New phosphosite-specific antibodies to unravel the role of GRK phosphorylation in dopamine D(2) receptor regulation and signaling

The dopamine D(2) receptor (D(2)R) is the target of drugs used to treat the symptoms of Parkinson’s disease and schizophrenia. The D(2)R is regulated through its interaction with and phosphorylation by G protein receptor kinases (GRKs) and interaction with arrestins. More recently, D(2)R arrestin-mediated signaling has been shown to have distinct physiological functions to those of G protein signalling. Relatively little is known regarding the patterns of D(2)R phosphorylation that might control these processes. We aimed to generate antibodies specific for intracellular D(2)R phosphorylation sites to facilitate the investigation of these mechanisms. We synthesised double phosphorylated peptides corresponding to regions within intracellular loop 3 of the hD(2)R and used them to raise phosphosite-specific antibodies to capture a broad screen of GRK-mediated phosphorylation. We identify an antibody specific to a GRK2/3 phosphorylation site in intracellular loop 3 of the D(2)R. We compared measurements of D(2)R phosphorylation with other measurements of D(2)R signalling to profile selected D(2)R agonists including previously described biased agonists. These studies demonstrate the utility of novel phosphosite-specific antibodies to investigate D(2)R regulation and signalling.