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. 2024 Jul 3;7:802. doi: 10.1038/s42003-024-06490-1

Fig. 2. β-arrestin2 recruitment to the muscarinic M2 and M5 acetylcholine receptors (M2R, M5R) is dependent on the ability of the GRK to be recruited to the membrane.

Fig. 2

a–d Halo-Tag-β-arrestin2 recruitment to M2R-NLuc (a, b) or M5R-NLuc (c, d) was measured in ΔQ-GRK cells in absence of GRKs (EV-transfected) and in presence of WT GRK2, GRK2-D110A, GRK2-R587Q, GRK2-D110A,R587Q or their respective membrane-tethered versions via a CAAX box (b, d). Normalized Δ net BRET change (%) upon stimulation with 100 µM of Acetylcholine (ACh) is shown for M2R (a, b) of n = 3 and for M5R (c, d) of n = 4 (except M5R: EV and WT GRK2 of n = 7; GRK2-D110A, GRK2-R587Q and GRK2-D110A,R587Q of n = 6; GRK2-D110A-CAAX of n = 3) independent experiments ± SEM, normalized to GRK2 (a, c) or GRK2-CAAX (b, d). The data measured in absence of GRKs (EV) is shown in both graphs each (a, b for M2R; c, d for M5R) for direct comparison. Statistical differences were tested using one-way ANOVA, followed by a Tukey’s test (ns not significant; *p < 0.05; **p < 0.01; *** p < 0.001; **** p < 0.0001). Detailed statistical results are provided in Supplementary Table 4 (M2R) and Supplementary Table 6 (M5R).