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. 2021 Apr 15;11:8288. doi: 10.1038/s41598-021-87417-2

Figure 2.

Figure 2

GRK2 and GRK3 mediate phosphorylation at Ser317/Thr318 and enhance β-arrestin-2 recruitment. (A) HEK293 cells stably expressing HA-hD2LR were stimulated with 1 µM quinpirole, 1 µM PMA or 10 µM forskolin for 10 min at 37 °C. Cell lysates were immunoblotted with anti-pSer317/Thr318 [5102] antibody. Blots were stripped and reprobed for D2R [5106] to confirm equal loading of the gel. Blots are representative, n = 3. (B) Cells described in (A) were pre-incubated with either vehicle (DMSO; control (−)) or the GRK2/3-specific inhibitor compound 101 (cmpd 101) at the indicated concentrations for 30 min at 37 °C, then treated with water (−) or 1 µM quinpirole for 10 min at 37 °C. Lysates were immunoblotted as described in (A). Blots are representative, n = 3. (C,D) Cells described in (A) were transfected with siRNAs targeting GRK2, GRK3, GRK2 and GRK3 (GRK2/3) or a scrambled control (SCR) (C) or with siRNAs targeting GRK5, GRK6, GRK5 and GRK6 (GRK5/6) or a scrambled control (SCR) (D). 72 h post-transfection, cells were stimulated with 1 µM quinpirole for 10 min at 37 °C and cell lysates were immunoblotted as described in (A). Blots were stripped and reprobed for D2R [5106] to confirm equal loading of the gel. Densitometry analysis, shown above the blots, was normalized to the signal obtained in SCR-transfected cells, which was set to 100%. Data are mean ± SEM from five to six independent experiments. (*p < 0.05 vs. SCR by one-way ANOVA with Tukey post-test). (E,F) β-arrestin2 recruitment to the D2R in the presence and absence of overexpressed GRK2. HEK 293 cells were transfected with cDNA encoding hD2LR-NLuc, YFP-β-arrestin2, and either GRK2 (E) or pcDNA3.1 control (F) as described in Methods. Transfected cells were then preincubated with either vehicle (DMSO) or 30 µM cmpd101 for 30 min at 37 °C before stimulation with increasing concentrations of quinpirole for 10 min at 37 °C. Data represents mean ± SEM from 3–4 separate experiments and are normalized to the maximal effect of quinpirole in the presence of GRK2 overexpression.