Biased Signaling Agonists Promote Distinct Phosphorylation and Conformational States of the Dopamine D3 Receptor

Biased agonists of G-protein-coupled receptors (GPCRs) have emerged as promising selective modulators of signaling pathways by offering therapeutic advantages over unbiased agonists to minimize side effects. The dopamine D3 receptor (D3R), a pivotal GPCR in the central nervous system, has gained significant attention as a therapeutic target for neurological diseases, including Parkinson's disease (PD), addiction, psychosis, depression, and anxiety. We have recently designed and tested SK609, a G-protein biased D3R selective agonist, and demonstrated its efficacy in reducing motor impairment and improving cognitive effects in a rodent model of PD. The molecular mechanism by which SK609 recruits G-protein but not β-arrestin pathways is poorly understood. Utilizing all-atom molecular dynamics simulations, we investigated the distinct conformational dynamics imparted by SK609 and the reference unbiased agonist Pramipexole (PRX). Results from these studies show that the flexibility of transmembrane 3 is key to unbiased signaling, with a ~30° and ~17° shift in tilt angle in the D3R-Gi and D3R-βarrestin2 complexes, respectively. Additionally, untargeted phosphoproteomics analysis reveals unique phosphorylation sites by SK609 and PRX in D3R. These results suggest that SK609 induces conformational changes and unique phosphorylation patterns that promote interactions with G-proteins and are not conducive for β-arrestin2 recruitment and signaling.