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

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 (∼80% of WT CB1R (CB1R-WT)). Moreover, this mutant showed an enhanced basal β-arrestin2 (β-arr2) recruitment. More strikingly, the double-mutant CB1R-D3.49A/R3.50A was biased toward β-arrs, as it gained a robustly increased β-arr1 and β-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 β-arrs in response to agonist stimulus, while still activating G-proteins, although at a reduced level (∼70% of CB1R-WT). Agonist-induced ERK1/2 activation of the CB1R mutants showed a good correlation with their β-arr recruitment ability but not with their G-protein activation or inhibition of cAMP accumulation. Our results suggest that G-protein activation and β-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 β-arr-mediated functions of CB1R.