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 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.

Key phosphorylation sites for robust β-arrestin2 binding at the MOR revisited

“…Finally, increasing evidence is starting to suggest that GRK5/6 tend to phosphorylate sites “proximal” to the plasma membrane within the C-terminus of a GPCR, while GRK2/3 are able to phosphorylate more “distal” sites (Kaufmann et al, 2022). Our data agrees with this “geometric” model, with Thr 370 and Ser 375 being “proximal” while Thr 376 and Thr 379 would be the “distal”, GRK2/3 sites.…”

“…PierceTM HA epitope tag antibody was obtained from Thermo Scientific (Rockford, IL, USA). The rabbit polyclonal phosphosite-specific µ-opioid receptor antibodies anti-pT370 (7TM0319B), anti-pT376 (7TM0319D), anti-pT379 (7TM0319E), anti-pS375 (7TM0319C) and anti-HA antibody (7TM000HA) were obtained from 7TM Antibodies (Jena, Germany) (Kaufmann et al, 2022). The secondary horseradish peroxidase (HRP)-linked anti-rabbit antibody was purchased from Cell Signaling (Frankfurt, Germany).…”

G protein–receptor kinases 5/6 are the key regulators of G protein–coupled receptor 35–arrestin interactions

“…The production of genome-edited cell lines lacking expression of one or more of the ubiquitously expressed GRK isoforms, GRK2, GRK3, GRK5, and GRK6 ( 13 ), also offers new approaches to functional reconstitution and definition of GRK selectivity. In addition, the development of both selective small molecule GRK inhibitors ( 14 , 18 ) and similarly, the development of phospho-site–specific antisera that detect sites of regulated or constitutive phosphorylation within GPCRs ( 5 , 6 , 9 , 27 , 28 ) have been providing new insights.…”

Phosphorylation Sites of the Gastric Inhibitory Polypeptide Receptor (GIPR) Revealed by Trapped-Ion-Mobility Spectrometry Coupled to Time-of-Flight Mass Spectrometry (TIMS-TOF MS)

“…MS1 spectra were taken from 100 to 1700 m/z using a TIMS-TOF Pro Q-TOF (Bruker Daltonics) operating in PASEF mode 26 with an ion mobility range (1/k 0 ) of 0.60−1.60 Vs/cm 2 . For tandem MS, the following parameters were used: number of PASEF MS/MS scans (10); total cycle time (1.16 s); target intensity (20 000); intensity threshold (2500); charge range (0−5); isolation width (2 m/z for m/z < 700 and 3 m/z for m/z > 700); collisional energy (20−59 eV). Precursor ions were excluded within a 0.4 min window and reconsidered if the intensity was 4-fold higher than the previous selection.…”

“…Techniques for probing GPCR phosphorylation include site-directed mutagenesis (Ser/Thr → Ala to eliminate potential phosphorylation sites; Ser/Thr → Glu to mimic phosphorylation in an immutable form), 32 P labeling, and use of phosphorylation-specific antibodies. , Mass spectrometry (MS) analysis has emerged as a powerful approach for determining the locations and abundances of phosphorylation sites. This technique does not require prior knowledge of sites, in contrast to mutagenesis or antibody-based strategies. − However, identifying phosphorylation sites within membrane proteins such as GPCRs via MS is challenging, because of protein size, low abundance, and hydrophobicity.…”