Article

Vav Family GEFs Link Activated Ephs to Endocytosis and Axon Guidance

Department of Molecular and Cell Biology, Harvard University, Cambridge, Massachusetts, United States
Neuron (Impact Factor: 15.05). 05/2005; 46(2):205-17. DOI: 10.1016/j.neuron.2005.03.019
Source: PubMed
ABSTRACT
Ephrin signaling through Eph receptor tyrosine kinases can promote attraction or repulsion of axonal growth cones during development. However, the mechanisms that determine whether Eph signaling promotes attraction or repulsion are not known. We show here that the Rho family GEF Vav2 plays a key role in this process. We find that, during axon guidance, ephrin binding to Ephs triggers Vav-dependent endocytosis of the ligand-receptor complex, thus converting an initially adhesive interaction into a repulsive event. In the absence of Vav proteins, ephrin-Eph endocytosis is blocked, leading to defects in growth cone collapse in vitro and significant defects in the ipsilateral retinogeniculate projections in vivo. These findings suggest an important role for Vav family GEFs as regulators of ligand-receptor endocytosis and determinants of repulsive signaling during axon guidance.

Full-text

Available from: Michael Z Lin
Neuron, Vol. 46, 205–217, April 21, 2005, Copyright ©2005 by Elsevier Inc. DOI 10.1016/j.neuron.2005.03.019
Vav Family GEFs Link Activated Ephs
to Endocytosis and Axon Guidance
Christopher W. Cowan,
1,5
Yu Raymond Shao,
1,2,5
spinal tract, and the establishment of visual topo-
graphic maps in the midbrain and tectum. RecentMustafa Sahin,
1,3
Steven M. Shamah,
1
Michael Z. Lin,
1,2
Paul L. Greer,
1,2
Sizhen Gao,
4
studies also indicate that Ephs can regulate the forma-
tion and functional properties of neuronal synapsesEric C. Griffith,
1
Joan S. Brugge,
4
and Michael E. Greenberg
1,
* (Henkemeyer et al., 2003; Kullander and Klein, 2002).
Thus, Ephs display extensive functional versatility, reg-
1
Neurobiology Program
Children’s Hospital and ulating numerous patterning and morphogenic pro-
cesses in the developing and mature nervous system.Departments of Neurology and Neurobiology
Harvard Medical School Much has been learned in recent years about the
mechanisms by which Ephs and their ephrin ligands300 Longwood Avenue
Boston, Massachusetts 02115 regulate Eph-dependent biological processes. The Ephs
(EphA1–8 and EphB1–4,6) are single-pass transmem-
2
Program in Biological and Biomedical Sciences
Harvard Medical School brane receptors with intrinsic tyrosine kinase activity.
Ephrins are membrane tethered as either transmem-240 Longwood Avenue
Boston, Massachusetts 02115 brane (ephrin-B1–3) or glycosylphosphatidyl inositol-
linked (ephrin-A1–5) ligands. Unlike secreted, diffusible
3
Department of Neurology
Children’s Hospital guidance cues, such as netrins or slits (Guan and Rao,
2003; Tessier-Lavigne and Goodman, 1996), membraneBoston, Massachusetts 02115
4
Department of Cell Biology bound ephrins bind to Ephs only upon cell-cell contact.
Ephrin binding to an Eph results in receptor clustering,Harvard Medical School
240 Longwood Avenue stimulation of the intrinsic tyrosine kinase activity, and
Eph autophosphorylation. This in turn initiates Eph-de-Boston, Massachusetts 02115
pendent forward signaling that promotes growth cone
attraction or repulsion.
Summary
With regard to axonal repulsion, this process has
been characterized most thoroughly by studying the ef-
Ephrin signaling through Eph receptor tyrosine ki-
fects of soluble ephrin on growth cone dynamics in cul-
nases can promote attraction or repulsion of axonal
ture (Drescher et al., 1995; Meima et al., 1997a; Meima
growth cones during development. However, the
et al., 1997b). Under these conditions, ephrin treatment
mechanisms that determine whether Eph signaling
induces a strong repulsion event termed growth cone
promotes attraction or repulsion are not known. We
collapse. Ephrin-induced growth cone collapse re-
show here that the Rho family GEF Vav2 plays a key
quires the activation of RhoA and Rac family of small
role in this process. We find that, during axon guid-
GTPases (Fournier et al., 2000; Jurney et al., 2002; Wahl
ance, ephrin binding to Ephs triggers Vav-dependent
et al., 2000). Rac activity is thought to promote internal-
endocytosis of the ligand-receptor complex, thus
ization of plasma membrane, whereas RhoA activity is
converting an initially adhesive interaction into a re-
critical for promoting contractility and disassembly of
pulsive event. In the absence of Vav proteins, ephrin-
the F-actin cytoskeleton. Eph activation of RhoA has
Eph endocytosis is blocked, leading to defects in
been shown recently to be mediated by the Rho family
growth cone collapse in vitro and significant defects
GEF, ephexin1, which in turn regulates growth cone col-
in the ipsilateral retinogeniculate projections in vivo.
lapse (Shamah et al., 2001; Sahin et al., 2005 [this issue
These findings suggest an important role for Vav fam-
of Neuron]). However, the mechanism by which Ephs
ily GEFs as regulators of ligand-receptor endocytosis
activate Rac and the role of Rac in mediating Eph-
and determinants of repulsive signaling during axon
dependent events during development are not yet
guidance.
known.
Axon guidance in vivo involves the cell contact-medi-
Introduction
ated interaction of membrane bound ephrins and Ephs.
The initial interaction between axon growth cone and
Eph family receptor tyrosine kinases (Ephs) regulate a
target cell results in an adhesion between the ephrin
and Eph; however, in many cases the contact subse-wide variety of biological processes in developing and
quently promotes repulsion of the axon. Therefore, the
adult organs (Flanagan and Vanderhaeghen, 1998; Kul-
Eph-expressing growth cone must overcome ephrin-
lander and Klein, 2002). Within the nervous system, Eph
Eph adhesion if axonal repulsion is to occur. One way
signaling mediates the initial sorting and positioning of
in which growth cones may convert the initial ephrin-
cells and axons during development. Eph signaling
Eph adhesion into repulsion is by Rac-dependent en-
regulates the migration pattern of neural crest cells,
docytosis, an atypical endocytic mechanism by which
the boundary formation between hindbrain segments
the ephrin-Eph complex and surrounding plasma mem-
(rhombomeres), the proper formation of the cortico-
brane are internalized into one cell. Two recent studies
showed that the endocytosis of the ephrin-Eph com-
*Correspondence: michael.greenberg@childrens.harvard.edu
5
These authors contributed equally to this work.
plex is required for the repulsion of ephrin-B- and
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206
EphB-expressing cells in culture (Marston et al., 2003;
Zimmer et al., 2003). In these experiments, the forward
ephrin-Eph endocytosis process requires Eph kinase
activity (Marston et al., 2003; Zimmer et al., 2003) and
subsequent Rac activity (Marston et al., 2003). Under
conditions where ephrin-Eph endocytosis is blocked,
the ephrin-Eph interaction results in cell-cell adhesion,
suggesting that activation of Rac-dependent endocyto-
sis of the ephrin-Eph complex may convert initial adhe-
sion into repulsive signaling. As such, ephrin-Eph endo-
cytosis may represent a critical point of regulation that
determines whether ephrin-Eph binding promotes ad-
hesion/attraction or repulsion. At present, the signaling
mechanisms by which ephrin-Eph complexes link to
Rac activation and endocytosis are not known, and it
is unclear whether ephrin-Eph endocytosis is critical for
axon guidance during development.
In this study, we investigated the molecular mecha-
Figure 1. Vav2 Interacts with the Intracellular Domain of EphA4 in
nisms that underlie ephrin-Eph endocytosis and the
the Yeast Two-Hybrid Assay
conversion of ephrin-Eph adhesion into repulsion. We
(A) Domain structures of the intracellular domain of EphA4 (EphA4-
show here that the Rho family GEF Vav2 plays a central
IC) bait protein and the interacting clone of Vav2 (Vav2-CT) iden-
role in these processes. In response to ephrin binding
tified in the yeast two-hybrid screen. SAM domain, sterile α motif
domain; PRD, proline-rich domain; SH3, Src homology 3 domain;
to Ephs, Vav2 is recruited to the intracellular domain of
SH2: Src homology 2 domain.
Ephs and becomes transiently activated. Vav proteins
(B) Characterization of Vav2-CT and EphA4-IC interaction in yeast.
are required for ephrin-Eph endocytosis and ephrin-
Wild-type Vav2-CT was coexpressed in the yeast two-hybrid assay
induced growth cone collapse, suggesting that Vav
with vector, wild-type, or various mutants of EphA4-IC. V635M, ki-
GEFs provide a molecular link between activated Ephs
nase-inactivating mutation; Y576F, Y596F, and Y602F, mutations of
and Rac-dependent endocytosis. Analysis of Vav2
−/−
juxtamembrane tyrosines; Y928F and Y937F, mutations of tyrosine
residues in SAM domain; PDZ, deletion of PDZ binding motif.
Vav3
−/−
mice revealed abnormal axon projections from
(C) Domain composition of Vav family proteins, adapted from
the retina to the thalamus, suggesting that Vav GEFs
Turner and Billadeau (2002). CH, calponin homology domain; AD,
may play an important role in ephrin-Eph endocytosis
acidic domain; DH, Dbl homology domain; PH, pleckstrin homology
and Eph-dependent repulsion in vivo. Taken together,
domain; ZF, zinc finger domain; P, proline-rich domain; SH3, Src
these findings suggest that activation of Vav GEFs
homology type 3 domain; SH2, Src homology type 2 domain.
switches Eph forward signaling from adhesion to repul-
sion by regulating ephrin-Eph endocytosis. As such,
Vav-dependent regulation of receptor endocytosis may
stream of Eph forward signaling. In neurons, ephrin
determine the biological response to ephrins and pos-
binding to an Eph induces receptor clustering and
sibly other axon guidance factors.
autophosphorylation of the highly conserved juxta-
membrane (JM) tyrosines (Y596 and Y602 of EphA4)
(Bartley et al., 1994; Davis et al., 1994; Ellis et al., 1996).Results
Eph tyrosine kinase-mediated autophosphorylation of
these JM residues generates docking sites for phos-The Rho Family GEF Vav2 Interacts
with the Intracellular Domain of EphA4 photyrosine binding proteins (Holland et al., 1997; Pan-
dey et al., 1995; Pandey et al., 1994; Stein et al., 1996;Using the autophosphorylated intracellular domain of
EphA4 (EphA4IC) as bait, we performed a yeast two- Stein et al., 1998; Zisch et al., 1998). Mutant forms of
Ephs lacking the intracellular domain or intrinsic kinasehybrid screen with a cDNA library prepared from em-
bryonic day 14 (E14) rat spinal cord and dorsal root activity fail to mediate ephrin-induced axonal repulsion
in vitro and in vivo (Dearborn et al., 2002; Kullander etganglia (DRG). In addition to the identification of the
RhoA-GEF ephexin1 (Shamah et al., 2001), we isolated al., 2001; Walkenhorst et al., 2000), suggesting that
ephrin binding to Eph induces kinase-dependent for-the Rho family GEF Vav2 as an EphA4-interacting pro-
tein (Figure 1A). Vav2 belongs to a subfamily of GEFs ward signaling to promote axonal repulsion. To deter-
mine if Vav2 interacts with Ephs in a kinase-dependent(Vav1, Vav2, and Vav3) that are evolutionarily conserved
from nematodes to mammals and play important roles manner, we tested whether Vav2 binds to kinase-
inactive mutants of EphA4. In both yeast (Figure 1B)in multiple aspects of cell signaling (Bustelo, 2001;
Turner and Billadeau, 2002). To test whether Vav2 in- and mammalian cells (Figure 2), Vav2 failed to interact
with a kinase-inactivated mutant of EphA4 (V635M; Fig-teracts with Ephs in mammalian cells, we performed
coimmunoprecipitations with full-length versions of ures 1B and 2C) or with EphA4 JM tyrosine mutants
that also lack Eph kinase activity (Y596F, Y602F, orVav2 and EphA4 or EphB2 overexpressed in HEK293T
cells. We observed that Vav2 interacted with either Y596F/Y602F; Figures 1B and 2C and data not shown),
suggesting that Vav2 interacts specifically with theEphA4 or EphB2 (Figures 2A and 2B), indicating that
Vav2 can bind to either EphA or EphB subclass re- ephrin-activated form of the Eph and might play a role
in kinase-dependent forward signaling.ceptors.
We next asked whether Vav2 might function down- The failure of Vav2 to interact with the JM tyrosine
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Role of Vav Family GEFs in Eph Forward Signaling
207
Figure 2. Vav2 and Ephs Interact in Mamma-
lian Cells
(A and B) Coimmunoprecipitation of Vav2
and EphA4 or EphB2 in HEK293T cells. Cells
were transfected with T7-tagged Vav2 and
Flag-tagged EphA4 or EphB2. Total cell ly-
sates (250 g) were immunoprecipitated
with anti-T7 antibody, then immunoblotted
with anti-Flag (Eph) or anti-T7 (Vav2) anti-
bodies. Whole-cell lysates (12.5 g) were
also blotted with anti-Flag or anti-T7 an-
tibody.
(C–E) Analysis of the Vav2 and EphA4 in-
teraction by coimmunoprecipitation. (C) T7-
tagged Vav2 was coexpressed with Flag-
tagged EphA4 (wt), EphA4-YF (Y602F), or
EphA4-KD (kinase dead: V635M); (D) T7-
tagged Vav2 was coexpressed with Flag-
tagged EphA4 (wt) or a JM tyrosine mutant
of EphA4 (EE: Y596E/Y602E); (E) Flag-
tagged EphA4 was coexpressed with T7-
tagged Vav2 (wt), Vav2-Y172F, or SH2-
Vav2. Total cell lysates (TCL) were blotted
with anti-Flag, anti-T7, or a p-Y172-specific
Vav2 antibody. The autoradiograph expo-
sures shown were chosen to illustrate rela-
tive loading and protein expression levels
rather than to indicate the degree of immu-
noprecipitation or coimmunoprecipitation from
the cell lysates.
mutants of EphA4 suggests a possible direct interac- phosphorylation-dependent manner, we sought to de-
termine whether activated Ephs might regulate the GEFtion with the phosphorylated JM tyrosines; however,
previous studies have shown that tyrosine to phenylala- activity of Vav2. A combination of biochemical, muta-
tional, and structural data indicate that phosphorylationnine mutation of the JM tyrosines also leads to a loss
of Eph kinase activity (Binns et al., 2000; Zisch et al., of conserved tyrosine residues in the acidic domain of
Vavs regulates the GDP/GTP exchange activity of Vav1998; Zisch et al., 2000). To test whether Vav2 directly
binds to the JM tyrosines, we tested the ability of Vav2 proteins (Aghazadeh et al., 2000; Crespo et al., 1997;
Lopez-Lago et al., 2000; Movilla and Bustelo, 1999;to bind to a mutant of EphA4 (Y596E/Y602E) that re-
tains normal tyrosine kinase activity but cannot be Schuebel et al., 1998). In the absence of tyrosine phos-
phorylation, an intramolecular interaction of the Vavphosphorylated at the JM tyrosines (Zisch et al., 2000).
We found that Vav2 interacted much more weakly with acidic domain with the catalytic Dbl homology (DH) do-
main blocks access to Rho family GTPases. This inhibi-the YE mutant than wild-type EphA4, suggesting that
Vav2 interacts directly with phosphorylated JM tyro- tory interaction is disrupted when Vav is phosphory-
lated at highly conserved tyrosines in the acidic domainsines of EphA4 (Figure 2D). The residual binding of Vav2
to the YE mutant may be mediated by the negative (e.g., Tyr174), resulting in active GDP/GTP exchange on
Rho family GTPases. As an activating mutation ofcharge of the glutamic acid (E), a substitution that is
often used to mimic phosphorylation. These findings Tyr174 on Vav1 has previously been shown to be suffi-
cient to induce near maximal GDP exchange activity,suggest that, upon ephrin binding to an Eph, the Eph
autophosphorylation of the JM tyrosines generates a we utilized a site-specific phospho-antibody against
this site on Vav2 (Tyr172) to determine if activateddocking site for Vav2 recruitment.
How then does Vav2 interact with the phosphorylated EphA4 induces tyrosine phosphorylation of this regula-
tory site on Vav2. While little tyrosine phosphorylationJM tyrosines? All the Vav family GEFs contain a SH2
phosphotyrosine binding domain in the C-terminal por- of Vav2 was detected in the absence of EphA4 ex-
pression, coexpression in HEK293T cells of Vav2 andtion of the protein. Therefore, we speculated that Vav2
might be recruited to the phosphorylated JM tyrosines wild-type EphA4 significantly increased Vav2 Tyr172
phosphorylation (Figure 2C). As with EphA4, the coex-of ephrin-activated Ephs via its SH2 domain. We tested
the ability of Vav2 containing a mutated SH2 domain pression of EphA7, EphB2, EphB3, or EphB4 with Vav2
led to significant Tyr172 phosphorylation (data not(SH2-Vav2) to interact with wild-type EphA4. We
found that, in contrast to wild-type Vav2, SH2-Vav2 shown), suggesting that multiple EphAs and EphBs are
capable of activating Vav2. However, it was uncleardoes not interact with EphA4 (Figure 2E). We conclude
that, once ephrin activates Eph and the JM tyrosines whether enhanced phosphorylation of Vav2 was depen-
dent on a physical association between the Vav2 andbecome autophosphorylated, Vav2 is recruited to the
ephrin-Eph complex and binds to Eph JM tyrosines via Eph or due to an indirect Eph-dependent signaling
event. Coexpression of wild-type EphA4 with the SH2-the Vav2 SH2 domain.
Having established that Vav2 and Ephs interact in a Vav2 mutant that failed to interact with the activated
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208
Figure 3. Vav2 Protein Expression in Neurons
(A) Western blotting with Vav2 antibodies in
whole-brain lysates from wild-type or Vav2
−/−
Vav3
−/−
mice, or from dissociated primary rat
neurons. Lysates represent total protein from
150,000 rat P6 retinal ganglion cells, 750,000
rat E17/18 cortical neurons, or 750,000 rat
E17/18 hippocampal neurons.
(B) Developmental expression profile of Vav2
or β-actin from whole-brain lysates (no cere-
bellum) at indicated embryonic (E) or postna-
tal (P) day (100 g/lane).
(C) Expression of Vav2-EGFP (green) or
nuclear staining (Hoechst, blue) in E17/18
cortical neurons at 3 days in culture (inset,
growth cone region of Vav2-EGFP-express-
ing neurons).
EphA4 did not result in Vav2 tyrosine phosphorylation pressed most highly during embryonic (E13–E18) and
early postnatal time points (P2–P6) but then declinesat Tyr172 (data not shown), indicating that Vav2 tyrosine
phosphorylation and activation require a direct interac- postnatally when synaptic proteins such as PSD-95 are
being upregulated (Figure 3B and data not shown).tion of Vav2 with the activated Eph. Similarly, coexpres-
sion of the EphA4 YE mutant with Vav2 resulted in de- Therefore, Vav2 protein is expressed highly in the brain
during the period when axon guidance is occurringcreased levels of binding (Figure 2D) as well as
decreased levels of Vav2 tyrosine phosphorylation in vivo.
To determine whether Vav2 is found in growth cones,(data not shown). We note that phosphorylation of Vav2
on Tyr172 was not necessary for binding to EphA4 (Fig- we used several different approaches. As our anti-Vav2
antibodies failed to show specific staining in culturedure 2E), suggesting that Vav2 phosphorylation is a con-
sequence of the interaction with EphA4, not the cause. wild-type neurons, we analyzed the localization of a
Vav2-EGFP fusion protein in cultured cortical and hip-Taken together, these experiments suggest that, when
Ephs become tyrosine autophosphorylated, the acti- pocampal neurons (Figure 3C and data not shown). We
detected Vav2-EGFP in growth cones (Figure 3C, inset),vated Eph interacts with Vav2 and triggers Vav2 tyro-
sine phosphorylation and activation. neurites, and the cell body, but not the nucleus, consis-
tent with previous reports of neuronal Vav2 localization
(Chauvet et al., 2003). To analyze Vav2 protein subcellu-
Endogenous Vav2 Is Transiently Activated by Ephrin
lar distribution a different way, we harvested distal ax-
Stimulation in Neurons
ons or cell bodies from rat DRG explants grown in com-
Having established that Vav2 binds to and is activated
partmentalized chambers. By Western blotting, Vav2
by Ephs, we sought to address three major questions:
was detected in both distal axons and cell bodies, al-
(1) is Vav2 expressed in neurons, (2) is Vav2 found in
though Vav2 was found to be most abundant in the cell
growth cones where Eph-dependent axon guidance
body fraction. In contrast, the nuclear proteins CBP
occurs, and (3) does ephrin stimulation of endogenous
and MECP2 were detected in the cell body fraction, but
Eph receptors induce the activation of Vav2? To deter-
not in the distal neurites (data not shown). Thus, Vav2
mine if Vav2 is expressed in neurons, we tested total
is present in a number of ephrin-responsive neurons,
brain and primary neuronal culture lysates by Western
is synthesized in the brain during the time when axon
blotting with an anti-Vav2 antibody. We detected an
guidance is occurring, and is detected in the growth
w95 kDa band that comigrates with recombinant Vav2
cones and distal neurites where ephrin-induced axon
and is absent in adult brain lysates obtained from
guidance occurs.
Vav2
−/−
Vav3
−/−
mice, suggesting that the 95 kDa band
Finally, we asked if Vav2 becomes tyrosine phosphor-
is a Vav family member (Figure 3A). By Western blotting,
ylated by ephrin binding to Ephs in neurons. To this
Vav2 was also detected in lysates from E17/18 cultured
end, primary rat cortical or striatal cultures were treated
rat cortical, striatal, or hippocampal neurons, as well as
with ephrin-A1 to specifically activate EphAs, ephrin-
rat and mouse RGCs isolated from postnatal day 6 (P6)
B1 to selectively stimulate EphBs, or an Fc control pro-
retinas (Figure 3A and data not shown). We conclude
tein. Cells were harvested at various times following
that Vav2 is expressed in a variety of embryonic and
stimulation, and Vav2 was immunoprecipitated. The
postnatal neurons. To determine whether Vav2 is ex-
activation state of Vav2 was assessed by Western blot-
pressed at an appropriate time to play a role in axon
ting using a general anti-phosphotyrosine monoclonal
guidance, we isolated rat brains over a wide range of
antibody (4G10) or the anti-Vav2 p-Tyr172 site-specific
embryonic and postnatal days during development
antibody. Upon ephrin-A1 stimulation, Vav2 became
(E13 to adult) and analyzed Vav2 protein expression by
Western blotting. We found that Vav2 protein is ex- phosphorylated rapidly and transiently, with Vav2 phos-
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Role of Vav Family GEFs in Eph Forward Signaling
209
development, we investigated whether Vav proteins
might also be necessary for proper axon guidance in
vivo. Since Vav2 and Vav3 are known to be expressed
in the brain, are closely related with respect to amino
acid sequence, and are both activated by tyrosine
phosphorylation (Movilla and Bustelo, 1999; Schuebel
et al., 1998), we speculated that a potential role for Vav2
or Vav3 in Eph-dependent signaling might be unclear in
the single knockout mice due to compensation by the
remaining Vav gene product. Therefore, we analyzed
the projection pattern of axons from retinal ganglion
cells (RGCs) to the dorsal lateral geniculate nucleus
(dLGN) in wild-type and Vav2
−/−
Vav3
−/−
mice. To visual-
ize Eph-dependent axon projections, we injected the
left and right eyes with either Alexa 488 (green)- or
Alexa 594 (red)-conjugated cholera toxin B (ChTxB)
subunits to anterogradely label the axon terminals in
the dLGN. As shown in Figure 5A, the wild-type contra-
lateral projections (green) occupy a large majority of the
dLGN, whereas the ipsilateral projections (red) occupy
a smaller, centrally located, and spatially segregated re-
gion of the dLGN. By casual observation, the ipsilateral
projections in Vav2
−/−
Vav3
−/−
mice appear to be re-
duced in number and ventrally shifted, and to display a
patchy pattern when compared to wild-type animals.
To analyze ipsilaterally projecting axons more care-
fully, we quantified the total signal intensity and av-
erage distribution for all ipsilateral projections to the
dLGN in both wild-type and Vav2
−/−
Vav3
−/−
mice. By
Figure 4. Ephrin Stimulation Activates Endogenous Vav2 in Neurons
(A) E17/18 cortical neurons were cultured for 4 days and then stim-
these analyses (Figure 5C, top), we observed a 55%
ulated with ephrin-A1-Fc or Fc (control) for the indicated times.
decrease in the total ipsilateral projection signal in the
Vav2 was immunoprecipitated from whole-cell lysates and then
blotted with phosphotyrosine antibody (4G10) (top panel) or the
Vav2
−/−
Vav3
−/−
mice as compared to wild-type mice, in-
p-Y172-specific Vav2 antibody (data not shown). Whole-cell lysates
dicating that Vav2
−/−
Vav3
−/−
mice have fewer total ipsi-
were also blotted with anti-Vav2, anti-phospho-Eph, or anti-EphA4
lateral projections. As this reduction could be due to
antibodies.
reduced labeling efficiency in the eye or reduced trans-
(B) E17/18 striatal neurons cultured for 4 days were stimulated with
port of the anterograde label to the nerve terminals of
5 g/ml clustered ephrin-A1-Fc. Cell lysates were immunoprecipi-
Vav2
−/−
Vav3
−/−
mice, we analyzed the contralateral pro-
tated with anti-Vav2 antibody or Protein A beads alone (control IP)
and blotted with anti-phosphotyrosine antibody or anti-Vav2 an-
jections to determine if there was a similar decrease
tibody.
in signal intensity in Vav2
−/−
Vav3
−/−
mice. However, we
(C) Cortical neurons at E17/18 + 4 DIV were stimulated with ephrin-
found that the total signal intensity of Vav2
−/−
Vav3
−/−
B1-Fc. Vav2 was immunoprecipitated and blotted with phosphoty-
contralateral projections was similar to that of the wild-
rosine antibody. Total cell lysates were blotted with anti-Vav2 or
type mice (Figure 5C, bottom), indicating that the signif-
site-specific antibodies against the phosphorylated JM tyrosines
icant decrease in the number of ipsilateral projections
of Ephs.
to the Vav2
−/−
Vav3
−/−
dLGN is not due to an overall de-
crease in labeling efficiency or to lower levels of
ChTxnB at dLGN nerve terminals.
phorylation detectable as early as 1 min following stim-
The number of ipsilateral projections in mutant and
ulation with ephrin-A1 (Figures 4A and 4B). The peak of
wild-type dLGNs was also quantified by generating bi-
Vav2 phosphorylation was between 2 and 5 min, and
nary images of ipsilateral and contralateral projections
then Vav2 phosphorylation declined quickly thereafter.
to the dLGN (Figure 5B). By this method of analysis, we
Similarly, ephrin-B1 stimulation induced the transient
also detected a decrease in the number of ipsilateral
phosphorylation of Vav2, but with delayed kinetics that
projections in Vav2
−/−
Vav3
−/−
mice relative to wild-type
mirrored the activation phase of the EphBs (Figure 4C).
mice (data not shown). By contrast, pixel occupancy
Treatment with Fc control did not induce Vav2 tyrosine
for the contralateral projections was similar in Vav2
−/−
phosphorylation at any of the time points analyzed.
Vav3
−/−
and wild-type animals. The decrease in ipsilat-
Taken together, these experiments suggest that Vav2
eral projections in the Vav2
−/−
Vav3
−/−
mice is similar in
is transiently tyrosine phosphorylated and activated in
magnitude to the reduction in ipsilateral projections ob-
neurons by Eph forward signaling under conditions that
served in the EphB1
−/−
and EphB1
−/−
EphB2
−/−
EphB3
−/−
induce Eph-dependent repulsion.
mice (Williams et al., 2003). The finding that there is a
reduction in ipsilateral axon projections in Vav-deficient
Abnormal Retinogeniculate Projections
mice indicates that Vav proteins are important for
in the Vav2
−/−
Vav3
−/−
Mice
proper axon targeting to the dLGN and raises the pos-
Since ephrin/Eph forward signaling induces the tran-
sibility that Ephs promote axon guidance via a Vav- and
sient phosphorylation of Vav2 in neurons, and Ephs are
Rac-dependent process.
In addition to the reduction of ipsilateral nerve ter-known to be critical for proper axon guidance during
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210
Figure 5. Retinogeniculate Projections Are Altered in the Vav2
−/−
Vav3
−/−
Mice
(A) Confocal images of contralateral (green) and ipsilateral (red) axon projections in the dorsal lateral geniculate nucleus (dLGN) from two
wild-type (top) and two Vav2
−/−
Vav3
−/−
(bottom) mice.
(B) Corresponding binary images of the ipsilateral projections from the wild-type (top) and Vav2
−/−
Vav3
−/−
(bottom) mice.
(C) Analysis of the total axon projections (ipsilateral and contralateral) between wild-type (blue) and Vav2
−/−
Vav3
−/−
(red) mice. Data represent
the mean of 11 wild-type and 12 Vav2
−/−
Vav3
−/−
mice ± SEM. The difference of total ipsilateral projections ([C], top) between the wild-type
and Vav mutant mice is statistically significant (Student’s t test; p < 0.001).
(D) Average distribution (mean ± SEM) of the ipsilateral projections along the long (dorsomedial to ventrolateral) and short (laterodorsal to
medioventral) axes. The data are plotted on a normalized axis (0 to 1) of the long and short axis of the dLGN as depicted on the inset
diagrams. D, dorsal; V, ventral; L, lateral; M, medial.
minals in the Vav2
−/−
Vav3
−/−
mice, the distribution of indicating that the ipsilateral axons in the Vav2
−/−
Vav3
−/−
mice that successfully project to the dLGN are abnor-ipsilateral projections appears to be more diffuse,
patchy, and skewed toward the ventrolateral region of mal in their targeting. This ventrolateral skewing of ipsi-
lateral projections in Vav-deficient animals may be thethe dLGN (Figures 5B and 5D). To compare the distribu-
tion of the ipsilateral projection pattern, we employed result of a defect in Eph signaling. Taken together, the
projection defects in the Vav2
−/−
Vav3
−/−
mice indicatea line scan technique to calculate the mean pixel inten-
sity of projection terminals in wild-type and Vav2
−/−
an important role for Vav family GEFs in the proper for-
mation of the ipsilateral retinogeniculate map and raiseVav3
−/−
mice along the long (dorsomedial [DM] to
ventrolateral [VL]) and short (laterodorsal [LD] to medio- the possibility that Vav proteins may regulate aspects
of ephrin/Eph-dependent axon guidance.ventral [MV]) axes. The mean pixel intensity for each
pixel line was averaged for 11 wild-type mice (blue line,
Figure 5D) and 12 Vav2
−/−
Vav3
−/−
mice (red line, Figure Vav Family GEFs Are Necessary for Ephrin-Induced
Growth Cone Collapse of RGCs5D). The distribution of axon projections along the short
axis (Figure 5D, right panel) appeared similar in wild- Axonal repulsion in vivo requires an initial adhesive in-
teraction between ephrins expressed on target cellstype and Vav2
−/−
Vav3
−/−
mice, indicating that Vav pro-
teins are dispensable for proper axon targeting along and Ephs expressed on guiding axonal growth cones.
Subsequent to ephrin-Eph binding, the Ephs clusterthis axis. However, the distribution of projections along
the long axis, which coincides with the orientation of and autophosphorylate their JM tyrosines, which in turn
stimulate signaling events that switch from adhesionthe ephrin-A gradient in wild-type mice, was distorted
in the Vav2
−/−
Vav3
−/−
mice (Figure 5D, left panel). Wild- to repulsion. One possible reason for abnormal Eph-
mediated axon guidance in the Vav-deficient micetype mice displayed a bell-shaped distribution of pro-
jections, with the center of the distribution just dorsal might be a defect in the initial ephrin-Eph adhesion
event. To address this possibility, we cultured dissoci-to the midline. In contrast, the Vav2
−/−
Vav3
−/−
mice
showed a distinct VL-shifted and sloping distribution, ated RGCs from wild-type and Vav-deficient mice and
Page 6
Role of Vav Family GEFs in Eph Forward Signaling
211
Figure 6. Vav Family GEFs Are Necessary for
Ephrin-A1-Induced Growth Cone Collapse of
Retinal Ganglion Cells
(A) Cultured wild-type and Vav2
−/−
Vav3
−/−
retinal ganglion cells (RGCs) were stimulated
with clustered ephrin-A1 or Fc control for 12
min, and then lysates were immunoblotted
with anti-phospho-Eph (top) or anti-EphA4-
specific (bottom) antibodies.
(B) Morphology of uncollapsed (top) and col-
lapsed (bottom) growth cones were visual-
ized with Texas red-labeled phalloidin (Mo-
lecular Probes).
(C) F-actin content and growth cone mor-
phology of wild-type (left) and Vav2
−/−
Vav3
−/−
(right) RGCs (phalloidin-Texas red).
(D) Growth cone collapse assay with wild-
type (open bars) and Vav2
−/−
Vav3
−/−
(closed
bars) RGCs. Cultured RGCs (P7, 2 DIV) were
stimulated with ephrin-A1, Semaphorin 3A
(Sema3A), or Fc control for 30 min prior to
fixation and phalloidin staining. Data repre-
sent the mean of three (Sema3A) or four
(ephrin-A1 and Fc) independent experi-
ments ± SEM. Asterisk marks significant dif-
ference between wild-type and mutant
RGCs (Student’s t test; p = 0.001).
incubated them with ephrin-A1-Fc or Fc at 4°C in order ephrin-A1 treatment when compared to the Fc control
(36% and 28%, respectively; Figure 6D, solid bars).to detect ephrin-Eph surface binding. We detected a
similar staining intensity and signal distribution of sur- Thus, Vav family GEFs play a key role in mediating
ephrin-dependent growth cone collapse.face bound ephrin (data not shown), indicating that
ephrin-Eph adhesion is normal in the RGCs of Vav2
−/−
The observation that there is a defect in ephrin-
induced growth cone collapse of Vav-deficient RGCsVav3
−/−
mice. We next asked whether Eph activation by
tyrosine autophosphorylation might be defective in the suggests that there is a specific deficit in Eph signaling
in these cells. However, we considered the alternativeVav2
−/−
Vav3
−/−
mice, thus resulting in defective Eph for-
ward signaling. However, stimulation of cultured RGCs possibility that Vav2
−/−
Vav3
−/−
RGCs might have an in-
trinsic defect in the ability to respond to many repulsivefrom either wild-type or Vav2
−/−
Vav3
−/−
mice with
ephrin-A1 led to a significant increase in JM tyrosine guidance factors. To address this possibility, we com-
pared the response of wild-type and Vav2
−/−
Vav3
−/−
autophosphorylation (Figure 6A), suggesting that axon
guidance defects in Vav-deficient mice are not likely RGCs to treatment with Semaphorin 3A (Sema3A), a
secreted ligand that stimulates growth cone collapsedue to abnormal ephrin-Eph adhesion or initial recep-
tor activation. through the neuropilin1/plexin-A1 receptor complex
(He and Tessier-Lavigne, 1997; Kolodkin et al., 1997;To investigate whether the axon guidance defects
observed in the Vav2
−/−
Vav3
−/−
mice might be due Kolodkin et al., 1992; Luo et al., 1993; Takahashi et al.,
1999; Tamagnone et al., 1999). We detected no signifi-to defects in Eph-dependent repulsion signaling, we
turned to a neuronal culture-based assay. Addition of cant difference in Sema3A-induced growth cone col-
lapse between wild-type and Vav2
−/−
Vav3
−/−
RGCs (Fig-ephrin-As to dissociated RGCs in culture induces a dra-
matic change in the morphology of axonal growth ure 6D). Taken together, these findings indicate that the
growth cone collapse deficit in Vav2
−/−
Vav3
−/−
RGCs re-cones that is characterized by the retraction of extend-
ing filopodia and the collapse of the growth cone (Fig- flects a specific defect in Eph-dependent signaling.
ure 6B). To determine if Vav family GEFs are required
for ephrin-induced growth cone collapse, we examined Vav Family GEFs Are Required
for Ephrin-Eph Endocytosisthe response of RGCs derived from wild-type and
Vav2
−/−
Vav3
−/−
mice to ephrin-A1 stimulation. Impor- We next explored the cellular mechanism by which Vav
proteins regulate ephrin-Eph-mediated growth conetantly, the overall cell morphology, number of neurites,
and growth cone morphology of cultured Vav2
−/−
Vav3
−/−
collapse. Vav proteins have been shown to activate
RhoA, Rac, and Cdc42 proteins in vitro. However,RGCs appeared similar to wild-type RGCs (Figure 6C).
After 2 days in culture, RGCs were stimulated with studies of Vav proteins in mammalian cells have re-
vealed that they induce membrane ruffles and lamelli-ephrin-A1 or Fc control for 30 min, then fixed and
scored for collapsed or uncollapsed growth cones. podia and strongly activate Rac family GTPases (Arthur
et al., 2004; Kawakatsu et al., 2005; Liu and Burridge,Ephrin-A1 treatment induced the collapse of a large
percentage of the growth cones in wild-type RGC cul- 2000; Marcoux and Vuori, 2003; Marignani and Carpen-
ter, 2001; Schuebel et al., 1998; Servitja et al., 2003;tures as compared to the Fc control treatment (61%
and 24%, respectively; Figure 6D, open bars). In con- Tamas et al., 2003). Since Rac activity has been shown
to be required for ephrin-Eph endocytosis (Marston ettrast, Vav2
−/−
Vav3
−/−
RGCs were poorly responsive to
Page 7
Neuron
212
Figure 7. Vav Family GEFs Are Required for
Ephrin-Eph Endocytosis
(A) Fluorescence images of Fc control or
ephrin-A1-Fc staining (red) of wild-type or
Vav2
−/−
Vav3
−/−
RGC distal neurites/growth
cones (green, DiO). “Surface” represents
RGCs blocked without 0.1% Triton X-100,
whereas “surface plus internal” represents
RGCs blocked in the presence of 0.1% Triton
X-100.
(B) Quantification of ephrin-A1 staining in-
tensity of nonpermeabilized or detergent-
permeabilized RGC distal neurite/growth
cones from wild-type (open bars) and Vav2
−/−
Vav3
−/−
(closed bars) mice after 30 min of
stimulation (see Experimental Procedures).
The difference between the nonpermeabi-
lized and permeabilized levels indicates the
internalized fraction of ephrin-A1. Data re-
present intensity quantification of a total of
25 to 47 growth cones for each condition
from three independent experiments ± SEM.
The amount of internalized ephrin-A1 in
Vav2
−/−
Vav3
−/−
RGCs was significantly dif-
ferent than that of wild-type neurons (ANOVA
test; p < 0.001).
(C) The endocytosis of transferrin by wild-type (open bars) and Vav2
−/−
Vav3
−/−
(closed bars) RGCs was determined following a 30 min
incubation with TRITC-labeled transferrin at 37°C (endocytosis permissive) or 4°C (endocytosis blocked). Internalized transferrin was quanti-
fied after acid/high-salt wash to remove surface bound ligand. Data represent a total of 32 to 36 neurons for each condition ± SEM.
al., 2003), we investigated the possibility that Vav-defi- cently labeled transferrin protein, we detected a similar
amount of endocytosis in cultured RGCs derived fromcient RGCs might be defective in ephrin-Eph endocyto-
sis. We compared the internalization of ephrin-A1/EphA wild-type or Vav2
−/−
Vav3
−/−
mice (Figure 7C), suggest-
ing that Vav proteins are required for endocytosis of thecomplexes in growth cones of cultured RGCs from
wild-type and Vav2
−/−
Vav3
−/−
mice (Figure 7). Briefly, ephrin-Eph complex, but not all endocytic processes.
Taken together, our observations that there are defectsRGC cultures were treated with ephrin-A1-Fc or Fc
control, then fixed and stained to detect the amount of in ephrin-induced growth cone collapse and ephrin-
Eph endocytosis in Vav-deficient RGCs suggest thatendocytosed ephrin-A1. Total ephrin-A1 staining (sur-
face bound + internalized) was determined under de- defects in axon guidance observed in Vav2
−/−
Vav3
−/−
mice might be due, at least in part, to a defect in endo-tergent permeabilizing conditions, whereas surface
bound ephrin-A1 was specifically measured under non- cytosis-dependent Eph repulsive signaling.
permeabilizing conditions. The endocytosed fraction of
ephrin-A1 represents the difference between the total Discussion
staining and the surface bound staining. In wild-type
mouse and rat RGCs, the amount of endocytosed In this study, we show that when ephrins bind to Ephs
the Rho family GEF, Vav2, is recruited to the phosphory-ephrin-A1 increased in a time-dependent manner (data
not shown). In contrast, no significant ephrin-Eph en- lated JM region of activated Ephs, and Vav2 becomes
transiently phosphorylated on tyrosine residues thatdocytosis was observed in the Vav-deficient RGCs
when compared with wild-type cells at any time tested stimulate its GEF activity. We observed retinogeniculate
axonal projection defects in the Vav2
−/−
Vav3
−/−
mice,(Figure 7B and data not shown). For both wild-type and
Vav2
−/−
Vav3
−/−
RGC cultures, the incubation with Fc indicating an important role for Vav proteins in neuronal
development and suggesting a possible role for Vavcontrol or no treatment failed to induce detectable en-
docytosis (Figure 7A). Thus, Vav family proteins are proteins in Eph-dependent axonal targeting in vivo. We
find that RGCs derived from Vav2
−/−
Vav3
−/−
mice failnecessary for internalization of the ephrin-Eph com-
plex, suggesting that the failure of Vav-deficient RGC to collapse their growth cones in response to ephrin-A
stimulation in culture. In addition, Vav proteins are nec-growth cones to collapse in response to ephrin treat-
ment may be due, at least in part, to defective ephrin- essary for ephrin-Eph endocytosis, a Rac-dependent
process that appears to be important for the processEph endocytosis.
To determine whether Vav2
−/−
Vav3
−/−
RGCs were of axonal repulsion. Taken together, our data suggest
that defects in Vav-dependent Eph signaling result ingenerally defective in receptor-dependent endocytosis
or whether the internalization defect was specific for axon targeting defects in vivo and that Vav proteins
promote the local and transient activation of a Rho fam-ephrin-Eph complexes, we analyzed the endocytosis of
transferrin in wild-type and Vav2
−/−
Vav3
−/−
neurons. ily GTPase to stimulate ephrin-Eph endocytosis, an im-
portant early step in axonal repulsion.Transferrin is a serum protein that delivers bound iron
atoms to the intracellular compartment of cells by con- As with any complex mouse phenotype, it is difficult
to link definitively the retinogeniculate defects in thestitutive receptor-mediated endocytosis. Using fluores-
Page 8
Role of Vav Family GEFs in Eph Forward Signaling
213
Vav2
−/−
Vav3
−/−
mice to a specific signaling pathway. naling, as well as to determine the fate of the absent
ipsilateral axons in the Vav2
−/−
Vav3
−/−
mice.While our data suggest that the retinogeniculate map
defects in the Vav2
−/−
Vav3
−/−
mice may be due to dis- In contrast to studies of ephrin-A-EphA endocytosis
and axonal repulsion, it is not yet clear whether ephrin-rupted Vav-dependent Eph repulsive signaling, it is im-
portant to note that Vav proteins are activated down- B-EphB endocytosis is necessary for repulsive signal-
ing. Although ephrin-B1 can stimulate forward endocy-stream of several growth factor receptors, immune cell
receptors, and adhesion molecules (Bustelo, 2000). De- tosis in cultured mouse neurons, and ephrin-B-EphB
endocytosis is required for cell-cell repulsion whenfective signaling downstream of one or several of these
various receptors could contribute to the disruption of EphB and ephrin-B are ectopically expressed in cul-
tured fibroblasts (Marston et al., 2003; Zimmer etthe retinogeniculate projection map in a number of
ways. These include effects on axon outgrowth, neurite al., 2003), Xenopus RGCs can undergo growth cone
collapse in the absence of forward ephrin-B-EphB en-pruning/branching, or gene expression. With regard to
axon outgrowth, we find that in culture the neurites docytosis (Mann et al., 2003). This suggests that
EphB-mediated repulsion can, at least under some cir-from wild-type and Vav2
−/−
Vav3
−/−
RGCs are similar in
length (C.W.C., unpublished data). In addition, axon cumstances, occur independently of endocytosis. As
these studies emphasize, it will be important to deter-projections to the contralateral dLGN in the Vav2
−/−
Vav3
−/−
mice appear normal (Figure 5C, bottom), sug- mine whether ephrin-B-EphB endocytosis occurs in
vivo during normal axon guidance and whether ephrin-gesting that there is not a general defect in axon out-
growth. We have also considered the possibility that B-EphB endocytosis is required for cell-cell detach-
ment and axonal repulsion.the altered retinogeniculate projections in Vav2
−/−
Vav3
−/−
mice could be due to changes in ephrin or Eph The reduction of projections to the dLGN in the
Vav2
−/−
Vav3
−/−
mice is not the only observed retinogen-expression levels. To begin to address this possibility,
we analyzed by semiquantitative RT-PCR the levels of iculate targeting defect. The remaining ipsilateral pro-
jections along the dorsomedial to VL axis of the dLGNseveral ephrin and Eph mRNAs in the brain and eye
of wild-type and Vav2
−/−
Vav3
−/−
mice. We observed no are also abnormally distributed (Figure 5C). In wild-type
mice, ephrin-A2 and ephrin-A5 are expressed in a gra-obvious differences between wild-type and Vav2
−/−
Vav3
−/−
mice in the mRNA levels of ephrins and Ephs dient along this axis with highest expression at the VL
region (Feldheim et al., 1998). In addition, ephrin-As arethat are known to contribute to retinogeniculate or
retinocollicular axon targeting (Figure S1 in the Supple- critical for RGC axon targeting along this axis. Specifi-
cally, ephrin-A5
−/−
and ephrin-A2
−/−
ephrin-A5
−/−
micemental Data available with this article online).
If Vav-dependent endocytosis is required for Eph re- display mistargeted projections along the plane of the
ephrin-A gradient (Feldheim et al., 2000; Feldheim etpulsive signaling during neuronal development, it is im-
portant to consider how disruption of Vav-dependent al., 1998; Frisen et al., 1998). Interestingly, the mis-
targeted ipsilateral projections in the Vav2
−/−
Vav3
−/−
Eph signaling might cause defects in axon guidance.
Recent studies are consistent with the possibility that mice were shifted toward the higher concentrations of
ephrin-A, raising the possibility that defective endocy-the observed reduction in ipsilateral projections in the
Vav2
−/−
Vav3
−/−
mice might be due in part to failure of tosis of the ephrin-A-EphA complex and/or repulsive
signaling may result in attraction/adhesion toward morethe ipsilateral axons to repel at the optic chiasm. Spe-
cifically, wild-type RGC axons arriving at the optic chi- ventrolateral positions (i.e., higher concentrations of
ephrin-A) in the dLGN. With our current data, we cannotasm encounter ephrin-B2-expressing glia. EphB1-posi-
tive axons from the ventrotemporal region of the retina firmly establish that the shift in ipsilateral projections in
Vav-deficient mice represents defective Eph signalingare repelled by ephrin-B2 at the optic chiasm and adopt
the ipsilateral projection path, whereas the vast major- or ephrin-Eph endocytosis; however, the fact that the
abnormal axonal projection pattern is specifically de-ity of axons that do not express EphB1 cross the optic
chiasm to establish the contralateral projection path fective in the axis of the ephrin-A gradient and that cul-
tured Vav-deficient RGCs are defective in ephrin-A-(Williams et al., 2003). In EphB1
−/−
and EphB1
−/−
EphB2
−/−
EphB3
−/−
mice, ipsilateral projections were induced growth cone collapse and endocytosis sug-
gests that Vav proteins may play a critical role in ephrin-decreased by 43 and 56 percent, respectively (Williams
et al., 2003). Similarly, we observed a 55 percent de- A/EphA-dependent repulsion in vivo.
Although there is now considerable evidence sug-crease in ipsilateral projections in the Vav2
−/−
Vav3
−/−
mice, raising the possibility that Vav proteins might play gesting that ephrin-Eph endocytosis plays an important
role in repulsive signaling, Flanagan and colleaguesan important role in EphB1-dependent ipsilateral guid-
ance at the optic chiasm. As we observed clear defi- have reported that the proteolytic cleavage of the
ephrin-A ligand is also required for ephrin-A-inducedcits in ephrin-A-induced endocytosis in Vav2
−/−
Vav3
−/−
mice, and both ephrin-A and ephrin-B induce Vav2 tyro- repulsion (Hattori et al., 2000). Upon ephrin-A-EphA bind-
ing, the metalloprotease Kuzbanian (Adam10) cleaves thesine phosphorylation, it is tempting to speculate that
the EphB1-positive axons may be defective in ephrin- ephrin-A ligand, thus providing a mechanism for dis-
rupting the ephrin-A-EphA adhesion event. Failure toB-induced endocytosis. If this is the case, then in the
Vav2
−/−
Vav3
−/−
mice the EphB1-positive axons may proteolyze ephrin-A resulted in delayed axonal repul-
sion, suggesting that ephrin-A cleavage is a criticalbind to the ephrin-B2-positive glia but fail to undergo
ephrin-Eph endocytosis and axonal repulsion. In the fu- step for normal repulsive signaling. In the future, it will
be important to explore the relationship between theture, it will be important to test whether Vav proteins
regulate ephrin-B-EphB endocytosis and repulsive sig- ephrin-A cleavage event and Rac-dependent ephrin-
Page 9
Neuron
214
Figure 8. A Model for How Vav and Ephexin
Family GEFs May Mediate Axonal Outgrowth
and Ephrin-Induced Growth Cone Repulsion
Eph endocytosis during the processes of Eph-medi- ligand-induced endocytosis of other cell surface re-
ceptors.ated axonal repulsion or attraction.
One question that our study raises is how Vav pro- Our findings suggest that Ephs promote repulsion by
orchestrating a series of distinct events and that acti-teins promote ephrin-Eph endocytosis. The growth
cone collapse and endocytosis assays demonstrate a vated Ephs engage different GEFs to affect the distinct
actin cytoskeletal changes necessary for repulsionrequirement for Vav proteins for these processes in cul-
tured neurons, but it is not yet clear how Vav proteins (Figure 8). Specifically, we find that both ephexin1 (Sha-
mah et al., 2001; Sahin et al., 2005) and Vav proteinsmediate these processes. Since local activation of a
Rho family GTPase appears to be necessary for endo- are necessary for ephrin-A-induced growth cone col-
lapse but that these GEFs appear to regulate distinctcytosis, we suspect that the GEF activity of Vav pro-
teins is required for endocytosis and growth cone aspects of repulsive signaling. Upon ephrin-Eph bind-
ing, Vav2 is recruited to the autophosphorylated Ephcollapse. In support of this idea, we find that overex-
pression of a dominant interfering form of Vav2 that and becomes rapidly and transiently activated, possi-
bly by Src family kinases (Marignani and Carpenter,lacks GDP/GTP exchange activity reduces the respon-
siveness of wild-type rat RGCs to ephrin-induced 2001; Schuebel et al., 1998), to promote local Rac-
dependent endocytosis of the ephrin-Eph complex andgrowth cone collapse (C.W.C. and M.S., unpublished
data). However, Vav proteins contain a number of addi- surrounding plasma membrane. Similarly, ephexin1 be-
comes tyrosine phosphorylated, resulting in a strongtional functional domains (Figure 1C) that may also play
a role in Vav-dependent Eph forward signaling. switch to RhoA activation, which is necessary for
F-actin disassembly and contractility. It will be worth-In addition to understanding the mechanism by
which Vav proteins promote ephrin-Eph endocytosis, while to understand the temporal and causal relation-
ship between endocytosis and repulsive signaling thatwe are also interested in understanding how this endo-
cytic event induces a repulsive response. Recent stimulates F-actin contractility and disassembly during
Eph-mediated axon guidance. If Rac-dependent endo-studies indicate that the endocytosis of ligand-receptor
complexes can determine signaling output. For exam- cytosis regulates RhoA-dependent repulsive signaling,
then regulation of ephrin-Eph endocytosis could deter-ple, endocytosis and retrograde transport of the acti-
vated neurotrophin receptor complex is required for mine whether ephrin-Eph binding promotes attraction/
adhesion or repulsion.transmission of the survival signal in neurons (Heerssen
et al., 2004; Kuruvilla et al., 2004; York et al., 2000; Taken together, the findings described here implicate
Vav family GEFs as critical regulators of Eph forwardZhang et al., 2000). Similarly, regulated endocytosis of
the epidermal growth factor (EGF) receptor (EGFR) con- signaling in vitro and in vivo. By regulating endocytosis
of the ephrin-Eph complex, Vav proteins may converttributes to productive EGF signaling, and dysregulation
of EGFR trafficking may contribute to cellular trans- an initially adhesive interaction of ephrin-Eph binding
to repulsive signaling. It remains to be determinedformation (Levkowitz et al., 1998). For ephrin-Eph endo-
cytosis, it will be interesting to learn whether the re- whether Vav proteins play more general roles as media-
tors of endocytic processes and how Vav proteinscruited factors and signaling outputs are different
between surface-localized Ephs and endocytic vesicle- might mediate these responses.
localized Ephs. Furthermore, as Vav proteins have been
found to be activated downstream of a number of
Experimental Procedures
growth factor receptors such as EGFR and PDGF-R (Liu
and Burridge, 2000; Moores et al., 2000; Pandey et al.,
Yeast Two-Hybrid Screening
2000; Tamas et al., 2003; Tamas et al., 2001), it will be
Briefly, mouse EphA4 (amino acids 570–986) was screened as bait
using an E14 rat spinal cord/DRG cDNA library consisting of 2 ×
important to examine the role of Vav family GEFs in the
Page 10
Role of Vav Family GEFs in Eph Forward Signaling
215
10
6
primary transformants as previously described (Shamah et al., Louis, MO), respectively. M.E.G. acknowledges the generous sup-
port of the F.M. Kirby Foundation to the Neurobiology Program of2001).
Children’s Hospital. This work was supported by a NRSA grant
(AG05870) from the National Institute of Aging (C.W.C.); a Lefler
Antibodies, DNA Constructs, and Coimmunoprecipitations
Foundation postdoctoral fellowship (C.W.C.); a Fu Foundation pre-
Details can be found in the Supplemental Data.
doctoral fellowship (Y.R.S.); National Institute of Child Health and
Human Development grant K08 HD01384 (M.S.); a NSF predoctoral
Generation of Vav2
−/−
Vav3
−/−
Mice
fellowship (P.L.G.); NIH grant HL059561 and ACS Research Profes-
Details can be found in the Supplemental Data.
sorship (J.S.B.); Mental Retardation Research Center grant
HD18655 and NIH grant NS43855 (M.E.G.); and a grant from Daiichi
Analysis of Retinogeniculate Projections
Pharmaceuticals (M.E.G.).
Mouse pups at postnatal day 14 were injected binocularly with
fluorescence-labeled Cholera Toxin B subunit (Alexa-488 or Alexa-
594; Molecular Probes) and allowed to recover for 36 hr. Dissected
Received: October 27, 2004
brains were fixed in 10% (v/v) formalin for 48 hr at 4°C. Coronal
Revised: February 15, 2005
sections (100 m) were imaged with a Zeiss confocal microscope
Accepted: March 22, 2005
with a 10× objective. Images were acquired from similar sections
Published: April 20, 2005
under blinded conditions. To normalize images for differences in
labeling efficiency, we adjusted the laser intensity such that peak
References
pixel values were just saturating. However, most images had peak
values that were nearly identical, allowing for identical settings for
Aghazadeh, B., Lowry, W.E., Huang, X.Y., and Rosen, M.K. (2000).
imaging and analysis. Contralateral and ipsilateral images were an-
Structural basis for relief of autoinhibition of the Dbl homology do-
alyzed using NIH ImageJ software. Background subtraction was
main of proto-oncogene Vav by tyrosine phosphorylation. Cell 102,
performed by 200 pixel rolling ball method as described (Torborg
625–633.
and Feller, 2004).
Arthur, W.T., Quilliam, L.A., and Cooper, J.A. (2004). Rap1 promotes
cell spreading by localizing Rac guanine nucleotide exchange
Growth Cone Collapse Assays
factors. J. Cell Biol. 167, 111–122.
Details can be found in the Supplemental Data.
Bartley, T.D., Hunt, R.W., Welcher, A.A., Boyle, W.J., Parker, V.P.,
Lindberg, R.A., Lu, H.S., Colombero, A.M., Elliott, R.L., and Guthrie,
Ephrin-Eph Endocytosis Assays
B.A. (1994). B61 is a ligand for the ECK receptor protein-tyrosine
Cultured RGCs (P6–P7) were incubated with 5 g/ml ephrin-A1-
kinase. Nature 368, 558–560.
Fc or Fc control, or 100 g/ml transferrin (tetramethylrhodamine
Binns, K.L., Taylor, P.P., Sicheri, F., Pawson, T., and Holland, S.J.
conjugated; Molecular Probes) for 3–30 min at 37°C or 4°C. Cells
(2000). Phosphorylation of tyrosine residues in the kinase domain
were washed three times with ice-cold D-PBS then fixed for 10 min
and juxtamembrane region regulates the biological and catalytic
with 4% PFA/2% sucrose in D-PBS at room temperature. For
activities of Eph receptors. Mol. Cell. Biol. 20, 4791–4805.
ephrin-A1 endocytosis assays, cells were blocked with 3% (w/v)
Bustelo, X.R. (2000). Regulatory and signaling properties of the Vav
BSA, 5% (v/v) goat serum, D-PBS in the presence or absence of
family. Mol. Cell. Biol. 20, 1461–1477.
0.1% (v/v) Triton X-100. Surface bound (unpermeabilized) or total
Bustelo, X.R. (2001). Vav proteins, adaptors and cell signaling. On-
(detergent permeabilized) ephrin-A1-Fc or Fc control was detected
cogene 20, 6372–6381.
using anti-human Fc conjugated with Cy3 (Jackson Immunore-
search Labs) at 1/300 dilution for 1 hr. Growth cone morphology
Chauvet, N., Prieto, M., Fabre, C., Noren, N.K., and Privat, A. (2003).
was visualized using DiO(C
6
) (Molecular Probes) at 500 ng/ml in
Distribution of p120 catenin during rat brain development: potential
D-PBS for 2–3 min. RGCs were imaged with a 60× oil objective lens
role in regulation of cadherin-mediated adhesion and actin cy-
using a Nikon (E600) fluorescence microscope. The distal-most 12
toskeleton organization. Mol. Cell. Neurosci. 22, 467–486.
microns of RGC neurites were identified by DiO staining and out-
Crespo, P., Schuebel, K.E., Ostrom, A.A., Gutkind, J.S., and Bus-
lined using NIH ImageJ software. Mean pixel intensity of the distal
telo, X.R. (1997). Phosphotyrosine-dependent activation of Rac-1
processes in the Cy3 channel was measured and adjusted for local
GDP/GTP exchange by the vav proto-oncogene product. Nature
background. For endocytosis of transferrin, RGCs were placed on
385, 169–172.
ice and washed twice with ice-cold D-PBS, twice with ice-cold 500
Davis, A., Sage, C.R., Dougherty, C.A., and Farrell, K.W. (1994).
mM NaCl/0.2N acetic acid solution (5 min incubation for first wash),
Microtubule dynamics modulated by guanosine triphosphate hy-
and then twice with ice-cold PBS to readjust pH to neutral. RGCs
drolysis activity of beta-tubulin. Science 264, 839–842.
were fixed in 4% PFA/2% sucrose in D-PBS at room temperature
Dearborn, R., Jr., He, Q., Kunes, S., and Dai, Y. (2002). Eph receptor
for 10 min and then stained with DiO. Internalized transferrin-TRITC
tyrosine kinase-mediated formation of a topographic map in the
was imaged and quantified from RGC cell bodies.
Drosophila visual system. J. Neurosci. 22, 1338–1349.
Drescher, U., Kremoser, C., Handwerker, C., Loschinger, J., Noda,
M., and Bonhoeffer, F. (1995). In vitro guidance of retinal ganglion
Supplemental Data
cell axons by RAGS, a 25 kDa tectal protein related to ligands for
The Supplemental Data include Supplemental Experimental Pro-
Eph receptor tyrosine kinases. Cell 82, 359–370.
cedures and one supplemental figure and can be found with this
article online at http://www.neuron.org/cgi/content/full/46/2/205/
Ellis, C., Kasmi, F., Ganju, P., Walls, E., Panayotou, G., and Reith,
DC1/.
A.D. (1996). A juxtamembrane autophosphorylation site in the Eph
family receptor tyrosine kinase, Sek, mediates high affinity interac-
tion with p59fyn. Oncogene 12, 1727–1736.
Acknowledgments
Feldheim, D.A., Vanderhaeghen, P., Hansen, M.J., Frisen, J., Lu, Q.,
Barbacid, M., and Flanagan, J.G. (1998). Topographic guidance la-
The authors would like to thank Linda Hu for assistance with anti-
bels in a sensory projection to the forebrain. Neuron 21, 1303–1313.
body purification; Louisa Mook and Renatta Knox for technical as-
Feldheim, D.A., Kim, Y.I., Bergemann, A.D., Frisen, J., Barbacid, M.,
sistance; Tae-Kyung Kim and Steven Flavell for assistance with de-
and Flanagan, J.G. (2000). Genetic analysis of ephrin-A2 and
velopmental expression data; Zhigang He for the gift of Sema3A
ephrin-A5 shows their requirement in multiple aspects of retinocol-
protein; Rosalind Segal for the DRG explant lysates; Sara Vasquez
licular mapping. Neuron 25, 563–574.
for technical assistance with neuronal cell cultures; and Ben Barres
Flanagan, J.G., and Vanderhaeghen, P. (1998). The ephrins and Eph
and Jeff Goldberg for RGC culture protocols. Vav2
−/−
and Vav3
−/−
receptors in neural development. Annu. Rev. Neurosci. 21, 309–345.
mice were generous gifts of Martin Turner (Babraham Institute,
Cambridge, UK) and Wojciech Swat (Washington University, St. Fournier, A.E., Nakamura, F., Kawamoto, S., Goshima, Y., Kalb,
Page 11
Neuron
216
R.G., and Strittmatter, S.M. (2000). Semaphorin3A enhances endo- sion-dependent activation of the Rac GTPase: a role for phosphati-
dylinositol 3-kinase and Vav2. Oncogene 22, 6100–6106.cytosis at sites of receptor-F-actin colocalization during growth
cone collapse. J. Cell Biol. 149, 411–422.
Marignani, P.A., and Carpenter, C.L. (2001). Vav2 is required for cell
Frisen, J., Yates, P.A., McLaughlin, T., Friedman, G.C., O’Leary,
spreading. J. Cell Biol. 154, 177–186.
D.D., and Barbacid, M. (1998). Ephrin-A5 (AL-1/RAGS) is essential
Marston, D.J., Dickinson, S., and Nobes, C.D. (2003). Rac-depen-
for proper retinal axon guidance and topographic mapping in the
dent trans-endocytosis of ephrinBs regulates Eph-ephrin contact
mammalian visual system. Neuron 20, 235–243.
repulsion. Nat. Cell Biol. 5, 879–888.
Guan, K.L., and Rao, Y. (2003). Signalling mechanisms mediating
Meima, L., Kljavin, I.J., Moran, P., Shih, A., Winslow, J.W., and
neuronal responses to guidance cues. Nat. Rev. Neurosci. 4, 941–
Caras, I.W. (1997a). AL-1-induced growth cone collapse of rat corti-
956.
cal neurons is correlated with REK7 expression and rearrangement
Hattori, M., Osterfield, M., and Flanagan, J.G. (2000). Regulated
of the actin cytoskeleton. Eur. J. Neurosci. 9, 177–188.
cleavage of a contact-mediated axon repellent. Science 289,
Meima, L., Moran, P., Matthews, W., and Caras, I.W. (1997b). Lerk2
1360–1365.
(ephrin-B1) is a collapsing factor for a subset of cortical growth
He, Z., and Tessier-Lavigne, M. (1997). Neuropilin is a receptor for
cones and acts by a mechanism different from AL-1 (ephrin-A5).
the axonal chemorepellent Semaphorin III. Cell 90, 739–751.
Mol. Cell. Neurosci. 9, 314–328.
Heerssen, H.M., Pazyra, M.F., and Segal, R.A. (2004). Dynein mo-
Moores, S.L., Selfors, L.M., Fredericks, J., Breit, T., Fujikawa, K.,
tors transport activated Trks to promote survival of target-depen-
Alt, F.W., Brugge, J.S., and Swat, W. (2000). Vav family proteins
dent neurons. Nat. Neurosci. 7, 596–604.
couple to diverse cell surface receptors. Mol. Cell. Biol. 20, 6364–
6373.
Henkemeyer, M., Itkis, O.S., Ngo, M., Hickmott, P.W., and Ethell,
I.M. (2003). Multiple EphB receptor tyrosine kinases shape den-
Movilla, N., and Bustelo, X.R. (1999). Biological and regulatory
dritic spines in the hippocampus. J. Cell Biol. 163, 1313–1326.
properties of Vav-3, a new member of the Vav family of oncopro-
teins. Mol. Cell. Biol. 19, 7870–7885.
Holland, S.J., Gale, N.W., Gish, G.D., Roth, R.A., Songyang, Z.,
Cantley, L.C., Henkemeyer, M., Yancopoulos, G.D., and Pawson,
Pandey, A., Lazar, D.F., Saltiel, A.R., and Dixit, V.M. (1994). Activa-
T. (1997). Juxtamembrane tyrosine residues couple the Eph family
tion of the Eck receptor protein tyrosine kinase stimulates phos-
receptor EphB2/Nuk to specific SH2 domain proteins in neuronal
phatidylinositol 3-kinase activity. J. Biol. Chem. 269, 30154–30157.
cells. EMBO J. 16, 3877–3888.
Pandey, A., Duan, H., and Dixit, V.M. (1995). Characterization of a
Jurney, W.M., Gallo, G., Letourneau, P.C., and McLoon, S.C. (2002).
novel Src-like adapter protein that associates with the Eck receptor
Rac1-mediated endocytosis during ephrin-A2- and semaphorin 3A-
tyrosine kinase. J. Biol. Chem. 270, 19201–19204.
induced growth cone collapse. J. Neurosci. 22, 6019–6028.
Pandey, A., Podtelejnikov, A.V., Blagoev, B., Bustelo, X.R., Mann,
Kawakatsu, T., Ogita, H., Fukuhara, T., Fukuyama, T., Minami, Y.,
M., and Lodish, H.F. (2000). Analysis of receptor signaling pathways
Shimizu, K., and Takai, Y. (2005). Vav2 as a Rac-GEF responsible
by mass spectrometry: identification of vav-2 as a substrate of the
for the nectin-induced, c-Src- and Cdc42-mediated activation of
epidermal and platelet-derived growth factor receptors. Proc. Natl.
Rac. J. Biol. Chem. 280, 4940–4947.
Acad. Sci. USA 97, 179–184.
Kolodkin, A.L., Matthes, D.J., O’Connor, T.P., Patel, N.H., Admon,
Sahin, M., Greer, P.L., Lin, M.Z., Poucher, H., Eberhart, J., Schmidt,
A., Bentley, D., and Goodman, C.S. (1992). Fasciclin IV: sequence,
S., Wright, T.M., Shamah, S.M., O’Connell, S., Cowan, C.W., et al.
expression, and function during growth cone guidance in the
(2005). Eph-dependent tyrosine phosphorylation of ephexin1 mod-
grasshopper embryo. Neuron 9, 831–845.
ulates growth cone collapse. Neuron 46, this issue, 179–184.
Kolodkin, A.L., Levengood, D.V., Rowe, E.G., Tai, Y.T., Giger, R.J.,
Schuebel, K.E., Movilla, N., Rosa, J.L., and Bustelo, X.R. (1998).
and Ginty, D.D. (1997). Neuropilin is a semaphorin III receptor. Cell
Phosphorylation-dependent and constitutive activation of Rho pro-
90, 753–762.
teins by wild-type and oncogenic Vav-2. EMBO J. 17, 6608–6621.
Kullander, K., and Klein, R. (2002). Mechanisms and functions of
Servitja, J.M., Marinissen, M.J., Sodhi, A., Bustelo, X.R., and Gut-
Eph and ephrin signalling. Nat. Rev. Mol. Cell Biol. 3, 475–486.
kind, J.S. (2003). Rac1 function is required for Src-induced trans-
formation. Evidence of a role for Tiam1 and Vav2 in Rac activation
Kullander, K., Mather, N.K., Diella, F., Dottori, M., Boyd, A.W., and
by Src. J. Biol. Chem. 278, 34339–34346.
Klein, R. (2001). Kinase-dependent and kinase-independent func-
tions of EphA4 receptors in major axon tract formation in vivo. Neu-
Shamah, S.M., Lin, M.Z., Goldberg, J.L., Estrach, S., Sahin, M., Hu,
ron 29, 73–84.
L., Bazalakova, M., Neve, R.L., Corfas, G., Debant, A., and
Greenberg, M.E. (2001). EphA receptors regulate growth cone dy-
Kuruvilla, R., Zweifel, L.S., Glebova, N.O., Lonze, B.E., Valdez, G.,
namics through the novel guanine nucleotide exchange factor
Ye, H., and Ginty, D.D. (2004). A neurotrophin signaling cascade
ephexin. Cell 105, 233–244.
coordinates sympathetic neuron development through differential
control of TrkA trafficking and retrograde signaling. Cell 118, 243–
Stein, E., Cerretti, D.P., and Daniel, T.O. (1996). Ligand activation of
255.
ELK receptor tyrosine kinase promotes its association with Grb10
and Grb2 in vascular endothelial cells. J. Biol. Chem. 271, 23588–
Levkowitz, G., Waterman, H., Zamir, E., Kam, Z., Oved, S., Langdon,
23593.
W.Y., Beguinot, L., Geiger, B., and Yarden, Y. (1998). c-Cbl/Sli-1 reg-
ulates endocytic sorting and ubiquitination of the epidermal growth
Stein, E., Huynh-Do, U., Lane, A.A., Cerretti, D.P., and Daniel, T.O.
factor receptor. Genes Dev. 12, 3663–3674.
(1998). Nck recruitment to Eph receptor, EphB1/ELK, couples li-
gand activation to c-Jun kinase. J. Biol. Chem. 273, 1303–1308.
Liu, B.P., and Burridge, K. (2000). Vav2 activates Rac1, Cdc42, and
RhoA downstream from growth factor receptors but not beta1
Takahashi, T., Fournier, A., Nakamura, F., Wang, L.H., Murakami,
integrins. Mol. Cell. Biol. 20, 7160–7169.
Y., Kalb, R.G., Fujisawa, H., and Strittmatter, S.M. (1999). Plexin-
neuropilin-1 complexes form functional semaphorin-3A receptors.
Lopez-Lago, M., Lee, H., Cruz, C., Movilla, N., and Bustelo, X.R.
Cell 99, 59–69.
(2000). Tyrosine phosphorylation mediates both activation and
downmodulation of the biological activity of Vav. Mol. Cell. Biol. 20,
Tamagnone, L., Artigiani, S., Chen, H., He, Z., Ming, G.I., Song, H.,
1678–1691.
Chedotal, A., Winberg, M.L., Goodman, C.S., Poo, M., et al. (1999).
Plexins are a large family of receptors for transmembrane,
Luo, Y., Raible, D., and Raper, J.A. (1993). Collapsin: a protein in
secreted, and GPI-anchored semaphorins in vertebrates. Cell 99,
brain that induces the collapse and paralysis of neuronal growth
71–80.
cones. Cell
75, 217–227.
Tamas, P., Solti, Z., and Buday, L. (2001). Membrane-targeting is
Mann, F., Miranda, E., Weinl, C., Harmer, E., and Holt, C.E. (2003).
critical for the phosphorylation of Vav2 by activated EGF receptor.
B-type Eph receptors and ephrins induce growth cone collapse
Cell. Signal. 13, 475–481.
through distinct intracellular pathways. J. Neurobiol. 57, 323–336.
Marcoux, N., and Vuori, K. (2003). EGF receptor mediates adhe- Tamas, P., Solti, Z., Bauer, P., Illes, A., Sipeki, S., Bauer, A., Farago,
Page 12
Role of Vav Family GEFs in Eph Forward Signaling
217
A., Downward, J., and Buday, L. (2003). Mechanism of epidermal
growth factor regulation of Vav2, a guanine nucleotide exchange
factor for Rac. J. Biol. Chem. 278, 5163–5171.
Tessier-Lavigne, M., and Goodman, C.S. (1996). The molecular biol-
ogy of axon guidance. Science 274, 1123–1133.
Torborg, C.L., and Feller, M.B. (2004). Unbiased analysis of bulk
axonal segregation patterns. J. Neurosci. Methods 135, 17–26.
Turner, M., and Billadeau, D.D. (2002). VAV proteins as signal inte-
grators for multi-subunit immune-recognition receptors. Nat. Rev.
Immunol. 2, 476–486.
Wahl, S., Barth, H., Ciossek, T., Aktories, K., and Mueller, B.K.
(2000). Ephrin-A5 induces collapse of growth cones by activating
Rho and Rho kinase. J. Cell Biol. 149, 263–270.
Walkenhorst, J., Dutting, D., Handwerker, C., Huai, J., Tanaka, H.,
and Drescher, U. (2000). The EphA4 receptor tyrosine kinase is nec-
essary for the guidance of nasal retinal ganglion cell axons in vitro.
Mol. Cell. Neurosci. 16, 365–375.
Williams, S.E., Mann, F., Erskine, L., Sakurai, T., Wei, S., Rossi, D.J.,
Gale, N.W., Holt, C.E., Mason, C.A., and Henkemeyer, M. (2003).
Ephrin-B2 and EphB1 mediate retinal axon divergence at the optic
chiasm. Neuron 39, 919–935.
York, R.D., Molliver, D.C., Grewal, S.S., Stenberg, P.E., McCleskey,
E.W., and Stork, P.J. (2000). Role of phosphoinositide 3-kinase and
endocytosis in nerve growth factor-induced extracellular signal-
regulated kinase activation via Ras and Rap1. Mol. Cell. Biol. 20,
8069–8083.
Zhang, Y., Moheban, D.B., Conway, B.R., Bhattacharyya, A., and
Segal, R.A. (2000). Cell surface Trk receptors mediate NGF-induced
survival while internalized receptors regulate NGF-induced differ-
entiation. J. Neurosci. 20, 5671–5678.
Zimmer, M., Palmer, A., Kohler, J., and Klein, R. (2003). EphB-
ephrinB bi-directional endocytosis terminates adhesion allowing
contact mediated repulsion. Nat. Cell Biol. 5, 869–878.
Zisch, A.H., Kalo, M.S., Chong, L.D., and Pasquale, E.B. (1998).
Complex formation between EphB2 and Src requires phosphoryla-
tion of tyrosine 611 in the EphB2 juxtamembrane region. Oncogene
16, 2657–2670.
Zisch, A.H., Pazzagli, C., Freeman, A.L., Schneller, M., Hadman, M.,
Smith, J.W., Ruoslahti, E., and Pasquale, E.B. (2000). Replacing two
conserved tyrosines of the EphB2 receptor with glutamic acid pre-
vents binding of SH2 domains without abrogating kinase activity
and biological responses. Oncogene 19, 177–187.
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  • Source
    • "First, Vav could downregulate the EGFR pathway by reducing the number of receptors available at the cell surface. In support of this model, it has been shown that mammalian Vav GEFs are required for endocytosis of both ephrin, during axon guidance (Cowan et al., 2005), and B cell antigen receptor, which is in many ways similar to receptor tyrosine kinase internalisation (Malhotra et al., 2009). Second, Vav could stimulate the transcription of negative regulators of the EGFR pathway, such as Kekkon. "
    [Show abstract] [Hide abstract] ABSTRACT: Organ shaping and patterning depends on the coordinated regulation of multiple processes. The Drosophila compound eye provides an excellent model to study the coordination of cell fate and cell positioning during morphogenesis. Here, we find that loss of vav oncogene function during eye development is associated with a disorganised retina characterised by the presence of additional cells of all types. We demonstrate that these defects result from two distinct roles of Vav. First, and in contrast to its well-established role as a positive effector of the EGF receptor (EGFR), we show that readouts of the EGFR pathway are upregulated in vav mutant larval eye disc and pupal retina, indicating that Vav antagonises EGFR signalling during eye development. Accordingly, decreasing EGFR signalling in vav mutant eyes restores retinal organisation and rescues most vav mutant phenotypes. Second, using live imaging in the pupal retina, we observe that vav mutant cells do not form stable adherens junctions, causing various defects, such as recruitment of extra primary pigment cells. In agreement with this role in junction dynamics, we observe that these phenotypes can be exacerbated by lowering DE-Cadherin or Cindr levels. Taken together, our findings establish that Vav acts at multiple times during eye development to prevent excessive cell recruitment by limiting EGFR signalling and by regulating junction dynamics to ensure the correct patterning and morphogenesis of the Drosophila eye. © 2015. Published by The Company of Biologists Ltd.
    Full-text · Article · Mar 2015 · Development
  • Source
    • "Investigation of Vav2 and Vav3 single or double knockout mice has provided more information about the functional roles of these proteins in the nervous system. In early developmental stages, Vav2 and Vav3 are found to mediate ephrin/Eph signaling regulated axon guidance of ipsilateral retinogeniculate projections [89]. Vav2 binds to activated EphA4 receptors and its Rac GEF activity was stimulated by ephrin-A1. "
    [Show abstract] [Hide abstract] ABSTRACT: Rho family GTPases, including RhoA, Rac1, and Cdc42 as the most studied members, are master regulators of actin cytoskeletal organization. Rho GTPases control various aspects of the nervous system and are associated with a number of neuropsychiatric and neurodegenerative diseases. The activity of Rho GTPases is controlled by two families of regulators, guanine nucleotide exchange factors (GEFs) as the activators and GTPase-activating proteins (GAPs) as the inhibitors. Through coordinated regulation by GEFs and GAPs, Rho GTPases act as converging signaling molecules that convey different upstream signals in the nervous system. So far, more than 70 members of either GEFs or GAPs of Rho GTPases have been identified in mammals, but only a small subset of them have well-known functions. Thus, characterization of important GEFs and GAPs in the nervous system is crucial for the understanding of spatiotemporal dynamics of Rho GTPase activity in different neuronal functions. In this review, we summarize the current understanding of GEFs and GAPs for Rac1, with emphasis on the molecular function and disease implication of these regulators in the nervous system.
    Full-text · Article · Mar 2015 · BioMed Research International
  • Source
    • "After washing three times with PBS, cells were fixed with 4% PFA/4% sucrose for 10 min at 25 C and then blocked/permeabilized with 5% BSA, 15% goat serum, and 0.3% Triton X-100 in PBS for 30 min. The remaining EphB-eB1 complexes were stained with a Cy5-goat a-human Fc antibody at 25 C for 30 min (Cowan et al., 2005; Deininger et al., 2008). Confocal images were then acquired using fixed parameters, and custom ImageJ macros were used to quantify EphB-eB1 staining intensity. "
    [Show abstract] [Hide abstract] ABSTRACT: The small GTPase Rac1 orchestrates actin-dependent remodeling essential for numerous cellular processes including synapse development. While precise spatiotemporal regulation of Rac1 is necessary for its function, little is known about the mechanisms that enable Rac1 activators (GEFs) and inhibitors (GAPs) to act in concert to regulate Rac1 signaling. Here, we identify a regulatory complex composed of a Rac-GEF (Tiam1) and a Rac-GAP (Bcr) that cooperate to control excitatory synapse development. Disruption of Bcr function within this complex increases Rac1 activity and dendritic spine remodeling, resulting in excessive synaptic growth that is rescued by Tiam1 inhibition. Notably, EphB receptors utilize the Tiam1-Bcr complex to control synaptogenesis. Following EphB activation, Tiam1 induces Rac1-dependent spine formation, whereas Bcr prevents Rac1-mediated receptor internalization, promoting spine growth over retraction. The finding that a Rac-specific GEF/GAP complex is required to maintain optimal levels of Rac1 signaling provides an important insight into the regulation of small GTPases.
    Full-text · Article · Jun 2014 · Developmental Cell
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