The CB₁ Cannabinoid Receptor Juxtamembrane C-Terminal Peptide Confers Activation to Specific G proteins in Brain

Abstract

Under reducing conditions of SDS-polyacrylamide gel electrophoresis, the CB₁ receptor exists in its monomeric form as well as in an SDS-resistant high molecular weight form that appears to be devoid of G proteins. The CB₁ cannabinoid receptor was immunoprecipitated from 3-[(3-cholamidopropyl)dimethylammonio]propanesulfonate-solubilized rat brain membranes using an antibody against the CB₁receptor N terminus. The CB₁ receptor was coimmunoprecipitated with its associated G proteins, specifically those of the Gα_i/o family, but not Gα_s, Gα_q, or Gα_z. The CB₁receptor-Gα_i/o complex existed in the absence of exogenous agonists, and the cannabinoid receptor agonist desacetyllevonantradol failed to alter the stoichiometry of the receptor-Gα_i/o interaction. Guanosine-5′-O-(3-thio)triphosphate could disrupt the interaction. A peptide derived from the CB₁ receptor juxtamembrane C-terminal domain, peptide CB₁401-417, autonomously activates G_i/o proteins. Peptide CB₁401-417 competitively disrupted the CB₁receptor association with Gα_o and Gα_i3 but not Gα_i1 or Gα_i2. This G protein specificity was also observed in detergent extracts from membranes of the frontal cortex, striatum, and cerebellum. Alternative peptides, including peptides from the CB₁ receptor third intracellular loop and the G protein activating peptide mastoparan-7, failed to promote uncoupling from Gα_o. A CB₂receptor juxtamembrane C-terminal peptide failed to disrupt the CB₁ receptor-Gα_o complex. These studies illustrate that the CB₁ receptor can exist as an SDS-resistant multimer. In 3-[(3-cholamidopropyl)dimethylammonio]propanesulfonate detergent, the CB₁ receptor exists in a complex with G proteins of the G_i/o family in the absence of exogenous agonists. Furthermore, this study provides the first description of domain specificity for interaction with a selective set of G proteins.