Molecular Characterization of Two Wamide Neuropeptide Signaling Systems in Mollusk Aplysia.

Neuropeptides with the C-terminal Wamide (Trp-NH2) are one of the last common ancestors of peptide families of eumetazoans and play various physiological roles. In this study, we sought to characterize the ancient Wamide peptides signaling systems in the marine mollusk Aplysia californica, i.e., APGWamide (APGWa) and myoinhibitory peptide (MIP)/Allatostatin B (AST-B) signaling systems. A common feature of protostome APGWa and MIP/AST-B peptides is the presence of a conserved Wamide motif in the C-terminus. Although orthologs of the APGWa and MIP signaling systems have been studied to various extents in annelids or other protostomes, no complete signaling systems have yet been characterized in mollusks. Here, through bioinformatics, molecular and cellular biology, we identified three receptors for APGWa, namely, APGWa-R1, APGWa-R2, and APGWa-R3. The EC50 values for APGWa-R1, APGWa-R2, and APGWa-R3 are 45, 2100, and 2600 nM, respectively. For the MIP signaling system, we predicted 13 forms of peptides, i.e., MIP1-13 that could be generated from the precursor identified in our study, with MIP5 (WKQMAVWa) having the largest number of copies (4 copies). Then, a complete MIP receptor (MIPR) was identified and the MIP1-13 peptides activated the MIPR in a dose-dependent manner, with EC50 values ranging from 40 to 3000 nM. Peptide analogs with alanine substitution experiments demonstrated that the Wamide motif at the C-terminus is necessary for receptor activity in both the APGWa and MIP systems. Moreover, cross-activity between the two signaling systems showed that MIP1, 4, 7, and 8 ligands could activate APGWa-R1 with a low potency (EC50 values: 2800-22,000 nM), which further supported that the APGWa and MIP signaling systems are somewhat related. In summary, our successful characterization of Aplysia APGWa and MIP signaling systems represents the first example in mollusks and provides an important basis for further functional studies in this and other protostome species. Moreover, this study may be useful for elucidating and clarifying the evolutionary relationship between the two Wamide signaling systems (i.e., APGWa and MIP systems) and their other extended neuropeptide signaling systems.

Identification of three elevenin receptors and roles of elevenin disulfide bond and residues in receptor activation in Aplysia californica.

Neuropeptides are ubiquitous intercellular signaling molecules in the CNS and play diverse roles in modulating physiological functions by acting on specific G-protein coupled receptors (GPCRs). Among them, the elevenin signaling system is now believed to be present primarily in protostomes. Although elevenin was first identified from the L11 neuron of the abdominal ganglion in mollusc Aplysia californica, no receptors have been described in Aplysia, nor in any other molluscs. Here, using two elevenin receptors in annelid Platynereis dumerilii, we found three putative elevenin GPCRs in Aplysia. We cloned the three receptors and tentatively named them apElevR1, apElevR2, and apElevR3. Using an inositol monophosphate (IP1) accumulation assay, we demonstrated that Aplysia elevenin with the disulfide bond activated the three putative receptors with low EC50 values (ranging from 1.2 to 25 nM), supporting that they are true receptors for elevenin. In contrast, elevenin without the disulfide bond could not activate the receptors, indicating that the disulfide bond is required for receptor activity. Using alanine substitution of individual conserved residues other than the two cysteines, we showed that these residues appear to be critical to receptor activity, and the three different receptors had different sensitivities to the single residue substitution. Finally, we examined the roles of those residues outside the disulfide bond ring by removing these residues and found that they also appeared to be important to receptor activity. Thus, our study provides an important basis for further study of the functions of elevenin and its receptors in Aplysia and other molluscs.

Characterization of an Aplysia vasotocin signaling system and actions of posttranslational modifications and individual residues of the ligand on receptor activity.

The vasopressin/oxytocin signaling system is present in both protostomes and deuterostomes and plays various physiological roles. Although there were reports for both vasopressin-like peptides and receptors in mollusc Lymnaea and Octopus, no precursor or receptors have been described in mollusc Aplysia. Here, through bioinformatics, molecular and cellular biology, we identified both the precursor and two receptors for Aplysia vasopressin-like peptide, which we named Aplysia vasotocin (apVT). The precursor provides evidence for the exact sequence of apVT, which is identical to conopressin G from cone snail venom, and contains 9 amino acids, with two cysteines at position 1 and 6, similar to nearly all vasopressin-like peptides. Through inositol monophosphate (IP1) accumulation assay, we demonstrated that two of the three putative receptors we cloned from Aplysia cDNA are true receptors for apVT. We named the two receptors as apVTR1 and apVTR2. We then determined the roles of post-translational modifications (PTMs) of apVT, i.e., the disulfide bond between two cysteines and the C-terminal amidation on receptor activity. Both the disulfide bond and amidation were critical for the activation of the two receptors. Cross-activity with conopressin S, annetocin from an annelid, and vertebrate oxytocin showed that although all three ligands can activate both receptors, the potency of these peptides differed depending on their residue variations from apVT. We, therefore, tested the roles of each residue through alanine substitution and found that each substitution could reduce the potency of the peptide analog, and substitution of the residues within the disulfide bond tended to have a larger impact on receptor activity than the substitution of those outside the bond. Moreover, the two receptors had different sensitivities to the PTMs and single residue substitutions. Thus, we have characterized the Aplysia vasotocin signaling system and showed how the PTMs and individual residues in the ligand contributed to receptor activity.

Identification of an allatostatin C signaling system in mollusc Aplysia.

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.