Stabilized D2R G-protein coupled receptor oligomers identify multi-state β-arrestin complexes

Abstract

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 {beta}-arrestin-2 ({beta}arr2) bound to predicted dimer models suggests a 2:2 receptor:{beta}arr2 stoichiometry, with the dimer favouring {beta}arr2 over Gi 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 {beta}arr2 with either faster or ligand-independent {beta}arr2 recruitment, increased internalization and reprogrammed regulation of ERK signaling. Through GPCR dimer-stabilization, we propose that D2R di/oligomerization has a role in {beta}arr2-biased signaling.