Identification of a G protein-coupled receptor for pheromone biosynthesis activating neuropeptide from pheromone glands of the moth Helicoverpa zea

Abstract

Pheromone biosynthesis-activating neuropeptide (PBAN), a peptide produced by the subesophageal ganglion, is used by a variety of moths to regulate pheromone production. PBAN acts directly on pheromone gland cells by using calcium and cAMP as second messengers. We have identified a gene encoding a G proteincoupled receptor (GPCR) from pheromone glands of the female moth Helicoverpa zea. The gene was identified based on sequence identity to a group of GPCRs from Drosophila that are homologous to neuromedin U receptors in vertebrates. The full-length PBAN receptor was subsequently cloned, expressed in Sf9 insect cells, and shown to mobilize calcium in response to PBAN. This response was dose-dependent (EC 50 ‫؍‬ 25 nM) with a maximum response at 300 nM and a minimal observable response at 10 nM. Four additional peptides produced by the PBAN-encoding gene were also tested for activity, and it was determined that three had similar activity to PBAN and the other was slightly less active. Peptides belonging to the same family as PBAN, namely pyrokinins, as well as the vertebrate neuromedin U peptide also induced a calcium response. We have identified a GPCR for the PBAN͞pyrokinin family of peptides with a known function of stimulating pheromone biosynthesis in female moths. It is related to several receptors from insects (Drosophila and Anopheles) and to neuromedin U and ghrelin receptors from vertebrates.

The pheromone biosynthesis activating neuropeptide (PBAN) receptor of Heliothis virescens: Identification, functional expression, and structure–activity relationships of ligand analogs

“…1). Comparisons of C-terminal intracellular regions of GPCRs indicate that PBANRs reported previously from Helicoverpa zea [3] and Bombyx mori [9] were highly similar to HevPBANR-A and HevPBANR-C, respectively (Fig. 1B). …”

“…In the distance tree, three moth PBANRs (HevPBANR, HezPBANR and BomPBANR) are grouped with Drosophila GPCRs CG8784 and CG8795 to produce the monophyletic 'PBANR clade' with a 97% bootstrap value. The PBANRclade is further supported by pharmacological profiles shared by its members: All receptors in this clade show marked sensitivity to pyrokinins/PBAN, which feature the FXPRLa Cterminal amino acid motif [3,9,21,26]. The PBANR clade is grouped with CG9918, mainly sensitive to CAP2b-3 and a diapause hormone (DH) analog that contains a Cterminal WFGPRLa motif [2,21].…”

“…All subtypes share the sequence extending from the N-terminus to the end of transmembrane domain 7 (TM 7), but not the C-terminal intracellular loop. Recently, two independent groups reported PBAN receptors from Helicoverpa zea (HezPBANR) [3] and Bombyx mori (BomPBANR) [9], respectively. Strikingly, we found that the HevPBANR-C subtype described here shares a considerable portion of the C-terminal intracellular loop with the BomPBANR, while the HezPBANR was quite similar to HevPBANR-A subtype (Fig.…”

“…Only the HevPBANR-C responded to PBAN under the conditions of our particular assay system. Intriguingly, the H. zea counterpart of HevPBANR-A known as HezPBANR (GenBank Accession number, AY319852) showed strong sensitivity to PBAN and its related peptides when expressed in Sf9 cells, a cell line derived from the Noctuid moth Spodoptera frugiperda [3], suggesting that subtype specific domains are involved in coupling receptors with their downstream intracellular signaling partners.…”

“…These findings suggested that the moth counterpart of CG8795 would likely encode a functional PBAN receptor. Indeed, moth orthologs of CG8795 have been identified as functional PBAN receptors in the moth Helicoverpa zea [3] and Bombyx mori [9].…”