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. Author manuscript; available in PMC: 2023 Oct 1.
Published in final edited form as: Br J Pharmacol. 2022 Jul 19;179(19):4692–4708. doi: 10.1111/bph.15900

Figure 3 – Analysis of β-agonist induced GRK5 phosphorylation and β-arrestin-mediated trafficking of the β2AR.

Figure 3 –

Kinetics of in vitro GRK5-mediated phosphorylation of β2AR in the presence of 50 μM ISO, RAC, DOB and HIG are shown as (A) a representative autoradiograph of 32P incorporation into β2AR and (B) time course of β2AR phosphorylation compared to ISO. Reaction rates were normalized to β2AR phosphorylation in the presence of ISO at 40 min of incubation and plotted as kinetic curves using GraphPad Prism. Data are mean ± SEM, n = 3. Internalization (C) and endosomal localization (D) of β2AR was assessed by BRET. β2AR-mediated internalization and endosomal localization were assessed by transfecting HEK293 cells with BRET donor β2AR-Rluc and acceptors Venus-Kras as membrane marker or Venus-Rab5 as early endosome marker. After 48 h, cells were stimulated with increasing concentrations of ISO, RAC, DOB or HIG (10−10 to 10−4 M) for 20 min. Signal generated by each condition was plotted and fitted using the three parameters non-linear regression curve fitting in GraphPad Prism to generate concentration/activity curves. Signal was normalized to the average maximal response of ISO as 100%. Data are mean ± SEM, n = 6.