Functional characterization of neuropeptides that act as ligands for both calcitonin-type and pigment-dispersing factor-type receptors in a deuterostome

The calcitonin (CT) family of related peptides exerts diverse physiological effects in mammals via two G-protein-coupled receptors: CTR and the CTR-like receptor CLR. Phylogenetic analysis of CT-type signaling has revealed the presence of CT-type peptides and CTR/CLR-type proteins in both deuterostome and protostome invertebrates. Furthermore, experimental studies have demonstrated that in the protostome <i>Drosophila melanogaster,</i> the CT-like peptide DH₃₁ can act as a ligand for a CTR/CLR-type receptor and a pigment-dispersing factor (PDF) receptor. Here, we investigated the signaling mechanisms and functions of CT-type neuropeptides in a deuterostome invertebrate, the sea cucumber <i>Apostichopus japonicus</i> (phylum Echinodermata). In <i>A. japonicus,</i> a single gene encodes two CT-type peptides (AjCT1 and AjCT2), and both peptides act as ligands for a CTR/CLR-type receptor (AjCTR) and two PDF-type receptors (AjPDFR1, AjPDFR2), but with differential activation of downstream cAMP/PKA, Gαq/Ca²⁺/PKC, and ERK1/2 signaling pathways. AjCT1/AjCT2-encoding transcripts were detected in the central nervous system and a variety of organ systems, and neuropeptide expression was visualized immunohistochemically using an antiserum to a starfish CT-type peptide (ArCT). In vitro pharmacological experiments demonstrated that AjCT1 and/or AjCT2 cause dose-dependent relaxation of longitudinal muscle and intestine preparations. Furthermore, in vivo pharmacological experiments, combined with gain- and loss-of-function experiments, revealed a potential physiological role for AjCT2/AjPDFR2 signaling in promoting feeding and growth in <i>A. japonicus</i>. To our knowledge, this is the first study to obtain evidence that CT-type peptides can act as ligands for both CTR/CLR-type and PDF-type receptors in a deuterostome. Moreover, it provides the first evidence for appetite-stimulating and growth-promoting effects of CT-type neuropeptides in bilaterians. Given the economic importance of <i>A. japonicus</i> as a foodstuff, the discovery of CT-type peptides as potential regulators of feeding and growth in this species may offer novel strategies for aquaculture applications.