Abstract
Ion transport peptide (ITP) and its alternatively spliced variant, ITP-like (ITPL), are insect peptides that belong to the crustacean hyperglycemic hormone family. These peptides modulate the homeostatic mechanisms for regulating energy metabolism, molting, and reproduction and are specifically conserved in ecdysozoans. Many of the details of the molecular mechanisms by which crustacean hyperglycemic hormone family peptides exert pleiotropy remain to be elucidated, including characterization of their receptors. Here we identified three Bombyx mori orphan neuropeptide G protein-coupled receptors (BNGRs), BNGR-A2, -A24, and -A34, as receptors for ITP and ITPL (collectively referred to as ITPs). BNGR-A2 and -A34 and BNGR-A24 respond to recombinant ITPs, respectively, with EC50 values of 1.1-2.6 × 10(-8) M, when expressed in a heterologous expression system. These three candidate BNGRs are expressed at larval B. mori tissues targeted by ITPs, with cGMP elevation observed after exposure to recombinant ITPs. ITPs also increased the cGMP level in B. mori ovary-derived BmN cells via membrane-bound and soluble guanylyl cyclases. The simultaneous knockdown of bngr-A2 and -A34 significantly decreased the response of BmN cells to ITP, whereas knockdown of bngr-A24 led to decreased responses to ITPL. Conversely, transient expression of bngr-A24 potentiated the response of BmN cells to ITPL. An in vitro binding assay showed direct interaction between ITPs and heterologously expressed BNGRs in a ligand-receptor-specific manner. Taken together, these data demonstrate that BNGR-A2 and -A34 are ITP receptors and that BNGR-A24 is an ITPL receptor in B. mori.
Highlights
Ion transport peptide (ITP) and ITP-like (ITPL) are crustacean hyperglycemic hormone family peptides in insects
Measurements of the cGMP levels in BmN cells following 30-min exposure to Recombinant ITP (rITP), recombinant ITPL (rITPL), and rITP-Gly confirmed the biological functionality of rITP and rITPL, rITP-Gly did not show distinct bioactivity at least at a final concentration of 100 nM (Fig. 1A)
In the present study, screening of receptors for ITPs from orphan Bombyx neuropeptide G protein-coupled receptors (BNGRs) revealed that BNGR-A2 and -A34 are ITP receptors, whereas BNGR-A24 is an ITPL receptor in B. mori (Fig. 8)
Summary
Ion transport peptide (ITP) and ITP-like (ITPL) are crustacean hyperglycemic hormone family peptides in insects. Ion transport peptide (ITP) and its alternatively spliced variant, ITP-like (ITPL), are insect peptides that belong to the crustacean hyperglycemic hormone family. These peptides modulate the homeostatic mechanisms for regulating energy metabolism, molting, and reproduction and are conserved in ecdysozoans. Further understanding of the molecular mechanisms underlying functional activation of CHH family receptors is crucial for elucidating the biological functions mediated by these ligands; advances have been largely impeded by the absence of an identified receptor from any species for these peptides. We discuss the intercellular cGMP-generating pathways utilized by the ITPs and their cognate receptors
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