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.

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Pheromone-biosynthesis-activating neuropeptide (PBAN) regulates sex pheromone production in many female moths. PBAN-like peptides, with common FXPRLamide C-terminals are found in other insect groups where they have other functions. The ubiquity and multifunctional nature of the pyrokinin/PBAN family of peptides suggests that the PBAN receptor proteins could also be present in a variety of insect tissues with alternative functions from that of sex pheromone biosynthesis. Previously we showed the presence of the PBAN-R in Helicoverpa armigera at the protein level. In the present study we confirm the similarities between the two Helicoverpa species: armigera and zea by (1) demonstrating the presence of the receptor protein in Sf9 cells, cloned to express the HezPBAN receptor, as compared with the endogenous receptor protein, previously shown in H. armigera pheromone glands, and (2) by identifying the nucleotide sequence of the PBAN-R from mRNA of H. armigera pheromone glands. Sequences of the two Helicoverpa spp. are 98% identical with most changes taking place in the 3'-end. We demonstrate the spatial distribution of the PBAN receptor protein in membranes of H. armigera brain (Br), thoracic ganglion (TG) and ventral nerve cord (VNC). We also demonstrate the presence and differential expression of the PBAN receptor gene (using reverse transcription-polymerase chain reaction and reverse transcription-quantitative real-time polymerase chain reaction, respectively) in the neural tissues (Br, TG and VNC) of adult H. armigera female moths as compared with its presence in pheromone glands. Surprisingly, the gene for the PBAN receptor is also detected in the male tissue homologous to the female pheromone gland, the aedeagus, although the protein is undetectable and PBAN does not induce physiological (pheromone production) or cellular (cyclic-adenosine monophosphate production) responses in this tissue. Our findings indicate that PBAN or PBAN-like receptors are present in the neural tissues and may represent a neurotransmitter-like function for PBAN-like peptides. In addition, the surprising discovery of the presence of the gene encoding the PBAN receptor in the male homologous tissue, but its absence at the protein level, launches opportunities for studying molecular regulation pathways and the evolution of these G protein coupled receptors (GPCRs).