Phosphoproteomic Analysis of Dopamine D2 Receptor Signaling Reveals Interplay of G Protein- and β‑Arrestin-Mediated Effects

Leveraging biased signaling of G protein-coupled receptors has been proposed as a promising strategy for the development of drugs with higher specificity. However, the consequences of selectively targeting G protein- or β-arrestin-mediated signaling on cellular functions are not comprehensively understood. In this study, we utilized phosphoproteomics to gain a systematic overview of signaling induced by the four biased and balanced dopamine D2 receptor (D₂R) ligands MS308, BM138, quinpirole, and sulpiride in an in vitro D₂R transfection model. Quantification of 14,160 phosphosites revealed a low impact of the partial G protein agonist MS308 on cellular protein phosphorylation, as well as surprising similarities between the balanced agonist quinpirole and the inverse agonist sulpiride. Analysis of the temporal profiles of ligand-induced phosphorylation events showed a transient impact of the G protein-selective agonist MS308, whereas the β-arrestin-preferring agonist BM138 elicited a delayed, but more pronounced response. Functional enrichment analysis of ligand-impacted phosphoproteins and treatment-linked kinases confirmed multiple known functions of D₂R signaling while also revealing novel effects, for example of MS308 on sterol regulatory element-binding protein-related gene expression. All raw data were deposited in MassIVE (MSV000089457).