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. 2017 Mar;360(3):424–433. doi: 10.1124/jpet.116.238436

Fig. 1.

Fig. 1.

Regulation of Gαi2-AGS4 interaction by the prototype chemokine receptor CXCR4. (A) Top panel: Schematic of BRET system along with representation of hypothesized agonist-induced regulation of Gαi2YFP/AGS4-Rluc BRET association by receptor activation. For simplification, AGS4-Rluc is shown interacting with a single Gαi2-YFP; however, AGS4 can bind multiple Gαi subunits simultaneously (Kimple et al., 2004; Oner et al., 2010b), which may be responsible for the robust BRET signals observed (Oner et al., 2010b). Bottom panel: Net BRET signals were obtained from HEK293 cells transfected with 2 ng of phRLucN3::AGS4 or 2 ng of phRLucN3::AGS4-Q/A-Rluc and 500 ng of pcDNA3::Gαi2-YFP. Cells were also transfected in the presence or absence of 500 ng pcDNA3::CXCR4. Vehicle (Tyrode’s solution) or CXCL12 (100 ng/ml) were added to cells as indicated, followed by fluorescence and luminescence readings as described in Materials and Methods. The CXCR4 antagonist AMD3100 (plerixafor, 1 µg/ml) was added 10 minutes before agonist stimulation as indicated. Cells were treated with pertussis toxin (PTX, 100 ng/ml) 18 hours before receptor stimulation where indicated. Data are expressed as means ± S.E.M. from three independent experiments with triplicate determinations (n = 9). *P < 0.0001 as compared with vehicle-treated control group as determined by one-way analysis of variance with Tukey’s post-hoc test. (B) Top panel: Schematic representing the BRET system used to measure effect of chemokine receptor activation on the proximity of Gαi-AGS4 complex to CXCR4-Venus. Bottom panel: HEK cells were transfected with 2 ng of phRLucN3::AGS4 or 2 ng phRLucN3::AGS4-Q/A along with 750 ng pIRESpuro3::CXCR4-Venus in the presence or absence of 750 ng pcDNA3::Gαi2 as indicated. Vehicle (Tyrode’s solution) or CXCL12 (100 ng/ml) was added to cells as indicated, followed by fluorescence and luminescence readings as described in Materials and Methods. The CXCR4 antagonist AMD3100 (plerixafor, 1 µg/ml) was added 10 minutes before agonist stimulation as indicated. Cells were treated with pertussis toxin (PTX, 100 ng/ml) 18 hours before receptor stimulation where indicated. Data are expressed as means ± S.E.M. from at least three independent experiments with triplicate determinations (n = 9). *P < 0.0001 as compared with vehicle treated control group as determined by one-way analysis of variance with Tukey’s post hoc test.