Abstract

The mechanism by which CB₁ cannabinoid receptors are coupled to the G_i/G_o class of G proteins was studied. A peptide representing the juxtamembrane carboxyl terminus robustly stimulated guanosine-5′-O-(3-thio)triphosphate binding. Peptides simulating subdomains of the third intracellular loop (IL3) activated minimally when present alone but produced additive effects when present in combination. Peptides representing the amino-side IL3 and the juxtamembrane carboxyl terminus autonomously inhibited adenylate cyclase, and this response was not significantly augmented or inhibited by peptides representing other intracellular domains. Site-directed antipeptide antibodies developed against the domains of the amino terminus, first extracellular loop, amino-side IL3, and juxtamembrane carboxyl terminus of CB₁ receptors failed to influence binding of [³H]CP-55940. However, IgG raised against the amino-side IL3 diminished the agonist-dependent inhibition of adenylate cyclase. These experiments suggest that the juxtamembrane carboxyl terminus is critical for G protein activation by CB₁ cannabinoid receptors and that the amino-side IL3 also may interact with G_i proteins leading to inhibition of adenylate cyclase.