Fig. 4.

Differential phosphorylation and β-arrestin recruitment by CCL19 and CCL21 are caused by strict specificity and bias among GRK isoforms for activated CCR7. (A) HEK-293 cells stably expressing CCR7-FLAG or CCR7-CFP/ß-arrestin2-YFP were transfected with control siRNA or siRNA specific for GRK2, GRK3, or GRK6. A representative immunoblot showing the effect of siRNA on GRK expression is shown. (B) Agonist-induced phosphorylation for each condition is expressed as a percentage of CCL19-mediated phosphorylation in control siRNA-treated cells. The mean ± SE from four to nine independent experiments is represented by the bar graph and demonstrates the effect of GRK expression on CCR7 bulk phosphorylation. (C) Cells were stimulated with 100 nM CCL19 or 100 nM CCL21, and the recruitment of ß-arrestin2 was measured by FRET and normalized to CCL19 responses in control transfected cells. The bar graft shows the effect of GRK expression on the integrated FRET response after 10 min in response to each ligand. Data represent the mean ± SE from three to eight independent experiments, each done in duplicate. Statistical significance was determined by paired two-tailed t tests. *, P < 0.05; **, P < 0.01; ***, P < 0.001).