized in Pacific abalone (Haliotis discus hannai). To address the functional evolutionary route of the invertebrate LFRFa and NPF signaling system, in this study, we identified cDNAs encoding LFRFa precursors and the sNPF receptor (Hdh-sNPFR) in Pacific abalone. Four LFRFa mature peptides with 6 or 7 amino acids were predicted: GSLFRFa, GGLFRFa, GTLFRFa, and GSTLFRFa. Hdh-sNPFR was identified as a classical rhodopsin-like GPCR and classified into a molluscan sNPFR group. In HEK293 cells, Hdh-sNPFR was mainly localized in the cell membranes and internalized in the cytoplasm following treatment with LFRFa peptides. Reporter assays demonstrated that LFRFa peptides inhibit forskolin-stimulated cAMP accumulation in Hdh-sNPFR-expressing HEK293 cells. LFRFa precursor and Hdh-sNPFR transcripts were more strongly expressed in the cerebral and pleural-pedal ganglia of Pacific abalone than in the peripheral tissues such as the ovary, gills, intestine, and hepatopancreas. The levels of LFRFa transcripts in the ovary, intestine, and hepatopancreas were significantly higher in mature female abalone than in immature females. Injection of LFRFa induced the egg release and spawning behavior of mature abalone, but suppressed food intake. These results suggest that LFRFa peptides are endogenous ligands for Hdh-sNPFR involved in food intake and reproduction through a Gαi-protein dependent signaling pathway.