Oligomerization of Frizzled and LRP5/6 protein initiates intracellular signaling for the canonical WNT/β-catenin pathway

Abstract

Upon binding to the canonical WNT glycoproteins, Frizzled family receptors (FZDs) and low-density lipoprotein receptor-related protein 5/6 (LRP5/6) undergo a series of polymerizations on the cell surface that elicit canonical WNT/β-catenin signaling. The hyperactivation of WNT/β-catenin signaling is the major cause of tumorigenesis, but the mechanism in tumors such as hepatoma remains unclear. Here, we observed that WNT3A manifested the hyperactivity in β-catenin-dependent signaling after binding to FZD's competitive inhibitory molecule secreted Frizzled-related protein 2 (SFRP2). To understand the mechanism of FZDs in the presence of SFRP2, we explored how FZDs can bind and activate the LRP5/6 signalosome independently of WNT glycoproteins. Our findings further revealed that oligomerizations of FZDs and LRP5/6 can integrate the cytoplasmic protein Dishevelled into the LRP5/6 signalosome, resulting in a robust activation of ligand-independent β-catenin signaling. We propose that besides WNT-bridged FZD-WNT-LRP5/6 protein complexes, the homo- and hetero-oligomerizations of WNT receptors may contribute to the formation of the LRP5/6 signalosome on the cell surface. Of note, we identified four highly expressed FZDs in the hepatoma cell line HepG2, all of which significantly promoted ligand-independent LRP5/β-catenin signaling. As FZDs are ectopically expressed in numerous tumors, our findings may provide a new perspective on tumor pathologies. Furthermore, the results in our study suggest that the composition and stoichiometry of FZDs and LRP5/6 within the LRP5/6 signalosome may tune the selection of bound WNT glycoproteins and configure downstream WNT/β-catenin signaling.

Keywords: Frizzled; LRP5/6; SFRP2; Wnt signaling; cancer; cell signaling; hepatoma; receptor endocytosis; signalosome; tumor development.

Figure 1.

Competitive binding of SFRP2 to WNT3A leads to hyperactivation of β-catenin signaling. A, schematic diagram showing the experimental procedure for determining the activity of SFRP2-embedded WNT3A proteins. Purified WNT3A proteins were premixed with vehicle (VC) or SFRP2 CM for 6 h and then subjected to coimmunoprecipitation or reporter assays, respectively. B, coimmunoprecipitation experiments determined the percentage of SFRP2-embedded WNT3A proteins. Western blot image is a representative of three independent experiments, and the percentage shown in the graph was measured by calculating the ratio of WNT3A proteins in the immunoprecipitations to the input. Error bars represent S.D. C, HEK293T cells transfected with TOPFlash were stimulated with free or SFRP2-embedded WNT3A for 6 h, and then luciferase activity was examined. TOP, β-catenin–responsive luciferase reporter TOPFlash. Error bars represent S.D. D, the activity of WNT3A/β-catenin signaling was determined in vehicle- or SFRP2 CM-pretreated cells. HEK293T cells transfected with TOPFlash were pretreated with vehicle or SFRP2 CM for 6 h, and then CM was withdrawn. The pretreated cells were subsequently stimulated by WNT3A for an additional 6 h. Error bars represent S.D. E, immunofluorescence experiments with a β-catenin antibody examined intracellular β-catenin accumulation. F, SFRP2 accelerated the accumulation of cytosolic β-catenin (β-cat) stimulated by WNT3A. The cytosolic β-catenin proteins were extracted as described under “Experimental procedures.” G, SFRP2 enhanced the sensitivity of cells responding to WNT3A. Error bars represent S.D. *, p < 0.05; **, p < 0.01; ns, not significant; Student's t test; n = 3. β-tub, β-tubulin; DAPI, 4′,6-diamidino-2-phenylindole.

Figure 2.

SFRP2 regulates the paracrine activity of WNT glycoproteins. A, analysis of binding affinities of SFRP2 and the soluble extracellular FZD5-N for WNT3A. SP, signal peptide; TM1–7, transmembrane helices 1–7; C-tail, carboxyl tail. B, SFRP2 suppressed the association of FZD5-N with WNT3A. C, the binding ratio of WNT3A to SFRP2 was determined in SFRP2 immunoprecipitation complexes. Briefly, an excessive dose of WNT3A proteins was preincubated with FLAG-tagged SFRP2 for 6 h, and then SFRP2 was pulled down by FLAG M2 antibodies. The standard protein concentration gradients of FLAG-tagged GST-fused proteins and WNT3A (25, 50, and 100 fmol) were used in Western blotting. D, coimmunoprecipitation experiments showed that the CRD was sufficient to bind to WNT3A proteins. E, the lack of the CRD completely and the lack of the NTR domain weakly inhibited SFRP2 promotion in WNT3A/β-catenin signaling. Error bars represent S.D. F, inhibition of endogenous noncanonical WNT signaling did not counteract SFRP2 promotion in WNT3A/β-catenin signaling. LGK974 was used at 1 μm. Transf. Wnt11, WNT11 plasmids were cotransfected with TOPFlash (TOP) reporter; VC, vector. Error bars represent S.D. G, SFRP2 promoted the paracrine activity of the diffused WNT3A proteins. Auto., autocrine; Para., paracrine; Rspo1, R-spondin 1–conditioned medium; LGRs, leucine-rich repeat–containing G-protein–coupled receptors. Error bars represent S.D. H, SFRP2 promoted the paracrine activity of WNT1 and WNT3A. WNT plasmids and TOPFlash reporter were transfected to HEK293T cells separately, and then the transfected cells were cocultured for 24 h. Error bars represent S.D. *, p < 0.05; **, p < 0.01, Student's t test; n = 3.

Figure 3.

Ligand-independent oligomerization of FZD5 and LRP5 activates β-catenin signaling. A, SFRP2 enhanced WNT3A/LRP5 signaling. HEK293T cells were cotransfected with TOPFlash (TOP) and LRP5 plasmids for 24 h, and then cells were stimulated with WNT3A with or without SFRP2. Error bars represent S.D. B, SFRP2 increased WNT3A-stimulated LRP6 phosphorylation (pLRP6) (phospho-Ser-1490) in HEK293T cells. C, SFRP2 and FZD5 additively augmented WNT3A/β-catenin signaling. Error bars represent S.D. D, FZD5 directly stimulated LRP5/β-catenin signaling. DKK1 was used at 200 ng/ml. E, inhibition of endogenous canonical WNT glycoproteins did not affect FZD5-activated LRP5/β-catenin signaling. Transf., transfected; Pro., protein. Error bars represent S.D. F, FZD3 and FZD4 stimulated LRP5/β-catenin signaling. Error bars represent S.D. G, endoplasmic reticulum chaperone MSED enhanced FZD5 regulation in LRP5/β-catenin signaling. Error bars represent S.D. H, coimmunoprecipitation of FLAG-tagged FZD5 with LRP5 in HEK293T cells. TCL, total cell lysates. I, coimmunoprecipitation of FLAG-tagged FZD5 with LRP6 in HEK293T cells. J, the assay of endogenous interaction between LRP5 and FZD5 was performed in human hepatoma HepG2 cells. K, WNT1 and WNT3A could not regulate the interaction between LRP5 and FZD5. L, overexpressed AXIN1 and GSK3β slightly increased the interaction between LRP5 and FZD5. *, p < 0.05; **p < 0.01, Student's t test; n = 3. VC, vector.

Figure 4.

The oligomerization of FZD5 and LRP5 is regulated by multiple sites. A, coimmunoprecipitation of FLAG-tagged FZD5-ECD with LRP5 or LRP6 in HEK293T cells, respectively. B, the CRD was dispensable for the interaction between FZD5-ECD and LRP5 or between FZD5-ECD and LRP6. C, LDLR repeats of LRP5 were required for the interaction between FZD5-ECD and LRP5. D, both FZD5-ECD and FZD5-loop-tail interacted with LRP5. TCL, total cell lysates; VC, vector; aa., amino acids.

Figure 5.

DVL family proteins are required for ligand-independent FZD–LRP5/6 axis. A, FZD5 activated LRP5/β-catenin signaling, relying on the LDLR repeats of LRP5. Error bars represent S.D. B, all of the structural modules of FZD5, except for the CRD, were required to activate LRP5/β-catenin signaling. Error bars represent S.D. C, immunofluorescence (IF) experiments showed the recruitment of DVL3 to the plasma membrane by FZD5. D, WT but not ECD-lacking FZD5 regulated the collaboration of DVL3 and LRP5 in β-catenin signaling. The inset shows the posttranslational modification of DVL3 in the presence of FZD5 overexpression. Error bars represent S.D. E, the CRD was dispensable for FZD5 regulation of DVL3 and LRP5 collaboration. Error bars represent S.D. F, FZD5 lacking its loop and carboxyl tail could not regulate DVL3 and LRP5 collaboration. Error bars represent S.D. G, immunofluorescence images show the recruitment of myristoylated (Myr) DVL3 to the plasma membrane. H, the recruitment of DVL3 to the plasma membrane by myristoylation did not mimic the role of the loop and carboxyl tail of FZD5 in regulating DVL3 and LRP5 collaboration. Error bars represent S.D. I, triple knockout (TKO) of DVL1, DVL2, and DVL3 led to suppression of FZD5-activated, but not MSED-enhanced or constitutively activate by deletion of P1E1–P4E4 repeats, LRP5/β-catenin signaling. Western blotting showed deficiencies of DVL2 and DVL3 expression in triple knockout cells. Error bars represent S.D. *, p < 0.05; **, p < 0.01, Student's t test; n = 3. DAPI, 4′,6-diamidino-2-phenylindole; VC, vector; TOP, TOPFlash.

Figure 6.

Ligand-independent FZD–LRP5/6 signaling may be involved in tumorigenesis. A, quantitative RT-PCR analyses for LRP5 mRNA in human tumor cells. Error bars represent S.D. B, quantitative RT-PCR analyses for LRP6 mRNA in human tumor cells. Error bars represent S.D. C, Western blot analyses for LRP5 and LRP6 protein contents in human tumor cells. D, quantitative RT-PCR analyses for FZD1–10 mRNA in human tumor cells. Error bars represent S.D. E, FZD3/4/5/9 regulated LRP5 and DVL3 collaboration in β-catenin signaling. Error bars represent S.D. F, immunofluorescence (IF) experiments show the recruitment of DVL3 to the plasma membrane by FZD3/4/5/9. G, DVL triple knockout (TKO) led to significant suppression of FZD9-activated LRP5/β-catenin signaling. Error bars represent S.D. H, treatment with GSK3β inhibitor CHIR99021, but not the WNT antagonist DKK1, affected β-catenin (β-cat) stabilization in HepG2 cells. *, p < 0.05; **, p < 0.01, Student's t test; n = 3. VC, vector; TOP, TOPFlash; β-tub, β-tubulin.

Figure 7.

The oligomeric state of receptors regulates the response of cells to WNT glycoproteins. A, the homodimerization of FZD5 with its N terminus and loop-tail sequence. B, WNT3A did not regulate the homodimerization of FZD5 with its N terminus. C, DVL3 did not regulate the homodimerization of FZD5 with its loop-tail sequence. D, FZD5 could augment WNT3A/β-catenin signaling in a dose-dependent manner. Error bars represent S.D. E, increase in FZD5 protein content enhanced cell response to WNT3A. Error bars represent S.D. F, overexpression of FZDs enhanced WNT3A/β-catenin signaling. Error bars represent S.D. G, LRP5 could augment WNT3A/β-catenin signaling in a dose-dependent manner. Error bars represent S.D. H, subtypes of canonical WNT glycoproteins adopted distinct stoichiometries of FZDs and LRP5/6 on the cell surface. Error bars represent S.D. *, p < 0.05; **, p < 0.01, Student's t test; n = 3. TCL, total cell lysates; VC, vector; TOP, TOPFlash; β-tub, β-tubulin.

Figure 8.

Receptor oligomerizations configure the canonical WNT/β-catenin signaling mechanism. A, CLTC deficiencies suppressed β-catenin signaling caused by APC knockdown. Error bars represent S.D. B, CLTC did not regulate β-catenin signaling caused by double knockdown of AXIN1 and AXIN2. Error bars represent S.D. C, CLTC did not regulate basal or constitutive activity of LRP5/β-catenin signaling. Error bars represent S.D. D, CLTC deficiencies suppressed FZD5-activated LRP5/β-catenin signaling. Error bars represent S.D. E, CLTC deficiencies did not affect the β-catenin signaling stimulated by Wnt1 and overexpressed LRP5. Error bars represent S.D. F, CLTC deficiencies did not affect the β-catenin signaling stimulated by WNT3A and overexpressed LRP5. Error bars represent S.D. G, knockdown of CAV1 suppressed the β-catenin signaling stimulated by WNT3A and overexpressed LRP5. Error bars represent S.D. H, DVL triple knockout (TKO), but not CLTC knockout (KO), suppressed the β-catenin signaling stimulated by WNT3A and overexpressed FZD5. I, the multiple modes of the canonical WNT/β-catenin signaling transduction according to receptor oligomerization. i shows the traditional model of canonical WNT/β-catenin signaling. ii shows ligand-independent FZD-mediated LRP5/6 signaling via clathrin-mediated endocytosis. iii shows the putative mechanism for LRP5/6-predominant receptor oligomerization in ligand-dependent β-catenin signaling, which may rely on caveolin-mediated endocytosis. iv shows the putative mechanism for FZD-predominant receptor oligomerization in ligand-dependent β-catenin signaling where clathrin-mediated endocytosis and caveolin-mediated endocytosis may both be dispensable. TCL, total cell lysates; VC, vector; TOP, TOPFlash; β-tub, β-tubulin; CK1α, casein kinase 1α; TCF/LEF, T-cell factor/lymphoid-enhanced binding factor; β-TrCP, β-transducin repeat–containing protein.