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

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

SUBMITTER: Kaufmann J 

PROVIDER: S-EPMC9646841 | biostudies-literature | 2022 Nov

REPOSITORIES: biostudies-literature