|LEE, J.M. - RIKEN INSTITUTE|
|MASTSUMOTO, S. - RIKEN INSTITUTE|
Submitted to: Insect Molecular Biology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 8/15/2011
Publication Date: 9/28/2011
Citation: Hull, J.J., Lee, J., Mastsumoto, S. 2011. Identification of specific sites in the third intracellular loop and carboxyl terminus of the Bombyx mori PBAN receptor crucial for ligand-induced internalization. Insect Molecular Biology. 20(6):801-811.
Interpretive Summary: The larval/caterpillar forms of many moth species are major agronomic pests. Because of increased pesticide resistance and ecological concerns regarding specificity, the continued viability of organosynthetic chemicals in pest control is dubious; consequently, the need for developing novel pest management strategies. The mating behaviors of most moth species are controlled by sex pheromones; thus disruption of sex pheromone production would be expected to negatively impact pest populations. Sex pheromone production is regulated by the interaction between a peptide termed pheromone biosynthesis activating neuropeptide (PBAN) and its cognate receptor, the PBAN receptor. Antagonists that block the pheromone producing effects of PBAN are currently under development; however, little is known about how the receptor is regulated. As part of the cell’s normal response to peptide binding, the respective receptor undergoes desensitization, a process that terminates the extracellular signal by removing the receptor from the cell surface. Once depleted of bound peptide, the receptor is recycled back to the cell surface where it can once again interact with its respective peptide ligand. The present study focused on identifying specific structural features of the PBAN receptor necessary for initiating the desensitization mechanism. A complete understanding of the mechanisms underlying the cellular response of desensitization will further facilitate the development of rationally-designed PBAN antagonists that not only block normal PBAN function but which also prevent the recycling of internalized receptor back to the cell surface.
Technical Abstract: Sex pheromone production in most moths is mediated by the pheromone biosynthesis activating neuropeptide receptor (PBANR). Similar to other rhodopsin-like G protein-coupled receptors, the silkmoth Bombyx mori PBANR (BmPBANR) undergoes agonist-induced internalization. Despite interest in developing PBANR antagonists, our understanding of the molecular mechanisms underlying PBANR regulation is limited. To address this deficiency, we examined the effect of phospholipase C inhibitors, site-directed point mutations, and N terminal truncations on the internalization of a fluorescent ligand, rhodamine red-PBAN (RR-PBAN). Phospholipase C inhibition significantly reduced RR-PBAN internalization as did Glu and Ala mutations of Arg263 and Arg263, potential G protein interaction sites. These results are consistent with G protein dependent internalization and suggest the involvement of a second messenger dependent protein kinase. Four potential protein kinase C sites are present in BmPBANR, two in intracellular loop 3 and two in the C terminus. Site-directed mutagenesis of the C terminal sites (Ser333 and Ser366) significantly impaired RR-PBAN internalization, whereas similar mutagenesis of the intracellular loop 3 sites had no effect. Truncation of the BmPBANR N terminus indicated that the first 27 residues, including two potential N-linked glycosylation sites, are not essential for cell surface trafficking or RR-PBAN internalization.