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- Title
A bead-based GPCR phosphorylation immunoassay for high-throughput ligand profiling and GRK inhibitor screening.
- Authors
Kaufmann, Johanna; Blum, Nina Kathleen; Nagel, Falko; Schuler, Anna; Drube, Julia; Degenhart, Carsten; Engel, Julian; Eickhoff, Jan Eicke; Dasgupta, Pooja; Fritzwanker, Sebastian; Guastadisegni, Maria; Schulte, Clemens; Miess-Tanneberg, Elke; Maric, Hans Michael; Spetea, Mariana; Kliewer, Andrea; Baumann, Matthias; Klebl, Bert; Reinscheid, Rainer K.; Hoffmann, Carsten
- Abstract
Analysis of agonist-driven phosphorylation of G protein-coupled receptors (GPCRs) can provide valuable insights into the receptor activation state and ligand pharmacology. However, to date, assessment of GPCR phosphorylation using high-throughput applications has been challenging. We have developed and validated a bead-based immunoassay for the quantitative assessment of agonist-induced GPCR phosphorylation that can be performed entirely in multiwell cell culture plates. The assay involves immunoprecipitation of affinity-tagged receptors using magnetic beads followed by protein detection using phosphorylation state-specific and phosphorylation state-independent anti-GPCR antibodies. As proof of concept, five prototypical GPCRs (MOP, C5a1, D1, SST2, CB2) were treated with different agonizts and antagonists, and concentration-response curves were generated. We then extended our approach to establish selective cellular GPCR kinase (GRK) inhibitor assays, which led to the rapid identification of a selective GRK5/6 inhibitor (LDC8988) and a highly potent pan-GRK inhibitor (LDC9728). In conclusion, this versatile GPCR phosphorylation assay can be used extensively for ligand profiling and inhibitor screening. A G protein-coupled receptors (GPCRs) phosphorylation assay for cell culture plates can be used for ligand profiling and inhibitor screening, as evidenced by the identification of two GRK inhibitor compounds.
- Subjects
IMMUNOASSAY; G protein coupled receptors; AFFINITY chromatography; CELL culture; PHOSPHORYLATION
- Publication
Communications Biology, 2022, Vol 5, Issue 1, p1
- ISSN
2399-3642
- Publication type
Article
- DOI
10.1038/s42003-022-04135-9