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ARS Home » Pacific West Area » Maricopa, Arizona » U.S. Arid Land Agricultural Research Center » Pest Management and Biocontrol Research » Research » Publications at this Location » Publication #320148

Research Project: Ecologically Based Pest Management in Western Crops Such as Cotton

Location: Pest Management and Biocontrol Research

Title: A class-A GPCR solubilized under high hydrostatic pressure retains its ligand binding ability

Author
item Katayama, Yukie - University Of Tokyo
item Suzuki, Tatsuya - University Of Tokyo
item Ebisawa, Tatsuki - University Of Tokyo
item Ohtsuka, Jun - University Of Tokyo
item Wang, Shipeng - University Of Tokyo
item Natsume, Ryo - Tokyo Denki University
item Lo, Yu-hua - Photon Factory, Institute Of Materials Structure Science
item Senda, Toshiya - Photon Factory, Institute Of Materials Structure Science
item Nagamine, Toshihiro - Advance Science Institute, Riken
item Hull, Joe
item Matsumoto, Shogo - Advance Science Institute, Riken
item Nagasawa, Hiromichi - University Of Tokyo
item Nagata, Koji - University Of Tokyo
item Tanokura, Masaru - University Of Tokyo

Submitted to: Biochimica et Biophysica Acta
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/17/2016
Publication Date: 6/21/2016
Publication URL: http://handle.nal.usda.gov/10113/62961
Citation: Katayama, Y., Suzuki, T., Ebisawa, T., Ohtsuka, J., Wang, S., Natsume, R., Lo, Y., Senda, T., Nagamine, T., Hull, J.J., Matsumoto, S., Nagasawa, H., Nagata, K., Tanokura, M. 2016. A class-A GPCR solubilized under high hydrostatic pressure retains its ligand binding ability. Biochimica et Biophysica Acta. 1858(9):2145–2151.

Interpretive Summary: Most female moths produce and release sex pheromones as a means of attracting male moths. The production of sex pheromones occurs in a specialized tissue in females referred to as the pheromone gland. Interactions between a neuropeptide termed pheromone biosynthesis activating neuropeptide (PBAN) and its receptor (PBAN receptor) trigger the production of sex pheromones in the pheromone gland. PBAN can be thought of as a key and the PBAN receptor as a lock that fits the PBAN key. If we understand the specific features of the lock that allow the key to fit with it, it becomes easier to design compounds that can block the lock, which will limit the production of sex pheromones, and thus diminish the female’s ability to attract males. To identify the specific structural features of the PBAN receptor, however, it is necessary to purify intact receptor from cells. In this study, varying techniques utilizing high pressure were assayed for their efficacy in extracting the PBAN receptor from cells. The results provide new conditions for extracting relatively large amounts of high quality receptor, which will ultimately make it easier to identify the specifics of how the PBAN key interacts with its lock.

Technical Abstract: The effect of high hydrostatic pressure (HHP) on the solubilization of a class-A G protein-coupled receptor, the silkmoth pheromone biosynthesis-activating neuropeptide receptor (PBANR), was investigated. PBANR was expressed in expresSF+ insect cells as a C-terminal fusion protein with EGFP. The membrane fraction was sub- jected to HHP treatment (200 MPa) at room temperature for 1–16 h in the presence of 0–2.0% (w/v) n-dodecyl- ß-D-maltopyranoside (DDM). The solubilization yield of PBANR-EGFP in the presence of 0.6% (w/v) DDM in- creased to ~1.5-fold after 1 h HHP treatment. Fluorescence-detection size-exclusion chromatography demon- strated that the PBANR-EGFP ligand binding ability was retained after HHP-mediated solubilization. The PBANR-EGFP solubilized with 1.0% DDM under HHP at room temperature for 6 h retained ligand binding ability, whereas solubilization in the absence of HHP treatment resulted in denaturation.