dcFRAP in combination with chemical cell surface crosslinking reveals FZD₄-Gα_12/13 complex formation. (A) HEK293T cells express fluorescently tagged FZD₄ predominantly in the cell membrane. Size bar = 10 µm. (B) dcFRAP experiments are done in cells cotransfected with fluorescently tagged FZD₄, Gα subunits, and untagged βγ subunits. Micrographs show FZD₄-GFP and Gα₁₂-mCherry before, shortly after, and about 100 seconds after the high-laser-power photobleaching in a region of interest (white lines). Surface proteins are chemically crosslinked (CL) by Sulfo-NHS-LC-LC-biotin and avidin. Size bar = 2 µm. (C–H) The figure includes a schematic presentation clarifying the experimental setup, a confocal micrograph showing HEK293T cells coexpressing FZD₄ with the respective Gα subunit (size bar = 10 µm), fluorescence intensity curves before (gray) and after (red) CL for both FZD₄ and the respective G protein and a bar graph summarizing the mobile fractions of FZD₄ and the Gα subunit under each experimental condition. Color code for mobile fractions (consistent throughout the manuscript): white, FZD₄ before CL; red hatched, FZD₄ after CL; gray, Gα before CL; gray + red hatched, Gα after CL. ***P < 0.001 (n = 3). Error bars provide the S.E.M. ns = not significant.

Nonreceptor control ensures that the mobile fraction of Gα_12/13-mCherry is not affected by chemical surface crosslinking in the absence of FZD₄. HEK293 cells expressing farnesylated GFP-KRAS, untagged βγ subunits, and N-terminally tagged Gα₁₂-mCherry were used for a dcFRAP assay using chemical surface crosslinking (CL) with Sulfo-NHS-LC-LC-biotin and avidin as described in Fig. 1. CL affected neither the mobile fraction of GFP-KRAS nor that of Gα₁₂-mCherry (A) or Gα₁₃-mCherry (B). The figure includes a schematic presentation clarifying the experimental setup, a confocal micrograph showing HEK293T cells coexpressing GFP-KRAS with the Gα₁₂-mCherry (A) or Gα₁₃-mCherry (B; size bars = 10 µm), fluorescence intensity curves before (gray) and after (red) CL for both GFP-KRAS and Gα_12/13-mCherry, and a bar graph summarizing the mobile fractions of GFP-KRAS and the Gα subunit under each experimental condition. The data verify that the decrease in G protein mobile fraction observed for Gα_12/13-mCherry in the presence of FZD₄-GFP is not evoked by CL of endogenously expressed receptors. Error bars provide the S.E.M. ns, not significant. ***P < 0.001. Bar graph summarizes measurements from at least four independent experiments, each including data from several individual cells.

FZD₄-Gα_12/13 complex dissociates upon WNT stimulation. dcFRAP experiments were performed in HEK293T cells expressing FZD₄-GFP and Gα₁₂- or Gα₁₃-mCherry. The mobile fractions of the two proteins were determined before and after CL, as well as 5 and 10 minutes after CL/WNT stimulation (all WNTs at 300 ng/ml). Kinetic analysis of the mobile fraction indicates dissociation of the receptor G protein complex upon WNT stimulation. (A and B) For WNT-5A, we investigated WNT-induced dissociation from FZD₄-GFP for Gα₁₂- or Gα₁₃-mCherry. (C–E) For the other WNTs (WNT-3A, -7A, 10B), only WNT-induced Gα₁₂-mCherry dissociation was measured. *P < 0.05; **P < 0.01; ***P < 0.001 (n = 3). Error bars provide the S.E.M. ns, not significant. Bar graph summarizes measurements from at least three independent experiments, each including data from several individual cells.

FRET analysis supports WNT-evoked FZD₄-Gα_12/13 complex dissociation. (A) FRET analysis performed in HEK293 cells expressing FZD₄-GFP, untagged βγ subunits, and either Gα₁₂- or Gα₁₃-mCherry indicates that FRET between GFP and mCherry decreased in response to WNT-7A stimulation (300 ng/ml; 5 minutes). FRET measurements were performed with photoacceptor bleaching in fixed cells. Light grey bars show FRET between GFP and mCherry in the absence of WNT stimulation. Dark grey bars show FRET upon WNT stimulation. Black bars show FRET efficiency at baseline in cells expressing myristoylated mCherry as negative control. The bar graph summarizes data from three independent experiments with a minimum of 27 ROIs from different cells analyzed per individual experiment and condition. Bars and error bars provide the mean ± S.E.M., respectively. *P < 0.05; **P < 0.001. (B) The experimental setup is illustrated schematically.

DVL does not play a central role for FZD₄-Gα_12/13 complex formation. (A and B) DVL1-FLAG, DVL2-MYC, and DVL3-FLAG were coexpressed in HEK293T cells. Cells that were used for dcFRAP were lysed afterward to assess DVL1, DVL2, and DVL3 levels in cellular lysates by immunoblotting using anti-FLAG or anti-MYC antibodies. α-Tubulin was used as the loading control. (B) dcFRAP experiments in cells coexpressing FZD₄-GFP and Gα₁₂-mCherry in the presence of DVL1, DVL2, or DVL3. Downregulation of DVL1, DVL2, and DVL3 by using pan-DVL siRNA did not affect FZD₄-Gα_12/13 assembly. Bar graphs summarize dcFRAP measurements from three independent experiments, each including data from several individual cells. Densitometry analysis of three independent experiments using panDVL siRNA and control indicated that DVL1, DVL2, and DVL3 were routinely reduced by 35–56% [values in percentage reduction in DVL1, DVL2, and DVL3 band intensity by panDVL siRNA compared with control siRNA (mean ± S.E.M.): DVL1 (40 ± 8%); DVL2 (56 ± 2%); DVL3 (35 ± 4%)]. For a graphical presentation of the DVL1, DVL2, and DVL3 levels in control and panDVL siRNA-treated cells (N = 3), see Supplemental Fig. 6. (C) Immunoblotting indicates reduced expression of the three endogenous DVL isoforms in HEK293T cells used for dcFRAP. (D) dcFRAP analysis in cells coexpressing FZD₄-GFP and Gα₁₂-mCherry shows no change in the mobile fraction of Gα₁₂-mCherry upon CL in cells with reduced levels of DVL. ***P < 0.001 (n = 3). Error bars provide the S.E.M. Bar graphs summarize measurements from three independent experiments, each including data from several individual cells.

C-terminal domain swapping between FZD₄ and FZD₆. (A) Schematic presentation of the exchange strategy of the C termini between FZD₄ and FZD₆, resulting in FZD_4-6 C-terminal tail and FZD_6-4 C-terminal tail. (B) The primary structure of human FZD₄ and FZD₆. Green highlights the conserved KTxxxW sequence involved in DVL binding on the presumptive helix 8. Bold marks the terminal PDZ ligand domain. dcFRAP experiments in HEK293T cells coexpressing FZD_4-6-GFP and Gα₁₂-mCherry, FZD_4-6-mCherry and Gα_i1-GFP, or FZD_4-6-mCherry and Gα_q-Venus (C) and FZD_6-4-GFP and Gα₁₂-mCherry, FZD_6-4-mCherry and Gα_i1-GFP, or FZD_6-4-mCherry and Gα_q-Venus (D) reveal predominant assembly with Gα₁₂ of the chimeric receptors. ***P < 0.001 (n = 3). Bar graphs show the mean ± S.E.M. PDZ, PSD-95/dics large/ZO-1 homologous.

WNT-induced dynamic mass redistribution depends on Gα_12/13 and FZD4-GFP. HEK293 wild-type (wt) cells and HEK293 cells lacking Gα_12/13 were stimulated with increasing amounts of WNT-5A (100, 300, and 1000 ng/ml). Changes in DMR were recorded over time. The apparent negative, WNT-5A–induced DMR responses in empty vector (A) or FZD₄-GFP–transfected (B) HEK293 cells were not observed in the absence of Gα_12/13 proteins (C and D). Experiments were done after overnight treatment with the porcupine inhibitor C59 (5 µM) at 37°C. Shown are representative traces (N = 3), buffer-corrected and measured in triplicates + S.E.M. See Supplemental Fig. 5 for expression levels of FZD₄-GFP and forskolin-induced DMR responses of wt and Gα_12/13-knockout (KO) HEK293 cells.

FZD₄ induces p115-RHOGEF-GFP membrane recruitment in a Gα_12/13- and WNT-dependent manner. (A, B, F, and I) HEK293 cells were cotransfected with combinations of FZD₄-Cerulean, Gα₁₂- or Gα₁₃-mCherry, and p115-RHOGEF-GFP and examined by live-cell confocal imaging. p115-RHOGEF-GFP showed an even cytosolic distribution when expressed alone or in combination with either Gα₁₂/Gα₁₃-mCherry or FZD₄-Cerulean (see Supplemental Fig. 2). The combination of FZD₄-Cerulean and either Gα₁₂- or Gα₁₃-mCherry increased p115-RHOGEF-GFP plasma membrane localization. Quantification of the FZD₄-Cerulean–dependent p115-RHOGEF-GFP recruitment was done by counting cells showing membranous versus cytosolic p115-RHOGEF-GFP distribution. Data from three independent experiments (>300 cells from several visual fields counted per condition for each individual experiment) are presented in the bar graphs (C and G). Data represent cells with a membranous p115-RHOGEF-GFP localization calculated as the percentage of total p115-RHOGEF-GFP–positive cells counted. In combination with the porcupine inhibitor C59 (5 µM; overnight treatment), FZD₄-Cerulean–induced and Gα₁₂- or Gα₁₃-mCherry–mediated p115-RHOGEF-GFP recruitment was significantly reduced. Error bars provide the S.E.M. ***P < 0.001 (N = 3). Cellular distribution profiles of p115-RHOGEF-GFP (green) and Gα₁₂ or Gα₁₃-mCherry (red) fluorescence intensity along a line drawn over a single cell shown in (A, B, F, and I) (see arrow) in either the absence or presence FZD₄-Cerulean are shown in (D, E, H, and J). Size bars = 10 µm.