Fig. 4: β-arrestin recruitment and ERK activation.
From: Computationally designed GPCR quaternary structures bias signaling pathway activation
a (Left) Schematic representation of the BRET-based ligand-induced β-arrestin-2 (βarr2) translocation assay. (Right) CXCL12-promoted βarr2 recruitment to CXCR4 measured by BRET in HEK293T cells transfected with CXCR4-RLuc, WT or mutant as indicated, and βarr2-YFP. BRET480-YFP between CXCR4-RLuc and βarr2-YFP was measured after the addition of coel-h (10 min) and CXCL12 (15 min). Data shown represent the mean ± SEM of at least three independent experiments and are represented as ΔBRET. b (Left) Phosphorylation at S324/5 of WT and W1955.34L CXCR4 promoted by stimulation with 200 nM CXCL12 for 30 min detected using an anti pS324/5 antibody (pCXCR4 indicates the CXCR4-S324/5 phosphorylation band; non-SP correspond to a non-specific band). (Right) Quantification of phosphorylation bands normalized as a function of the intensity of the total HA-CXCR4 detected using an anti-HA antibody. Shown in the inset is the fold increase in phosphorylation over basal levels. Data shown represent the mean ± SEM of three independent experiments. Statistical significance was assessed using unpaired t test. #p = 0.001, ##p < 0.0001, n.s. not significant p > 0.05. c (Left) Schematic representation of ERK activation by CXCR4. (Right) ERK phosphorylation in U87 stably expressing equivalent levels of WT and W1955.34L CXCR4 induced by stimulation with 10 nM CXCL12 for the indicated times was monitored by HTRF. CXCR4 mutations predicted to stabilize the open-dimer or the closed-dimer conformation are annotated with a blue or red dimer symbol, respectively. Data shown represent the mean ± SEM of at least three independent experiments.
