Fig. 3

LPA₁ inhibits CXCL12-induced Gα_i activation, β-arrestin recruitment, and ligand binding to CXCR4. A Schematic diagram of heterotrimeric G protein activation by BRET. B HEK293A cells were transfected with Myc-CXCR4 alone or together with Flag-LPA₁ in the presence of Gα_i1-Rluc8, Gβ₃, and Gγ₉-GFP2 (at a 1:1:1 DNA ratio), and CXCL12-induced Gα_i1 activation was measured. C HEK293A cells were transfected with Flag-LPA₁ alone or together with Myc-CXCR4 in the presence of Gα_i1-Rluc8, Gβ₃, and Gγ₉-GFP2 (at a 1:1:1 DNA ratio), and LPA-induced Gα_i1 activation was measured. Data represent the mean ± SEM of n = 3 to 4 independent experiments performed in triplicate. D Schematic diagram of β-arrestin recruitment to GPCR by BRET. E, F Ligand-induced β-arrestin1/2 BRET assay was performed in HEK293A cells. Vehicle or CXCL12 (50 nM)-induced ΔBRET increase between CXCR4-Rluc8 and mCitrine-β-arrestin1 (E) or mCitrine-β-arrestin2 (F) was measured. Data from n = 3 independent experiments (performed in triplicate) are presented as one site binding model. G PRESTO-Tango assay was performed for the recruitment of β-arrestin to CXCR4. Data represent the mean ± SEM of n = 4 independent experiments performed in duplicate. H Ligand binding assay was performed in HEK293A cells expressing Gluc-CXCR4 alone (Left panel) or together with Flag-LPA₁ (Right panel). TZ14011-AF488 was used as a acceptor. IT1t (20 μM) was pretreated for the nonspecific BRET signal. The specific BRET signal was defined as the difference between the total BRET signal and the nonspecific BRET signal. I BRET-based competition binding experiments with CXCL12. After treatment with 10 nM TZ14011-AF488, unlabeled CXCL12 (0 to 3.3 μM) were applied for competition. Data represent the mean ± SEM of n = 3 to 4 independent experiments performed in duplicate. Statistical significance was tested by two-way ANOVA followed by Bonferroni’s multiple comparison test. *P < 0.05; ****P < 0.0001; ns, not significant