BNGR-A25L and -A27 are two functional G protein–coupled receptors for CAPA periviscerokinin neuropeptides in the silkworm Bombyx mori

Abstract

CAPA peptides, such as periviscerokinin (PVK), are insect neuropeptides involved in many signaling pathways controlling, for example, metabolism, behavior, and reproduction. They are present in a large number of insects and, together with their cognate receptors, are important for research into approaches for improving insect control. However, the CAPA receptors in the silkworm (<i>Bombyx mori</i>) insect model are unknown. Here, we cloned cDNAs of two putative CAPA peptide receptor genes, BNGR-A27 and -A25, from the brain of <i>B. mori</i> larvae. We found that the predicted BNGR-A27 ORF encodes 450 amino acids and that one BNGR-A25 splice variant encodes a full-length isoform (BNGR-A25L) of 418 amino acid residues and another a short isoform (BNGR-A25S) of 341 amino acids with a truncated C-terminal tail. Functional assays indicated that both BNGR-A25L and -A27 are activated by the PVK neuropeptides <i>Bom</i>-CAPA-PVK-1 and -PVK-2, leading to a significant increase in cAMP-response element-controlled luciferase activity and Ca²⁺ mobilization in a G_q inhibitor-sensitive manner. In contrast, BNGR-A25S was not significantly activated in response to the PVK peptides. Moreover, <i>Bom</i>-CAPA-PVK-1 directly bound to BNGR-A25L and -A27, but not BNGR-A25S. Of note, CAPA-PVK-mediated ERK1/2 phosphorylation and receptor internalization confirmed that BNGR-A25L and -A27 are two canonical receptors for <i>Bombyx</i> CAPA-PVKs. However, BNGR-A25S alone is a nonfunctional receptor but serves as a dominant-negative protein for BNGR-A25L. These results provide evidence that BNGR-A25L and -A27 are two functional G_q-coupled receptors for <i>Bombyx</i> CAPA-PVKs, enabling the further elucidation of the endocrinological roles of <i>Bom</i>-CAPA-PVKs and their receptors in insect biology.