Structural determinants in the second intracellular loop of the human cannabinoid CB1 receptor mediate selective coupling to Gs and Gi

BACKGROUND AND PURPOSE The cannabinoid CB1 receptor is primarily thought to be functionally coupled to the Gi form of G proteins, through which it negatively regulates cAMP accumulation. Here, we investigated the dual coupling properties of CB1 receptors and characterized the structural determinants that mediate selective coupling to Gs and Gi. EXPERIMENTAL APPROACH A cAMP-response element reporter gene system was employed to quantitatively analyze cAMP change. CB1/CB2 receptor chimeras and site-directed mutagenesis combined with functional assays and computer modelling were used to determine the structural determinants mediating selective coupling to Gs and Gi. KEY RESULTS CB1 receptors could couple to both Gs-mediated cAMP accumulation and Gi-induced activation of ERK1/2 and Ca2+ mobilization, whereas CB2 receptors selectively coupled to Gi and inhibited cAMP production. Using CB1/CB2 chimeric receptors, the second intracellular loop (ICL2) of the CB1 receptor was identified as primarily responsible for mediating Gs and Gi coupling specificity. Furthermore, mutation of Leu-222 in ICL2 to either Ala or Pro switched G protein coupling from Gs to Gi, while to Ile or Val led to balanced coupling of the mutant receptor with Gs and Gi. CONCLUSIONS AND IMPLICATIONS The ICL2 of CB1 receptors and in particular Leu-222, which resides within a highly conserved DRY(X)5PL motif, played a critical role in Gs and Gi protein coupling and specificity. Our studies provide new insight into the mechanisms governing the coupling of CB1 receptors to G proteins and cannabinoid-induced tolerance.