Mutations in the 'DRY' motif of the CB1 cannabinoid receptor result in biased receptor variants.

Abstract

The role of the highly-conserved 'DRY' motif in the signaling of the CB1 cannabinoid receptor (CB1R) was investigated by introducing single, double and triple alanine mutations into this site of the receptor. We found that the CB1R-R3.50A mutant displays a partial decrease in its ability to activate heterotrimeric Go proteins (~85% of wild-type CB1R (CB1R-WT)). Moreover, this mutant showed impaired beta-arrestin binding in response to agonist stimulus, although its basal beta-arrestin binding was enhanced. More strikingly, the double mutant CB1R-D3.49A/R3.50A was biased toward beta-arrestins, as it gained a robustly increased beta-arrestin1 and beta-arrestin2 binding ability compared to the wild-type receptor, while its G protein activation was decreased. In contrast, the double mutant CB1R-R3.50A/Y3.51A proved to be G protein-biased, as it was practically unable to recruit beta-arrestin2 in response to agonist stimulus, while still activating G proteins, although at a reduced level (~75% of CB1R-WT). Agonist-induced ERK1/2 activation of the CB1R mutants showed good correlation with their beta-arrestin binding ability but not with their G protein activation or inhibition of cAMP accumulation. Our results suggest that G protein-activation and beta-arrestin-binding of the CB1R are mediated by distinct receptor conformations and the conserved 'DRY' motif plays different roles in the stabilization of these conformations, thus mediating both G protein- and beta-arrestin2-mediated functions of CB1R.