Biochemical characterization of Wnt-frizzled interactions using a soluble, biologically active vertebrate Wnt protein - PubMed (original) (raw)
Biochemical characterization of Wnt-frizzled interactions using a soluble, biologically active vertebrate Wnt protein
J C Hsieh et al. Proc Natl Acad Sci U S A. 1999.
Abstract
Biochemical studies of Wnt signaling have been hampered by difficulties in obtaining large quantities of soluble, biologically active Wnt proteins. In this paper, we report the production in Drosophila S2 cells of biologically active Xenopus Wnt8 (XWnt8). Epitope- or alkaline phosphatase-tagged XWnt8 proteins are secreted by concentrated S2 cells in a form that is suitable for quantitative biochemical experiments with yields of 5 and 0.5 mg per liter, respectively. Conditions also are described for the production in 293 cells of an IgG fusion of the cysteine-rich domain (CRD) of mouse Frizzled 8 with a yield of 20 mg/liter. We demonstrate the use of these proteins for studying the interactions between soluble XWnt8 and various Frizzled proteins, membrane anchored or secreted CRDs, and a set of insertion mutants in the CRD of Drosophila Frizzled 2. In a solid phase binding assay, the affinity of the XWnt8-alkaline phosphatase fusion for the purified mouse Frizzled 8-CRD-IgG fusion is approximately 9 nM.
Figures
Figure 1
Secretion of biologically active XWnt8-myc by S2 cells. (A) Immunoblot using anti-myc mAb. (Left) Lanes: 1, 32 μl of 40-fold concentrated conditioned medium from 293 cells transfected with XWnt8-myc; 2, 32 μl of 4-fold concentrated conditioned medium from untransfected S2 cells; 3, 32 μl of 4-fold concentrated conditioned medium from S2 cells transfected with XWnt8-myc; 4, 32 μl of 80-fold concentrated conditioned medium from S2 cells transfected with XWnt8-myc-AP. (Right) Dilution series of a purified fusion protein used as an immunoblot standard; the fusion protein contains the myc epitope flanked by the E. coli maltose binding protein at the amino terminus and an ≈15-kDa segment of a ser/thr protein phosphatase at the carboxyl terminus (57), a configuration that eliminates problems in quantitation that might arise from proteolytic cleavage of an epitope fused to either of the two termini. Molecular mass standards are 194, 120, 87, 64, 52, 39, 26, and 21 kDa. (B) XWnt8-myc stabilizes β-catenin in C57MG cells at room temperature, and this activity is enhanced by heparin. Conditioned medium from untransfected S2 cells (−) or XWnt8-myc transfected S2 cells (+) in the presence of the indicated concentration of heparin was applied to C57MG cells at either 23 or 37°C as indicated. Cytosolic proteins were prepared, and β-catenin was visualized with mAb 15B8. To control for equal loading, HSP-70 was visualized with mAb BRM-22 (Sigma). Molecular mass standards are 194, 120, 87, 64, 52, and 39 kDa.
Figure 2
Binding of XWnt8-AP to full-length Frizzled proteins and the corresponding GPI-anchored CRDs on the surface of transfected COS cells. (A) Schematic diagram showing the location of the CRD (filled ball) in a full-length Frizzled protein and in a CRD-myc-GPI construct. Horizontal lines represent the membrane, and zigzag lines represent the lipid component of GPI. N, amino terminus; C, carboxyl terminus. (B and C) XWnt8-AP binding and surface localization assays for three Frizzled proteins that show undetectable binding (mfz6), intermediate binding (mf7), and strong binding (mfz8). (B) Light microscopy of representative samples of COS cells transiently transfected with the indicated full-length Frizzled (left column) or the corresponding Frizzled CRD-myc-GPI construct (center and right columns). Live cells were stained with XWnt8-AP (left and center columns) or with anti-myc mAb and a fluorescent secondary antibody (right column). The cells remained intact during the binding reaction as determined by the failure of the anti-myc mAb to bind myc-tagged Dishevelled (a cytoplasmic protein) under these conditions; fixation and permeabilization with acetone and paraformaldehyde before incubation with the anti-myc mAb led to intense staining of myc-tagged Dishevelled (data not shown). (C) PI-PLC release of GPI-anchored CRDs from the surface of live cells. Transfected COS cells were incubated in the absence (−) or presence (+) of PI-PLC, and, after centrifugation, the released protein was recovered from the supernatant (S) and the cells were recovered from the pellet (P). Proteins were resolved by SDS/PAGE and were visualized with anti-myc mAb immunoblotting. Molecular mass standards are 194, 120, 87, 64, 52, 39, 26, and 21 kDa.
Figure 3
Binding of XWnt8-AP to tripeptide insertion mutants within the Dfz2 CRD. For the wild type and for each mutant, the experiments were performed with both full-length Dfz2 and the corresponding GPI-anchored CRDs displayed on the surface of transfected COS cells. (A) Alignment of CRDs from a subset of Frizzled proteins showing the locations of the 23 gly-ser-gly insertion mutations. (B) Light microscopy of representative samples of COS cells transiently transfected with the indicated full-length Dfz2 (left column) or the corresponding Dfz2 CRD-myc-GPI construct (center and right columns). Examples are shown for wild-type Dfz2, Dfz2 mutant 14 (128GSG129), which shows strong binding and Dfz2 mutant 17 (141GSG142), which shows no detectable binding. Live cells were stained with XWnt8-AP (left and center columns) or with anti-myc mAb and a fluorescent secondary antibody (right column), as described for Fig. 2. (C) PI-PLC release of GPI-anchored Dfz2 CRDs from the surface of live cells, as described for Fig. 2. Molecular mass standards are 194, 120, 87, 64, 52, 39, 26, and 21 kDa.
Figure 4
Binding of XWnt8-myc and XWnt8-AP to mfz8CRD-IgG. (A) Coomassie-stained gel with mfz8CRD-IgG (lane 1) or the IgG fusion partner alone (consisting of the hinge and Fc regions) (lane 2) secreted from transfected 293 cells and purified by using protein A Sepharose. Molecular mass standards are 194, 120, 87, 64, 52, 39, 26, 21, and 15 kDa. (B) Binding efficiency of XWnt8-myc with IgG or mfz8CRD-IgG prebound to protein A Sepharose beads. Equal fractions of the bound (B), unbound (U), or starting material (1X) were analyzed by SDS/PAGE and anti-myc immunoblotting. A 2-fold dilution series of the starting material is shown at left. Greater than 90% of XWnt8-myc was bound to mfz8CRD-IgG, with little or no binding to IgG. Molecular mass standards are 194, 120, 87, 64, 52, 39, 26, and 21 kDa. (C) Solid phase binding of XWnt8-AP to IgG (circles) or mfz8CRD-IgG (squares). The rate of hydrolysis of p-nitrophenyl phosphate (measured as the rate of change in absorbance at 405 nm) is plotted against XWnt8-myc concentration. Inset shows a Scatchard plot of the binding data fit to a single straight line, which yields a calculated _K_d of 8 nM. B, bound; F, free.
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