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. 2022 Jan 25;13:487. doi: 10.1038/s41467-022-28056-7

Fig. 3. GRK-subtype-dependent β-arrestin functionalities.

Fig. 3

a, Schematic representation of the HiBiT-based AT1R-internalization assay. N-terminally HiBiT-fused AT1R was expressed in the parent or the GRK-deficient cell lines and a cell-surface AT1R level upon ligand stimulation was measured by complementation with the recombinant LgBiT protein added to the conditioned media. b, Luminescent kinetics of the AT1R-internalization assay. The indicated cells were stimulated with 10 nM Ang II, 100 nM TRV027 or 10 nM Ang II pretreated with 1 µM YM-254890. At every measurement time point, a ligand-stimulated count was normalized to a vehicle count. c, Quantifications of the AT1R-internalization assay. Bars and error bars represent mean and SEM, respectively, of 5 independent experiments with each performed in duplicate. d, AlphaLISA-based pERK measurement. AT1R-expressing cells were pretreated with or without the PKC inhibitor, Go6983 (3 µM), and stimulated with the indicated ligand for 30 min. e–g, Schematic illustration (e), luminescent kinetics (f) and quantification (g) of the tail-hanging β-arrestin-recruitment assay. AT1R-Sm and Lg-β-arrestin1 with the truncation in the finger-loop region (∆FLR) were expressed in the parent cells and treated with the indicated conditions. Shaded regions denote SEM (n = 3–5; b, f). Cell lines used were ∆GRK2/3, CL2; ∆GRK5/6, CL2; ∆GRK2/3/5/6, CL2 (a–c). In Fig. 3b, f, lines and shaded regions represent mean and SEM, respectively, of 3 independent experiments with each performed in duplicate. In Fig. 3c, d, g, bars and error bars represent mean and SEM, respectively, of 3 independent experiments with each performed in duplicate. In Fig. 3c, d, g, * and ** represent P < 0.05 and 0.01, respectively, with two-tailed multiple t-test (d) or one-way ANOVA (g) or two-way (c) ANOVA followed by the Dunnett’s test for multiple comparison analysis (with reference to the vehicle stimulation (c) or the Ang II stimulation (g)). ns, not significantly different between the groups. See Supplementary statistics data file for additional statistics and exact P values.