Location: Pest Management and Biocontrol ResearchTitle: Identification of functionally important residues in the silkmoth pheromone biosynthesis-activating neuropeptide receptor, an insect ortholog of the vertebrate Neuromedin U Receptor Author
|Lee, Jae Min|
Submitted to: Journal of Biological Chemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/21/2014
Publication Date: 7/4/2014
Publication URL: http://handle.nal.usda.gov/10113/59079
Citation: Kawai, T., Nagata, K., Guo, L., Liu, D., Suzuki, T., Katayama, Y., Hayakawa, K., Lee, J., Nagamine, T., Hull, J.J., Matsumoto, S., Nagasawa, H., Tanokura, M. 2014. Identification of functionally important residues in the silkmoth pheromone biosynthesis-activating neuropeptide receptor, an insect ortholog of the vertebrate Neuromedin U Receptor. Journal of Biological Chemistry. 27:19150-19163. Interpretive Summary: Adult females of most moths produce and release volatile chemical signals called sex pheromones as a means of attracting males. The sex pheromones are produced in the female pheromone gland in response to an internal chemical signal termed the pheromone biosynthesis activating neuropeptide (PBAN). PBAN turns on sex pheromone production by binding to a receptor, termed the PBAN receptor (PBANR), which is expressed at the surface of pheromone-producing cells. This interaction can be thought of in terms of a “lock” (the receptor) and a “key” (the peptide). Because of its importance in driving the mating behaviors of many moth pests, significant research has gone into understanding how the PBAN “key” fits into the PBANR “lock” with the goal of building a better key that either fits the lock but fails to turn on pheromone production or prevents the natural key from binding. To achieve this goal, it is crucial to understand how and where the key contacts the lock. We generated a three-dimensional model of the silkmoth PBANR by utilizing the spatial coordinates of other receptors as a template. Structures that are the same among multiple PBANRs and PBANR-like receptors were mapped onto that model in order to get a three-dimensional image of a potential binding pocket (i.e., where the key inserts into the lock). A number of components (amino acids) of the pocket identified as potential contact points were changed (mutated) to examine their effects on PBANR function. In total, 11 mutations affected both PBANR binding and subsequent function, suggesting that the mutated amino acids represent true contact points. The location and identity of these amino acids was compared with other receptors, including a human receptor for a peptide that slightly resembles PBAN. The proposed PBAN binding pocket was similar to the binding pocket of a number of other receptors, suggesting a common mechanism of activation. Not all peptide keys fit our model, indicating that multiple binding pockets and mechanisms exist to accommodate a diversity of peptide keys.
Technical Abstract: The biosynthesis of sex pheromone components in many lepidopteran insects is regulated by interactions between pheromone biosynthesis-activating neuropeptide (PBAN) and the PBAN receptor (PBANR), a class-A G-protein-coupled receptor (GPCR). To identify functionally important amino acid residues in the silkmoth PBANR, a series of 27 alanine substitutions were generated using a PBANR chimera C-terminally fused with EGFP. The PBANR mutants were expressed in Sf9 insect cells and their ability to bind and be activated by a core PBAN fragment (C10PBAN^R2K) were monitored. Among the 27 mutants, 23 localized to the cell surface of transfected Sf9 cells, while the other four remained intracellular. Reduced binding relative to wildtype was observed with 17 mutants and decreased Ca 2+ mobilization responses were observed with 12 of the mutants. Ala substitution of E95, E120, N124, V195, F276, W280, F283, R287, Y307, T311, and F319 affected both binding and Ca 2+ mobilization. The most pronounced effects were observed with the E120A mutation. A molecular model of PBANR indicated that the functionally important PBANR residues map to the second, third, sixth and seventh transmembrane helices, implying that the same general region of class-A GPCRs recognizes both peptidic and non-peptidic ligands. Docking simulations suggest similar ligand-receptor recognition interactions for PBAN-PBANR and the orthologous vertebrate pair - neuromedin U (NMU) and NMU receptor (NMUR). The simulations highlight the importance of two glutamate residues, E95 and E120 in silkmoth PBANR and E117 and E142 in human NMUR1, in the recognition of the most functionally critical regions of the ligands, the C-terminal residue and amide.