Identification of an allatostatin C signaling system in mollusc Aplysia

Abstract

Neuropeptides, as pervasive intercellular signaling molecules in the CNS, modulate a variety of behavioral systems in both protostomes and deuterostomes. Allatostatins are neuropeptides in arthropods that inhibit the biosynthesis of juvenile hormones. Based on amino acid sequences, they are divided into three different types in arthropods: allatostatin A, allatostatin B, allatostatin C. Allatostatin C (AstC) was first isolated from Manduca sexta, and it has an important conserved feature of a disulfide bridge formed by two cysteine residues. Moreover, AstC appears to be the ortholog of mammalian somatostatin, and it has functions in common with somatostatin, such as modulating feeding behaviors. The AstC signaling system has been widely studied in arthropods, but minimally studied in molluscs. In this study, we seek to identify the AstC signaling system in the marine mollusc Aplysia californica. We cloned the AstC precursor from the cDNA of Aplysia. We predicted a 15-amino acid peptide with a disulfide bridge, i.e., AstC, using NeuroPred. We then cloned two putative allatostatin C-like receptors and through NCBI Conserved Domain Search we found that they belonged to the G protein-coupled receptor (GPCR) family. In addition, using an inositol monophosphate 1 (IP1) accumulation assay, we showed that Aplysia AstC could activate one of the putative receptors, i.e., the AstC-R, at the lowest EC50, and AstC without the disulfide bridge (AstC') activated AstC-R with the highest EC50. Moreover, four molluscan AstCs with variations of sequences from Aplysia AstC but with the disulfide bridge activated AstC-R at intermediate EC50. In summary, our successful identification of the Aplysia AstC precursor and its receptor (AstC-R) represents the first example in molluscs, and provides an important basis for further studies of the AstC signaling system in Aplysia and other molluscs.

AI protein structure prediction-based modeling and mutagenesis of a protostome receptor and peptide ligands reveal key residues for their interaction

“…4 and S5 ). In these experiments, we did not need to cotransfect with a promiscuous Gα q protein (see ( 9 , 10 , 11 , 89 )) to elicit IP1 accumulation upon ALK peptide stimulation, suggesting that ALKR can associate with native Gα q proteins in CHO-K1 cells. All endogenous ALK peptides could activate ALKR in a dose-dependent manner ( Fig.…”

“…Neuropeptides primarily act on G protein-coupled receptors (GPCRs) to influence a variety of behaviors and physiological processes, including feeding, locomotion, and reproduction. Although a growing number of neuropeptides and their receptors have been characterized ( 6 , 7 , 8 , 9 , 10 , 11 ), molecular-level details of how peptide ligands engage their receptors remain poorly understood in many cases. Ideally, this information is obtained through high-resolution structures of a GPCR in a bound state with its peptide ligand ( e.g.…”

“…However, few have explored the contributions of specific residues or other properties of ligands to receptor activity based on the structure of a protostome’s receptor that has no known homologs in deuterostomes (vertebrates and some invertebrates), partly because a protein structure cannot be obtained using a homology modeling approach. In the present work, we utilize a molluscan model system, Aplysia californica ( 11 , 27 , 28 , 29 , 30 , 31 , 32 , 33 , 34 , 35 , 36 , 37 , 38 , 39 , 40 , 41 , 42 , 43 , 44 , 45 , 46 , 47 , 48 , 49 , 50 , 51 , 52 , 53 , 54 , 55 , 56 , 57 , 58 , 59 ), to study this issue using Aplysia leucokinin peptides (ALKs) ( 60 ) and their receptor. In particular, recent successful efforts ( 14 ) have been made to predict protein structure based on the amino acid sequence of a protein, particularly template-free modeling ( 61 ) using artificial intelligence (AI) deep machine learning algorithms such as Robetta ( 62 ) and AlphaFold ( 63 ).…”

Functional characterization of a neuropeptide receptor exogenously expressed in Aplysia neurons

“…Consequently, it has been challenging to study peptide signaling systems in vertebrates. Thus, relatively simple model systems, such as Aplysia [3-28], with identifiable neurons in well-defined neural circuits [3-10, 12, 14-17, 21, 22, 24-36] have been used in these studies. Earlier studies in model systems have focused on identifying neuropeptides and their bioactivity [2, 34, 35, 37-47].…”

“…Earlier studies in model systems have focused on identifying neuropeptides and their bioactivity [2, 34, 35, 37-47]. Recently, growing genetic information in model systems is becoming available and has facilitated studying of both neuropeptides and their receptors [36, 48-52]. In the latter studies [49, 50], a common approach is to express putative GPCRs in a cell line, and test activity of potential ligands on the receptor.…”