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. 2015 Mar 13;290(11):6789-98.
doi: 10.1074/jbc.M114.612648. Epub 2015 Jan 20.

Systematic mapping of WNT-FZD protein interactions reveals functional selectivity by distinct WNT-FZD pairs

Jacomijn P Dijksterhuis  1 , Bolormaa Baljinnyam  2 , Karen Stanger  3 , Hakki O Sercan  2 , Yun Ji  2 , Osler Andres  2 , Jeffrey S Rubin  2 , Rami N Hannoush  4 , Gunnar Schulte  5
Affiliations

Systematic mapping of WNT-FZD protein interactions reveals functional selectivity by distinct WNT-FZD pairs

Jacomijn P Dijksterhuis et al. J Biol Chem. .

Abstract

The seven-transmembrane-spanning receptors of the FZD1-10 class are bound and activated by the WNT family of lipoglycoproteins, thereby inducing a complex network of signaling pathways. However, the specificity of the interaction between mammalian WNT and FZD proteins and the subsequent signaling cascade downstream of the different WNT-FZD pairs have not been systematically addressed to date. In this study, we determined the binding affinities of various WNTs for different members of the FZD family by using bio-layer interferometry and characterized their functional selectivity in a cell system. Using purified WNTs, we show that different FZD cysteine-rich domains prefer to bind to distinct WNTs with fast on-rates and slow off-rates. In a 32D cell-based system engineered to overexpress FZD2, FZD4, or FZD5, we found that WNT-3A (but not WNT-4, -5A, or -9B) activated the WNT-β-catenin pathway through FZD2/4/5 as measured by phosphorylation of LRP6 and β-catenin stabilization. Surprisingly, different WNT-FZD pairs showed differential effects on phosphorylation of DVL2 and DVL3, revealing a previously unappreciated DVL isoform selectivity by different WNT-FZD pairs in 32D cells. In summary, we present extensive mapping of WNT-FZD cysteine-rich domain interactions complemented by analysis of WNT-FZD pair functionality in a unique cell system expressing individual FZD isoforms. Differential WNT-FZD binding and selective functional readouts suggest that endogenous WNT ligands evolved with an intrinsic natural bias toward different downstream signaling pathways, a phenomenon that could be of great importance in the design of FZD-targeting drugs.

Keywords: 32D Cells; Disheveled; Frizzled; Functional Selectivity; LDL Receptor-related Protein 6; Myeloid Cell; Receptor; WNT Pathway; WNT Signaling; β-Catenin (B-catenin).

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Figures

FIGURE 1.
FIGURE 1.
Binding affinities of various WNTs for various FZD-CRDs as measured by bio-layer interferometry. A, representative binding curves of WNT-3A and WNT-5A binding to FZD CRDs. B, summary of WNT-FZD CRD binding data. Tabulated are the binding values of four different WNTs and various FZD CRD-Fc proteins represented as follows: −, no binding; +, very weak binding (>100 nm); ++, weak binding (40–100 nm); +++, intermediate binding (10–40 nm); and ++++, strong binding (< 10 nm). NQ indicates weak binding, not quantifiable due to the narrow response signal window in the binding curves. WNT-7A/9B/10B/11 and FZD10 showed no detectable binding to any of the chosen FZD CRDs or WNTs in this study, respectively (data not shown).
FIGURE 2.
FIGURE 2.
32D myeloid progenitor cell line expresses little or no endogenous FZDs but does express LRP5/6 co-receptors. A, the bar graph presents the FZD expression profile (FZD1–10) of 32D cells (hatched gray bars) and primary microglia cells (white bars) determined by qPCR (n ≥ 3). Error bars represent S.E. GAPDH was used as a housekeeping gene for normalization. By comparing FZD expression in primary microglia cells (previously published by Halleskog et al. (21)) and 32D cells, it becomes apparent that FZD levels in 32D cells are very low. B, WNT co-receptor profile in 32D cells determined by RT-PCR. LRP5 and LRP6 (but neither RYK nor ROR1/2) are expressed in 32D cells. The negative control consisted of 32D cell cDNA prepared without reverse transcriptase; mouse tail genomic DNA or cDNA from GL261 cells (RYK) was used as a positive control. C, shown are comparative PCR expression profiles of 32D and L929 cells, including WNTs, FZDs, LRPs, DKKs, and SFRPs. The table summarizes the average Ct values of two biological replicates. The following genes were not included on the PCR array: Wnt9b, Wnt10b, Fzd9, Fzd10, Ryk, Ror1, and Ror2. D, immunoblotting for HA-tagged FZDs in 32D cells expressing FZD2, FZD4, or FZD5. β-Actin was used as a loading control. The molecular masses of FZDs were determined with Bio-Rad software.
FIGURE 3.
FIGURE 3.
WNT-3A evokes a dose-dependent β-catenin stabilization in mouse 32D/FZD2/4/5 cells. A, the bar graphs summarize immunoblot experiments quantifying β-catenin levels in lysates from parental mouse 32D and 32D/FZD2/4/5 cells stimulated for 2 h with 0, 3, 10, 30, 100, 300, and 1000 ng/ml WNT-3A, -4, -5A, or -9B. B, representative immunoblots showing β-catenin levels in response to increasing WNT concentrations. β-Actin was used as a loading control in all the experiments. Error bars indicate standard S.E. (n ≥ 3). *, p < 0.05; **, p < 0.01.
FIGURE 4.
FIGURE 4.
WNT-3A induces dose-dependent phosphorylation of LRP6 in mouse 32D cells. A, the bar graphs show phosphorylation of LRP6 (P-LRP6) in lysates from 32D/FZD2/4/5 cells stimulated for 2 h with PBS and 3, 10, 30, 100, 300, and 1000 ng/ml WNT-3A. WNT-4, -5A, and -9B did not evoke phospho-LRP6 in any of the 32D/FZD cell lines (data not shown). B, representative immunoblots for phospho-LRP6. β-Actin was used as a loading control in all experiments. *, p < 0.05; **, p < 0.01. Densitometry data from n ≥ 3 independent experiments were used for graphical summary.
FIGURE 5.
FIGURE 5.
WNT-3A, -4, and -5A induce dose-dependent phosphorylation and electrophoretic mobility shift of DVL2 (PS-DVL2) in mouse 32D/FZD cells. A, the bar graphs show PS-DVL2 in lysates from 32D/FZD2/4/5 cells stimulated for 2 h with PBS and 3, 10, 30, 100, 300, and 1000 ng/ml WNT-3A, -4, and -5A. Densitometry data ratios (i.e. the quotient of the values from the upper (PS-DVL) and lower DVL bands) were normalized to the unstimulated control values. Basal PS-DVL2 was not detectable (ND) in the parental 32D cells. B, representative immunoblots showing the detection of PS-DVL2. ▶, DVL2; ▷, PS-DVL2. β-Actin was used as a loading control in all the experiments. Error bars indicate standard S.E. (n ≥ 3). *, p < 0.05; **, p < 0.01; ***, p < 0.001.
FIGURE 6.
FIGURE 6.
WNT-3A, -4, and -5A induce dose-dependent phosphorylation and shift of DVL3 (PS-DVL3) in mouse 32D/FZD cells. A, the bar graphs show phosphorylation of DVL3 (PS-DVL3) in lysates from 32D/FZD2/4/5 cells stimulated for 2 h with PBS and 3, 10, 30, 100, 300, and 1000 ng/ml WNTs. Densitometry data ratios (i.e. the quotient of the values from the upper (PS-DVL) and lower DVL bands) were normalized to the unstimulated control values. Basal PS-DVL2 was not detectable (ND) in the parental 32D cells. B, representative immunoblots for detection of PS-DVL3. ▶, DVL3; ▷, PS-DVL3. β-Actin was used as a loading control in all the experiments. Error bars indicate standard S.E. (n ≥ 3). *, p < 0.05; **, p < 0.01; ***, p < 0.001.
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