Fig. 6
From: Agonist-induced dimer dissociation as a macromolecular step in G protein-coupled receptor signaling
Relationship between FZD6 dimerization and phosphorylation of ERK1/2. a Mouse lung epithelial (MLE-12) cells were stimulated with WNT-5A (300 ng ml−1) for 0, 2.5, 5, 10, 20 min. Cell lysates were analyzed for P-ERK1/2 and β-actin by immunoblotting. Additional information in Supplementary Fig. 6. b Bar graph summarizes three independent experiments. Densitometry data were normalized to the unstimulated control. P = 0.0037; F (4, 15) = 6.212. *P < 0.01; **P < 0.001 (one-way ANOVA with Fisher’s least significant difference (LSD) post hoc analysis). c Generation of a FZD6 +/− MLE-12 line using the CRISPR/Cas9 system resulted in decreased basal P-ERK1/2. d CRISPRa-mediated enhancement of FZD6 expression led to increased P-C-RAF and P-ERK1/2. Additional information in Supplementary Fig. 6. SNAP-FZD6 wt- and SNAP-FZD6 dimer mutant-induced ERK1/2 phosphorylation in the absence e and presence f of the porcupine inhibitor C59 (5 µM, overnight) were probed by immunoblotting for the SNAP-tag, P-ERK1/2, total ERK1/2 and β-actin. g The bar graph summarizes densitometry data of P-ERK1/2 normalized to total ERK1/2 from 6 different experiments. P = 0.0005; F (2, 15) = 13.08. ***P < 0.001. Empty vector transfection was used as mock. h Increasing amounts of plasmid of either SNAP-FZD6 wt or SNAP-FZD6 dimer mutant in the presence of C59 show a dose-dependent increase in P-ERK1/2 only with the dimer mutant. Data from three independent experiments for the dimer mutant were fit to a linear least-squares model (slope = 0.40 ± 0.10; R 2 = 0.5943) and this line is shown superimposed. The ascending slope in the case of the dimer mutant, but not wt is significantly different from non-zero (P = 0.0012). Error bars give s.e.m. i Based on our experimental data, we propose a signaling paradigm where FZD6 dimers confer the inactive receptor species, which can dynamically produce monomeric species showing a higher degree of activation, which feed into ERK1/2 signaling. This dissociation-dependent activation can be evoked by agonist (WNThigh) or mutation of the dimer interface (symbolized by red stars). WNTlow conditions can be achieved in the presence of C59, which reduces FZD6 wt signaling
