New phosphosite-specific antibodies to unravel the role of GRK phosphorylation in dopamine D2 receptor regulation and signaling

Mann, Anika; Keen, Alastair C.; Mark, Hanka; Dasgupta, Pooja; Javitch, Jonathan A.; Canals, Meritxell; Schulz, Stefan; Robert Lane, J.

Abstract

The dopamine D2 receptor (D2R) is the target of drugs used to treat the symptoms of Parkinson’s disease and schizophrenia. The D2R is regulated through its interaction with and phosphorylation by G protein receptor kinases (GRKs) and interaction with arrestins. More recently, D2R 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 D2R phosphorylation that might control these processes. We aimed to generate antibodies specific for intracellular D2R phosphorylation sites to facilitate the investigation of these mechanisms. We synthesised double phosphorylated peptides corresponding to regions within intracellular loop 3 of the hD2R 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 D2R. We compared measurements of D2R phosphorylation with other measurements of D2R signalling to profile selected D2R agonists including previously described biased agonists. These studies demonstrate the utility of novel phosphosite-specific antibodies to investigate D2R regulation and signalling.

Citation

Mann, A., Keen, A. C., Mark, H., Dasgupta, P., Javitch, J. A., Canals, M., Schulz, S., & Robert Lane, J. (2022). New phosphosite-specific antibodies to unravel the role of GRK phosphorylation in dopamine D2 receptor regulation and signaling. Scientific Reports, 11(1), Article 8288. https://doi.org/10.1038/s41598-021-87417-2

• Journal Article Type: Article
• Acceptance Date: Mar 25, 2021
• Online Publication Date: Apr 15, 2021
• Publication Date: Apr 15, 2022
• Deposit Date: Nov 19, 2021
• Publicly Available Date: Nov 19, 2021
• Journal: Scientific Reports
• Electronic ISSN: 2045-2322
• Publisher: Nature Publishing Group
• Peer Reviewed: Peer Reviewed
• Volume: 11
• Issue: 1
• Article Number: 8288
• DOI: https://doi.org/10.1038/s41598-021-87417-2
• Keywords: Multidisciplinary
• Public URL: https://nottingham-repository.worktribe.com/output/6738627
• Publisher URL: https://www.nature.com/articles/s41598-021-87417-2