Figure 4

BRET ² analysis of protein-protein interactions in the Wnt/β-catenin signaling pathway. Panel A, Lef/Tcf-sensitive gene activation in HEK 293 cells in response to Wnt3a. HEK 293 cells were co-transfected with either Rfz1 and M50 or Rfz1-Rluc and M50. HEK 293 cells were stimulated with Wnt3a (20 ng/ml) for 8 hr. Lef/Tcf-sensitive transcription activity was determined. Panel B, HEK 293 cells stably expressing Rfz1 were transiently co-transfected with Axin-Rluc and/or Dvl2-GFP2, Dvl2-Rluc, GSK3-GFP2, β-cat-GFP2, β-cat-Rluc as indicated for 48 h and then treated with Wnt3a (20 ng/ml) for 15 min. BRET ratios were measured by addition of DeepBlue C (5 μM) in cells co-expressing Rluc and GFP2 fusion proteins. Results are expressed as the mean ± S.E. of at least three independent experiments. Panel C,the β-adrenergic agonist isoproterenol (Iso) stimulates the interaction between GPCR and β-arrestin2. HEK293 cells were transiently transfected with β₂AR-Rluc and β-arrestin2-GFP2 for 48 h, and then treated with Iso (10 μM) or Iso plus the β-adrenergic antagonist propranolol (Pro, 10 μM) for 0 to 60 min. Panel D, Iso stimulates the interaction between GPCR and the heterotrimeric G protein subunit Gβ₁. HEK293 cells were transiently transfected with β₂AR-Rluc, Gβ₁-GFP₂, Gα_s, and Gγ₂ for 48 h, and then treated with agonist Iso (10 μM) or Iso plus antagonist Pro (10 μM) for 0 to 30 min. BRET ratios (GFP2/Rluc activity) were measured by addition of DeepBlue C (5 μM) in cells coexpressing Rluc and GFP2 fusion proteins. Results are expressed as the mean ± S.E. of three independent experiments. Note that isoproterenol stimulates GPCR interaction with G-protein first (peaks within 5 min of agonist) and later leads to association of GPCR with β-arrestin2 (peaks at 30 min). Both sets of protein-protein interactions are blocked by simultaneous addition of propranolol with agonist.