Stabilized D2R 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 (D₂R) homomer identified distinct combinations of substitutions likely to stabilize 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 D₂R mutant homomers exhibited greater stability. The mutant D₂R homomers also exhibited bias towards recruitment of the GPCR adaptor protein βarr2 with either faster or ligand-independent βarr2 recruitment, increased internalization and reprogrammed regulation of ERK signaling. Through GPCR dimer-stabilization, we propose that D₂R di/oligomerization has a role in βarr2-biased signaling.