title: Identification and characterization of receptors for ion transport peptide (ITP) and ITP-like (ITPL) in the silkworm Bombyx mori.
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.
authors.affiliation: Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan.
authors.forename: Chiaki
authors.initials: C
authors.lastname: Nagai
authors.affiliation: Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan.
authors.forename: Hideaki
authors.initials: H
authors.lastname: Mabashi-Asazuma
authors.affiliation: Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan.
authors.forename: Hiromichi
authors.initials: H
authors.lastname: Nagasawa
authors.affiliation: Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan. Electronic address: shinjin@k.u-tokyo.ac.jp.
authors.forename: Shinji
authors.initials: S
authors.lastname: Nagata
mesh_terms: D000818:Animals; D060829:Arthropod Proteins / Q000737:chemistry*; D012831:Bombyx; D006152:Cyclic GMP / Q000737:chemistry; D019476:Insect Proteins / Q000737:chemistry*; D007447:Invertebrate Hormones / Q000737:chemistry*; D017136:Ion Transport; D008024:Ligands; D009419:Nerve Tissue Proteins / Q000737:chemistry*; D009479:Neuropeptides / Q000737:chemistry*; D016366:Open Reading Frames; D027361:Organic Anion Transporters / Q000737:chemistry*; D010455:Peptides / Q000737:chemistry; D010802:Phylogeny; D011485:Protein Binding; D043562:Receptors, G-Protein-Coupled / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry; D015398:Signal Transduction; D013329:Structure-Activity Relationship; D014018:Tissue Distribution
chemical_list: D060829:Arthropod Proteins; D019476:Insect Proteins; D007447:Invertebrate Hormones; D008024:Ligands; D009419:Nerve Tissue Proteins; D009479:Neuropeptides; D027361:Organic Anion Transporters; D010455:Peptides; D043562:Receptors, G-Protein-Coupled; D011994:Recombinant Proteins; C015947:hyperglycemic hormone, crustacean; D006152:Cyclic GMP
keywords: Bombyx mori; Cyclic GMP (cGMP); Deorphanization; G Protein-coupled Receptor; G Protein-coupled Receptor (GPCR); Guanylyl Cyclase; Hormone Receptor; Insect; Neuropeptide; Peptide Hormone