Fig. 1
The dampening effect of US28 expression on CXCR4 - induced G protein-dependent signaling is controlled by the DRY motif. a Changes in agonist-induced cAMP concentrations after activation of CXCR4 were monitored in presence or absence of US28wt or US28 mutants. Cells were treated with CXCL12 at indicated concentrations and 10 μM forskolin for 15 min before measurement. BRET ratios were normalized on signal from mock-cotransfected cells stimulated with 100 nM CXCL12 (0%) or vehicle (100%). Curves represent means ± SEM of at least three independent experiments performed in triplicates (n = 3–8). b Agonist-induced recruitment of Gαi1 to CXCR4 in presence or absence of US28 or US28 mutants. BRET was measured in HEK293T cells cotransfected with CXCR4-Rluc8, Gαi1-91mVenus, Gβ1, Gγ2 and mock, US28wt or US28 mutants 2 min after addition of 100 nM CXCL12 or vehicle. ΔBRET was calculated by subtracting BRET ratios of vehicle-treated cells from BRET ratios of cells treated with 100 nM CXCL12 for each individual transfection. Columns represent means ± SEM of three independent experiments performed in quadruplicates. Statistical analysis was performed using one-way ANOVA with Dunnett’s post hoc test. *P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant