The role of the highly conserved 'DRY' motif in the signaling of the CB 1 cannabinoid receptor (CB 1 R) was investigated by inducing single-, double-, and triple-alanine mutations into this site of the receptor. We found that the CB 1 R-R3.50A mutant displays a partial decrease in its ability to activate heterotrimeric G o proteins (w80% of WT CB 1 R (CB 1 R-WT)). Moreover, this mutant showed an enhanced basal b-arrestin2 (b-arr2) recruitment. More strikingly, the double-mutant CB 1 R-D3.49A/R3.50A was biased toward b-arrs, as it gained a robustly increased b-arr1 and b-arr2 recruitment ability compared with the WT receptor, while its G-protein activation was decreased. In contrast, the double-mutant CB 1 R-R3.50A/Y3.51A proved to be G-protein-biased, as it was practically unable to recruit b-arrs in response to agonist stimulus, while still activating G-proteins, although at a reduced level (w70% of CB 1 R-WT). Agonist-induced ERK1/2 activation of the CB 1 R mutants showed a good correlation with their b-arr recruitment ability but not with their G-protein activation or inhibition of cAMP accumulation. Our results suggest that G-protein activation and b-arr binding of the CB 1 R 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 b-arr-mediated functions of CB 1 R.