Heparan sulfate regulates ephrin-A3/EphA receptor signaling.
ABSTRACT Increasing evidence indicates that many signaling pathways involve not only ligands and receptors but also various types of coreceptors and matrix components as additional layers of regulation. Signaling by Eph receptors and their ephrin ligands plays a key role in a variety of biological processes, such as axon guidance and topographic map formation, synaptic plasticity, angiogenesis, and cancer. Little is known about whether the ephrin-Eph receptor signaling system is subject to such additional layers of regulation. Here, we show that ephrin-A3 binds to heparan sulfate, and that the presence of cell surface heparan sulfate is required for the full biological activity of ephrin-A3. Among the ephrins tested, including ephrin-A1, -A2, -A5, -B1, and -B2, only ephrin-A3 binds heparin or heparan sulfate. Ephrin-A3-dependent EphA receptor activation is reduced in mutant cells that are defective in heparan sulfate synthesis, in wild-type cells from which cell surface heparan sulfate has been removed, and in the hippocampus of conditional knockout mice defective in heparan sulfate synthesis. Ephrin-A3-dependent cell rounding is impaired in CHO cells lacking heparan sulfate, and cortical neurons lacking heparan sulfate exhibit impaired growth cone collapse. In contrast, cell rounding and growth cone collapse in response to ephrin-A5, which does not bind heparan sulfate, are not affected by the absence of heparan sulfate. These results show that heparan sulfate modulates ephrin/Eph signaling and suggest a physiological role for heparan sulfate proteoglycans in the regulation of ephrin-A3-dependent biological processes.
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ABSTRACT: A recent genome-wide association study indicated that rs11098403, a single nucleotide polymorphism in the vicinity of NDST3, was strongly associated with the risk of schizophrenia in Caucasians. However, this relation has not been validated in other populations or ethnic groups. Herein, we conducted a case-control study to investigate the association of rs11098403 polymorphism with the schizophrenia risk in a Han Chinese population comprising 440 schizophrenia patients and 450 control subjects. For the first time, we showed that the minor allele (G) of rs11098403 is closely associated with a reduced risk of schizophrenia (OR=0.614; 95% CI: 0.453-0.833; P=0.002; Power=0.832). Meanwhile, the G allele of rs11098403 seemed to reduce the schizophrenia risk via a dominant manner (GG+AG vs. AA, OR=0.526; 95% CI: 0.374-0.74; P<0.001). Furthermore, this association was further confirmed using an independent replication sample containing 267 schizophrenia patients and 400 control subjects with a Han Chinese descent (OR=0.652; 95% CI: 0.469-0.907; P=0.011; Power=0.772). Taken together, these findings demonstrate a significant association between rs11098403 and schizophrenia risk in Han Chinese, confirming the data that previously obtained from Caucasians.Neuroscience Letters 08/2014; · 2.06 Impact Factor
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ABSTRACT: Heparanase is a β-D-endoglucuronidase that cleaves heparan sulfate (HS), facilitating degradation of the extracellular matrix (ECM) and the release of HS-bound biomolecules including cytokines. The remodeling of the ECM by heparanase is important for various physiological and pathological processes, including inflammation, wound healing, tumour angiogenesis and metastasis. Although heparanase has been proposed to facilitate leukocyte migration through degradation of the ECM, its role in inflammation by regulating the expression and release of cytokines has not been fully defined. In this study, the role of heparanase in regulating the expression and release of cytokines from human and murine immune cells was examined. Human peripheral blood mononuclear cells treated ex vivo with heparanase resulted in the release of a range of pro-inflammatory cytokines including IL-1β, IL-6, IL-8, IL-10 and TNF. In addition, mouse splenocytes treated ex vivo with heparanase resulted in the release of IL-6, MCP-1 and TNF. A similar pattern of cytokine release was also observed when cells were treated with soluble HS. Furthermore, heparanase-induced cytokine release was abolished by enzymatic-inhibitors of heparanase, suggesting this process is mediated via the enzymatic release of cell surface HS fragments. As soluble HS can signal through the Toll-like receptor (TLR) pathway, heparanase may promote the upregulation of cytokines through the generation of heparanase-cleaved fragments of HS. In support of this hypothesis, mouse spleen cells lacking the key TLR adaptor molecule MyD88 demonstrated an abolition of cytokine release after heparanase stimulation. Furthermore, TLR4-deficient spleen cells showed reduced cytokine release in response to heparanase treatment, suggesting that TLR4 is involved in this response. Consistent with these observations, the pathway involved in cytokine upregulation was identified as being NF-κB-dependent. These data identify a new mechanism for heparanase in promoting the release of pro-inflammatory cytokines that is likely to be important in regulating cell migration and inflammation.PLoS ONE 10/2014; 9(10):e109596. · 3.53 Impact Factor
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ABSTRACT: Asparagine-linked glycosylation is an endoplasmic reticulum co- and post- translational modification that enables the transit and function of receptor tyrosine kinase (RTK) glycoproteins. To gain insight into the regulatory role of glycosylation enzymes on RTK function, we investigated shRNA and siRNA knockdown of mannose phosphate isomerase (MPI), an enzyme required for mature glycan precursor biosynthesis. Loss of MPI activity reduced phosphorylation of FGFR family receptors in U-251 and SKMG-3 malignant glioma cell lines and also resulted in significant decreases in FRS2, Akt, and MAPK signaling. However, MPI knockdown did not affect ligand-induced activation or signaling of EGFR or MET RTKs, suggesting that FGFRs are more susceptible to MPI inhibition. The reductions in FGFR signaling were not caused by loss of FGF ligands or receptors, but instead were caused by interference with receptor dimerization. Investigations into the cellular consequences of MPI knockdown showed that cellular programs driven by FGFR signaling, and integral to the clinical progression of malignant glioma, were impaired. In addition to a blockade of cellular migration, MPI knockdown also significantly reduced glioma cell clonogenic survival following ionizing radiation. Therefore our results suggest that targeted inhibition of enzymes required for cell surface receptor glycosylation can be manipulated to produce discrete and limited consequences for critical client glycoproteins expressed by tumor cells. Furthermore, this work identifies MPI as a potential enzymatic target for disrupting cell surface receptor-dependent survival signaling and as a novel approach for therapeutic radiosensitization.PLoS ONE 10/2014; 9(10):e110345. · 3.53 Impact Factor
Heparan sulfate regulates ephrin-A3/EphA
Fumitoshi Irie*, Misako Okuno*, Kazu Matsumoto*, Elena B. Pasquale†, and Yu Yamaguchi*‡
*Sanford Children’s Health Research Center and†Cancer Center, Burnham Institute for Medical Research, 10901 North Torrey Pines Road, La Jolla, CA 92037
Communicated by Erkki Ruoslahti, Burnham Institute for Medical Research, Santa Barbara, CA, February 8, 2008 (received for review August 3, 2007)
Increasing evidence indicates that many signaling pathways in-
volve not only ligands and receptors but also various types of
coreceptors and matrix components as additional layers of regu-
lation. Signaling by Eph receptors and their ephrin ligands plays a
key role in a variety of biological processes, such as axon guidance
and topographic map formation, synaptic plasticity, angiogenesis,
and cancer. Little is known about whether the ephrin-Eph receptor
signaling system is subject to such additional layers of regulation.
Here, we show that ephrin-A3 binds to heparan sulfate, and that
the presence of cell surface heparan sulfate is required for the full
biological activity of ephrin-A3. Among the ephrins tested, includ-
ing ephrin-A1, -A2, -A5, -B1, and -B2, only ephrin-A3 binds heparin
or heparan sulfate. Ephrin-A3-dependent EphA receptor activation
is reduced in mutant cells that are defective in heparan sulfate
has been removed, and in the hippocampus of conditional knock-
out mice defective in heparan sulfate synthesis. Ephrin-A3-depen-
dent cell rounding is impaired in CHO cells lacking heparan sulfate,
and cortical neurons lacking heparan sulfate exhibit impaired
growth cone collapse. In contrast, cell rounding and growth cone
collapse in response to ephrin-A5, which does not bind heparan
sulfate, are not affected by the absence of heparan sulfate. These
results show that heparan sulfate modulates ephrin/Eph signaling
and suggest a physiological role for heparan sulfate proteoglycans
in the regulation of ephrin-A3-dependent biological processes.
proteoglycan ? cell adhesion ? growth cone collapse
more heparan sulfate chains are covalently attached to a variety
of core proteins (1). Heparin is a specialized form of heparan
sulfate synthesized exclusively by connective tissue mast cells,
whereas heparan sulfate is expressed in many cell types. The
negatively charged sulfate groups of heparan sulfate mediate
interactions with a variety of proteins. Increasing evidence
indicates that heparan sulfate acts as an integral component of
a number of morphogen and growth factor signaling pathways by
interacting with these molecules. For example, fibroblast growth
factors, Wnts, Sonic hedgehog, bone morphogenetic proteins,
and neuregulins bind to heparan sulfate and are functionally
modulated by it (2, 3). Molecules involved in axon pathfinding,
such as netrin-1, Slit, and semaphorins, are other major targets
of regulation by heparan sulfate (4). We have used genetic
ablation of the Ext1 gene, which encodes a glycosyltransferase
essential for heparan sulfate synthesis (5, 6), to demonstrate the
physiological significance of the interactions of some of these
molecules with heparan sulfate (7–9).
The Eph receptors form the largest family of receptor tyrosine
kinases. Together with their membrane-bound ligands, the
ephrins, they are involved in bidirectional signaling between two
interacting cells (10, 11). Signals generated by the engagement of
ephrin ligands to Eph receptors generally result in repulsive
responses, such as retraction of the cell periphery and cell
rounding, and growth cone collapse in neurons. These repulsive
responses are thought to be the basis for the function of the
ephrin-Eph system in axon guidance.
eparan sulfate is a class of sulfated glycosaminoglycans. It
occurs as heparan sulfate proteoglycans, in which one or
The function of many important morphogens and guidance
molecules has been shown to involve accessory molecules as
coreceptors or as a mechanism to control the topological dis-
tribution of the guidance molecules (7, 8, 12–14). Curiously,
there is little information on whether ephrin-Eph signaling is
influenced by heparan sulfate or other accessory regulatory
molecules. In this study, we examined whether ephrin-Eph
signaling is modulated by heparan sulfate. Our results surpris-
ingly reveal that ephrin-A3 uniquely binds heparan sulfate.
Ephrin-A3-mediated EphA receptor activation and biological
activities are attenuated in cells lacking heparan sulfate. Cortical
neurons deficient in heparan sulfate synthesis, isolated from
conditional Ext1 knockout mice (7), exhibit greatly impaired
growth cone collapse in response to ephrin-A3, as do wild-type
cortical neurons from which cell surface heparan sulfate has
been removed by heparitinase digestion. These results provide
evidence for an involvement of heparan sulfate in ephrin/Eph
receptor signaling and suggest that heparan sulfate modulates
ephrin-A3-dependent biological processes.
binding of various ephrins to heparan sulfate, we generated a
These recombinant ephrins all included the entire extracellular
domain, up to the last amino acid before the stretch of hydro-
phobic amino acids that serve as either the signal for GPI-anchor
attachment (in the case of the A ephrins) or the transmembrane
domain (in the case of the B ephrins), followed by the FLAG
epitope. Culture supernatants from transfected 293T cells con-
taining the secreted ephrin ectodomains were applied to hepa-
rin-Sepharose and eluted by a stepwise increase in NaCl con-
centration. Only ephrin-A3 bound to heparin (Fig. 1A). None of
the other ephrins examined (ephrin-A1, -A2, -A5, -B1, -B1, or
-B2) nor the ectodomains of several Eph receptors (EphA2, -A3,
-A4, or -B2) exhibited detectable binding to heparin. Elution of
ephrin-A3 from heparin-Sepharose required 0.4–0.8 M NaCl.
This suggests that ephrin-A3 binds with an affinity similar to
those of several known heparin-binding factors, such as Sonic
hedgehog, Wnt1, the Ig-like domain-containing isoform of neu-
regulin 1, and various chemokines (15–18).
To further confirm the significance of the above results, we
performed two additional binding experiments. First, we exam-
ined binding of full-length untagged ephrins to heparin. Cell
lysates from 293T cells that had been transfected with full-length
untagged ephrin-A3 or -A5 were applied to heparin-Sepharose,
and eluted ephrin-A3 and -A5 were detected with the respective
antibodies. Consistent with the results obtained with FLAG-
Author contributions: F.I., E.B.P. and Y.Y. designed research; F.I. and M.O. performed
research; E.B.P. contributed new reagents/analytic tools; K.M. analyzed data; and F.I. and
Y.Y. wrote the paper.
The authors declare no conflict of interest.
‡To whom correspondence should be addressed. E-mail: firstname.lastname@example.org.
This article contains supporting information online at www.pnas.org/cgi/content/full/
© 2008 by The National Academy of Sciences of the USA
August 26, 2008 ?
vol. 105 ?
no. 34 ?
tagged ectodomains, the native form of ephrin-A3 bound to
heparin, whereas that of ephrin-A5 did not (Fig. 1B). Second, we
examined whether ephrin-A3 binds not only to heparin but also
to heparan sulfate. Heparin is synthesized exclusively in con-
nective tissue mast cells (19) and, therefore, binding to heparin
does not necessarily imply the biological significance of the
interaction in other cell type. Binding of soluble forms of ephrins
and Eph receptors to heparan sulfate was examined in an
ELISA-type assay. As shown in Fig. 2, ephrin-A3 displayed
receptor autophosphorylation were analyzed in cells lacking heparan sulfate
expression. (A) Experiments using CHO cells. (Left) Comparison between
wild-type (WT) and pgsD-677 mutant (pgsD-677) cells. pgsD-677 cells show
reduced EphA2 phosphorylation in response to ephrin-A3 (compare lane 3
and lane 4). (Right) Removal of cell surface heparan sulfate by heparitinase
treatment reduces EphA2 autophophorylation in wild-type CHO cells (com-
pare lane 9 and lane 10). (H’ase) heparitinase treated cells; (Cont) mock
treated cells. Note that phosphorylation in response to ephrin-A5 was not
affected in either pgsD-677 cells or wild-type cells treated with heparitinase
(lanes 5, 6, 11, and 12). (B) Experiments using cortical neurons. (Left) Compar-
ison between wild-type (WT) and Ext1 null (Ext1-KO) cortical neurons. Ext1
null neurons show reduced EphA4 phosphorylation in response to ephrin-A3
(compare lanes 3 and 4). (Right) Removal of cell surface heparan sulfate by
heparitinase treatment also reduces EphA4 autophophorylation in wild-type
cortical neurons (compare lane 9 and lane 10). (H’ase) heparitinase treated
cells; (Cont) mock treated cells. Note that the response to ephrin-A5 was not
affected in either Ext1 null neurons or wild-type neurons treated with hep-
aritinase (lanes 5, 6, 11, and 12). Bar graphs below each blot represent
fold-changes in the intensity of immunoreactive bands. Error bars represent
standard deviation of the relative intensity from three independent experi-
ments. (PY-EphA2) tyrosine phosphorylated EphA2; (PY-EphA4) tyrosine
tyrosine phosphorylation in vivo. Phosphorylation of endogenous EphA4 in
the hippocampus is compared between CaMKII-Cre;Ext1flox/flox(lane 2) and
control mice (lane 1). Bar graphs represent fold-changes in the intensity of
Ephrin-A3-dependent EphA signaling is impaired in cells defective in
proteins to heparin-Sepharose. FLAG-tagged ectodomains of the indicated
ephrins and Eph receptors were expressed in 293T cells and the culture
supernatants (Sample) were incubated with heparin-Sepharose. Unbound
materials were collected (Pass) and bound materials were eluted with indi-
cated final concentrations of NaCl. Each fraction was analyzed by immuno-
and ephrin-A5 to heparin-Sepharose. Lysates of 293T cells transfected with
full-length ephrin-A3 or ephrin-A5 were incubated with heparin-Sepharose.
After washing with 0.2 M NaCl, bound materials were eluted with 1.2 M NaCl
(Bound). Each fraction was analyzed by immunoblotting with anti-ephrin-A3
or anti-ephrin-A5 antibody.
Ephrin-A3 binds heparin. (A) Binding of soluble ephrin and Eph
receptors with heparan sulfate were examined by an ELISA-type assay. Puri-
fied recombinant ectodomains of ephrins and Eph receptors fused to Fc and
a hexahistidine tag (R&D Systems) were coated on copper-treated wells.
Biotinylated heparan sulfate was incubated in these wells, and binding was
measured by colorimetric enzyme reaction. Error bars represent standard
deviation from quadruplicate wells.
Ephrin-A3 binds heparan sulfate. Interactions of ephrins and Eph
www.pnas.org?cgi?doi?10.1073?pnas.0801302105Irie et al.
specific binding to heparan sulfate, whereas all other A and B
ephrins and EphA2 and -B2 did not show detectable binding.
Abrogation of Cellular Heparan Sulfate Expression Impairs Ephrin-A3-
Induced EphA Signaling.Engagementwithephrin-Aligandscauses
autophosphorylation of Eph receptors on tyrosine residues,
which is the first step leading to activation of downstream
signaling pathways (11). We therefore examined whether lack of
heparan sulfate affects ephrin-A3-dependent EphA receptor
tyrosine phosphorylation using two cell types that endogenously
express different EphA receptors. First, we analyzed EphA2
phosphorylation in wild-type CHO cells and their heparan
sulfate-deficient mutant pgsD-677 (Fig. 3A, lanes 1–6). pgsD-677
cells (20), a subline of CHO cells, lack the ability to synthesize
heparan sulfate due to the disruption of Ext1, which encodes a
glycosyltransferase essential for heparan sulfate synthesis (5, 6).
Treatment with ephrin-A3–Fc induced substantial phosphory-
lation of EphA2 in wild-type CHO cells (lane 3) but much
weaker phosphorylation in pgsD-677 cells (lane 4). In contrast,
ephrin-A5 induced similar levels of EphA2 phosphorylation in
wild-type and pgsD-677 cells (lanes 5 and 6). We further
examined whether enzymatic elimination of cell surface heparan
sulfate by heparitinase treatment affects EphA2 autophosphor-
ylation (Fig. 3A, lanes 7–12). This treatment also reduced
ephrin-A3-dependent EphA2 phosphorylation (lane 10) but had
no effect on ephrin-A5-dependent phosphorylation (lanes 11
Second, we examined signaling in cortical neurons, which
express EphA4 and undergo growth cone collapse in response to
ephrin-A3 and -A5. Ext1-null and wild-type cortical neurons
were prepared from embryonic day (E)15.5 Nestin-Cre;Ext1flox/
flox (7) and control embryos. We have previously demonstrated
that heparan sulfate expression is abrogated in these neurons. In
wild-type neurons, treatment with ephrin-A3 induced strong
EphA4 phosphorylation (Fig. 3B, lane 3). In Ext1-null neurons,
EphA4 phosphorylation in response to ephrin-A3 was signifi-
cantly reduced (lane 4). Heparitinase treatment of wild-type
neurons also reduced EphA4 phosphorylation (compare lanes 9
and 10). However, loss of heparan sulfate, either by genetic
ablation of Ext1 or heparitinase treatment, did not affect ephrin-
A5-induced EphA4 phosphorylation (compare lanes 5 and 6 and
lanes 11 and 12).
Finally, we examined the role of heparan sulfate in ephrin-A3
signaling in vivo. Ephrin-A3 is the predominant ephrin-A ligand
expressed in the adult hippocampus and has been proposed to
play a major role in the activation of endogenous EphA4 in this
brain structure (21). If heparan sulfate is physiologically involved
in ephrin-A3 signaling, the phosphorylation level of EphA4
should be reduced in mice lacking heparan sulfate in the
hippocampus. To test this, we generated conditional Ext1 knock-
out mice using the CaMKII-Cre2834 transgene (22). [Condi-
tional Ext1 knockout mice driven by Nestin-Cre could not be
used because they die at birth (7).] The CaMKII-Cre2834
transgene drives forebrain-specific recombination only after
birth and is particularly efficient in the hippocampus [ref. 22; see
also supporting information (SI) Fig. S1]. Consistent with a
physiological role of heparan sulfate, tyrosine phosphorylation
response to ephrin-A3. (A) Morphological changes in response to ephrin-A3
and -A5 were examined in wild-type CHO cells (a–c), heparitinase-treated
wild-type CHO cells (d–f), and pgsD-677 mutant CHO cells (g–i). Cells were
(c, f, and i), and then stained with rhodamine-phalloidin. Ephrin-A3-treated
wild-type CHO cells show almost complete cell rounding (b). In contrast, both
heparitinase-treated wild-type cells and pgsD-677 mutant cells maintain a
certain level of spreading and stress fibers, although they undergo partial cell
retraction (e and h). The effects of ephrin-A5 were not influenced by the
restores the ephrin-A3-mediated cell rounding response in pgsD-677 cells.
Myc-tagged Ext1 cDNA was transfected in pgsD-677 cells and cells were
Fc (a, d, and g). Cells were double-labeled with rhodamine-phalloidin (a–c)
and anti-Myc antibody (d–f) to visualize cell morphology and to identify cells
Cells lacking heparan sulfate exhibit impaired cell rounding in
Note that ephrin-A3 induces cell rounding in pgsD-677 cells expressing trans-
fected Ext1 (indicated by arrowheads in b, e, h), whereas nontransfected cells
in the same culture does not exhibit cell rounding (indicated by an arrow in b,
e, and h). Expression of Ext1 does not affect cell rounding induced by eph-
bar, 10 ?m.) (C) Quantitative analysis of cell rounding. See SI Text for details
bars) ephrin-A3; (shaded bars) ephrin-A5. Data represent mean ? SD (n ? 4).
*, P ? 0.02;**, P ? 0.01;***, P ? 0.001.
Irie et al.
August 26, 2008 ?
vol. 105 ?
no. 34 ?
of EphA4 was significantly reduced in mutant hippocampal
tissue compared with wild-type tissue (Fig. 3C).
Ephrin-A3-Induced Cell Rounding Is Impaired in CHO Cells Lacking
Heparan Sulfate. Activation of ephrin-A/EphA signaling induces
a variety of cellular responses, one of which is retraction of the
cell periphery and cell rounding (23, 24). Wild-type CHO cells
underwent robust cell rounding in response to ephrin-A3 (Fig.
4Ab). In comparison, the cell-rounding response of pgsD-677
mutant cells was significantly impaired. Although ephrin-A3-
cell periphery, they remained substantially more spread than
ephrin-A3-treated wild-type CHO cells (Fig. 4Ah). Similarly,
heparitinase-treated wild-type CHO cells remained more spread
than untreated cells after ephrin-A3 stimulation (Fig. 4Ae).
However, cell retraction and rounding in response to ephrin-A5
were similar in heparan sulfate-deficient cells (both hepariti-
nase-treated wild-type cells and pgsD-677 cells) and wild-type
cells (compare Fig. 4A c, f, and i; Fig. 4C).
To confirm that the impaired response of pgsD-677 cells to
ephrin-A3 was due to the lack of heparan sulfate, rather than
other nonspecific changes in these cells, we examined whether
transfection of Ext1 restores normal cell rounding in response to
ephrin-A3 (Fig. 4B). pgsD-677 cells transfected with Ext1 un-
derwent robust cell rounding (indicated by arrowheads in Fig. 4B
b, e, and h), whereas nontransfected cells in the same culture did
not (indicated by arrows in Fig. 4B b, e, and h). Thus, the
impaired cell rounding response of pgsD-677 cells is a direct
consequence of the lack of heparan sulfate. Expression of Ext1
did not make any difference in the cell rounding response to
ephrin-A5 (Fig. 4Bi; Fig. 4C).
Ephrin-A3-Induced Growth Cone Collapse Is Impaired in Cortical
Neurons Lacking Heparan Sulfate. To gain insight into the role of
heparan sulfate in ephrin-A3-mediated axon pathfinding, we
examined growth cone collapse in Ext1-null and heparitinase-
treated cortical neurons. Ephrin-A3 potently induced growth
cone collapse in E15.5 wild-type neurons (Fig. 5Aa), causing the
complete collapse of ?70% of the growth cones (Fig. 5B, WT,
black bar). In contrast, growth cone collapse was significantly
impaired in Ext1-null neurons (Fig. 5Ac). Although some shrink-
age occurred in many growth cones, complete collapse was
observed much less frequently in Ext1-null neurons (Fig. 5B,
Ext1-KO, black bar). The decrease in the number of collapsed
growth cones in Ext1-null neurons is not due to faster recovery
of previously collapsed growth cones, because a time course
study demonstrated that Ext1-null neurons exhibit impaired
growth cone collapse at any time points between 5 and 30 min
of ephrin-A3 stimulation (data not shown). Impairment of
growth cone collapse was also observed in heparitinase-treated
wild-type neurons (Fig. 5Ab; Fig. 5B, WT ? H’ase, black bar),
whereas the absence of HS did not affect growth cone collapse
in response to ephrin-A5 (Fig. 5B, shaded bars). Finally, trans-
fection of Ext1 restored ephrin-A3-dependent growth cone
collapse in Ext1-null neurons (Fig. 5Ad; Fig. 5B, Ext1-KO ?
Ext1-myc, black bar). Together with the results of the cell
retraction and rounding assay, these results demonstrate that cell
biological activity of ephrin-A3.
In this study, we demonstrate the interaction of ephrin-A3 with
heparin and heparan sulfate and its biological significance. Eph-
rin-A3 binds not only to heparin, which is expressed only by
connective tissue mast cells, but also to heparan sulfate, which is
expressed broadly in developing tissues. The affinity of the inter-
for which unambiguous genetic evidence has demonstrated the
physiological relevance of the interaction with heparan sulfate
(25–27). The biological significance of the interaction is supported
by functional assays with cultured CHO cells and cortical neurons
and by the phosphorylation analysis of endogenous EphA4 in the
adult hippocampus of conditional Ext1 knockout mice. Although it
was somewhat surprising to find that binding to heparan sulfate is
unique for ephrin-A3 among the ephrin-A and -B ligands, the
neurons lacking heparan sulfate. (A) Gallery of images of five representative
growth cones from cultures of E15.5 cortical neurons treated with ephrin-A3–Fc
or control Fc. (a) wild-type neurons, (b) wild-type neurons treated with hepariti-
nase, (c) Ext1-null neurons, (d) Ext1-null neurons transfected with Ext1. (Scale
growth cone collapse. WT, wild-type neurons; WT ? H’ase, wild-type neurons
treated with heparitinase; Ext1-KO, Ext1-null neurons; Ext1-KO ? Ext1-myc,
Ext1-null neurons successfully transfected with Ext1-myc. Open bars, control Fc;
black bars, ephrin-A3; shaded bars, ephrin-A5. See SI Text for details of quanti-
**, P ? 0.002;***, P ? 0.01. Note that Ephrin-A3-induced growth cone collapse
is inhibited in both heparitinase-treated wild-type neurons and Ext1-null neu-
rons. Expression of Ext1 restores growth cone collapse in mutant neurons to a
level similar to wild-type neurons.
Growth cone collapse in response to ephrin-A3 is inhibited in cortical
www.pnas.org?cgi?doi?10.1073?pnas.0801302105Irie et al.
specificity observed in our biological experiments is consistent with
the specificity of the binding.
Our results differ from those obtained in a carbohydrate
microarray-based screening of heparin-binding proteins recently
reported by Shipp and Hsieh-Wilson (28). In those experiments,
ephrin-A1-Fc and -A5-Fc were found to bind to heparin coated
on microarrays. Ephrin-A3 was not included in their study, and
the interaction of ephrin-A1-Fc and -A5-Fc with heparan sulfate
was not examined. We do not know the reason for this discrep-
ancy, although it might be due to the difference in assay systems.
In any event, our data demonstrate that ephrin-A1-Fc and
-A5-Fc do not bind to heparan sulfate (see Fig. 2), and that the
biological effects of ephrin-A5 are not affected by the ablation
of cellular heparan sulfate (see Figs. 4 and 5).
The specific interaction of ephrin-A3 with heparan sulfate
suggests that ephrin-A3 binds heparan sulfate via a region
unique to this ephrin. The ectodomain of A-type ephrins consists
of an N-terminal receptor-binding domain and a C-terminal
juxtamembrane linker region. The N-terminal domain is highly
conserved (50–60% amino acid identity among different A-type
ephrins; ?90% between human and mouse within ephrin-A
orthologs). In contrast, the sequence of the C-terminal region is
different in different A-type ephrins but highly conserved across
species (100% amino acid identity between human and mouse
ephrin-A3 or -A5), suggesting that this juxtamembrane linker
region may be responsible for heparan sulfate binding. Consis-
tent with this, truncated ephrin-A3 lacking the entire juxtamem-
brane region does not bind to heparin (unpublished data).
However, this region does not contain any recognizable se-
region has not led to unambiguous identification of specific
residues that are essential for heparin-binding (unpublished
data). Identification of the location of the heparin-binding site
may require detailed analyses of the 3D structure of this region
of ephrin-A3 in complex with heparin or heparan sulfate.
The features of the ephrin-A3–heparan sulfate interaction
bear some interesting similarities to the interaction of neuregulin
isoforms containing an Ig-like domain with heparan sulfate (29).
Like the ephrins, neuregulins function mainly as membrane-
anchored ligands for receptor tyrosine kinases, but they can also
function as proteolytically shed, soluble ligands (30). ADAM
family metalloproteinases have been implicated in the shedding
of neuregulins (31). Interestingly, A-type ephrins are also
cleaved by ADAM family metalloproteases (32). Although
regulated ephrin-A cleavage upon EphA receptor binding has
recently attracted much attention, other studies have demon-
strated constitutive release of A-type ephrins from cells (33–35)
and suggest possible long-range activities of released A-type
ephrins (36). Although the physiological significance of these
shedding events remains to be determined, these observations
suggest the possibility that heparan sulfate serves to capture
released ephrin-A3 on the cell surface. Such a mechanism may
act to increase the local concentration of ephrin-A3 on the cell
surface and/or to cluster the secreted monomeric ephrin-A3.
Thus, when bound to cell surface heparan sulfate, secreted
ephrin-A3 may still activate EphA receptors through a novel cell
In conclusion, we demonstrate the specific interaction be-
tween ephrin-A3 and heparan sulfate and its effect on ephrin-
A3/EphA signaling in vitro and in vivo. Ephrin-A3 has been
shown to function in the guidance of various types of axons in the
developing nervous system (37–39) and in the control of den-
dritic spine morphology (21). Ephrin-A3 has also been proposed
to play a role in promoting the growth of pancreatic cancer cells
(40). Heparan sulfate is expressed in cell types and locations
relevant to these events (41–43). Our results suggest that ephrin-
A3/EphA signaling in these diverse biological settings may be
controlled not only by the ligand-receptor interaction but also by
an additional layer of regulation involving heparan sulfate.
Binding and Phosphorylation Assays. Binding of ephrins to heparin and hepa-
ran sulfate was examined by heparin-Sepharose chromatography and an
ELISA-type assay with biotinylated heparan sulfate, respectively. Ephrin-A-
tation/immunoblotting assay in wild-type and the psgD-677 heparan sulfate-
deficient CHO cells (20) and in primary cortical neurons from wild-type and
Nestin-Cre;Ext1flox/floxconditional knockout (7) embryos.
Assays for Cell Rounding and Growth Cone Collapse. Cell rounding in response
to ephrin-A treatment was examined by using wild-type and pgsD-677 CHO
cells as described in ref. 23. Growth cone collapse in response to ephrin-A
treatment was examined by using wild-type and Ext1 null cortical neurons.
Morphological changes were scored by an observer blinded to experimental
For detailed description of experimental procedures, see SI Text.
ACKNOWLEDGMENTS. We thank Dr. J. Esko for providing Ext1 cDNA and
psgD-677 cells. This work was supported by National Institutes of Health
Grants R01 NS49641 (to Y.Y.) and P01 HD25938 (to Y.Y. and E.B.P.) and by a
grant from the Mizutani Foundation for Glycoscience (to Y.Y.).
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