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 G(i)-, but also to G(s)-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 G(s)- and not a G(i)-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 G(s) activity. This may reflect a preference of this ligand for an activated receptor state associated with the G(i) coupling form and underscores the potential for developing therapeutics that selectively activate one pathway over another.