Fig. 4
From: Agonist-induced dimer dissociation as a macromolecular step in G protein-coupled receptor signaling
Absence of a role for DVL and G proteins in FZD6 dimer formation. a–d Analogous to data shown in Fig. 1c, d, we investigated the effect of V5-FZD6-mCherry surface crosslinking (CL) on the mobility of FZD6-GFP in the presence of a, b pan-DVL siRNA (P < 0.0001; t = 6.854; df = 53; N = 33 ROIs before CL; N = 22 after CL from three independent experiments) and c, d overexpressed DVL2-MYC (P < 0.0001; t = 6.243; df = 77; N = 36 ROIs before CL; N = 43 after CL from three independent experiments). e, f dcFRAP experiments in combination with chemical surface crosslinking show that the FZD6 dimer mutant is still capable of assembling in an inactive state with DVL2-GFP (P < 0.0001; t = 5.173; df = 74; N = 15 ROIs before CL; N = 61 after CL from three independent experiments), but not with Gαi1-GFP (P = 0.5646; t = 0.5801; df = 48; N = 29 ROIs before CL; N = 21 after CL from three independent experiments). Error bars give s.e.m.; ***P < 0.001; ns non significant (two-tailed t-test). g V5-FZD6-mCherry and FZD6-GFP expressed in HEK293 cells showed distinct co-localization predominantly in the membrane. The FZD6-R511C mutation (predominantly localized to lysosomes and unable to interact with heterotrimeric G proteins16, 25) forced the wt FZD6 to be retained intracellularly suggestive of dimer formation. Co-expression of the FZD6-R511C mutant together with the dimer mutant of FZD6 did not result in co-localization. Due to the lack of dimerization, the FZD6 dimer mutant showed surface expression in the presence of FZD6-R511C. Size bar—10 µm
