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 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 CB₂receptors with G_i, 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 G_i, HU210, WIN55,212, and anandamide all elicited maximal activation, whereas Δ⁹-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 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.