Discovery of sea urchin NGFFFamide receptor unites a bilaterian neuropeptide family

Abstract

Neuropeptides are ancient regulators of physiology and behaviour, but reconstruction of neuropeptide evolution is often difficult owing to lack of sequence conservation. Here, we report that the receptor for the neuropeptide NGFFFamide in the sea urchin Strongylocentrotus purpuratus (phylum Echinodermata) is an orthologue of vertebrate neuropeptide-S (NPS) receptors and crustacean cardioactive peptide (CCAP) receptors. Importantly, this has facilitated reconstruction of the evolution of two bilaterian neuropeptide signalling systems. Genes encoding the precursor of a vasopressin/oxytocin-type neuropeptide and its receptor duplicated in a common ancestor of the Bilateria. One copy of the precursor retained ancestral features, as seen in highly conserved vasopressin/oxytocin-neurophysin-type precursors. The other copy diverged, but this took different courses in protostomes and deuterostomes. In protostomes, the occurrence of a disulfide bridge in neuropeptide product(s) of the precursor was retained, as in CCAP, but with loss of the neurophysin domain. In deuterostomes, we see the opposite scenario-the neuropeptides lost the disulfide bridge, and neurophysin was retained (as in the NGFFFamide precursor) but was subsequently lost in vertebrate NPS precursors. Thus, the sea urchin NGFFFamide precursor and receptor are 'missing links' in the evolutionary history of neuropeptides that control ecdysis in arthropods (CCAP) and regulate anxiety in humans (NPS).

Keywords: NGFFFamide; evolution; neuropeptide; neuropeptide-S; receptor; sea urchin.

Figure 1.

NG peptides are ligands for the S. purpuratus NPS/CCAP-type receptor. Dose–response curves for Ca²⁺ responses evoked by the NG peptides NGFFFamide (S. purpuratus), NGFFYamide (A. rubens) and NGIWYamide (A. japonicus) in CHO-K1 cells expressing the S. purpuratus NPS/CCAP-type receptor. Each point (±s.e.m.) represents mean values from at least two independent experiments performed in at least triplicate. Dose–response data are shown as relative (%) to the highest value (100% activation) after normalization to the maximum Ca²⁺ response. The log EC₅₀ values (±s.e.m.) are NGFFFamide: −9.38 ± 0.09; NGFFYamide: −8.25 ± 0.04; NGIWYamide: −6.44 ± 0.04.

Figure 2.

The NG peptide/NPS signalling system in deuterostomes. (a) Phylogenetic diagram comparing the structural organization of neuropeptide-S (NPS) and NG peptide precursors in deuterostomes. Signal peptides are shown in blue, neuropeptides are shown in red (bounded by dibasic cleavage sites in green), and neurophysin domains are shown in purple. (b) Neighbour-joining tree (including bootstrap values out of 1000) showing NPS-type receptors in deuterostomes, with the hypothetical phylogenetic position of an as yet unidentified NPS-type receptor in the brittle star O. victoriae represented by the dashed line. (c) Alignment of neuropeptide(s) derived from the NPS/NG peptide precursors shown in (a); these neuropeptides are proven (underlined) or candidate (not underlined) ligands for the corresponding receptors shown in (b). The conserved NG motif is highlighted in yellow and the numbers in parentheses represent the number of copies of the peptide in the precursor. H. sap, Homo sapiens; B. flo, Branchiostoma floridae; S. kow, Saccoglossus kowalevskii; S. pur, Strongylocentrotus purpuratus; A. jap, Apostichopus japonicus; A. rub, Asterias rubens; O. vic, Ophionotus victoriae; A. med, Antedon mediterranea.

Figure 3.

Evolution of NPS/NG peptide/CCAP-type and vasopressin/oxytocin-type signalling systems in the bilateria. (a) Schematic showing how duplication of a vasopressin (VP)/oxytocin (OT)-type neuropeptide precursor in a common ancestor of the bilateria gave rise to the highly conserved precursors of VP/OT-type neuropeptides and the divergent precursors of NPS, NG peptides and CCAP-type peptides in extant bilaterians. Signal peptides are shown in blue, neuropeptides are shown in red (with the presence of a disulfide bridge labelled with an asterisk), dibasic cleavage sites are shown in green and neurophysin domains are shown in purple. (b) Schematic showing how duplication of a receptor in a common ancestor of the bilateria (white-filled receptor symbol) gave rise to NPS/NG peptide/CCAP-type receptors and VP/OT-type receptors in deuterostomian and protostomian species. Receptors where the molecular identity of the cognate ligand has been determined are shown as a black-filled receptor symbols and receptors where the molecular identity of the cognate ligand remains to be proven are shown as grey-filled receptor symbols. Where multiple isoforms of a receptor type occur in a species, this is shown as n = x. (c) Alignment of NPS/NG peptide/CCAP-type neuropeptides and VP/OT-type neuropeptides that are derived from the precursors shown in (a), which are proven or candidate ligands for the receptors shown in (b). An NG motif, which is highlighted in yellow, is a conserved feature of NPS, NG peptides and some CCAP-type peptides. A pair of cysteine residues (underlined), which form a disulfide bridge, are a conserved feature of CCAP-type peptides and VP/OT-type peptides. A TG motif and two phenylalanine (F) residues, which are conserved in sub-sets of peptides, are shown in purple. H. sap, Homo sapiens; B. flo, Branchiostoma floridae; S. kow, Saccoglossus kowalevskii; S. pur, Strongylocentrotus purpuratus; C. tel, Capitella teleta; L. gig, Lottia gigantea; T. cas, Tribolium castaneum; L. sta, Lymnaea stagnalis; C. ele, Caenorhabditis elegans. References: 1. [3]; 2. this paper; 3. [34]; 4. [–37]; 5. [38]; 6. [39]; 7. [40]; 8. [21,41].