title: Identification of specific sites in the third intracellular loop and carboxyl terminus of the Bombyx mori pheromone biosynthesis activating neuropeptide receptor crucial for ligand-induced internalization.
abstract: Sex pheromone production in most moths is mediated by the pheromone biosynthesis activating neuropeptide receptor (PBANR). Using fluorescent Bombyx mori PBANR (BmPBANR) chimeras to study PBANR regulation, we previously showed that BmPBANR undergoes rapid ligand-induced internalization, that the endocytotic motif resides between residues 358-367 of the BmPBANR C terminus, and that the internalization pathway is clathrin-dependent. Here, we sought to expand our understanding of the molecular mechanisms underlying BmPBANR function and regulation by transiently expressing a series of fluorescent BmPBANR chimeric constructs in cultured Spodoptera frugiperda (Sf9) cells and assaying for internalization of a fluorescently labelled ligand. Pharmacological inhibition of phospholipase C significantly reduced internalization, suggesting that BmPBANR regulation proceeds via a conventional G-protein-dependent pathway. This was further supported by impaired internalization following site-directed mutagenesis of R263 and R264, two basic residues at the transmembrane 6 intracellular junction that are thought to stabilize G-protein coupling via electrostatic interactions. Ala substitution of S333 and S366, two consensus protein kinase C sites in the C terminus, likewise impaired internalization, as did RNA interference-mediated knockdown of Sf9 protein kinase C. N-terminal truncations of BmPBANR indicate that the first 27 residues are not necessary for cell surface trafficking or receptor functionality.
authors.affiliation: Molecular Entomology Laboratory, RIKEN Advanced Science Institute, Wako, Saitama, Japan. joe.hull@ars.usda.gov
authors.forename: J J
authors.initials: JJ
authors.lastname: Hull
authors.forename: J M
authors.initials: JM
authors.lastname: Lee
authors.forename: S
authors.initials: S
authors.lastname: Matsumoto
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D012831:Bombyx / Q000235:genetics / Q000378:metabolism*; D004252:DNA Mutational Analysis; D006031:Glycosylation; D019476:Insect Proteins / Q000235:genetics / Q000378:metabolism*; D008024:Ligands; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010675:Pheromones / Q000096:biosynthesis*; D011487:Protein Conformation; D034622:RNA Interference; D018013:Receptors, Neuropeptide / Q000235:genetics / Q000378:metabolism*; D010738:Type C Phospholipases / Q000037:antagonists & inhibitors
chemical_list: D019476:Insect Proteins; D008024:Ligands; D010675:Pheromones; D018013:Receptors, Neuropeptide; D010738:Type C Phospholipases