Figure 3.

GRK2 activity and tyrosine phosphorylation status is key for inhibiting CXCR4-mediated Gi1 activation. Normalized FRET ratio/dose–response curves were obtained as detailed in Methods in HEK293 cells expressing CXCR4, Gi1 sensor, and either wild-type GRK2 (panel A), inactive GRK2 (GRK2-K220R) (panel B), or a mutant GRK2 unable to be phosphorylated at the specific tyrosine residues 13, 86, and 92 (GRK2-Y3F) (panel C). Cells were challenged with 0.001 nM to 1 μM CXCL12 as in previous figures. Data are reported as mean ± SEM of quadruplicate determinations in paired FRET assays representative of n = 11 (A) or 4 (B, C) independent experiments. In these particular experiments, the EC₅₀ values for CXCL12 were 2.8 nM (endogenous GRK2) and 9 nM (overexpression of GRK2) in panel A and 13 nM (endogenous GRK2), 27 nM (overexpression of GRK2-K220R), and 17 nM (overexpression of GRK2 Y3F) in panels B and C.