Binding of Endogenous Cannabinoid Ligand 2-Arachidonoylglycerol to Cannabinoid Receptor CB2 via the Lipid Matrix

Abstract

Endogenous cannabinoid ligand, 2-arachidonoylglycerol (2-AG), binds to and activates cannabinoid type-2 receptor CB2, controlling a variety of immune and hematopoietic cellular functions. Due to the high lipophilicity of cannabinoid ligands, there have been extensive discussions that after nonspecific incorporation into membrane, approach of the ligands to the binding pocket of CB2 may take place via lateral diffusion in lipid matrix. While previously reported microsecond-long molecular dynamics simulations showed molecular details in favor of this mechanism, no in-situ experimental evidence for the binding event has yet been made. Here we report on 2H NMR results that show exchange of deuterated 2-AG-d5 between the ligand binding site on CB2 and the surrounding lipid matrix at physiological conditions. Purified, recombinant CB2 was functionally reconstituted into liposomes containing four-fold molar excess of 2-AG-d5, and its deuterium resonances measured over the temperature range of 5-20°C. When the membranes contained CB2, the ligand resonances shifted upfield by 0.5 ppm and broadened significantly. Despite the excess amount of ligand, only one set of time-averaged resonances of the ligand was observed. The result suggests that 2-AG undergoes exchange between receptor-bound and free states on millisecond timescale. The resonance linewidth decreased with decreasing temperature as predicted from reduced exchange rates. The receptor binding was specific since only well-resolved signal of 2-AG in lipid matrix was observed when the binding pocket was blocked with a high affinity synthetic agonist CP-55,940. The experiments contrast to our previous experiments on CP-55,940 binding, where the free state of 2H-labeled CP-55,940-d6 in lipid matrix showed a highly-resolved signal under much slower exchange rate. The present work emphasizes the critical role of lipid matrix to provide a pathway and to regulate approach of lipophilic cannabinoid ligands to the receptor binding pocket.