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 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.

Cannabinoid Receptors in the Central Nervous System: Their Signaling and Roles in Disease

“…Investigating signaling from biased CB 1 receptors such as S426A/S430A and the DRY mutant (Asp-Arg-Tyr) together with the identification of biased ligands and the crystal structure of CB 1 receptors should provide important tools to elucidate the mechanisms and roles of CB 1 receptor signaling (Gyombolai et al, 2015; Delgado-Peraza et al, 2016; Hua et al, 2016). …”

Functional anatomy of the full length CXCR4-CXCL12 complex systematically dissected by quantitative model-guided mutagenesis

“…The conserved DRY motif in the intracellular region of TM3 participates directly in G protein coupling (58) and is known to modulate G protein signaling by class A GPCRs (59,60). Mutation of the central Arg(3.50) residue of the DRY motif in other GPCRs (61,62), including CCR5 (63), has been shown to produce receptor bias wherein G protein signaling is depleted but arrestin interactions are preserved or enhanced. Consistent with these findings, the CXCR4 R134A mutation almost completely abrogated Gαi recruitment ( Fig.…”

Functional selectivity at G-protein coupled receptors: Advancing cannabinoid receptors as drug targets

“…Biochemical/mutation data with cannabinergic ligands and information on the structures of other class-A GPCRs have been used to identify candidate residues potentially involved in ligand functional selectivity at CBRs. Similar to other GPCRs, double mutation of D213 3.49 and R214 3.50 in the highly conserved aspartic acid-arginine-tyrosine (“DRY”) motif that plays a pivotal role in regulating GPCR conformational/activity states caused CB1R to bias towards β-arrestin signaling and away from G-protein activation without significant loss in binding affinity of cannabinoid agonists [85]. It was also observed that C355 6.47 of the CWxP motif was critical for binding of CP55940 and other classical cannabinoids that are known to induce receptor internalization through β-arrestin-mediated pathways [85].…”

“…Similar to other GPCRs, double mutation of D213 3.49 and R214 3.50 in the highly conserved aspartic acid-arginine-tyrosine (“DRY”) motif that plays a pivotal role in regulating GPCR conformational/activity states caused CB1R to bias towards β-arrestin signaling and away from G-protein activation without significant loss in binding affinity of cannabinoid agonists [85]. It was also observed that C355 6.47 of the CWxP motif was critical for binding of CP55940 and other classical cannabinoids that are known to induce receptor internalization through β-arrestin-mediated pathways [85]. However, WIN55212, an aminoalkylindole derivative that does not induce receptor internalization, maintained high affinity for CB1R, even when C355 6.47 had been mutated.…”