Cell–cell signaling via Eph receptors and ephrins
Juha-Pekka Himanen1, Nayanendu Saha1and Dimitar B Nikolov
Eph receptors are the largest subfamily of receptor tyrosine
kinases regulating cell shape, movements, and attachment.
The interactions of the Ephs with their ephrin ligands are
restricted to the sites of cell–cell contact since both molecules
are membrane attached. This review summarizes recent
advances in our understanding of the molecular mechanisms
underlining the diverse functions of the molecules during
development and in the adult organism. The unique properties
of this signaling system that are of highest interest and have
been the focus of intense investigations are as follows: (i) the
signal is simultaneously transduced in both ligand-expressing
cells and receptor-expressing cells, (ii) signaling via the same
molecules can generate opposing cellular reactions depending
two subclasses with promiscuous intrasubclass interactions,
but rarely observed intersubclass interactions.
Structural Biology Program, Memorial Sloan-Kettering Cancer Center,
1275 York Avenue, New York, NY 10065, USA
Corresponding author: Nikolov, Dimitar B (email@example.com)
1Juha Pekka-Himanen and Nayanendu Saha contributed equally to this
Current Opinion in Cell Biology 2007, 19:534–542
This review comes from a themed issue on
Cell to cell contact and extracellular matrix
Edited by Lawrence Shapiro and Barry Honig
0955-0674/$ – see front matter
# 2007 Elsevier Ltd. All rights reserved.
The Eph receptors and their ephrin ligands control a
diverse array of cell–cell interactions in the nervous and
vascular systems, as well as in specialized epithelia
(reviewed in [1–4]). Upon ephrin binding, the tyrosine
kinase domain of theEphs isactivated,initiating‘forward’
signaling in the receptor-expressing cells. At the same
time, signals are also induced in the ligand-expressing
cells, a phenomenon referred to as ‘reverse’ signaling 
(Figure 1). Both the Ephs and the ephrins are divided into
two subclasses — A and B — based on their affinities for
each other and on sequence conservation  (http://eph-
nomenclature.med.harvard.edu/). In general, the A-sub-
to the A-class ephrins (ephrin-A1–ephrin-A6), while the
EphB subclass receptors (EphB1–EphB6) interact with
both Ephs and ephrins are membrane bound, their inter-
action occurs only at sites of cell–cell contact (Figure 1). It
is thought that in the absence of cell–cell interactions, the
molecules exist in loosely associated clusters (microdo-
mains) within their respective plasma membranes, which
become much more compact upon Eph/ephrin complex
formation, generating clearly defined signaling centers at
the cell–cell interfaces [7,8]. The past several years have
brought significant advances in our understanding of the
molecular mechanisms of action of Eph receptors and
ephrins, and these are the subjects of this review.
New insights into functions outside the
The Ephs and ephrins were initially identified as axon
guidance molecules mediating neuronal repulsion during
CNS development [2,3], but it was soon discovered that
they also regulate cell–cell communication in a variety of
other tissues and cell types [1,9]. While their best-studied
roles outside the nervous system are those during de-
velopment of the vascular system, recent studies reveal
the importance of Ephs and ephrins in the immune
system, bone, stem cells, epithelial cells, and in the
development and metastasis of many tumors.
Overwhelming evidence documents that the Eph recep-
tors,in conjunction with their ligands, control blood vessel
formation. Ephrin-A1, the first identified family member,
was cloned from human umbilical vein endothelial cells
(HUVECs)  and is implicated in regulating vascular
morphogenesis and angiogenesis [11,12]. EphB4, EphB2,
and EphB3 and their ephrin-B2 and ephrin-B1 ligands
were also shown to direct the formation of the circulatory
system . Most significantly, ephrin-B2 is expressed in
arteries, whereas its receptor, EphB4 — in veins, thus
defining their boundaries during development . In
addition, EphB2 and ephrin-B2 mediate the interactions
between the vessel endothelial cells and the adjacent
mesenchymal cells [13,14]. An important new study 
reveals that ephrin-B2 is also required for normal associ-
ation between the blood-vessel endothelial cells and the
supporting pericytes and vascular smooth muscle cells
(mural cells). Defective mural-cell coverage is associated
with the poorly organized and leaky vasculature seen in
tumors or other human diseases. The authors further
suggest that ephrin-B2 has some cell–cell-contact-inde-
pendent functions  during these events.
Many Ephs and ephrins, including EphA1, EphA2,
EphA3, EphA4, EphB2, EphB3, EphB4, and ephrin-A1
are overexpressed in a variety of cancers where they
Current Opinion in Cell Biology 2007, 19:534–542 www.sciencedirect.com
exhibit mostly, but not exclusively, tumor-promoting
properties [14,16]. Indeed, the founding Eph member,
EphA1, was isolated from a hepatoma cell line  and is
Many Ephs seem to be predominantly expressed in
metastatic cell lines as compared to the primary tumor
and their expression levels often correlate with the grade
of the tumor malignancy and invasiveness. EphB4 is
expressed in all examined breast carcinoma cell lines
 and its role in cancer is reviewed in . EphB4 is
particularly interesting because it has both tumor-sup-
pressing and tumor-promoting activities, which are
affected via different molecular mechanisms: the tumor
suppression seems to be a result of EphB4-dependent
downregulation of Crk that lead to inhibition of cell
mobility and invasion, as well as facilitation of apoptosis;
the tumor promoting properties seem to result mainly
from the angiogenesis-promoting EphB4 activity .
recently shown to regulate insulin secretion  by
directingthe communications betweenthe insulin secret-
ing b cells of the pancreas. These cells are aggregated in
pancreatic islets, where cell–cell contacts inhibit basal
insulin secretion but enhance glucose-stimulated insulin
secretion, thus contributing to glucose homeostasis
during fasting and feeding. It is now clear that EphA
forward signaling inhibits insulin secretion, whereas
ephrin-A reverse signaling stimulates it.
A recent study also shows that B-class Ephs and ephrins
mediate the activities of osteoclasts, which degrade bone,
and osteoblasts, which form bone, to maintain bone
ephrin-B2, while osteoblasts express the corresponding
EphB4 receptor. Reverse signaling through ephrin-B2
into osteoclast precursors suppresses osteoclast differen-
tiation, while forward signaling through EphB4 into
osteoblasts enhances it — thereby maintaining bone
Structure studies of the Eph/ephrin
Biochemical and X-ray crystallographic investigations
have generated abundant structural information about
the interacting domains of the Ephs, ephrins, and their
complex  indicated that the proteins form a tetra-
meric, ring-like assembly in which two receptor and two
ligand molecules interact via two distinct interfaces
(Figure 2). Such an architectural arrangement would
explain how cell–cell contact results in the rearrangement
of both ligands and receptors — initiating signals in both
interacting cells. As illustrated in Figure 2, one of the
Eph/ephrin interfaces is very extensive and is responsible
for the high-affinityligand-receptor
whereas the second interface is smaller and is responsible
for the assembly of the EphB–ephrin-B dimers into the
circular tetramers [4,20].
Cell–cell signaling via Eph receptors and ephrins Saha, Himanen and Nikolov535
Activation of bidirectional Eph/ephrin signaling. The current model of signaling initiation involves an initial 1:1 high-affinity interaction between
ligands and receptors, followed by tetramerization, oligomerization and clustering of the molecules at the sites of cell–cell contact . This brings
about phosphorylation of the cytoplasmic Eph (as well as B-class ephrin) domains and downstream signaling initiation . Kinase activation is
controlled by the phosphorylation of residues in the activation loop and the juxtamembrane segment, which affect the interlobe (subdomain)
dynamics of the kinase domain [31–35]. The minimal ligand-binding domain of the receptor is in blue and the ephrin receptor-binding domain is
in pink. The kinase and SAM (sterile a-motif) domain is in green and the phosphate groups are in orange.
Current Opinion in Cell Biology 2007, 19:534–542
EphB2, and EphB4. Consistent with targeting the ephrin-binding site, the
higher affinity peptides antagonize ephrin binding to the EphB receptors
and the authors even optimize an EphB4-binding peptide with affinity
comparable with that of the natural ligand, ephrin-B2.
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EphB4/Ephrin-B2 antagonist peptide complex reveals the
determinants for receptor specificity. Structure 2006,
In this study, the authors report the crystal structure of the EphB4
receptor in complex with a highly specific antagonistic peptide identified
in , documenting that the peptide binds in the ephrin-binding surface
channel of the receptor. Isothermal titration calorimetry reveals an inter-
esting thermodynamic discrepancy between ephrin-B2 binding ,
which is an entropically driven process, and peptide binding, which is
an enthalpically driven process.
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In this study, the authors use truncated versions of EphA3, single-amino
acid point mutants of ephrinA5 and fluorescence resonance energy
transfer technology to uncover a cis interaction between EphA3 and
ephrinA5 that is independent of the established ligand-binding domain of
EphA3 and involves, instead its FNIII region. This interaction is inhibitory
blocking EphA3 activation and reducing the cellular response to A-
ephrins presented in trans.
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The authors document for the first time that that ephrins are functionally
cleaved by the metalloprotease ADAM10, a process essential for dis-
rupting Eph/ephrin cell contacts in vivo.
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of the ADAM10/EphA3/ephrin-A5(A2) interactions and suggest a simple
mechanism, which ensures that only Eph-bound ephrins are recognized
and cleaved to allow separation of the interacting cells. This is also the
first study to document cleavage in trans (on the surface of the opposing
cell) by an ADAM family metalloprotease.
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In this study, the authors show that EphB stimulates a metalloproteinase
cleavage of ephrinB2, producing a carboxy-terminal fragment that is
further processed by PS1/gamma-secretase to produce an intracellular
peptide, which binds Src and inhibits its association with the inhibitory
kinase Csk. This is the first compelling evidence that the combined action
of a-secretase activity and g-secretase activity can mediate ephrin
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In this study, the authors show that cell contact-induced EphB–ephrinB
complexes are endocytosed during the retraction of cells and neuronal
growth cones. The observed endocytosis, which is sufficient to promote
cell detachment, occurs in a bidirectional manner and involves full-length
receptor and ligand complexes.
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directional endocytosis terminates adhesion allowing contact
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The authors show endocytosis of activated Eph receptors and their
bound, full-length ephrinB ligands in heterologous, nonneuronal cells.
They also observe that both the internalization of the receptor–ligand
complexes and the subsequent cell retraction events are dependent on
actin polymerization, which in turn is dependent on Rac signalling within
the receptor-expressing cells.
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endocytosis of ephrinBs regulates Eph–ephrin contact
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Cell to cell contact and extracellular matrix
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