Figure 2: LRP6 and Frz8 interact with each other in living cells.

(a) Schematic diagram of LRP6 and Frz8 deletion mutants. (b–i) Interaction between LRP6 and Frz8 as assessed by BRET saturation curves and competition assays. In saturation assays, HEK293 cells were co-transfected with a constant DNA concentration of LRP6-Rluc8 (b, black line and h) or LRP6-ΔN-Rluc8 (b, red line) and increasing DNA concentration of Frz8-GFP2 (b) or Frz8-ΔN-GFP2 (h). In competition assays, constant plasmids of LRP6-Rluc8 with Frz8-GFP2 or Frz8-ΔN-GFP2 were co-expressed with an increasing amount of untagged Frz8 (c), Frz8-ΔN (i), full-length LRP6 (d), LRP6-ΔC (e) or LRP6-ΔN plasmid (f). The BRET, total luminescence and total fluorescence were measured 48 h after transfections. The BRET levels are plotted as a function of the ratio of [receptor-GFP2]/[receptor-Rluc8] used as an index for the concentration of receptor-GFP2 constructs expressed. The results are expressed as the mean±s.d. of at least three independent experiments carried out in triplicates. The curves were fitted using a non-linear regression equation assuming a single binding site (GraphPad Prism). Of note, the BRET activity induced by a constant amount of LRP6-Rluc8 and Frz8-GFP2 was not affected by stimulation with Wnt3a (g), and co-transfection of LRP6-Rluc8 with Frz8-ΔN-GFP2 also increased the BRET ratio (h), which was inhibited by untagged Frz8-ΔN (i).