Fig. 2: In cellulo assays confirm wildtype-like behavior of GRK2 S670A.

a Schematic depiction of the split luciferase complementation assay (SLC)³³. Agonist activation of the Gα_q-coupled muscarinic acetylcholine receptor M1 (M1mAChR or mAChR1) leads to the dissociation of the G protein leading to Gα_q-phospholipase C-β (PLCβ) interaction. The Gα_q subunit is tagged with the smaller fragment (orange) of the click-beetle luciferase whereas the larger fragment (red) is fused to PLC-β3. Using this split luciferase sensor, we monitored mAChR1-mediated Gα_q-PLCβ interaction in ∆Q-GRK cells in the presence of GRK2 WT, GRK2 S670A and in absence of GRKs (empty vector (EV)-transfected). Data are analysed in ACh-concentration dependent (b), mean of n = 3 independent experiments and time-dependent (c) manner as relative light units (RLU), mean of n = 4 independent experiments. Error bars represent the standard error of the mean (SEM). Individual data points for each experiment are shown in lighter colors. For concentration-dependent analysis data points were averaged over a time frame from 25 to 30 min (3 timepoints) post substrate addition. Time-dependent analysis is depicted for an ACh-concentration of 10 µM. d Schematic representation of the performed NanoBret β-arrestin (βarr) recruitment assay³⁴. Agonist activation of the NanoLuciferase (NanoLuc)-tagged GPCR results in phosphorylation of the receptor by GRKs and subsequent recruitment of the Halo-Tag-βarr fusion protein. The recruitment-induced change in proximity of NanoLuc and Halo-Tag increases measured BRET ratios, enabling both the agonist concentration-dependent as well as the time-dependent analysis of βarr recruitment. e GRK-dependent Halo-Tag-βarr2 recruitment to the β₂AR-NanoLuc upon stimulation with isoprenaline (Iso) in ∆GRK2/3/5/6 HEK293 knockout cells (∆Q-GRK). Cells were co-transfected with either EV, wildtype GRK2 (GRK2 WT) or GRK2 S670A. BRET data are presented as normalized BRET ratio, mean of n  =  4 independent experiments  ±  SEM. f GRK-dependent Halo-Tag-βarr2 recruitment to the β₂AR-NanoLuc shown over time at a stimulation level of 100 nM isoprenaline for n  =  4 independent experiments ±  SEM. Individual data points for each experiment are shown in lighter colors.