A bead-based GPCR phosphorylation immunoassay for high-throughput ligand profiling and GRK inhibitor screening

Kaufmann, J.; Blum, N. K.; Nagel, F.; Anna Schuler, A. S.; Drube, J.; Degenhart, C.; Engel, J.; Eickhoff, J. E.; Dasgupta, P.; Fritzwanker, S.; Guastadisegni, M.; Schulte, C.; Miess-Tanneberg, E.; Maric, H. M.; Spetea, M.; Kliewer, A.; Baumann, M.; Klebl, B.; Reinscheid, R. K.; Hoffmann, C.; Schulz, S.

doi:10.1101/2022.07.25.501346

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bioRxiv · 10.1101/2022.07.25.501346

A bead-based GPCR phosphorylation immunoassay for high-throughput ligand profiling and GRK inhibitor screening

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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 agonists 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.

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Kaufmann, J., Blum, N. K., Nagel, F., Anna Schuler, A. S., Drube, J., Degenhart, C., Engel, J., Eickhoff, J. E., Dasgupta, P., Fritzwanker, S., Guastadisegni, M., Schulte, C., Miess-Tanneberg, E., Maric, H. M., Spetea, M., Kliewer, A., Baumann, M., Klebl, B., Reinscheid, R. K., Hoffmann, C., Schulz, S.. 2022-07-25. A bead-based GPCR phosphorylation immunoassay for high-throughput ligand profiling and GRK inhibitor screening. https://doi.org/10.1101/2022.07.25.501346

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