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.
Highlights
Allatostatins are a group of neuropeptides in arthropods that are released in the retrocerebral corpora allata, and there inhibit the biosynthesis of juvenile h ormones[1,2]
For the precursor, searching “Aplysia allatostatin C” in NCBI returned two entries: a predicted sequence (Fig. 1A), and an Allatostatin C (AstC) precursor deposited in 2010 (Fig. 1B), which is likely based on an early large-scale sequencing project[55]
To determine whether the three putative G protein-coupled receptor (GPCR) might be related to AstC receptors, we blasted each sequence in NCBI in species where more protein sequences have been studied, i.e., Caenorhabditis elegans, Drosophila melanogaster, Danio rerio and Mus musculus
Summary
Identifying a precursor for AstC and predicting peptides in Aplysia. To identify putative precursor and receptors for Aplysia AstC, we began with a bioinformatic analysis. To determine whether the three putative GPCRs might be related to AstC receptors, we blasted each sequence in NCBI in species where more protein sequences have been studied, i.e., Caenorhabditis elegans, Drosophila melanogaster, Danio rerio and Mus musculus. XM_005095082.3), named somatostatin receptor type 2-like receptor in NCBI, a number of sequences named somatostatin receptors or AstC receptors with low E-values came up at these searches in several invertebrate and vertebrate species (Supplementary Table 5), suggesting that this protein might be related to AstC receptors. We generated a phylogenetic tree of Aplysia AstC-R with AstC-Rs from selected species in arthropods, lophotrochozoa and some somatostatin receptors in vertebrates (Fig. 6, see Supplementary Table 6 for information on these sequences). The peptides with the disulfide bridge but having different variations of amino acid sequences from four other molluscan species had log[EC50] at intermediate values (Fig. 7F,G), indicating that both the disulfide bridge and amino acid sequences play some roles in the receptor activity
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