Proc. Natl. Acad. Sci. USA
Vol. 96, pp. 3546–3551, March 1999
Biochemical characterization of Wnt-Frizzled interactions using a
soluble, biologically active vertebrate Wnt protein
JEN-CHIH HSIEH*, AMIR RATTNER*†, PHILIP M. SMALLWOOD*†, AND JEREMY NATHANS*†‡§
*Department of Molecular Biology and Genetics,‡Departments of Neuroscience and Ophthalmology, and†Howard Hughes Medical Institute, Johns Hopkins
University School of Medicine, Baltimore, MD 21205
Contributed by Jeremy Nathans, January 20, 1999
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
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 ?9 nM.
Biochemical studies of Wnt signaling have
The Wnt proteins define a large family of extracellular signaling
molecules found throughout the animal kingdom. Wnts play a
central role in central nervous system, renal, placental, and limb
development in vertebrates; early embryonic cell fate decisions
and cell migration?polarity in Caenorhabditis elegans; and seg-
refs. 1–3). Wnts and the Wnt signaling pathway also have been
in Wnt genes have been identified in human cancers, recent
studies have shown that increased activity of the Wnt signaling
pathway, mediated by stabilization of ?-catenin, is an important
aspect of carcinogenesis in human melanomas and colorectal
Current evidence indicates that the Frizzled family of integral
membrane proteins act as Wnt receptors. The founding member
of this family was identified in Drosophila based on its role in
tissue polarity in the adult cuticle (8). Many additional family
members have been identified in both vertebrates and inverte-
brates, and all have at the amino terminus a conserved extracel-
(CRD), that spans ?120 amino acids and contains 10 invariant
cysteines, followed by seven putative membrane spanning do-
mains (9–11). The identification of Frizzled proteins as Wnt
receptors arose from experiments in cell culture showing that
Frizzled-2, a second Frizzled family member from Drosophila,
Armadillo (the Drosophila orthologue of ?-catenin) (12). Addi-
between pairs of Wnt and Frizzled genes in both embryonic and
larval development in C. elegans (13–15), from RNA injection
experiments in Xenopus embryos in which a mammalian Frizzled
protein was shown to alter the subcellular localization (16) or to
change the developmental effects of a coexpressed Wnt (17, 18),
and most recently from genetic studies in Drosophila that show
that Frizzled and Frizzled-2 function redundantly in the embryo
as Wg receptors (refs. 19–21; P. Bhanot, R. Nusse, J.N., and K.
experiments also provide evidence for specificity in Wnt and
Frizzled interactions: some, but not all, Frizzled proteins confer
Wg binding in transfected cells (12), and axis induction by
Xenopus Wnt5A (XWnt5A) is conferred by only one of seven
Frizzled proteins examined (17).
with the discovery in vertebrates of a family of secreted proteins
that possess a CRD resembling those present in the Frizzled
proteins; the members of this family are referred to as secreted
Frizzled related proteins (sFRP) (22–28). Among Frizzled pro-
teins, the CRD contains some or all of the Wnt-binding deter-
minants because it is both necessary and sufficient for conferring
presumably via the CRD, when either protein is artificially
one sFRP, sFRP-3?FRZB, is localized to Spemann’s organizer,
and injection of RNA encoding any of several sFRPs inhibits the
bioactivity of coinjected XWnt8 (22–24, 27, 28). These and other
of Wnt-Frizzled binding.
Despite the advances noted above, the biochemical and struc-
tural properties of Wnts and their interactions with Frizzled and
sFRP proteins and with extracellular matrix molecules remain
a low efficiency of secretion and a propensity to adhere to the
thus far have only been produced in small quantities and in
impure form. For example, biologically active Wg has been
produced in the conditioned medium of transfected Drosophila
Schneider (S2) cells (33), and biologically active Wnt-1 (34) and
Wnt-5A (35) have been produced in the conditioned medium of
been used for quantitative binding or structural studies.
In this paper, we report the production in Drosophila S2 cells
of biologically active XWnt8. Epitope- or alkaline phosphatase-
tagged XWnt8 proteins are secreted in a form that is suitable for
quantitative biochemical experiments. Conditions also are de-
scribed for the production of an IgG fusion of the CRD of mouse
of these proteins for studying the interactions between soluble
XWnt8 and various Frizzled proteins, membrane-anchored and
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accordance with 18 U.S.C. §1734 solely to indicate this fact.
PNAS is available online at www.pnas.org.
Abbreviations: AP, human placental alkaline phosphatase; CRD,
cysteine-rich domain; ECM, extracellular matrix; fz, Frizzled; GPI,
glycophosphatidylinositol; PI-PLC, phosphoinositide–phospholipase
C; sFRP, secreted Frizzled-related protein; Wg, Wingless; X-phos,
human, m, mouse, D, Drosophila; X, Xenopus.
§To whom reprint requests should be addressed at: 805 PCTB, 725
North Wolfe Street, Johns Hopkins University School of Medicine,
Baltimore, MD 21205. e-mail: email@example.com.
secreted CRDs, and a set of insertion mutants in the CRD of
Drosophila Frizzled 2.
MATERIALS AND METHODS
terminal myc epitope tag (EQKLISEEDL) inserted between
amino acids 339 and 340 (36). The XWnt8—human placental
alkaline phosphatase (AP) construct contains the coding region
of XWnt8-myc, without the carboxyl-terminal 19 amino acids,
fused upstream of the AP catalytic domain in a modified version
of pAPtag2, a vector from which the alkaline phosphatase signal
sequence has been removed (37). Glycophosphatidylinositol
(GPI)-anchored and myc-tagged CRDs were constructed as
described in ref. 26 by using a modified pCIS vector (38) in which
a DNA segment encoding a myc epitope (39) flanked by glycine
spacers (GGGMEQKLISEEDLNGGG) is followed by a seg-
ment encoding the carboxyl-terminal 40 amino acids of decay-
plasmids, PCR products engineered to contain an optimal Kozak
sequence around the initiator ATG and encompassing the indi-
cated codons were inserted in-frame and upstream of the myc-
GPI cassette: Drosophila fz2 (Dfz2), 1–270 (12); mfz3, 1–157;
mfz4, 1–181; human fz5 (hfz5), 1–172; mfz6, 1–153; mfz7, 1–185;
mfz8, 1–173 (11). Insertion of three codons (5?GGTTCCGGA
encoding gly-ser-gly and containing a unique BspEI site) into
each of 23 locations in the Dfz2 CRD was accomplished by PCR
amplification with pairs of mutagenic and flanking primers
followed by replacement of the wild-type DNA segment with the
either the BstEII site at codon 171 or the PstI site at codon 270.
The mfz8CRD-IgG fusion was generated by fusing codons 1–173
of mfz8 upstream of the hinge region of the human IgG heavy
chain gene (41). All DNA segments generated by PCR were
sequenced to rule out spurious mutations.
Production of XWnt8-myc and XWnt8-AP in S2 Cells. Con-
ditioned medium containing XWnt8-myc or XWnt8-AP was
produced in S2 cells stably transfected with the corresponding
cDNA under the control of a metallothionein promoter. For
protein production, cells were concentrated 4-fold and were
incubated at 25°C for 24 hr in serum-free Drosophila Expression
System medium (Invitrogen) containing 0.5 mM CuSO4. Control
medium was produced by CuSO4induction of untransfected S2
cells. The media were centrifuged at 100,000 ? g at 4°C for 1 hr
to remove aggregates and were concentrated 20-fold by ultrafil-
Production of mfz8CRD-IgG Fusion Protein. Mfz8CRD-IgG
and IgG were produced in 293 cells that were transiently trans-
fected by using Lipofectamine (GIBCO?BRL). One day after
transfection, cells were transferred to serum-free DMEM?F-12,
and the secreted protein was harvested after an additional 24 hr.
Control conditioned medium was obtained from untransfected
293 cells. For some experiments, mfz8CRD-IgG and IgG were
affinity purified by using protein A Sepharose (Amersham Phar-
macia) with a low pH elution. Protein concentrations were
estimated by visual comparison with a BSA standard after
SDS?PAGE and Coomassie blue staining.
?-Catenin Stabilization Assay. C57MG cells were grown in
35-mm wells in DMEM supplemented with 10% fetal bovine
of the assay. Cells were washed once with serum-free DMEM?
of S2-conditioned medium that was diluted with an equal volume
Sigma) was added as indicated. After a 3-hr incubation, the
conditioned medium was removed, and cells were harvested by
incubation at 4°C for 5 min in PBS containing 5 mM EDTA and
were collected by centrifugation. The cells were resuspended and
mM NaCl?5 mM EDTA?0.5 ?g/ml leupeptin?0.5 ?g/ml aproti-
nin?0.5 ?g/ml antipain), followed by centrifugation at 800 ? g for
2 min. The resulting supernatant was further centrifuged at
100,000 ? g for 30 min at 4°C, and the proteins in the high speed
supernatant (containing cytoplasmic ?-catenin) were separated
in a SDS?7.5% polyacrylamide gel and were immunoblotted by
using anti-?-catenin mAb 15B8 (Sigma).
Phosphoinositide–Phospholipid C (PI-PLC) Release of GPI-
Anchored Proteins. COS cells in 35-mm wells were transfected
with 2 ?g of plasmid DNA and 4 ?l of FuGene 6 transfection
reagent (Boehringer Mannheim). Twenty-four hours after trans-
fection, the cells were harvested, were centrifuged at 800 ? g for
2 min, and were resuspended in 100 ?l of PBS containing 5 mM
EDTA and 10 units of PI-PLC (Boehringer Mannheim). After a
1-hr incubation at 37°C with gentle rotation, the supernatant
containing the released proteins was separated from the cells by
centrifugation at 800 ? g for 2 min. The cell pellet was resus-
pended in 100 ?l of solublization buffer (20 mM Hepes, pH
7.2?150 mM NaCl?1 mM EGTA?1 mM EDTA?10% glycer-
ol?1% Triton X-100), was incubated on ice for 10 min, and was
centrifuged at 4°C for 2 min at 800 ? g to remove nuclei and cell
Binding of XWnt8-AP and Antibodies to Transfected Cells.
For staining under nonpermeablized condition, live transfected
COS cells growing on coverslips were incubated at room tem-
perature for 1 hr with anti-myc mAb 9E10 (39) diluted 1:1,000 in
were washed three times with DMEM?F12 and were fixed in 2%
methanol-free formaldehyde?PBS or were washed three times
with binding buffer (0.5 mg?ml BSA in Hank’s balanced salt
acetone, 3% formaldehyde in 20 mM Hepes (pH 7.0), and were
cells were incubated at room temperature with Texas Red-
conjugated anti-mouse antibody (Vector Laboratories) diluted
1:200 in 5% goat serum in PBS, followed by three washes in PBS.
150 mM NaCl were incubated at 65°C for 100 min, were washed
once in AP buffer (0.1 M Tris?HCl, pH 9.4?0.1 M NaCl?5 mM
NaCl) and were stained at room temperature overnight with
5-bromo-4-chloro-3-indolyl-phosphate (X-phos) (165 ?g?ml)?
nitroblue tetrazolium (NBT) (330 ?g?ml) in AP buffer. For
anti-myc staining under permeabilized condition, the transfected
cells were fixed in 60% acetone, 3% formaldehyde in 20 mM
Hepes (pH 7.0) before antibody incubation.
Solution Binding Assay. Twelve micrograms of IgG or
XWnt8-myc-conditioned medium. The beads were separated by
centrifugation into bound and unbound fractions and then were
washed over 10 minutes at 4°C with three changes of Hank’s
balanced salt solution with 20 mM Hepes (pH 7.0).
Solid Phase Binding Assay. One-hundred microliters of 293
cell conditioned medium containing 2 ?g of Mfz8CRD-IgG or
IgG alone was used to coat each well of a 96-well tray at 4°C
overnight. The unoccupied sites in the wells were blocked at 4°C
for 4 hr with 200 ?l of 2 mg?ml BSA in wash buffer [Hank’s
balanced salt solution with 20 mM Hepes (pH 7.0)]. After three
incubation at 4°C for 20 hr, the wells were washed five times with
200 ?l of wash buffer. Two-hundred microliters of 150 mM
p-nitrophenyl phosphate in 1 M diethanolamine (pH 9.8), 1 mM
MgCl2was added to each well, and the change in absorbance at
405 nm was measured.
Production of XWnt8-myc and XWnt8-AP in S2 Cells. Our
attempts to produce a soluble vertebrate Wnt protein for bio-
chemical experiments have focused on XWnt8 because Moon
and colleagues (36, 42) have observed that addition of a myc
Biochemistry: Hsieh et al.Proc. Natl. Acad. Sci. USA 96 (1999)3547
epitope tag near the XWnt8 carboxyl terminus, a construct
referred to here as XWnt8-myc, is compatible with biological
activity in Xenopus embryos. As an expression system, we have
chosen Drosophila Schneider (S2) cells, which, as noted above,
have been used successfully to produce bioactive Wg (33) as well
as a variety of other secreted vertebrate proteins (43). Fig. 1A
shows that S2 cells can secrete both XWnt8-myc and a fusion of
phosphatase (XWnt8-AP). A comparison of the anti-myc immu-
noblot signals from XWnt8-myc and XWnt8-AP and from a
myc-tagged derivative of the Escherichia coli maltose binding
protein indicates that XWnt8-myc and XWnt8-AP are secreted
from concentrated S2 cells with yields up to ?5 and ?0.5
The biological activity of XWnt8-myc was tested by applying
S2-conditioned medium containing XWnt8-myc or control S2
conditioned medium to C57MG cells and monitoring the stabi-
lization of cytoplasmic ?-catenin (Fig. 1B; ref. 44). In these
incubation was performed at 23°C but little or no stabilization at
37°C. XWnt8-myc activity was enhanced by the inclusion of
sensitivity of XWnt8-myc activity on C57MG cells could reflect
S2 cells or a temperature-dependence of ?-catenin turnover in
C57MG cells. XWnt8-myc is also active in stabilizing Armadillo
in Drosophila clone 8 cells, an imaginal disc cell line, but the
similarly prepared S2 conditioned medium containing Wg (data
not shown). XWnt8-myc produced in transiently transfected 293
cells was secreted with an efficiency ?10-fold lower than that
observed for S2 cells (Fig. 1A) and was inactive in the ?-catenin
Cell Surface Binding of XWnt8-AP to Frizzled Proteins and
Frizzled CRDs. The XWnt8-AP fusion protein described above
provides a convenient and quantitative probe to investigate the
containing XWnt8-AP was incubated at room temperature with
live COS or 293 cells and subsequently was visualized with a
X-phos?NBT tetrazolium histochemical reaction, little or no
cell-associated AP activity was observed, indicating that this
fusion protein does not bind significantly to either plasma mem-
brane proteins or the ECM produced by these two cell lines. This
behavior is in contrast to that of Wg produced by S2 cells, which
of Wg to cell surface Frizzled proteins only has been observed
after heparitinase pretreatment to decrease ECM binding (12).
Transient transfection of COS or 293 cells with Dfz2, mfz4, hfz5,
mfz7, mfz8, and Xfz8 conferred cell surface XWnt8-AP binding
whereas transfection with mfz3 and mfz6 did not, a pattern
ref. 12). Binding experiments with control conditioned medium
from untransfected S2 cells shows no alkaline phosphatase stain-
ing of COS cells transfected with Dfz2, mfz4, or hfz5, indicating
that the observed Frizzled binding activity derives from XWnt8-
AP. In these experiments, absence of XWnt8-AP binding could
stability, folding, or surface localization of the Frizzled protein.
Therefore, to measure the surface localization of each receptor
protein in a uniform manner and also to assess the binding of
XWnt8-AP to the various Frizzled CRDs in the absence of the
Frizzled membrane spanning domains, we constructed a set of
myc-tagged and GPI-anchored derivatives of each CRD (Fig. 2).
Incubation of XWnt8-AP with transfected cells expressing
CRD-myc-GPI constructs from Dfz2, mfz3, mfz4, hfz5, mfz6,
Immunoblot using anti-myc mAb. (Left) Lanes: 1, 32 ?l of 40-fold
concentrated conditioned medium from 293 cells transfected with
XWnt8-myc; 2, 32 ?l of 4-fold concentrated conditioned medium from
untransfected S2 cells; 3, 32 ?l of 4-fold concentrated conditioned
medium from S2 cells transfected with XWnt8-myc; 4, 32 ?l of 80-fold
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 ?-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
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.
Secretion of biologically active XWnt8-myc by S2 cells. (A)
different Frizzled and CRD-myc-GPI proteins based on
X-phos?NBT staining of live, transiently transfected COS cells
Semiquantitative assessment of XWnt8-AP binding to
1. Dfz2 (66GSG67)
2. Dfz2 (68GSG69)
3. Dfz2 (73GSG74)
4. Dfz2 (80GSG81)
5. Dfz2 (85GSG86)
6. Dfz2 (89GSG90)
7. Dfz2 (92GSG93)
8. Dfz2 (96GSG97)
9. Dfz2 (102GSG103)
10. Dfz2 (106GSG107)
11. Dfz2 (111GSG112)
12. Dfz2 (114GSG115)
13. Dfz2 (126GSG127)
14. Dfz2 (128GSG129)
15. Dfz2 (130GSG131)
16. Dfz2 (137GSG138)
17. Dfz2 (141GSG142)
18. Dfz2 (144GSG145)
19. Dfz2 (152GSG153)
20. Dfz2 (160GSG161)
21. Dfz2 (165GSG166)
22. Dfz2 (172GSG173)
23. Dfz2 (174GSG175)
The three amino acid insertion mutants are referred to by the codon
numbers flanking the gly-ser-gly insertion. For example, 66GSG67
refers to the insertion of the nine nucleotides encoding gly-ser-gly
between codons 66 and 67. ??, strong binding; ?, weak binding; ?,
undetectable binding. ND, not determined.
3548 Biochemistry: Hsieh et al.Proc. Natl. Acad. Sci. USA 96 (1999)
binding as seen with the corresponding full-length Frizzled pro-
teins. When live cells transfected with each CRD-myc-GPI
(Fig. 2B and data not shown). As an independent measure of
plasma membrane localization of the CRD-myc-GPI proteins,
PI-PLC to cleave the GPI anchor and release the surface-
accessible protein into the medium. Anti-myc immunoblotting of
the CRD-myc-GPI proteins showed that, in each case, three
major electrophoretic species are observed, presumably reflect-
ing different patterns of glycosylation. The protein released by
PI-PLC treatment corresponds to the lowest mobility species,
suggesting that this species is the only one present at the cell
surface. The cell surface CRD-myc proteins were released by
PI-PLC treatment with yields that range from 10 to 30% of the
total CRD-myc-GPI protein, suggesting that in each case a
significant fraction of the CRD protein is able to fold correctly
and move through the endoplasmic reticulum–Golgi-plasma
membrane pathway (Fig. 2C and data not shown; ref. 45).
for binding, these experiments demonstrate that XWnt8-AP
binds efficiently to the CRDs of Dfz2, mfz4, hfz5, mfz7, and mfz8
but binds poorly or not at all to the CRDs of mfz3 and mfz6.
XWnt8-AP Binding to Insertion Mutants of Dfz2. To begin to
define the regions of the CRD that mediate Wnt binding, we
measured the binding of XWnt8-AP to live cells that had been
transfected with a series of 23 insertion mutants in the CRD of
Dfz2 (Fig. 3A). Insertional mutagenesis was performed with a
three-codon cassette that codes for gly-ser-gly, an insertion that
would be predicted to sterically disrupt binding if located at any
point on the ligand-binding surface. Failure to bind XWnt8-AP
also could reflect a defect in the folding or stability of the CRD.
To minimize the probability that the tripeptide insertion might
amino acid from any cysteine and within those regions that are
most hydrophilic. Given the predicted flexibility of the gly-ser-gly
tripeptide (46), we presume that most insertions within surface
loops would be compatible with a correctly folded CRD.
Each of the 23 insertion mutants was studied in the context of
the full-length Dfz2 protein and in a CRD-myc-GPI construct.
After transfection, the efficiency of plasma membrane localiza-
tion was determined for each CRD-myc-GPI mutant by immu-
nostaining of live cells with anti-myc mAbs and by PI-PLC
digestion of live cells followed by immunoblotting as described
above (Fig. 3 B and C). Of interest, all 23 mutants resemble
wild-type Dfz2-CRD-GPI in showing robust cell surface immu-
nostaining and significant release of the low mobility electro-
of the total Dfz2-CRD protein), indicative of a high degree of
plasma membrane localization. On the assumption that passage
through the endoplasmic reticulum–Golgi-plasma membrane
pathway reflects correct folding (45), this result implies that the
native CRD structure can tolerate insertions at each of these
positions. This observation raises the possibility that the CRD
may be a relatively extended structure with a high surface to
Different Dfz2 insertion mutants differ markedly in
XWnt8-AP binding when assayed on the surface of live trans-
fected cells (Table 1 and Fig. 3B). Mutants 15 and 18
(130GSG131 and 144GSG145) bind XWnt8-AP when they are
present in the context of the full-length Dfz2 sequence but fail to
bind as GPI-anchored CRDs, suggesting the possibility that
stabilizing interactions may occur between the membrane-
embedded domain of the Frizzled protein and the CRD or the
CRD-Wnt complex. Among the Dfz2CRD-GPI mutants, 5 bind
weakly, and 14 fail to bind. Binding and nonbinding mutants are
distributed throughout the length of the CRD with nonbinding
mutants showing significant clustering.
Quantitative Binding of XWnt8-AP to mfz8CRD-IgG. The
binding of soluble XWnt8-AP to membrane-anchored CRDs
suggests that these proteins also might interact if the CRD is
produced as a secreted molecule. To test this idea, we produced
the mfz8 CRD as an amino-terminal fusion with a human IgG
heavy chain (mfz8CRD-IgG) in transfected 293 cells. In this
expression system, mfz8CRD-IgG accumulates to ?20 mg?liter
and can be purified to apparent homogeneity with protein A
Sepharose (Fig. 4A).
In an initial experiment, we determined the efficiency of
coprecipitation of XWnt8-myc with mfz8CRD-IgG or with the
IgG backbone alone (Fig. 4B). This experiment indicates that at
corresponding GPI-anchored CRDs on the surface of transfected COS
in a full-length Frizzled protein and in a CRD-myc-GPI construct.
Horizontal lines represent the membrane, and zigzag lines represent the
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
conditions; fixation and permeabilization with acetone and paraformal-
dehyde before incubation with the anti-myc mAb led to intense staining
of myc-tagged Dishevelled (data not shown). (C) PI-PLC release of
were incubated in the absence (?) or presence (?) of PI-PLC, and, after
centrifugation, the released protein was recovered from the supernatant
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.
Binding of XWnt8-AP to full-length Frizzled proteins and the
Biochemistry: Hsieh et al. Proc. Natl. Acad. Sci. USA 96 (1999)3549
bind mfz8CRD-IgG. These reagents then were used to develop
a solid phase enzyme-linked binding assay in which varying
concentrations of soluble XWnt8-AP were incubated with im-
4C). Four independent experiments using either crude condi-
tioned medium containing mfz8CRD-IgG, which is ?80% pure,
or Protein A Sepharose purified mfz8CRD-IgG yielded similar
results. When redrawn as a Scatchard plot and fit to a single line,
the binding data indicate an average affinity of 9 ? 2 nM (n ?
4). However, close examination of the Scatchard plots suggests
that two or more classes of sites may better represent the binding
data; under an assumption of two sites, the estimated affinities of
the two sites would be predicted to differ by ?10-fold.
Production of Soluble Wnt and Frizzled Derivatives. Produc-
ing soluble, biochemically well behaved Wnts has been a long-
standing goal in this field but has been problematic. Producing
secreted Frizzled or sFRP CRDs, the domain implicated in Wnt
binding, either alone or as fusion proteins with IgG or AP, also
has proven difficult because most are poorly secreted (A.R.,
J.-C.H., and J.N., unpublished data). The several examples de-
and host–vector systems for which we have observed good yields
of apparently native proteins. In the case of XWnt8, this could
reflect the presence in S2 cells of putative chaperones such as the
porcupine protein (47, 48), as yet uncharacterized proteins in the
temperature (23°C) at which S2 cells are grown. The efficient
secretion of mfz8CRD-IgG from transfected 293 cells—?20
mg?liter—may reflect a serendipitous choice of the fusion point
between the CRD and the IgG heavy chain in this construct.
Structural and Functional Mapping of the CRD. The analysis
of 23 tripeptide insertion mutants in the Dfz2 CRD identifies a
set of discontinuous segments along the linear sequence where
insertion results in loss of XWnt8-AP binding. These are pre-
sumed to be sites of direct or close contact with the XWnt8-AP
ligand. Other regions where tripeptide insertion fails to diminish
XWnt8-AP binding can be eliminated as sites of close contact.
The loss of XWnt8-AP binding caused by several tripepeptide
insertions in the carboxyl-terminal two-thirds of the CRD is at
odds with the results of Lin et al. (49) in which coimmunopre-
sFRP-3 derivatives, including deletion derivatives lacking large
parts of the CRD. This discrepancy may be related to the
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
Binding of XWnt8-AP to tripeptide insertion mutants within
Coomassie-stained gel with mfz8CRD-IgG (lane 1) or the IgG fusion
partner alone (consisting of the hinge and Fcregions) (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 pre-
bound to protein A Sepharose beads. Equal fractions of the bound (B),
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
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 Kdof 8 nM. B, bound; F, free.
3550 Biochemistry: Hsieh et al. Proc. Natl. Acad. Sci. USA 96 (1999)
significant endoplasmic reticulum retention that is characteristic Download full-text
of both Wnts and sFRPs, which suggests that much of the
association observed between these proteins in cotransfected
cells represents aggregation of misfolded proteins. These con-
flicting data serve to emphasize the importance for binding
Specificity of Wnt-CRD Binding. The available data for Wnt-
bind to multiple Frizzled and?or sFRP targets. For example, a
Drosophila Wnt, Wg, binds with similar efficiency to a subset of
mammalian Frizzled proteins and to Drosophila Frizzled and
with similar efficiency to Drosophila fz2 and the same set of
mammalian Frizzled proteins to which Wg binds.
semiquantitative cell surface binding assays do not reveal small
but biologically significant differences in affinity. This possibility
can be investigated by extending the quantitative enzyme-linked
determining the binding affinities of additional pairs of Wnts and
CRD-IgG fusions. It is also possible that additional factors
and?or narrow the specificity of their interactions. These factors
might include heparin, other ECM components, coreceptors,
plasma membrane lipids, and additional secreted proteins. The
well known affinity of Wnts for heparin and other ECM com-
ponents (e.g., ref. 31), the ability of heparin and other ECM
components to modulate Wnt signaling in vitro (ref. 50 and this
report), the affinity of the carboxyl-terminal domain of at least
two of the sFRPs for heparin (ref. 22; A.R., J.-C.H., and J.N.,
unpublished work), and the recent discovery of Wnt inhibitory
factor-1, a secreted Wnt-binding protein that lacks a CRD (51).
In vivo evidence for the importance of the ECM in Wnt signaling
comes from the effects of mutations in proteoglycan biosynthetic
genes in Drosophila (52–54).
Despite the evidence for additional cofactors, the in vitro
binding experiments with mfz8CRD-IgG show that affinities of
?10 nM are obtained with the CRD alone. These experiments
imply that the Frizzled transmembrane domains, plasma mem-
brane lipids, ECM, and putative coreceptors do not play an
obligatory role in Wnt-Frizzled binding. In particular, the solid
phase binding assay shows that heparin does not alter the affinity
stabilization induced by XWnt8-myc treatment of C57MG cells.
These data suggest that the role of heparin in Wnt signal
transduction may not involve a direct effect on receptor–ligand
binding, a possibility that would be in contrast to the direct role
of heparin in fibroblast growth factor dimerization and receptor
binding (55, 56). We note that the experiments reported here do
not rule out the possibility that other proteins in the S2 condi-
tioned medium may associate with XWnt8 and participate in the
Wnt, Frizzled, and sFRP proteins are all members of large
identified in mammals, and several Wnt and Frizzled genes have
been identified in Drosophila and in C. elegans. The broad and
overlapping patterns of expression seen for many of these genes
raises the possibility of multiple Wnt-Frizzled and Wnt-sFRP
interactions. There is currently little quantitative information
regarding the affinity and specificity of interactions among indi-
vidual members of these protein families in vitro, and, in most
cases, it is not yet known which family members interact in a
biologically meaningful manner in vivo. The in vitro interactions
observed among several Wnt, Frizzled, and sFRP proteins from
diverse vertebrate and invertebrate species indicate that the
relevant protein–protein interfaces have been conserved during
evolution and suggests that many pairs of family members may
have the potential to interact.
The authors thank Dr. Randy Moon for the XWnt8-myc plasmid; Dr.
Peter Klein for Xfz8; Dr. Brian Seed for the human IgG cDNA; Dr. John
Flanagan for the AP cDNA; Dr. Ingrid Caras for the decay activating
factor cDNA; and Drs. Phil Beachy and Dan Leahy for comments on the
manuscript. This work was supported by the Howard Hughes Medical
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