Fig. 2

FZD₆ dimer interface is formed by TM4 and TM5. a–c Presents the homology model of the FZD₆ homodimer interactions involving residues F337^4.55f, A344^4.62f, N346^4.64f, D365^5.41f, R368^5.44f and Y369^5.45f. Rainbow coloring, N-terminus—blue; C-terminus—red. Main interactions involve TM4 (green) and TM5 (yellow). a Upper panel: from intracellular side; lower panel: from extracellular side. b side view and c extracellular view with zoom on dimer interface. Residues are shown as sticks in b and c. d Space filling model of the FZD₆ dimer and each monomer (side view). Dimer interface area (ca 1,150 Ų) is colored in dark gray. Yellow/magenta identify residues important for dimer interaction as demonstrated by site-directed mutagenesis. For more details, see Supplementary Figs. 2 and 3. e, f dcFRAP analysis of V5-FZD₆-mCherry and the FZD₆-GFP dimer mutant showed that the mobile fraction of the dimer mutant is not affected by biotin-anti-V5/avidin surface crosslinking indicating a reduced ability to form dimers. Only data for FZD₆-GFP in the absence (gray) and presence (red) of CL are shown. P = 0.1438; t = 1.482; df = 57. N = 23 ROIs before CL; N = 36 after CL from three independent experiments. For analysis of additional mutations see Supplementary Fig. 4. g, h A corresponding FZD₆-GFP dimer control mutant, where mutations preserving side chain chemical properties were introduced, still interacts with the wt V5-FZD₆-mCherry as analyzed by dcFRAP. P < 0.0001; t = 4.839; df = 153. N = 75 ROIs before CL; N = 80 ROIs after CL from three independent experiments. i FCS experiments measuring the translational diffusion time (τ _D) of FZD₆-GFP and the dimer mutant FZD₆-GFP in the presence of the porcupine inhibitor C59 (5 µM, overnight). Data are presented as a scatter dot plot. P < 0.0001; t = 4.552; df = 29. N = 17 cells expressing FZD₆-GFP; N = 14 cells expressing FZD₆-GFP dimer mutant from three independent experiments. Error bar gives s.e.m. ***P < 0.001 (two-tailed t-test)