ABSTRACT
The role of the highly conserved 'DRY' motif in the signaling of the CB1 cannabinoid receptor (CB1R) was investigated by inducing 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 (â¼80% of WT CB1R (CB1R-WT)). Moreover, this mutant showed an enhanced basal ß-arrestin2 (ß-arr2) recruitment. More strikingly, the double-mutant CB1R-D3.49A/R3.50A was biased toward ß-arrs, as it gained a robustly increased ß-arr1 and ß-arr2 recruitment ability compared with the WT 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 ß-arrs in response to agonist stimulus, while still activating G-proteins, although at a reduced level (â¼70% of CB1R-WT). Agonist-induced ERK1/2 activation of the CB1R mutants showed a good correlation with their ß-arr recruitment ability but not with their G-protein activation or inhibition of cAMP accumulation. Our results suggest that G-protein activation and ß-arr 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 ß-arr-mediated functions of CB1R.