Figure 4.

Effect of tyrosine kinase modulators on the ability of GRK2 to regulate CXCR4/Gi coupling. Normalized FRET ratio/dose–response curves were obtained as detailed in Methods in HEK293 cells expressing CXCR4 and the Gi1 sensor as well as wild-type GRK2 where indicated. Cells were challenged with 0.001 nM to 1 μM CXCL12 under different conditions. (A) Preincubation for 1 h with 5 μM PP2 (a Src kinase inhibitor) does not alter the response to CXCL12 alone or prevents the decreased Gi1 activation upon GRK2 overexpression. Data are reported as mean ± SEM of quadruplicate determinations in paired FRET assays representative of n = 2 independent experiments. In this particular experiment, the EC₅₀ values for CXCL12 stimulation were 13 nM (control), 9.9 nM (after PP2 preincubation), 23 nM (overexpressed GRK2), and 24 nM (after PP2 preincubation with overexpressed GRK2). (B) EGF does not further affect CXCL12-mediated Gi1 activation under conditions of GRK2 overexpression. Data are reported as mean ± SEM of quadruplicate determinations in paired FRET assays representative of n = 6 independent experiments. In this particular experiment, the EC₅₀ values were 9 nM (CXCL12 challenge under conditions of overexpression of GRK2 alone) and 2 nM (CXCL12 plus EGF challenge).