Reconstitution of Human Cannabinoid 2 Receptor into Nanodiscs for Mass Spectrometric Studies

Abstract

Cannabinoid 2 receptor (CB2) belongs to the Class A family of G-protein coupled receptors and is expressed predominantly in the periphery and to a lower extent in the CNS. CB2 is an attractive target for pain management and other neurological and peripheral inflammatory disorders. CB2 upon activation by endogenous or synthetic ligands, modulate a diverse set of downstream signaling pathways. These responses are dictated by the structural conformation of CB2 following ligand binding. Therefore, detailed structural studies of CB2 in lipid bilayer membranes in the presence of the cannabinergic ligands are vital for the development of effective drugs. Here, we report the reconstitution of functional, purified human CB2 into novel lipid bilayer membrane mimic (nanodiscs), for structural studies. Using a baculovirus expression system, we expressed functional, full-length human CB2. The radioligand [3H] CP55940 binding assays revealed a Bmax of ~500 pmol/g and KD of 0.68 nM. We were able to purify, using immunoaffinity chromatography, a functional receptor with yields of ~200 ug/L of culture. The receptor was reconstituted into POPC/POPG nanodiscs and separated from empty nanodiscs using additional purification steps. CB2-incorporated nanodiscs were further evaluated using SDS-PAGE, western blotting, radioligand binding assay and proteomic mass spectrometric (MS) analysis. The CB2-nanodisc preparation would further be studied using hydrogen deuterium exchange MS, to elucidate protein structure and dynamics upon ligand binding. The work was supported by grants DA9158 and DA3801 to AM.