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

Authors: KATAYAMA, YUKIE - University Of Tokyo; SUZUKI, TATSUYA - University Of Tokyo; EBISAWA, TATSUKI - University Of Tokyo; OHTSUKA, JUN - University Of Tokyo; WANG, SHIPENG - University Of Tokyo; NATSUME, RYO - Tokyo Denki University; LO, YU-HUA - Photon Factory, Institute Of Materials Structure Science; SENDA, TOSHIYA - Photon Factory, Institute Of Materials Structure Science; NAGAMINE, TOSHIHIRO - Advance Science Institute, Riken; Hull, Joe; MATSUMOTO, SHOGO - Advance Science Institute, Riken; NAGASAWA, HIROMICHI - University Of Tokyo; NAGATA, KOJI - University Of Tokyo; 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.