NPF activates a specific NPF receptor and regulates food intake in Pacific abalone Haliotis discus hannai

Abstract

Neuropeptides function through G protein-coupled receptors (GPCRs) with high specificity, implying a significant degree of neuropeptide-GPCR coevolution. However, potential neuropeptide signaling systems in non-chordates are relatively elusive. We determined the specificity of the neuropeptide F (Hdh-NPF) signaling system with a cognate receptor (Hdh-NPFR) in the Pacific abalone, Haliotis discus hannai. Phylogenetic and exon–intron arrangement analyses of bilaterian NPF and the chordate ortholog NPY with their receptor sequences revealed a likely common ancestor, and Hdh-NPFR was similar to the NPYR2 subtype among the NPYR1, NPYR2, and NPYR5 subtypes. Among four Hdh-NPFR-related receptors, Hdh-NPFR specifically responded to Hdh-NPF peptide, supported by the dose–response luciferase reporter curve, intracellular Ca2+ mobilization, and phosphorylation of ERK1/2 and its inhibition with a protein kinase C inhibitor. Peptide fragmentations and shuffling of Hdh-NPF with human NPY could not activate the cellular response of Hdh-NPFR. Three-dimensional in silico modeling suggested that interaction of Hdh-NPF C-terminal amino acids with the extracellular loops of Hdh-NPFR is critical for Hdh-NPFR activation. In vivo injection of Hdh-NPF peptide increased food consumption, and knockdown of Hdh-NPF expression decreased food consumption in Pacific abalone. These findings provide evidence for co-evolution of the NPF/Y ligand-receptor system, enabling further research on mollusk orexigenic neuropeptides.

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

“…Recently, a unique 22-aa-long vertebrate PrRP-type peptide was identified as a potent ligand for an sNPF/PrRP-type receptor in the starfish Asterias rubens [ 23 ], suggesting that sNPF-related peptides co-evolved with diversified sNPF receptors in the deuterostomian phylum Echinodermata. The invertebrate sNPF peptide sequences are distinct from the bilaterian NPF/Y sequences, which range in length from 18 to 40 aa with the C-terminal RXRF/Yamide motif [ 7 , 26 ]. Here, we identified an LFRFa precursor including GSLFRFa, GGLFRFa, GTLFRFa, and GSTLFRFa in the Pacific abalone.…”

“…The N-linked glycosylation and intracellular phosphorylation sites were predicted by the NetNGlyc and NetPhos servers, respectively ( https://services.healthtech.dtu.dk/ ). To generate phylogenetic trees, aa sequences of invertebrate LFRFa/sNPF-related precursors ( S1 Table ) and sNPF/NPF/NPY-related receptors ( S2 Table ) were retrieved from the literature [ 7 , 23 , 39 , 40 ] and the NCBI databases. In total, 29 neuropeptide precursors and 95 receptors were aligned and automatically trimmed as described previously [ 7 ].…”

“…To generate phylogenetic trees, aa sequences of invertebrate LFRFa/sNPF-related precursors ( S1 Table ) and sNPF/NPF/NPY-related receptors ( S2 Table ) were retrieved from the literature [ 7 , 23 , 39 , 40 ] and the NCBI databases. In total, 29 neuropeptide precursors and 95 receptors were aligned and automatically trimmed as described previously [ 7 ]. The trimming contained a total of 54 and 215 residues for neuropeptide precursors and receptors, respectively, which were used to generate the maximum-likelihood phylogenetic tree with W-IQ server v1.6.12 [ 41 ].…”

“…The abalone were starved for two days, placed in individual containers, and refed for one day before the experiment. On the day of the experiment, kelp pieces were divided into two equal parts, blotted, and weighed to obtain the wet mass (g) before and after the feeding period as previously reported [ 7 ]. Abalone were weighed prior to each assay (n = 8 per group) and 350 μL of mollusk saline (13 g HEPES, 25.66 g NaCl, 0.82 g KCl, 1.69 g CaCl 2 , 10.17 g MgCl 2 , 2.56 g Na 2 SO 4 , 1.0 L dH 2 O; pH 7.2) including LFRFa peptides or RFa (2.5 μg/g BW) was injected into the adduct muscle sinus using a 26-gauge needle.…”

“…In previous studies, we explored neuropeptides and their signaling systems in the Pacific abalone, Haliotis discus hannai , through transcriptome and proteome analyses [ 5 , 6 ]. Recently, we revealed the presence of the orexigenic neuropeptide F (NPF) signaling system in Pacific abalone [ 7 ].…”