Repertoires of G protein-coupled receptors for Ciona -specific neuropeptides

Abstract

Neuropeptides play pivotal roles in various biological events in the nervous, neuroendocrine, and endocrine systems, and are correlated with both physiological functions and unique behavioral traits of animals. Elucidation of functional interaction between neuropeptides and receptors is a crucial step for the verification of their biological roles and evolutionary processes. However, most receptors for novel peptides remain to be identified. Here, we show the identification of multiple G protein-coupled receptors (GPCRs) for species-specific neuropeptides of the vertebrate sister group, Ciona intestinalis Type A, by combining machine learning and experimental validation. We developed an original peptide descriptor-incorporated support vector machine and used it to predict 22 neuropeptide–GPCR pairs. Of note, signaling assays of the predicted pairs identified 1 homologous and 11 Ciona-specific neuropeptide–GPCR pairs for a 41% hit rate: the respective GPCRs for Ci-GALP, Ci-NTLP-2, Ci-LF-1, Ci-LF-2, Ci-LF-5, Ci-LF-6, Ci-LF-7, Ci-LF-8, Ci-YFV-1, and Ci-YFV-3. Interestingly, molecular phylogenetic tree analysis revealed that these receptors, excluding the Ci-GALP receptor, were evolutionarily unrelated to any other known peptide GPCRs, confirming that these GPCRs constitute unprecedented neuropeptide receptor clusters. Altogether, these results verified the neuropeptide–GPCR pairs in the protochordate and evolutionary lineages of neuropeptide GPCRs, and pave the way for investigating the endogenous roles of novel neuropeptides in the closest relatives of vertebrates and the evolutionary processes of neuropeptidergic systems throughout chordates. In addition, the present study also indicates the versatility of the machine-learning–assisted strategy for the identification of novel peptide–receptor pairs in various organisms.

The ventral peptidergic system of the adult ascidian Ciona robusta (Ciona intestinalis Type A) insights from a transgenic animal model

“…Papillae are thought to assist in passing the mucus sheet produced in the endostyle across the pharyngeal wall from the ventral to the dorsal side, and the mucus sheet containing food is folded into a mucus cord by the dorsal lamina 26 . Given that genes encoding Ci-CT, Ci-LF-8 receptor, Ci-YFV-1 receptor, and Ci-YFV-3 receptor, are expressed in the pharynx 12,46 , multiple peptides may be involved in regulating the food transport function of the papillae.…”

“…Regarding the expression of neuropeptides and their receptors, homologous peptide and receptor genes of vertebrates including Ci-TK 7 , cionin (a cholecystokinin/gastrin homolog peptide) 16 , and Ci-GALP 13,46 , are known to be expressed in the alimentary system. In particular, in situ hybridization showed that the Ci-TK mRNA is expressed in the intestinal epithelial layers 7 .…”

“…In particular, in situ hybridization showed that the Ci-TK mRNA is expressed in the intestinal epithelial layers 7 . Furthermore, genes encoding the receptors for Ciona-specific www.nature.com/scientificreports www.nature.com/scientificreports/ peptides, Ci-LF-8 receptor and Ci-YFV-3 receptor, are also expressed in the stomach and intestine 46 . Although the localization of these peptides and receptors has yet to be rigorously investigated, the broad distribution of Kaede-positive cells in the stomach and intestine suggests that certain neuropeptides, including Ci-TK, cionin, Ci-GALP, Ci-LF-8, and Ci-YFV-3, participate in regulating all functions of the stomach and intestine.…”

“…Although the localization of these peptides and receptors has yet to be rigorously investigated, the broad distribution of Kaede-positive cells in the stomach and intestine suggests that certain neuropeptides, including Ci-TK, cionin, Ci-GALP, Ci-LF-8, and Ci-YFV-3, participate in regulating all functions of the stomach and intestine. Interestingly, these peptide and receptor genes are expressed also in the neural complex 13,46 , suggesting that the peptidergic systems regulate the brain-stomach and brain-gut axis in Ciona as well as in vertebrates 51 . Recently, gastrin-releasing peptide (GRP) gene was identified in the two amphioxus species, other invertebrate chordates 52,53 .…”

Comparative Aspects of Structure and Function of Cnidarian Neuropeptides

“…However, no receptors for the neuropeptides remain to be identified in Hydra and cnidarians. Recently, Shiraishi and coworkers developed the machine-learning-assisted strategy for the identification of novel peptide-receptor pairs (87). As they indicate the multiplicity of use of the strategy, it is worth to use the strategy for increasing the receptor (especially GPCR) repertoire as many as possible on Hydra and cnidarians.…”