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.