Involvement of the Carboxyl Terminus of the Third Intracellular Loop of the Cannabinoid CB1 Receptor in Constitutive Activation of Gs

: The human cannabinoid receptor CB1 functionally couples primarily to Gi‐, but also to Gs‐mediated pathways to modulate intracellular cyclic AMP (cAMP) levels. To probe the features of the receptor that may be involved in promoting interactions with one G protein type over another, we generated the L341A/A342L mutant CB1 receptor. The double mutation involved the swap in position of two adjacent residues in the carboxyl‐terminal segment of the third intracellular loop of CB1. This resulted in partial constitutive activation of the receptor and an agonist‐independent enhancement in cAMP levels. Characterization following treatment with either pertussis or cholera toxin indicated that the constitutive activity is selective for a Gs‐ and not a Gi‐mediated pathway. Treatment with the CB1‐specific inverse agonist SR141716A inhibited the basal accumulation of cAMP in the presence of pertussis toxin, establishing that the effect is CB1 mediated. The binding of the agonist CP‐55,940 to the L341A/A342L receptor was not markedly different from that for the wild‐type receptor despite the constitutive Gs activity. This may reflect a preference of this ligand for an activated receptor state associated with the Gi coupling form and underscores the potential for developing therapeutics that selectively activate one pathway over another.