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

The dopamine D receptor (DR) is the target of drugs used to treat the symptoms of Parkinson's disease and schizophrenia. The DR is regulated through its interaction with and phosphorylation by G protein receptor kinases (GRKs) and interaction with arrestins. More recently, DR 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 DR phosphorylation that might control these processes. We aimed to generate antibodies specific for intracellular DR phosphorylation sites to facilitate the investigation of these mechanisms. We synthesised double phosphorylated peptides corresponding to regions within intracellular loop 3 of the hDR 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 DR. We compared measurements of DR phosphorylation with other measurements of DR signalling to profile selected DR agonists including previously described biased agonists. These studies demonstrate the utility of novel phosphosite-specific antibodies to investigate DR regulation and signalling.