Stabilised D ₂ R G-protein coupled receptor oligomers identify multi-state β-arrestin complexes

The G-protein coupled receptor (GPCR) superfamily directs central roles in many physiological and pathophysiological processes via diverse and complex mechanisms. GPCRs can exhibit signal pleiotropy via formation of di/oligomers both with themselves and other GPCRs. A deeper understanding of the molecular basis and functional role of oligomerization would facilitate rational design of activity-selective ligands. A structural model of the D2 dopamine receptor (D ₂ R) homomer identified distinct combinations of substitutions likely to stabilise protomer interactions. Molecular modelling of β-arrestin-2 (βarr2) bound to predicted dimer models suggests a 2:2 receptor:βarr2 stoichiometry, with the dimer favouring βarr2 over Gαi coupling. A combination of biochemical, biophysical and super-resolution, single molecule imaging approaches demonstrated that the D ₂ R mutant homomers exhibited greater stability. The mutant D ₂ R homomers also exhibited bias towards recruitment of the GPCR adaptor protein βarr2 with either faster or ligand-independent βarr2 recruitment, increased internalization and reprogrammed ERK signaling compared to D ₂ R WT. Through GPCR dimer-stabilisation, we demonstrate that D ₂ R di/oligomerization has a role in βarr2-biased signaling.