Isolation, Purification and Sequence Determination of Pheromonotropic-Receptors

Abstract

Moths constitute a major group of pest insects in agriculture. Pheromone blends are utilised by a variety of moth species to attract conspecific mates, which is under circadian control by the neurohormone, PBAN (pheromone-biosynthesis-activating neuropeptide). Our working hypothesis was that, since the emission of sex-pheromone is necessary to attract a mate, then failure to produce and emit pheromone is a potential strategy for manipulating adult moth behavior. The project aimed at identifying, characterising and determining the sequence of specific receptors responsible for the interaction with pheromonotropic neuropeptide/s using two related moth species: Helicoverpa armigera and H. lea as model insects. We established specific binding to a membrane protein estimated at 50 kDa in mature adult females using a photoaffinity-biotin probe for PBAN. We showed that JH is required for the up-regulation of this putative receptor protein. In vitro studies established that the binding initiates a cascade of second messengers including channel opening for calcium ions and intracellular cAMP production. Pharmacological studies (using sodium fluoride) established that the receptor is coupled to a G-protein, that is, the pheromone-biosynthesis-activating neuropeptide receptor (PBAN-R) belongs to the family of G protein-coupled receptor (GPCR)'s. We showed that PBAN-like peptides are present in Drosophila melanogaster based on bioassay and immunocytochemical data. Using the annotated genome of D. melanogaster to search for a GPCR, we found that some were similar to neuromedin U- receptors of vertebrates, which contain a similar C-terminal ending as PBAN. We established that neuromedin U does indeed induce pheromone biosynthesis and cAMP production. Using a PCR based cloning strategy and mRNA isolated from pheromone glands of H. zea, we successfully identified a gene encoding a GPCR from pheromone glands. The full-length PBAN-R was subsequently cloned and expressed in Sf9 insect cells and was shown to mobilize calcium in response to PBAN in a dose-dependent manner. The successful progress in the identification of a gene, encoding a GPCR for the neurohormone, PBAN, provides a basis for the design of a novel battery of compounds that will specifically antagonize pheromone production. Furthermore, since PBAN belongs to a family of insect neuropeptides with more than one function in different life stages, this rationale may be extended to other physiological key-regulatory processes in different insects.