Fig. 5: Effects of P6.43F mutants in FZDs on DVL2 recruitment and WNT-induced signaling.
From: Residue 6.43 defines receptor function in class F GPCRs
a Schematic presentation of the DVL2 bystander BRET assay. b DVL2 recruitment to FZD4, FZD5, FZD6, and FZD7. Bystander BRET ratio changes (between Nluc-DVL2 and Venus-KRas) were assessed in ΔFZD1–10 HEK293 cells in the presence of overexpressed wild-type and mutated SNAP-tagged FZD4, FZD5, FZD6 and FZD7. The data were normalized to the receptor surface expression levels and are shown as mean ± SEM of four independent experiments. Data were analyzed for each wild-type/mutant pair using paired two-tailed t-test. *P < 0.05, **P < 0.01, ns not significant. P = 0.0146 for FZD5 wild-type vs. mutant and P = 0.0027 for FZD6 wild-type vs. mutant. c Refined bystander BRET between the Nluc-tagged DEP domain of DVL2 and Venus-KRas, where the BRET values are plotted over the signal for the surface-expressed SNAP-FZD6 (black) or SNAP-FZD6 P6.43F (gray). Data show basal DEP recruitment in dependence of the surface-expressed receptor construct in the presence of C59. Data are shown as mean ± SD of four independent experiments. d WNT-3A (1000 ng/ml)-induced TOPflash signaling in ΔFZD1–10 HEK293 cells in the presence of overexpressed wild-type and mutated SNAP-tagged FZD4, FZD5, FZD6, and FZD7. TOPflash data are normalized to the vehicle control for each individual receptor isoform and mutant. Data were analysed using one-way ANOVA with Fisher’s LSD test. **P < 0.01, ***P < 0.001. P = 0.0030 for FZD4 wild-type vs. mutant and P = 0.0004 for FZD5 wild-type vs. mutant. Data are presented as mean ± SEM of four independent experiments. e Schematic presentation of the TOPflash transcriptional reporter assay. See Supplementary Fig. 6e for the cell-surface expression data of the SNAP-tagged receptors. Source data are provided as a Source Data file.
