Fig. 9

Roles of GRK2 and GRK6 in the desensitization of β2 adrenoceptors and its transactivation of EGFRs. A–D Cells were treated with 10 μM of isoproterenol for 5 min and washed/rechallenged to induce desensitization. A Con-KD and GRK2-KD HEK-293 cells were transfected with β2AR. ##p < 0.01 when the ISO/GRK2-KD group was compared with its vehicle-treated group; ***p < 0.001 when the ISO/Con-KD group was compared with its vehicle-treated group (n = 3). B Con-KD and GRK6-KD HEK-293 cells were transfected with β2AR. ***p < 0.001 when the ISO/Con-KD group was compared with other experimental groups (n = 3). C Con- and GRK2-KD HEK-293 cells were transfected with EGFR-GFP and FLAG-β2AR. **p < 0.01 compared with other groups of the Con-KD and GRK2-KD cells (n = 3). Cell lysates from Con-KD and GRK2-KD cells were immunoblotted with antibodies against GRK2 and β-actin. The knockdown efficiency of arrestin2 and arrestin3 was about 85%. D Con-KD and GRK6-KD HEK-293 cells were transfected with EGFR-GFP and FLAG-β2AR. ##p < 0.01 compared with other groups of the Con-KD and GRK2-KD cells (n = 3). Cell lysates from Con-KD and GRK6-KD cells were immunoblotted with antibodies against GRK6 and β-actin. The knockdown efficiency of GRK6 was about 95%. E Con-KD and GRK2-KD HEK-293 cells were transfected with β2AR. Cells were pretreated with either vehicle or 200 nM of AG1478 for 30 min, followed by treatment with 10 μM of isoproterenol for 2 min to induce ERK activation. The ratios of ISO(+)/ISO(−) were 2.3 ± 0.45 (Veh/Con-KD), 0.95 ± 0.32 (AG1478/Con-KD), 3.5 ± 0.75 (Veh/GRK2-KD), and 3.3 ± 0.60 (AG1478/GRK2-KD). AG1478/Con-KD group was significantly different from the Veh/Con-KD group (p < 0.05, n = 3) and the groups of GRK2-KD cells (p < 0.01, n = 3). F Con-KD and GRK6-KD HEK-293 cells were transfected with β2AR. Cells were treated as in E. The ratios of ISO(+)/ISO(−) were 2.2 ± 0.34 (Veh/Con-KD), 0.97 ± 0.26 (AG1478/Con-KD), 2.5 ± 0.45 (Veh/GRK2-KD), and 2.05 ± 0.37 (AG1478/GRK2-KD). AG1478/Con-KD group was significantly different from other groups (p < 0.05, n = 3)