Structural determinants in the second intracellular loop of the human cannabinoid CB 1 receptor mediate selective coupling to G s and G i

Abstract

BACKGROUND AND PURPOSE The cannabinoid CB 1 receptor is primarily thought to be functionally coupled to the G i form of G proteins, through which it negatively regulates cAMP accumulation. Here, we investigated the dual coupling properties of CB 1 receptors and characterized the structural determinants that mediate selective coupling to G s and G i.EXPERIMENTAL APPROACH A cAMP-response element reporter gene system was employed to quantitatively analyze cAMP change. CB 1/CB 2 receptor chimeras and site-directed mutagenesis combined with functional assays and computer modelling were used to determine the structural determinants mediating selective coupling to G s and G i.KEY RESULTS CB 1 receptors could couple to both G s-mediated cAMP accumulation and G i-induced activation of ERK1/2 and Ca 2+ mobilization, whereas CB 2 receptors selectively coupled to G i and inhibited cAMP production. Using CB 1/CB 2 chimeric receptors, the second intracellular loop (ICL2) of the CB 1 receptor was identified as primarily responsible for mediating G s and G i coupling specificity. Furthermore, mutation of Leu-222 in ICL2 to either Ala or Pro switched G protein coupling from G s to G i, while to Ile or Val led to balanced coupling of the mutant receptor with G s and G i.CONCLUSIONS AND IMPLICATIONS The ICL2 of CB 1 receptors and in particular Leu-222, which resides within a highly conserved DRY(X) 5PL motif, played a critical role in G s and G i protein coupling and specificity. Our studies provide new insight into the mechanisms governing the coupling of CB 1 receptors to G proteins and cannabinoid-induced tolerance. © 2010 The Authors. British Journal of Pharmacology © 2010 The British Pharmacological Society.