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 protein-coupled 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 (EC50 = 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 PBAN/pyrokinin 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.

Fig. 1.

Amounts of pheromone found in isolated pheromone glands of H. zea after incubation with the indicated amounts of various peptides. NmU, porcine neuromedin U; MM, myomodulin. Isolated glands were incubated with the peptides for 30 min and then extracted, and pheromone titers were determined. Bars represent the amount of pheromone per gland + SEM (n > 4). Different letters at the top of each bar indicate statistically different treatments (one-way analysis of variance, P < 0.05).

Fig. 2.

cDNA and deduced amino acid sequence of the H. zea PBAN-R. Nucleotides are numbered starting at the first ATG codon in the ORF, and the first in-frame stop codon is indicated by *. The putative polyadenylation signal is underlined twice. The deduced amino acid sequence is indicated with the predicted transmembrane (TM) domains underlined and labeled TM1–TM7. Putative extracellular N-glycosylation sites are marked with ‡. The nucleotide sequences used for primer location in obtaining the ORF of the full-length clone are underlined. The nucleotide sequences corresponding to the degenerate primers used in the first round of PCR are shown in bold and underlined.

Fig. 3.

Alignment of H. zea PBAN-R with a PRXamide receptor from D. melanogaster (Dm CG8795), a putative GPCR from A. gambiae (Ag EBIP1526), and with two vertebrate receptors [neuromedin U receptor, GenBank accession no. NM_010341 (NmU-R), and Ghrelin receptor, GenBank accession no. U60178]. Identical amino acids are highlighted in dark gray and conserved amino acids in light gray for three or more sequences. The transmembrane domains for the H. zea PBAN-R are indicated by double dashes above the aligned sequences. Dashed lines indicate spaces to optimize alignment.

Fig. 4.

Phylogenetic tree for 14 GPCRs from insects, a nematode, and mammals that had the highest sequence similarity to H. zea PBAN-R. The tree was made with paup 4.0b10 by using the neighbor-joining distance method. Numbers indicate bootstrapping values in 1,000 replications. Dm, D. melanogaster; Ag, A. gambiae; Ce, C. elegans. Accession nos.: CG8795, AF522190; CG8784, AF522189; CG9918, AF522191; CG14575, AF522193; CG5911a, AF505863; CG5911b, AF505864; agCP3006, EAA08454; ebiP1526, EAA06972; ebiP4093, EAA03575; ebiP3295, EAA08403; NmU-R, NM_010341; ghrelin-R, U60178; Ce orphan, T15816.

Fig. 5.

Dose–response profiles of the effect of synthetic H. zea PBAN on the H. zea PBAN-R- and D. melanogaster CG8795-expressing Sf9 cells. Maximum fluorescence readings were obtained by treating cells with ionomycin after challenge with peptides. The y axis indicates the peak fluorescence value relative to the ionomycin treatment. Values represent the mean ± SD of at least 10 cells per ligand concentration.