Agonist Selective Regulation of G Proteins by Cannabinoid CB1and CB2Receptors

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Abstract
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 Gi and Go. Activation of CB1 receptors produced a high-affinity saturable interaction for both Gi and Go. Agonist stimulation of CB2 receptors also resulted in a high-affinity saturable interaction with Gi. In contrast, CB2 receptors did not interact efficiently with Go. G protein activation was then examined with a diverse group of ligands. For the interaction of CB2receptors with Gi, HU210 was the only compound tested that demonstrated maximal activation. In contrast, WIN55,212 (64%), anandamide (42%), and Δ9-tetrahydrocannabinol (Δ9-THC) (44%) all initiated submaximal levels of G protein activation. For CB1 receptor-catalyzed activation of Gi, HU210, WIN55,212, and anandamide all elicited maximal activation, whereas Δ9-THC (56 ± 6%) caused only partial Gi activation. In contrast, only HU210 effected maximal CB1 stimulation of Go, with anandamide, WIN55,212, and Δ9-THC all stimulating between 60 and 75% compared with HU210. These data demonstrate that different agonists induce different conformations of the CB1receptor, 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.

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