Agonist selective regulation of g proteins by cannabinoid CB1 and CB2 receptors

We have examined the ligand regulation and G protein selectivity of the human cannabinoid CB1 and CB2 receptors by an in situ reconstitution technique directly measuring G protein activation. Membranes from Spodoptera frugiperda cells expressing CB1 and CB2 receptors were chaotrope extracted to denature endogenous GTP-binding proteins. The ability of the receptors to catalyze the GDP-GTP exchange of each G protein was then examined with purified bovine brain G(i) and G(o). Activation of CB1 receptors produced a high-affinity saturable interaction for both G(i) and G(o). Agonist stimulation of CB2 receptors also resulted in a high-affinity saturable interaction with G(i). In contrast, CB2 receptors did not interact efficiently with G(o). G protein activation was then examined with a diverse group of ligands. For the interaction of CB2 receptors with G(i), HU210 was the only compound tested that demonstrated maximal activation. In contrast, WIN55,212 (64%), anandamide (42%), and Delta(9)-tetrahydrocannabinol (Delta(9)-THC) (44%) all initiated submaximal levels of G protein activation. For CB1 receptor-catalyzed activation of G(i), HU210, WIN55,212, and anandamide all elicited maximal activation, whereas Delta(9)-THC (56 +/- 6%) caused only partial G(i) activation. In contrast, only HU210 effected maximal CB1 stimulation of G(o), with anandamide, WIN55,212, and Delta(9)-THC all stimulating between 60 and 75% compared with HU210. These data demonstrate that different agonists induce different conformations of the CB1 receptor, which in turn can distinguish between different G proteins. Our data thus demonstrate agonist-selective G protein signaling by the CB1 receptor and suggest that therapeutic agents may be designed to regulate individual G protein-signaling pathways selectively.