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.