FIGURE 6.
GRK2 involvement in ETA desensitization. A, for expression control, HEK-293 cells were transiently transfected with ETA-WT and with the indicated GRK2 construct. Control cells were transfected exclusively with ETA-WT. After 24 h, cells were lysed, and the expression of GRK2/3 was investigated in a Western blot experiment using a GRK2/3-specific antibody (upper) and by densitometric analysis (lower). Bars represent the mean ± S.D. of three independent experiments. No significant differences in GRK2 expression could be observed. B, HEK-293 cells were transiently transfected with constructs of ETA-WT or ETA-6PD. Cotransfection was performed with 5 ng of plasmid DNA/cm2 of culture surface coding for WT or mutant GRK2 as indicated. No GRK2 cotransfection was performed with control cells. The cells were stimulated with 100 nm ET-1 for 30 min. IP1 accumulation was quantified as described under “Experimental Procedures.” Data were collected in triplicate. From each data point, the basal value obtained with non-stimulated control cells was subtracted to obtain PLC activity above the basal level (PLCnet). Inhibition of PLC activity is given in percent (means ± S.D.) and calculated as follows: 100 × (PLCnet of control cells − PLCnet of GRK-transfected cells)/(PLCnet of control cells). Results were compared with the values obtained with control cells using one-way ANOVA and Bonferroni correction. C, High level cotransfection using 25 ng of plasmid DNA/cm2 of culture surface encoding GRK2 variants. The experimental conditions and data processing are analogous to those described for B. D, knockdown of GRK2 using siRNA. HEK-293 cells were transfected with ETA-WT and control siRNA or GRK2-specific siRNA. After cell lysis, the expression of GRK2/3 was investigated in a Western blot experiment (upper) and by densitometric analysis (lower). Bars represent the mean ± S.D. of three independent experiments. Statistical analysis was performed using Student's t test. E, HEK-293 cells were transfected with ETA-WT or ETA-6PD and 75 nm control siRNA (control cells) or GRK2-specific siRNA. After 48 h, the cells were stimulated with 100 nm ET-1 for 30 min. IP1 accumulation was quantified as described under “Experimental Procedures.” From each data point, the basal value obtained with non-stimulated control cells was subtracted to obtain PLCnet. Bars represent the additional signal intensity obtained by treatment with GRK2-specific siRNA compared with control cells given in percent (means ± S.D.) and calculated as follows: 100 × ((PLCnet/PLCnet of control cells) − 1). Data were collected in triplicate. Statistical analysis was performed using Student's t test. *, p < 0.001; **, p < 0.01.