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EMBO Rep. 2003 August; 4(8): 813–818. Published online 2003 July 25. doi: 10.1038/sj.embor.embor902. | PMCID: PMC1326342 |
Copyright © 2003, European Molecular Biology Organisation Scientific Report Integrin-α3 mediates binding of Chordin to the cell surface and
promotes its endocytosis Juan Larraín,2 Carlos Brown,1 and Eddy M. De Robertis1a 1Howard Hughes Medical Institute, and Department of Biological Chemistry, University of California, Los Angeles, California 90095-1662, USA 2Present address: Department of Cellular and Molecular Biology, Centre for Cell Regulation and Pathology, Pontificia Universidad Católica de Chile, Alameda 340, Santiago, Chile Received April 14, 2003; Revised June 16, 2003; Accepted June 17, 2003. Dorsoventral patterning in animal development is regulated by a
morphogenetic gradient of Bone morphogenetic protein signalling, which is
established by a set of proteins that are conserved from Drosophila to
vertebrates. These include Chordin (Chd)/Short gastrulation, Xolloid/Tolloid
and Twisted gastrulation. Here, we report the identification of a cell-surface
component of this morphogenetic pathway. Prompted by the observation that Chd
protein bound to the surface of certain cell lines with subnanomolar affinity,
we identified two cell-surface proteins that bind to Chd, one of which
corresponds to Integrin-α3. Integrin-α3 and Chd are co-expressed in
the Xenopus embryo. Transfection of Integrin-α3 increased the
binding of Chd to the cell surface, which was competed by an excess of soluble
Integrin-α3. After binding to the cell surface, Chd was translocated into
intracellular endocytic compartments in a temperature-dependent manner. We
propose that Integrin-α3 may regulate the concentration of Chd protein in
the extracellular space by endocytosis. Embryonic patterning arises through the generation of morphogen
gradients during early development. Dorsoventral patterning of fly and
vertebrate embryos has been well studied, and is one of the paradigms for
understanding morphogen function ( Freeman & Gurdon,
2002). The establishment of an extracellular gradient of Bone
morphogenetic protein (BMP) determines cell-fate decisions along the
dorsoventral axis. A gradient of this morphogen is generated in the
extracellular space by the action of a set of evolutionarily conserved secreted
proteins, including Chordin (Chd)/Short gastrulation (Sog), Xolloid
(Xld)/Tolloid (Tld) and Twisted gastrulation (Tsg; De
Robertis et al., 2000). The Drosophila Chd homologue,
Sog, is a key component in the generation of embryonic BMP/Decapentaplegic
(Dpp) gradients ( Eldar et al., 2002). Sog
can diffuse along the dorsoventral axis, and its diffusion and extracellular
concentration levels are enhanced by a dynamin mutation that blocks
endocytosis ( Srinivasan et al., 2002). Chd
and Sog encode secreted proteins of ~120 kDa that contain four
cysteine-rich repeats (CRs). The CR modules bind directly to BMP, preventing
its binding to the BMP receptor ( Piccolo et al.,
1996; Larraín et al.,
2000). The anti-BMP activity of Chd is controlled by two cofactors,
Tld and Tsg. Tld is a zinc metalloproteinase that inactivates Chd by
proteolytic cleavage at sites located just after CR1 and CR3 ( Piccolo et al., 1997; Scott et
al., 1999). Tsg is an evolutionarily conserved secreted protein
that forms ternary complexes with BMP4 and Chordin that are unable to signal in
the absence of Tld. Tsg binding also makes Chd a better substrate for cleavage
by Tld, and competes the anti-BMP activity of Chd proteolytic fragments. Thus,
Tsg promotes BMP activity in the presence of Tld ( Oelgeschläger et al., 2000; Ross et al., 2001; Larraín
et al., 2001). This work was initiated in an attempt to identify other components of
the Chd/BMP/Tsg/Tld regulatory pathway. It was found that Chd binds to the cell
surface of various tissue-culture cell lines. To identify the molecular basis
of this interaction, we used affinity-chromatography Chd columns and found that
two cell membrane proteins bound to Chd. One of them was identified as the
transmembrane receptor Integrin-α3. An excess of soluble purified
Integrin-α3 prevented the binding of Chd to the cell surface. The
cell-surface interaction between Chd and Integrin-α3 was followed by
endocytosis at 37 °C. Chd and Integrin-α3 are coordinately expressed
in the dorsal mesoderm of Xenopus embryos. We propose that the levels of
Chd in the extracellular space may be regulated by endocytosis through
Integrin-α3. During screening for novel Chd-interacting proteins using a
secretion-trap expression cloning method ( Davis et
al., 1996), we noticed that non-transfected COS-7 cells bound
detectable amounts of a Chd–alkaline-phosphatase fusion protein
(Chd–AP). When binding was quantified using a biochemical fluorescent AP
assay, Chd–AP bound to COS-7 cells in amounts significantly higher than
background levels (). As negative controls, we
used a secreted version of AP (s-AP) and a Tsg–AP fusion protein, neither
of which bound to COS-7 cells in detectable amounts (; and data not shown). We were able to saturate the binding of
Chd–AP to COS-7 cells at 4 °C using increasing concentrations of
Chd–AP protein, with an estimated dissociation constant
( Kd) of 0.24 nM (). This binding
was specific, as it was competed by excess purified mouse Chd protein (). Chd–AP binding was lost from COS-7 cells that were pretreated
with 0.1% or 1% Triton X-100 (); these amounts of
detergent are known to solubilize integral plasma membrane proteins, but not
extracellular matrix (ECM) components ( Mehta et al.,
1985). This suggested that Chd binds to a cell membrane protein. To
visualize binding to the cell surface, 10T1/2 fibroblasts were incubated at 4
°C with a protein consisting of Chd fused to the Fc fragment of human IgG
(Chd–Fc), fixed, and stained with a fluorescent anti-human IgG.
Chd–Fc binding was detected in membrane ruffles, filopodia and structures
reminiscent of focal-adhesion plaques, particularly at the leading edge of the
cell (). Chd–Fc co-localized with actin
filaments stained by fluorescent phalloidin, indicating that some Chd binding
sites overlap with focal-adhesion plaques (,
note yellow staining where the Chd and actin signals overlap). These results
show that Chd can bind with high affinity ( Kd in the
subnanomolar range) to the cell surface in cultured cell lines. To identify the cell-surface proteins that bind to Chd, we prepared a
Chd affinity-chromatography column. Chd–Fc and secreted Fc (s-Fc) fusion
proteins were produced in S2 cells, purified with protein A, and covalently
crosslinked to protein-A–agarose beads using dimethylpimelimidate (DMP).
Cell-surface proteins from COS-7 cells were biotinylated (using EZ-link-Biotin;
Pierce), solubilized with 0.1% Triton X-100 (as in ), and loaded on Chd–Fc or s-Fc affinity columns. Bound proteins
were eluted with SDS and visualized by performing a
streptavidin–horseradish-peroxidase (SA–HRP) immunoblot. As shown
in , lane 3, the Chd–Fc column bound two
proteins that migrated in non-reducing SDS gels with apparent molecular weights
of 170 and 200 kDa. Neither protein bound to the s-Fc control column (, lane 2). The molecular weight of these proteins and the localization
of the Chd–Fc staining on focal-adhesion plaques () suggested integrins as candidate cell-surface receptors for
Chd. Integrins constitute a large transmembrane receptor family of at least
24 distinct heterodimers, and were originally described as one of the main
components of focal-adhesion plaques in fibroblasts ( Hynes,
2002). Among them, Integrin-α3 stood out as an attractive
candidate because it is co-expressed with Chd in the dorsal blastopore lip and
its derivatives (the notochord and chordoneural hinge) and is coordinately
upregulated with Chd in embryos that are dorsalized by lithium chloride
treatment (; Sasai
et al., 1994; Whittaker & DeSimone,
1991; Meng et al., 1997). As Chd
and Integrin-α3 are coordinately regulated in the dorsal mesoderm, it
seemed possible that they might function in the same biochemical pathway.
Furthermore, Integrin-α3 migrates with an apparent molecular weight of
170 kDa in non-reducing SDS gels ( Shang et al.,
2001). For these reasons, we tested whether Integrin-α3 is one
of the Chd cell-surface receptors that is expressed in COS-7 cells. An
anti-Integrin-α3 antibody immunoprecipitated a biotinylated protein with
a molecular weight similar to the smaller band bound by the Chd–Fc column
(, compare lanes 3 and 5). Furthermore, the
170-kDa protein purified by the Chd–Fc affinity matrix was specifically
recognized by the anti-Integrin-α3 antibody in immunoblots (, lane 3). Thus, one of the cellsurface proteins that
interact with Chd corresponds to Integrin-α3. The nature of the 200-kDa
protein remains unknown; one possibility is that it corresponds to
Integrin-β1, the heterodimerization partner of Integrin-α3, but this
seems unlikely as the expected molecular weight of Integrin-β1 in
non-reducing gels is 110 kDa ( Shang et al.,
2001). We next tested whether the binding of Chd to COS-7 cells could be
mediated by Integrin-α3. First, an excess of recombinant purified
Integrin-α3 blocked the binding of Chd–AP to COS-7 cells (). Second, we found that the amount of Chd–AP bound to the
different cell lines correlated with the endogenous levels of Integrin-α3
expression (). P19 cells, which express low levels
of Integrin-α3 bound approximately six times less Chd–AP than did
COS-7 cells, which express higher levels of Integrin-α3 protein (, inset). 293T cells, which do not express detectable
levels of Integrin-α3, did not bind Chd–AP (). Third, to test whether Integrin-α3 was sufficient for binding
of Chd to the cell surface, 293T cells were transfected with a full-length
human integrin-α3 complementary DNA and stained with Chd–AP. We
found that 293T cells transfected with integrin-α3 bound Chd–AP to
the cell surface (). We conclude from these
experiments that Integrin-α3 mediates the binding of Chd to the cell
surface. Recent studies have highlighted the importance of receptor-mediated
endocytosis in the formation of morphogen gradients in the embryo ( Lander
et al., 2002; Seto et al.,
2002; Vincent & Dubois, 2002). For
example, BMP/Dpp diffusion can be regulated by endocytosis after binding to its
signalling receptor in imaginal discs ( Entchev et al.,
2000), and the secreted protein Dickkopf regulates
Wingless/Int-related (Wnt) signalling by inducing the rapid removal of the Wnt
co-receptor lipoprotein-receptor-related protein 6 (LRP6) by endocytosis,
together with the transmembrane protein Kremen ( Mao et
al., 2002). Integrins are transmembrane glycoproteins that link
the ECM to intracellular cytoskeletal and signalling networks, which, in
concert, regulate cell adhesion, migration, cell–cell communication,
proliferation and survival ( Kreidberg, 2000). One
of the functions described for integrins is their ability to mediate the
internalization of ECM components, adenoviruses and apoptotic cells through
endocytosis ( Memmo & McKeown-Longo, 1998;
Li et al., 2001; Hanayama
et al., 2002). To determine whether Integrin-α3 regulates extracellular levels
of Chd through endocytosis, we analysed the cellular localization of
Chd–Fc in COS-7 cells using a fluorescent probe that marks endocytic
vesicles ( Entchev et al., 2000; Texas Red
Dextran; molecular weight 3,000 Da). When incubated at 4 °C, the
Chd–Fc signal was detected on the cell surface (). To allow internalization, cells were shifted to 37 °C
for 30 min. After this treatment, Chd–Fc colocalized with Texas Red
Dextran in punctate intracellular vesicles (; note yellow spots in the inset in ). When COS-7 cells were transfected with full-length
integrin-a3 cDNA and stained with Chd–AP, the amount of
intracellular Chd–AP was greatly increased compared with untransfected
COS-7 cells (data not shown). These results indicate that binding of Chd to
Integrin-α3 on the surface of COS-7 cells results in its endocytosis and
removal from the extracellular space in a temperature-dependent manner. Recently, a gradient of the Sog protein has been directly visualized
in the Drosophila embryo and modified using the temperature-sensitive
shibire ( shits) endocytosis mutant ( Srinivasan et al., 2002). Interestingly, at the
nonpermissive temperature, blockage of endocytosis increases the extracellular
levels of Sog and enhances its diffusion towards the dorsal pole of the fly
embryo ( Srinivasan et al., 2002). Thus, the
Sog gradient is regulated and maintained by endocytosis. Chd and
Integrin-α3 are co-expressed during Xenopus development.
Integrin-α3 may limit Chd diffusion by endocytosis and removal from the
extracellular space or, alternatively, may facilitate transcytosis, regulating
the shape of a hypothetical Chd gradient. The experiments presented here show
that Chd can bind to the cell surface through Integrin-α3 in cell
culture, and that this binding can result in the removal of Chd from the
extracellular space. Previous work has shown that endocytosis constitutes an
important step in the formation of morphogenetic gradients ( Entchev et al., 2000; Lander et
al., 2002; Seto et al., 2002;
Vincent & Dubois, 2002). A possible functional
interaction between Chd and Integrin-α3 during Xenopus embryonic
development is independently supported by recent observations in
Drosophila ( Araujo et al., 2003).
Genetic interactions have been detected between sog and several
integrin-coding genes ( myospheroid, multiple edematous wing and
scab) during the formation of wing veins in pupal development. In
addition, in co-immunoprecipitation experiments, it was found that α-PS1
integrin, which is encoded by multiple edematous wing, binds to the Sog
protein ( Araujo et al., 2003).
Interestingly, Drosophila α-PS1 integrin is most similar to
vertebrate integrin-α3. We propose that the interaction between
Integrin-α3 and Chordin provides another evolutionarily conserved
mechanism for modulating BMP/Dpp signalling in the extracellular space. Affinity chromatography and biotinylation. Chd–Fc and s-Fc fusion proteins were produced in stable
Drosophila S2 cell lines. For purification of Fc fusion proteins, 1 l of
Drosophila Serum-free Medium (Invitrogen) containing the secreted
proteins was bound to 1 ml of protein-A–sepharose, and the column was
washed with 50 ml of 0.01 M sodium phosphate, pH 8.0, 150 mM NaCl. Proteins
bound to protein A were eluted with five column-volumes of 0.1 M glycine, pH
3.0, and fractions of 1 ml were collected and neutralized in tubes containing
50 μl of 1 M Tris-HCl, pH 9.0. The proteins purified in this way were bound
to protein-A–agarose beads (~0.5 mg protein per ml of wet beads) and
crosslinked using DMP (Pierce; Harlow & Lane,
1988). For biotinylation, COS-7 cells were grown to confluency,
washed twice with ice-cold PBS, and incubated on ice for 30 min with 1.0 mg
ml −1 EZ-link sulpho-NHS-biotin (Pierce) in PBS. The
reaction was stopped by washing cells twice with ice-cold PBS and incubating
them for 30 min with 0.1 M glycine in PBS. Two 100-mm plates of cells were
lysed with lysis buffer (10 mM Tris pH 7.5, 0.15 M NaCl, 10% glycerol, 0.1%
Triton X-100; with complete protease inhibitor cocktail, EDTA-free (Roche
Biochemicals)). Chd–Fc and s-Fc beads were incubated for 16 h at 4 °C
with cell lysates in lysis buffer supplemented with 2 mM of each of the
following divalent cations: Mg 2+, Ca 2+ and
Mn 2+. Beads were washed four times with ice-cold lysis buffer,
and bound proteins were eluted with SDS loading buffer and analysed by
immunoblotting using SA–HRP (Pierce) at a dilution of 1 in 4,000. Cell-surface binding. Cells were grown to 80% confluence in 24-well plates, washed twice
with 1 ml of ice-cold HBSS containing 0.5 mg ml −1 BSA and
20 mM Hepes, pH 7.0, and incubated for 2 h on ice with 250 μl of AP fusion
protein ( Flanagan & Cheng, 2000). Cells were
then washed on ice six times with 1 ml of ice-cold HBSS and extracted with 100
μl of 10 mM Tris-HCl, pH 7.5, containing 1% Triton X-100. Endogenous AP
activity was inactivated by incubation of the plate at 65 °C for 1 h. AP
activity resulting from fusion proteins bound to the cells was detected by a
fluorescent assay in 96-well FluoroNunc microplates. Cell extracts (25 μl)
were mixed with 25 μl of 2× assay buffer containing 0.2 mM
4-methylumbelliferyl-phosphate (Sigma) in 0.1 M diethanolamine–HCl, pH
9.8, 1 mM MgCl 2. After 1 h of incubation at 37 °C, the reaction
was stopped with 200 μl of 0.5 M glycine–NaOH. Fluorescence was read
at 465 nm (emission) on an HTS 7000 Plus Bio Assay plate-reader (Perkin Elmer).
To equalize loading, protein concentration was measured using a Lowry-modified
colorimetric assay compatible with detergents (DC Protein Assay; BioRad). Cell staining and immunohistochemistry. For histochemical staining, cells were grown to 80% confluence in
six-well plates and incubated with the AP fusion proteins for 2 h at 25 °C.
Cells were washed for 5 min on ice, four times with 2 ml of ice-cold HBSS and
once with 2 ml of AP buffer (0.1 M Tris-HCl, pH 9.5, 0.1 M NaCl). To inactivate
endogenous AP, 1 mM levamisole (Sigma) was included in the last two washes. To
develop AP activity, cells were incubated at 25 °C with 2 ml of a NBT
(nitroblue tetrazolium chloride)/BCIP (5-bromo-4-chloro-3-indolyl-phosphate)
solution filtered using a 0.2-μm filter (Roche Biochemicals) until positive
purple cells were seen ( Oelgeschläger et al.,
2000; Flanagan & Cheng, 2000). For immunohistochemistry, COS-7 or 10T1/2 fibroblast cells were
grown on coverslips coated with poly-L-lysine until 80% confluency was reached,
incubated with Chd–Fc for 2 h on ice, and washed three times with
ice-cold PBS. Some samples were incubated on ice and others at 37 °C for 30
min in PBS containing 0.5 mM Texas-Red-dextran (lysine fixable; molecular
weight 3,000 Da; Molecular Probes) to mark endocytic vesicles ( Entchev et al., 2000). Cells were fixed with 4%
paraformaldehyde in PBS for 20 min and permeabilized with 0.1% Triton X-100 for
10 min. Fluorescence was visualized after incubation with an anti-human IgG
antibody conjugated to Alexa Fluor® 488 (1:200 dilution; Molecular Probes).
For actin staining, Alexa Fluor® 568 phalloidin (1:200 dilution; Molecular
Probes) was used after cell permeabilization. We thank E. Bier for communicating results before publication, S.
Millard and K.-W. Zhao for help with cell staining and fluorescent AP assays,
and O. Wessely, C. Coffinier, H. Lee and L. Fuentealba for reviewing the
manuscript. J.L. was a Pew Latin American Fellow. C.B. is supported by a
National Institutes of Health (NIH) Minority Supplement. This work was
supported by NIH grant R37 HD21502-17 and the Howard Hughes Medical Institute,
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