Download Nectins and nectin-like molecules: roles in contact inhibition of cell

REVIEWS
Nectins and nectin-like molecules:
roles in contact inhibition of cell
movement and proliferation
Yoshimi Takai*, Jun Miyoshi‡, Wataru Ikeda* and Hisakazu Ogita*
Abstract | Nectins and nectin-like molecules (Necls) are immunoglobulin-like transmembrane
cell adhesion molecules that are expressed in various cell types. Homophilic and heterophilic
engagements between family members provide cells with molecular tools for intercellular
communications. Nectins primarily regulate cell–cell adhesions, whereas Necls are involved
in a greater variety of cellular functions. Recent studies have revealed that nectins and
NECL‑5, in cooperation with integrin αvβ3 and platelet-derived growth factor receptor,
are crucial for the mechanisms that underlie contact inhibition of cell movement and
proliferation; this has important implications for the development and tissue regeneration of
multicellular organisms and the phenotypes of cancer cells.
Adherens junction
This junction comprises two
types of cell adhesions: cell–extracellular matrix and cell–cell. In the context of
this article, ‘adherens junction’
refers to the latter. Adherens
junctions contain classical
cadherins and catenins that
are attached to cytoplasmic
actin filaments and
mechanically connect two
apposing cells.
*Division of Molecular
and Cellular Biology,
Department of Biochemistry
and Molecular Biology,
Kobe University Graduate
School of Medicine,
Kobe 650‑0017, Japan.
‡
Department of Molecular
Biology, Osaka Medical
Center for Cancer and
Cardiovascular Diseases,
Osaka 537‑8511, Japan.
Correspondence to Y.T.
e-mail:
[email protected]
doi:10.1038/nrm2457
Nectins and nectin-like molecules (Necls) are immunoglobulin (Ig)-like cell adhesion molecules (CAMs)
that have recently been shown to be essential contributors to the formation of cell–cell adhesions and novel
regulators of cellular activities, including cell polar­
ization, differentiation, movement, proliferation and
survival1,2. The nectin and Necl families comprise four
and five members, respectively (TABLE 1). The four members of the nectin family are ubiquitously expressed and
have two or three splice variants. Nectin‑1 and nectin‑2
were initially isolated as receptors for α‑herpesvirus
and were called PRR1 (also known as HVEC) and
PRR2 (also known as HVEB), respectively 3,4. They
were renamed nectins from the Latin word necto, which
means ‘to connect’ (Ref. 5).
Nectins regulate the formation of various types
of cell–cell junctions, such as adherens junctions (AJs)
between neighbouring epithelial cells and fibroblasts,
Sertoli cell–spermatid junctions in the testes and puncta
adherentia junctions in the nervous system. Nectins
are also involved in the establishment of apical–basal
polarity at cell–cell adhesion sites and the formation of
tight junctions in epithelial cells.
Necls are ubiquitously expressed and have a greater
variety of functions than nectins. NECL-1 and NECL-4
mediate axo–glial interactions, Schwann cell different­
iation and myelination6–8. NECL-2 acts as a tumour
suppressor and a regulator for immune surveillance, and
NECL-5 is involved in the enhancement of cell movement and proliferation9,10. Although the physiological
roles of NECL-3 are currently unknown, Necls seem
to be crucial for morphogenesis and differentiation in
many cell types. Whether the functions of each Necl
family member are unique or whether they overlap with
other Necl family members remains unanswered.
NECL-5 is particularly notable among Necls because
of its unique expression profiles. NECL-5 was originally
identified as human poliovirus receptor (PVR; also
known as CD155)11,12 and as rodent TAGE4, which is
overexpressed in rodent colon carcinoma13,14. NECL‑5
expression is very low in most adult organs, but is
abundant in the developing or regenerating liver15,16. In
addition, NECL-5 is overexpressed in transformed cells
and promotes the cell cycle17,18. Thus, NECL-5 seems
to be an oncofetal protein that functions in embryonic
development and cancer progression.
Cell culture studies have demonstrated an important
role for nectins and NECL-5 in contact inhibition of cell
movement and proliferation (BOX 1). When moving and
proliferating cells form contacts with each other, they
promote the formation of AJs and the cessation of cell
movement and proliferation19,20. This phenomenon has
been known as ‘contact inhibition of cell movement and
proliferation’ for over half a century, although its underlying mechanism is not understood. Contact inhibition
of cell movement and proliferation is also necessary for
proper cell differentiation and survival.
The functions of nectins and Necls are highly correl­
ated with those of other well-known transmembrane
molecules, such as cadherins, integrins and growth
nature reviews | molecular cell biology
volume 9 | AUGUST 2008 | 603
© 2008 Macmillan Publishers Limited. All rights reserved.
REVIEWS
Table 1 | Nectin and Necl family members
Member
Old nomenclature
Function
Knockout mouse phenotype
Nectin‑1
PRR1, HVEC
Cell–cell adhesion molecule
Receptor for α‑herpes virus (HSV‑1, HSV‑2 and pseudorabies
virus) entry into cells.
(Defects in humans: cleft lip/palate-ectodermal dysplasia
syndrome, also known as Zlotogora–Ogur syndrome)
Microphthalmia, skin abnormalities
and abnormal mossy-fibre trajectories
in the hippocampus
Nectin‑2
PRR2, HVEB
Cell–cell adhesion molecule
Receptor for α‑herpesvirus entry into cells
Male-specific infertility
Nectin‑3
PRR3
Cell–cell adhesion molecule
Male-specific infertility
Microphthalmia and abnormal mossyfibre trajectories in the hippocampus
Nectin‑4
–
Cell–cell adhesion molecule
Overexpressed in breast carcinoma
–
NECL‑1
TSLL1, SynCAM3
Cell–cell adhesion molecule with neural tissue-specific
expression: localized at contact sites between axons and glial
cells or Schwann cells but not at synaptic junctions
–
NECL‑2
IGSF4, RA175, SglGSF, TSLC1,
SynCAM1
Cell–cell adhesion molecule that is localized on the
basolateral membranes in epithelial cells
Involved in spermatogenesis and synapse formation
Tumour suppressor in lung carcinoma
Male-specific infertility
NECL‑3
Similar to NECL3, SynCAM2
Putative cell–cell adhesion molecule
–
NECL‑4
TSLL2, SynCAM4
Cell–cell adhesion molecule
Mediates axo-glial interaction, Schwann cell differentiation
and myelination
Possible involvement in tumour suppression
–
NECL-5
TAGE4, PVR, CD155
Receptor for poliovirus
Overexpressed in various carcinomas
Enhancement of cell movement and proliferation (in
cooperation with integrin αvβ3 and PDGF receptor)
–
CAM, cell adhesion molecule; HSV, herpes simplex virus; HVE, herpesvirus entry; PDGF, platelet-derived growth factor; PRR, poliovirus receptor-related protein;
PVR, poliovirus receptor.
Tight junction
The most apical intercellular
junction, which functions as a
selective (semi-permeable)
diffusion barrier between
individual cells and as a fence
to prevent the intermingling of basolateral cell-surface
molecules with apical
molecules. Tight junctions are
identified as a belt-like region
in which two lipid-apposing
membranes lie close together.
Focal complex
A small (<0.5 µm diameter)
immature cell–extracellular
matrix junction that is
observed at the peripheral
region of the leading edge of
moving cells.
Focal adhesion
A mature cell–extracellular
matrix junction that associates
with integrin signalling factors,
filamentous-actin-binding
proteins and actin stress fibres.
factor receptors. Cadherins are single-membranespanning CAMs that constitute a family with over 100
members21,22. Homophilic engagements of the classical
cadherins, such as E-cadherin (epithelial), N-cadherin
(neuronal) and VE-cadherin (vascular endothelial), can
stabilize nectin-based cell–cell contacts to form AJs in
epithelial cells, fibroblasts and endothelial cells, respectively. Integrins are the CAMs that are involved in
cell–extracellular matrix (ECM) junctions, and are composed of heterodimers of α and β subunits23. To date, 18
α-subunits and 8 β-subunits have been identified, and
24 different combinations have been reported. Integrins
have important roles in the formation of focal complexes
and focal adhesions — essential structures for cell movement and proliferation. Many reports have demonstrated
physical and functional associations between CAMs and
growth factor receptors24–26. Here, we describe how nectins
and NECL-5 regulate cell–cell adhesions in cooperation
with cadherins, integrins and platelet-derived growth
factor (PDGF) receptor, and how they are involved in
contact inhibition of cell movement and proliferation.
Adhesion properties of nectins and Necls
The nectin and Necl molecules share common domains,
including an extracellular region with three Ig-like
loops, a transmembrane segment and a cytoplasmic
tail1 (FIG. 1a). Many proteins that directly interact with
nectins and Necls at their cytoplasmic region have
been identified: nectins interact with the filamentous
(F)-actin-binding protein afadin and the cell polarity
protein partitioning defective-3 (PAR3), whereas Necls
interact with scaffolding proteins, such as membraneassociated guanylate kinase (MAGUK) and Band4.1
family members, or with the motor-related protein
TCTEX1 (Refs 6,27–30) (FIG. 1a).
Nectins and Necls are classified by whether they can
bind afadin; nectins bind afadin, whereas Necls do not.
The direct binding between nectin and afadin is mediated by the conserved C‑terminal motif of nectins and
the PDZ domain of afadin1. Afadin was originally identified as an F‑actin-binding protein that localized at AJs
and had a structure that is similar to the AF‑6 gene
product31 — an ALL1 fusion partner that is involved in
acute myeloid leukaemias32. Afadin has multiple domains
and several alternative splicing sites in the C‑terminal
region. Four splicing variants have been identified31,33;
hereafter, afadin refers to the longest variant.
Two nectin or Necl molecules on the surface of the
same cell first form cis-dimers, and then this is followed
by trans-dimerization of the cis-dimers on apposing cells.
This results in the formation of cell–cell adhesions (FIG. 1b).
Nectins and cadherins cooperatively function to establish
AJs. In contrast to cadherins, nectins promote cell–cell contacts by forming homophilic or hetero­philic trans-dimers.
Heterophilic interactions have been detected between
nectin‑1 and nectin‑3, between nectin‑2 and nectin‑3,
604 | AUGUST 2008 | volume 9
www.nature.com/reviews/molcellbio
© 2008 Macmillan Publishers Limited. All rights reserved.
REVIEWS
Box 1 | Contact inhibition of cell movement and proliferation
Normal cells
Cell movement,
cell proliferation
Cell–cell adhesion
natural killer cells can recognize its partners, nectin‑1
and NECL-5, which are both expressed on target cells,
and stimulates cytotoxicity of natural killer cells39. The
CRTAM–NECL-2 inter­action promotes the cyto­toxicity
of natural killer cells and interferon‑γ secretion by CD8+
T cells, and is involved in natural killer cell-mediated
rejection of tumours that express NECL-2 (Ref. 40).
Therefore, these Ig-like molecules are mostly correlated
with the immunosurveillance network to distinguish
and eliminate tumour cells from normal cells.
Contact inhibition
Transformed cells
Cell movement,
cell proliferation
Disrupted
cell–cell adhesion
Loss of contact inhibition
The concepts of ‘contact inhibition of cell movement’
(Ref. 20)| Molecular
and ‘contact
Nature Reviews
Cellinhibition
Biology
of cell proliferation’ (Ref. 19) represent two sides of the same coin. Contact inhibition of
cell movement, originally described in fibroblasts, is the phenomenon of a cell ceasing
to migrate in the same direction after contact with another cell103,104. This concept has
been extended to include the immobilization of cells when they form cell–cell
adhesions, as demonstrated in epithelial wound healing105,106. Now, the term contact
inhibition of cell movement is used quite broadly107,108. By contrast, it is not clear
whether contact inhibition of cell proliferation depends on cell contact; in fact, there is
compelling evidence that it does not109,110. Therefore, downregulation of mitosis in
confluent cells is also called ‘density-dependent inhibition of mitosis’ (Ref. 111). The
contact inhibition of cell movement and proliferation is crucially important in
organogenesis as well as in wound healing. Although there are several reports that cell
adhesion molecules are involved in contact inhibition10,94,95, the mechanism for this is not
fully understood. In cancer cells, the mechanism of contact inhibition is usually
disrupted, resulting in uncontrolled cell movement and sustained cell proliferation (see
figure). Loss of contact inhibition of cell movement and proliferation allows cancer cells
to facilitate the invasion of neighbouring tissues and metastasis to remote organs.
PDZ domain
A protein–protein interaction
domain that was first found in
postsynaptic density protein95 (PSD95), Discs-large (DLG)
and zona occludens-1 (ZO1).
and between nectin‑1 and nectin‑4 (FIG. 1c). It is noteworthy that the heterophilic trans-interactions of nectins
are stronger than the homophilic trans-interactions34.
The extracellular regions of Necls, except for those of
NECL-4 and NECL‑5, form homophilic and heterophilic
interactions in trans with each other, whereas NECL-4 and
NECL-5 only form heterophilic interactions in trans with
other members of the nectin and Necl families5–8,30,34–37
(FIG. 1c). These trans-interactions contribute to many types
of cell–cell adhesions and contacts.
As well as the trans-interactions among the nectin and
Necl family members, these proteins form hetero­philic
interactions in trans with other Ig-like molecules, including CD226 (also known as DNAX accessory molecule-1;
DNAM1), CD96 (also known as Tactile) and class-I-MHCrestricted T-cell-associated molecule (CRTAM)38–40
(FIG. 1c). These molecules are mainly expressed in lympho­
cytes, such as cytotoxic T cells and natural killer cells,
and regulate immune responses. The trans-interaction
of CD226 on natural killer cells with either nectin‑2 or
NECL-5 on target cells enhances the natural killer cellmediated lysis of target cells38. CD96 that is expressed on
Role of nectins in cell–cell adhesion
Two types of CAMs — nectins and cadherins — localize
at AJs and have essential cooperative roles in the formation of AJs in various cell types, including epithelial cells
and fibroblasts. Necls do not necessarily localize at AJs,
although some Necl family members, such as NECL-1,
NECL-2 and NECL-4, accumulate at cell–cell adhesion
sites and participate in the connection of adjacent cells in
several organs, including the testes and the nervous system. However, whether nectins and Necls have cooperative
roles in cell–cell adhesions remains unknown.
Formation of AJs by nectins and cadherins. Nectins localize strictly at AJs in both epithelial cells and fibroblasts1.
Studies with many cultured cell lines have revealed that
nectins initiate the formation of AJs before cadherins
start to form cell–cell adhesions2. Once the initial cell–cell
contacts are formed between two neighbouring cells by
nectins, cadherins are recruited to these contact sites,
resulting in the formation of strong cell–cell adhesions.
The nectin and cadherin systems are then physically
associated with one another to establish AJs. Typical lines
of evidence for this role of nectins are as follows: first,
the dissociation constant (Kd) between nectin molecules
(2 nM for nectin‑1 and nectin‑3, and 360 nM for nectin‑2
and nectin‑3) is much lower than that between cadherin
molecules (~80 µM)37,41. Thus, the interactions between
nectins are more favourable than those between cadherins.
Second, in Madin–Darby canine kidney (MDCK) and
NIH3T3 cells, an inhibitor of nectin-based cell–cell adhesions, NEF3, prevents the formation of cadherin-based
AJs42. NEF3 is an engineered recombinant protein that
includes the extracellular fragment of nectin‑3 fused to
human IgG Fc and is designed to block the intercellular
interactions of endogenous nectins.
Cadherins are the major CAMs at AJs21,22. They directly
bind β‑catenin at their C‑terminal tail and p120ctn at their
juxtamembrane region21,43 (FIG. 2). β‑Catenin, in turn,
interacts with α‑catenin, which also binds to α‑actinin
and vinculin, whereas p120ctn regulates the adhesion activity and stability of the cadherins. α‑Catenin, α‑actinin
and vinculin are F‑actin-binding proteins that anchor
cadherins to the actin cytoskeleton. Cadherins, α‑catenin,
β‑catenin and p120ctn are widely distributed along the lateral plasma membrane as well as being present in AJs in
epithelial cells, whereas α‑actinin and vinculin localize
strictly at AJs and focal adhesions. The functional implication of this differential distribution of these molecules in
epithelial cells remains elusive, but all of these molecules
colocalize at AJs in fibroblasts.
nature reviews | molecular cell biology
volume 9 | AUGUST 2008 | 605
© 2008 Macmillan Publishers Limited. All rights reserved.
REVIEWS
a
Nectins
Afadin
S–S
RA1
Necls
S–S
S–S
RA2 FHA
S–S
S–S
PAR3
E/A-X-Y-V
DIL
S–S
b
TM
Nectin-1
Nectin-3
PDZ
PR1
PR2
F-actin
PR3
TM
NECL-1
NECL-2
MAGUK family (PALS2, DLG3, CASK)
Band4.1 family (DAL1)
NECL-5
TCTEX1
cis-Dimerization
trans-Interaction
Nectin
or
Necl
Monomer
Ig-like
loop
Plasma
membrane
cis-Dimer
transDimer
First
Second
Third
c
?
Nectin-4
CD96/Tactile
Necl-5
?
CD226/DNAM1
Necl-4
Nectin-1
Nectin-3
Nectin-2
Necl-3
Necl-1
Necl-2
CRTAM
?
?
Figure 1 | Molecular structures and modes of interaction of nectins, Necls and afadin.
a | Nectins and nectin-like (Necl) molecules contain three
Ig-like
loops
in their extracellular
Nature
Reviews
| Molecular
Cell Biology
region, a single transmembrane (TM) segment and a cytoplasmic tail. The nectin family
members possess a consensus motif of four amino acids (E/A‑X-Y‑V; X represents any
amino acid) at the C terminus that interacts with the adaptor protein afadin, which in
turn interacts with filamentous (F)-actin to connect nectins to F-actin. Direct binding
between nectins and afadin is conducted through the C‑terminal motif of nectins and the
PDZ (postsynaptic density protein-95, Discs-large, zona occludens-1) domain of afadin
and links nectins to the actin cytoskeleton. Nectin-1 and nectin-3 also bind partitioning
defective-3 (PAR3), a member of the PAR complex. Necls are structurally similar to nectins
but do not directly bind afadin. However, NECL-1 and NECL-2 interact with scaffolding
proteins, such as membrane-associated guanylate kinase (MAGUK) and Band4.1 family
members, and NECL-5 binds the motor-related protein TCTEX1. b | Two nectin and Necl
molecules of the same plasma membrane first form cis-dimers, and then this is followed by
the formation of a trans-interaction between the first Ig-like loops of cis-dimers located on
apposing cells. c | Nectins, Necls and other Ig-like molecules (CD96 (also known as Tactile),
CD226 (also known as DNAX accessory molecule-1; DNAM1) and class-I-MHC-restricted
T-cell-associated molecule (CRTAM)) form homophilic (looped arrows) and heterophilic
(double-headed arrows) interactions in trans with each other. NECL-4 and NECL-5 are
unable to form homophilic interactions. DIL, dilute domain; FHA, forkhead-associated
domain; RA, Ras-association domain; PR, Pro-rich domain.
Afadin, α‑catenin and their binding proteins are
involved in the association between nectins and cadherins that forms AJs44–48 (FIG. 2). Disruption of afadin
by knockout or knockdown techniques inhibits the
formation of cadherin-based AJs49,50. Disruption of
α‑catenin inhibits the formation of cadherin-based AJs,
but not that of nectin-based cell–cell adhesions51. These
results have provided the additional lines of evidence
that nectins contribute to the initiation of the formation
of cadherin-based AJs. Afadin and α‑catenin not only
directly interact with one another45,46, but also indirectly
interact through the F‑actin-binding proteins ponsin,
vinculin and α-actinin, and the adaptor proteins afadinand α-actinin-binding protein (ADIP) and LIM
domain only protein-7 (LMO7)44,47,48. All of these mole­
cules colocalize with afadin and α‑catenin at AJs. It is
unknown how or when these molecules associate with
afadin and α‑catenin during the formation of AJs.
A model has recently been proposed in which the
binding of α‑catenin to the E‑cadherin–β-catenin complex and F actin is mutually exclusive52–54. This model
seems to be in conflict with results from previous studies that show that α‑catenin functions as a connector
between cadherins and the actin cyto­skeleton at cell–cell
adhesions55,56. Furthermore, the model seems to underestimate the importance of cell adhesion systems other
than the cadherin–catenin complex. Although there
are no data available, the nectin–afadin complex might
support the α‑catenin-mediated linkage of cadherins to
the actin cytoskeleton, because afadin can interact with
both α‑catenin and F‑actin.
Intracellular signalling induced by nectins. The transinteraction of nectins at initial cell–cell contact sites
first induces the activation of a Tyr kinase, Src57 (FIG. 3a).
Activated Src then induces the activation of the small
G protein RAP1 through the adaptor protein Crk and
C3G (the guanine nucleotide-exchange factor (GEF)
for RAP1), and phosphorylates FRG (the GEF for
the small G protein CDC42) and VAV2 (the GEF for the
small G protein Rac) on Tyr57–59. Activated RAP1 activ­
ates phosphorylated FRG, resulting in the activation of
CDC42 and the formation of filopodia. Activated CDC42
enhances the activation of phosphorylated VAV2 and
eventually induces the activation of Rac and the form­
ation of lamellipodia. Protrusions, such as filopodia and
lamellipodia, which are usually formed in moving cells,
are also formed in immobilized cells by these signalling
pathways and contribute to the formation of cell–cell
junctions; filopodia increase the number of contact sites
between apposing cells, whereas lamellipodia efficiently
close the gaps between these contact sites60–62.
Role of integrin αvβ3 in nectin-induced signalling.
There is also crosstalk between cell–cell and cell–ECM
junctions63,64. Integrins positively or negatively regulate
the formation and stability of cell–cell junctions. For
instance, during embryonic development, integrins
promote epithelial cell remodelling by reducing the
interaction of cell–cell adhesion molecules at AJs65.
However, integrins induce the functional polarization
of cells and reinforce the cadherin-based AJs66. Nectin‑1
and nectin‑3, but not nectin‑2, physically associate with
integrin αvβ3 at cell–cell adhesion sites, an association that is essential for the nectin-induced activation
606 | AUGUST 2008 | volume 9
www.nature.com/reviews/molcellbio
© 2008 Macmillan Publishers Limited. All rights reserved.
REVIEWS
a
b
Nectin
Cadherin
tn
20 c
p1
n
ct
20
Rac
β-Catenin
Afadin
F-actin
bundles
p1
Plasma
membrane
Ponsin
Nectin
CDC42
Afadin
Ponsin
α-Catenin
IQGAP1
c
d
tn
in
Vinculin
20 c
p1
Ponsin
Rac
ADIP
LMO7
n
ct
ctin
α-A
Afadin
Nectin
20
ADIP
LMO7
n
ct
20 c
Cadherin
p1
20
p1
Nectin
p1
tn
Cadherin
ctin
α-A
in
Vinculin
Afadin
Ponsin
AJ
Small G protein
A monomeric GTP-binding
protein with a molecular mass
of 20–30 kDa that has intrinsic
GTPase activity. It has two
interconvertible forms: a GDP-bound inactive form and a GTP-bound active form. The
GTP-bound form interacts with
and activates several effector
proteins that mediate
downstream signalling events.
Filopodium
A thin cellular protrusion that is formed by bundle-type
reorganization of filamentous
actin through the activation of
CDC42.
Lamellipodium
A broad and flat cellular
protrusion that is formed by
meshwork-type reorganization
of filamentous actin through
the activation of Rac.
Figure 2 | Dynamic reorganization of the actin cytoskeleton in the formation of adherens junctions. a | The first step of
Nature Reviews | Molecular Cell Biology
the reorganization of the actin cytoskeleton begins with the primordial cell–cell contact that is initiated by the nectin–
afadin complex. At this stage, afadin binds ponsin, which is involved in the connection between the nectin–afadin and
cadherin–catenin complexes (as shown in panel c). b | The second step involves the nectin-induced activation of the small G
proteins Rac and CDC42 by several filamentous (F)‑actin-binding proteins, such as IQ-motif-containing GTPase-activating
protein-1 (IQGAP1). This is important for the recruitment of the cadherin–catenin complex to nectin-based cell–cell
adhesion sites. At this stage, cadherins do not trans-interact with each other, but form a complex with p120ctn, β‑catenin
and α‑catenin. c | The third step is induced by several connector complexes (ponsin–vinculin, the complex comprised of
α-actinin and afadin- and α-actinin-binding protein (ADIP), and the complex comprised of α-actinin and LIM domain only
protein-7 (LMO7)) that associate with F-actin and the cadherin–catenin complex and that link the nectin–afadin complex
to the cadherin–catenin complex. Moreover, afadin interacts directly with α‑catenin, but this interaction does not seem to
be strong. At this stage, the adhesion activity of cadherins is increased and the trans-interaction of cadherins occurs.
d | The fourth step is induced by cadherin-mediated activation of Rac, which is involved in the inhibition of endocytosis of
cadherins and contributes to the stabilization of the trans-interaction of cadherins at adherens junctions (AJs).
of Src, which in turn is crucial for the formation of
cadherin-based AJs57,67 (FIG. 3a).
Although nectins interact in cis both with the
active and with the inactive forms of integrin αvβ3,
the active form of integrin αvβ3 is essential for the
nectin-induced activation of Src, because Src activation is suppressed by the inhibition of integrin αvβ3.
Signalling from integrin αvβ3 to Src is mediated by
protein kinase C and focal adhesion kinase68. During
the initial stage of the formation of AJs, nectins interact in cis with the active form of integrin αvβ3, which
is then gradually converted into the inactive form as
the AJs are established. As nectins do not interact with the
other integrins that have thus far been tested, such as
integrin α5 and integrin β1, the signalling mechanism
seems to be specific to integrin αvβ3.
The role of actin in AJ formation. In the formation of
AJs, there are at least four sequential steps of dynamic
reorganization of the actin cytoskeleton (FIG. 2). The
first step is induced by afadin and its interacting protein
ponsin, which are directly and indirectly associated with
nectins, respectively. The second step is induced by the
nectin-mediated activation of CDC42 and Rac through
F‑actin-binding proteins, such as IQ-motif-containing
GTPase-activating protein-1 (IQGAP1). The third step is
induced by the F‑actin-binding proteins that are directly
or indirectly associated with nectins and cadherins, and
the fourth step is induced by the cadherin-induced
activation of Rac69.
The trans-interactions of the extracellular regions of
the nectin or cadherin molecules are essential for their
respective cell–cell adhesions, but alone they are not
nature reviews | molecular cell biology
volume 9 | AUGUST 2008 | 607
© 2008 Macmillan Publishers Limited. All rights reserved.
REVIEWS
a
Cadherin
Plasma membrane
β-Catenin
Reorganization of the
actin cytoskeleton
α-Catenin
Nectin-based
cell–cell adhesion
Afadin
PtdIns4P
P
P
PtdIns(4,5)P2
VAV2
P
FRG
Phosphorylation
PIPKIγ90
Integrin
αvβ3
(active)
CDC42
Rac
Nectin
Src
Crk
C3G
RAP1
P P
Talin
PKC
FAK
Extracellular matrix
b
Apical surface
β-Catenin
Cadherin
α-Catenin
PTPµ
Afadin
Nectin
AJ
PI3K
PDGF
receptor
AKT
Apoptosis
PDGF
PtdIns4P
Integrin
αvβ3
(inactive)
P
PIPKIγ90
Talin
Nectin-mediated inactivation
of integrin αvβ3 after the
establishment of AJs
Extracellular matrix
Figure 3 | Nectin-induced intracellular signalling during and after the formation of adherens junctions. a | During the
Nature Reviews | Molecular Cell Biology
formation of adherens junctions (AJs), nectins associate with integrin αvβ3, which is activated by binding of talin. Talin is a
molecule that directly connects integrins to the actin cytoskeleton and activates integrins intracellularly. The binding of
talin to integrin αvβ3 is enhanced by phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2), which is generated by phosphatidylinositol phosphate kinase type Iγ90 (PIPKIγ90). Signalling from integrin αvβ3 to Src is mediated by protein kinase C
(PKC) and focal adhesion kinase (FAK), and nectins and activated integrin αvβ3 cooperatively induce the activation of Src.
Activated Src then induces the activation of RAP1 through Crk and C3G (the guanine nucleotide-exchange factor (GEF) for
RAP1), and phosphorylates both FRG (the GEF for the small G protein CDC42) and VAV2 (the GEF for the small G protein
Rac) on Tyr. Activated RAP1 activates phosphorylated FRG, resulting in the activation of CDC42. Activated CDC42 also
enhances the activation of phosphorylated VAV2 and eventually induces the activation of Rac. These intracellular signalling
pathways are essential for the formation of AJs. b | After the formation of AJs, integrin αvβ3 is inactivated through the
nectin-induced activation of the protein Tyr phosphatase PTPµ and the consequent dephosphorylation and suppression of
PIPKIγ90. This is important for the stabilization of AJs, because the prolonged activation of integrin αvβ3 tends to disrupt
the formation of AJs. The nectin–afadin complex is thought to couple to the platelet-derived growth factor (PDGF)
receptor-mediated activation of the phosphoinositide 3-kinase (PI3K)–AKT signalling pathway, and this coupling might
have a role in preventing apoptosis and enhancing cell survival.
608 | AUGUST 2008 | volume 9
www.nature.com/reviews/molcellbio
© 2008 Macmillan Publishers Limited. All rights reserved.
REVIEWS
sufficient for the formation of AJs. The association of
these CAMs with the actin cytoskeleton is also required
for the clustering of CAMs, which eventually strengthens their adhesion activity. Both ‘outside-in’ signalling
(which is initiated by the trans-interactions of the extracellular regions of nectins or cadherins) and ‘inside-out’
signalling (which reinforces the adhesion activity of
these CAMs) facilitate the formation of AJs, as described
for the formation of cell–ECM junctions by the integrin
system70.
Nectin‑3–PDGF receptor binding and cell survival. Cells
continue to survive even after they become confluent
and establish cell–cell junctions. A number of reports
have demonstrated physical and functional associations between CAMs and growth factor receptors24–26.
Consistently, nectin‑3 associates with PDGF receptor
at cell–cell adhesion sites, and this association might
suppress apoptosis and contribute to PDGF-induced
cell survival71. Afadin also has an anti-apoptotic effect,
as shown in a study that used embryoid bodies that
had been derived from afadin–/– embryonic stem cells.
The phosphoinositide 3‑kinase (PI3K)–AKT signalling
pathway downstream of PDGF receptor seems to be
involved in the nectin‑3- and afadin-mediated prevention of apoptosis (FIG. 3b). Although AKT phosphorylates
many proteins that regulate apoptosis, such as B-cell
lymphoma protein-2 antagonist of cell death (BAD),
glycogen synthase kinase-3β (GSK3β) and inhibitor of
κB kinase (IKK), and exerts an anti-apoptotic effect72, it
is unclear how the nectin–afadin complex and PDGF
receptor use the signalling pathway downstream of
AKT for PDGF-induced cell survival. Further studies
are needed to address these issues and to certify the
importance of nectin and afadin for cell survival.
Peripheral ruffle
A membrane ruffle that
localizes at peripheral regions
of the cell, such as the leading
edge of moving cells.
Membrane ruffles are formed
by lamellipodia that have lifted
from the substratum along
which they previously
extended.
NECL-5 and cell movement
Prior to the formation of cell–cell contacts and junctions
in which nectins and afadin are primarily involved, cells
move in response to chemoattractants, such as PDGF73.
During cell movement, cells polarize and form the
leading edge to move in the direction of higher concentrations of the chemoattractants73. The morphology
of the leading edge is dynamically regulated by special
structures, including protrusions such as filopodia and
lamellipodia, peripheral ruffles, focal complexes and focal
adhesions74. Nectins are crucial for the formation of cell–
cell junctions, but they are not observed at the leading
edge. Instead, of the nectin and Necl family members,
NECL-5 is preferentially accumulated at the leading
edge75. NECL-5, in cooperation with PDGF receptor
and integrin αvβ3, has a pivotal role in the dynamics of
the leading edge76,77.
Ternary complex formation at the leading edge. It is
well known that growth factor receptors and integrins
synergistically participate in the regulation of various
intracellular signalling pathways24. It has been shown
that PDGF enhances cell migration when cells are
sparsely plated on a dish that is coated with vitronectin
(a ligand for integrin αvβ3), and that PDGF receptor is
co-immunoprecipitated with integrin αvβ3, indicating
the physical and functional association of PDGF receptor
with integrin αvβ3 (Ref. 78). Although the activation of
PDGF receptor and of integrin αvβ3 is important for cell
movement, NECL-5 has recently been proven to enhance
PDGF-receptor-induced and integrin-αvβ3-induced
signalling for cell movement, and to be essential for the
formation of leading-edge structures76,77,79 (FIG. 4).
NECL-5, PDGF receptor and integrin αvβ3 can form
any combination of the binary heterodimeric complex in
cis76,77. When a PDGF gradient induces directional cell
movement, NECL-5 preferentially regulates the interaction between PDGF receptor and integrin αvβ3 by
forming a ternary heterodimeric complex at the leading
edge. This ternary complex contributes to the long-term
activation of Rac locally at the leading edge, downstream
of PDGF receptor and integrin αvβ3. This results in the
persistent formation of lamellipodia and peripheral
membrane ruffles, which usually appear at the tip of
lamellipodia and are free from the extracellular matrix
(ECM), towards the higher concentration of PDGF76.
Although NECL-5, PDGF receptor and integrin
αvβ3 colocalize at peripheral ruffles at the leading edge
of moving cells, only the NECL-5–integrin αvβ3 complex localizes at focal complexes, and only integrin αvβ3
localizes at focal adhesions. The functional significance
of this differential localization is poorly understood, but
PDGF receptor is likely to be internalized and dissociated
from NECL-5 and integrin αvβ3 following the binding
of PDGF. This PDGF receptor internalization does not
affect the relocalization of the remaining NECL-5–
integrin αvβ3 complex, through which the attachment
of peripheral ruffles to the ECM occurs, resulting in the
formation of new focal complexes. NECL-5 is then dissociated from integrin αvβ3 during the transformation
of focal complexes to focal adhesions80. Focal adhesions
formed in this way are necessary to generate the sufficient driving force for cell movement — they support
the cell body. Thus, PDGF receptor and integrin αvβ3
are not the only proteins that have a crucial role in the
formation of leading-edge structures: NECL-5 also has
a role. These structures are involved in the extension of
cell protrusions and the generation of traction in the
direction of cell movement, and thus they eventually
facilitate directional cell movement.
Signalling at the leading edge. Leading-edge structures
are formed by the reorganization of the actin cyto­
skeleton, which is regulated by the actions of the Rho
family of small G proteins. Lamellipodia and ruffles are
formed by the action of Rac, filopodia are formed by the
action of CDC42, and focal complexes are formed by
the actions of Rac and CDC42 (Ref. 81). The formation
of these leading-edge structures, with the exception of
focal adhesions, is inhibited by the action of Rho. Focal
complexes are transformed into focal adhesions by the
inactivation of CDC42 and Rac and by the activation
of Rho82,83. Following the stimulation of NIH3T3 cells
by PDGF, RAP1 is locally activated at the leading edge by
the NECL-5–PDGF receptor–integrin αvβ3 complex79
(FIG. 4). This local activation of RAP1 is crucial for the
nature reviews | molecular cell biology
volume 9 | AUGUST 2008 | 609
© 2008 Macmillan Publishers Limited. All rights reserved.
REVIEWS
a
Cell movement
Nucleus
PDGF receptor
Actin
filaments
RAP1 GTP
PDGF
NECL-5
C3G
Integrin αvβ3
RAP1 GDP
Crk
Extracellular matrix
FA
b
Peripheral ruffle
Lamellipodium
SPA1
GTP
RAP1
VAV2
GTP
Rac
GTP
RAP1
Afadin
GDP
Rac
GTP
RAP1
ARAP1
GDP
RhoA
GTP
RhoA
FX
c
FA
Lamellipodium
GTP
RAP1
VAV2
GDP
Rac
FX
GTP
Rac
Early FA
ROCK
Internalized
PDGF
receptor
ARAP1
GTP
RhoA
GDP
RhoA
Late FA
Figure 4 | Leading-edge dynamics regulated by the NECL-5–integrin αvβ3–PDGF
receptor complex. a | After platelet-derived growth factor (PDGF) stimulation, RAP1 is
Nature Reviews | Molecular Cell Biology
locally activated at the leading edge by the nectin-like
protein-5 (NECL-5)–PDGF
receptor–integrin αvβ3 complex through Crk and C3G (the guanine nucleotide-exchange
factor (GEF) for RAP1). RAP1 that is activated in this way is involved in the development of
peripheral ruffles and the extension of the leading edge to the next stage. b | Activated
RAP1 binds to VAV2 (the GEF for Rac) and induces the activation of Rac, which promotes
the formation of lamellipodia, peripheral ruffles and focal complexes (FXs) at the leading
edge. Activated RAP1 also binds to afadin, which prevents the Rap GTPase-activating
protein (GAP) SPA1 from inactivating RAP1. Moreover, activated RAP1 induces the
inactivation of RhoA by binding to and activating the RhoGAP ARAP1. The RAP1-induced
activation of Rac is important for the development and maintenance of peripheral ruffles
and lamellipodia and drives cells to move forwards. However, the RAP1-induced
inactivation of RhoA through ARAP1 inhibits the transformation of FXs to focal adhesions
(FAs), preserving the flexibility of the leading edge for cell movement. c | When PDGF
receptor is downregulated from the cell surface by endocytosis, the activation of RAP1
stops and RAP1 is inactivated. The inactivation of RAP1 inactivates Rac by inhibiting VAV2
function, but also activates RhoA by blocking ARAP1 function. ARAP1 cannot bind to the
inactive form of RAP1, and free ARAP1 does not have RhoGAP activity. Activated RhoA
dissociates NECL-5 from FXs through Rho kinase (ROCK) and thereby enhances the
transformation of FXs to FAs. At this stage, peripheral-ruffle formation transiently pauses
because the RAP1–Rac pathway becomes inactivated. However, the RhoA–ROCK
pathway is activated and contributes to the transformation of FXs to FAs; these form the
firm adhesions between cells and the extracellular matrix and are necessary to generate
sufficient driving force for cell movement. The processes shown in a–c are repeated
during cell movement in response to PDGF.
formation of leading-edge structures that are associated
with the activation of Rac, the inactivation of RhoA and
the recruitment of afadin to the leading edge. Afadin that
is localized at the leading edge does not bind to nectins.
Afadin, in addition to the Rho family proteins and RAP1,
is essential for the formation of leading-edge structures
and directional cell movement.
Afadin regulates PDGF-induced intracellular signalling during the formation of the leading edge. Afadin is
phosphorylated at Tyr1237 by Src, which is activated by
the PDGF receptor, and then afadin binds to the Srchomology-2 (SH2) domain of the protein Tyr phosphatase
SHP2 (Ref. 84). The binding of afadin increases the phosphatase activity of SHP2 and prevents hyperactivation
of the Ras–ERK signalling pathway. Afadin and SHP2
are preferentially involved in forming the leading edge,
but are not involved in cell proliferation, at least during
the short period of PDGF stimulation. Afadin also acts
as a positive regulator of RAP1 activation, by blocking
the function of signal-induced proliferation associated
protein-1 (SPA1), a GTPase-activating protein (GAP)
for RAP1 (Y. Rikitake et al., unpublished observations).
PDGF sig­nalling is downregulated by the endocytosis
of PDGF receptor from the cell surface, and consequently
RAP1 becomes inactive. Inactivated RAP1 cannot
increase the activity of a RhoGAP, such as ARAP1, and
the activation of RhoA occurs at the leading edge. Rho
kinase, which is downstream of RhoA, is then activated
and thereby induces the dissociation of NECL-5 from
focal complexes, resulting in enhanced transformation
of focal complexes to focal adhesions80. After this series
of signalling events in response to PDGF is completed,
PDGF receptor is newly recruited to the leading edge and
again forms a complex with NECL-5 and integrin αvβ3,
starting a new cycle of activation and inactivation of small
G proteins. Thus, the NECL-5–PDGF receptor–integrin
αvβ3 complex mediates the cyclical activation and inactivation of these small G proteins, which are crucial for the
dynamic regulation of the leading-edge formation that is
necessary for directional cell movement.
Reorientation of the microtubule network is also
necessary for directional cell movement. In this process,
growing (pioneer) microtubules develop into leadingedge protrusions and search for membrane cues together
with plus-end-tracking proteins , such as dynein and
dynactin, which localize at the plus ends of growing
microtubules85. As NECL-5 binds to the dynein light
chain component TCTEX1 (Refs 27,86), NECL-5 is
a candidate membrane cue for the regulation of the
microtubule-network reorientation for directional cell
movement.
Nectins and Necls in contact inhibition
NECL-5 is a notable member of the Necl family because
it promotes cell movement and cell proliferation 18,75.
Importantly, the downregulation of NECL-5 from the
cell surface contributes to the induction of contact
inhibition. Nectins and NECL-5 concertedly modulate the sequential events of cell–cell contact, and thus
have roles in contact inhibition of cell movement and
proliferation.
610 | AUGUST 2008 | volume 9
www.nature.com/reviews/molcellbio
© 2008 Macmillan Publishers Limited. All rights reserved.
REVIEWS
SH2 domain
(Src-homology‑2 domain). A protein motif that recognizes
and binds sequences that have
been phosphorylated on Tyr
and thereby has a key role in
relaying cascades of signal
transduction.
Plus-end-tracking protein
A common designation for the
proteins that accumulate at the
plus end of microtubules.
Clathrin
The main component of the
surface of clathrin-coated
vesicles, which are involved in
membrane transport in the
endocytic pathway.
Cell–cell adhesion by NECL-5, nectins and afadin.
NECL-5 localizes at the leading edge of moving cells and
enhances cell movement75,76,79, whereas nectins induce
cell–cell adhesion1. During the early stage of cell–cell
adhesion formation, before nectin-mediated cell–
cell adhesion begins, cell–cell contact is initiated by the
heterophilic trans-interaction of NECL-5 at the leading
edge with nectin‑3 on the adjacent cell surface. This
occurs when individually moving cells collide with each
other10 (FIG. 5). In this process, afadin, which localizes
at the leading edge, can have a role in the recruitment
of nectin‑3 to the leading edge, because afadin binds
nectin‑3 (Ref. 1). Thus, NECL-5 interacts in trans with
nectin‑3 following the initial cell–cell contact.
However, the trans-interaction of NECL-5 with
nectin‑3 is transient, and NECL-5 is downregulated from
the cell surface by endocytosis in a clathrin-dependent
manner10. The decreased levels of NECL-5 lead to the disruption of the ternary complex, which contains NECL-5,
PDGF receptor and integrin αvβ3, and the reduction of
cell movement by inhibiting the signals that are initiated by PDGF receptor and integrin αvβ3, as described
above. It cannot be completely excluded that the transinteraction of NECL-5 with CAMs other than nectin‑3
might induce the downregulation of NECL-5 from the
cell surface. However, this possibility is unlikely because
knockdown of nectin‑3 does not cause this downregulation after the formation of cell–cell adhesion. Nectin‑3
that has dissociated from NECL-5 is retained on the cell
surface and subsequently interacts in trans with nectin‑1,
the most likely member of the nectin family to interact in
trans with nectin‑3 because the Kd value between nectin‑1
and nectin‑3 is the lowest of any of the combinations of
the nectin family members (see above)37. Similar to the
recruitment of nectin‑3 to the leading edge for its transinteraction with NECL-5, afadin also contributes to the
recruitment of nectin‑1 to the leading edge, causing
the trans-interaction between nectin‑1 and nectin‑3.
Once a nectin-based cell–cell adhesion is formed,
afadin further recruits α‑catenin by direct binding and/or
reorganization of the actin cytoskeleton. α‑Catenin can
be free in the cytosol or can be associated with cadherins
through β‑catenin. The direct and indirect association of
afadin with α‑catenin promotes nectin- and cadherinbased formation of AJs. Thus, afadin might determine
the site of nectin-based cell–cell adhesions after the initial
cell–cell contacts, although definitive evidence for this
role has not been obtained. Similar to afadin, α‑catenin
might also participate in the determination of the site
of cadherin-based cell–cell adhesion. As a consequence of
the functions of nectins, cadherins and their related
proteins, firm cell–cell junctions are established, limiting cell movement and proliferation for the long-term
maintenance of cell–cell junctions in normal cells.
Inactivation of integrin αvβ3 by nectins. After the establishment of AJs, integrin αvβ3 is inactivated but continues
to colocalize with nectins at AJs67,68. This in­activation
is beneficial for the maintenance of AJs, because the
sustained activation of integrin αvβ3 renders cells
highly motile, which tends to disrupt cell–cell junctions.
Integrin αvβ3 is activated by the binding of talin — which
directly connects the integrin to the actin cytoskeleton
— to the cytoplasmic tail of the β3 subunit, because this
interaction changes the intracellular conformation of
integrin αvβ3 to increase its affinity for its extracellular
ligands87.
The binding of talin to integrin αvβ3 is enhanced
by an increased amount of phosphatidylinositol-4,5bisphosphate (PtdIns(4,5)P2)88, which is generated by
phosphatidylinositol phosphate kinases (PIPKs) such as
PIPK type Iγ90 (PIPKIγ90). The activation of PIPKIγ90
is correlated with its phosphorylation state. The protein
Tyr phosphatase PTPµ effectively dephosphorylates
PIPKIγ90, and thus cancels the PIPKIγ90-dependent
activ­ation of integrin αvβ3 by blocking its interaction
with talin89. All members of the nectin family can potentially interact with PTPµ through their extracellular
regions, and the trans-interactions of nectins enhance its
phosphatase activity, leading to a decrease in the phosphorylation of PIPKIγ90 (FIG. 3b). In this way, nectins
function in the inactivation of integrin αvβ3 at AJs. As
well as the cell–cell contact-mediated downregulation
of NECL-5, this nectin-induced inactivation of integrin
αvβ3 provides an additional mechanism for contact
inhibition of cell movement.
Contact inhibition of cell proliferation and NECL‑5.
NECL-5 regulates cell proliferation by enhancing the
growth-factor-induced activation of the signalling pathway that includes Ras, Raf, MEK (mitogen-activated
protein kinase (MAPK)–extracellular signal-regulated
kinase (ERK) kinase) and ERK18. This shortens the period
of the G1 phase of the cell cycle owing to the modulation
of cell-cycle regulators. Sprouty is a negative regulator of
growth-factor-induced cell proliferation90,91, although it
was originally identified as an antagonist of the fibroblast
growth factor signalling that patterns apical branching
of the Drosophila melanogaster airways92. When sprouty
is phosphorylated on Tyr by Src in response to growth
factors, it inhibits the growth-factor-induced activation of
Ras signalling at a site upstream of Ras and downstream
of the growth factor receptors91. Binding of growth factors to their receptors induces the activation of both Ras
and Src, but Ras signalling is activated and sprouty is
in­activated during cell proliferation. NECL-5 interacts
with sprouty2 and prevents sprouty2 from being phosphorylated on Tyr by Src93 (FIG. 5). Thus, NECL-5 prolongs
the growth-factor-induced activation of cell proliferation
signalling through the inhibition of sprouty2, although
sprouty2 might not be the sole partner of NECL-5.
However, when NECL-5 is downregulated from the
cell surface by endocytosis (triggered by the trans-interaction of NECL-5 with nectin‑3) sprouty2 is released
from NECL-5 and is phosphorylated by Src, leading to
the inhibition of PDGF-induced activation of Ras. This
inhibition might further suppress de novo synthesis of
NECL-5. These signalling pathways seem to correlate
with the mechanisms that underlie contact inhibition
of cell proliferation, although the mechanisms of contact inhibition are complex and other CAMs, such as
E‑cadherin and CD44, might be involved94,95.
nature reviews | molecular cell biology
volume 9 | AUGUST 2008 | 611
© 2008 Macmillan Publishers Limited. All rights reserved.
REVIEWS
a Leading edge of moving cell
b Initial cell–cell contact
Nectin
Cell membrane
Cadherin
NECL-5
Rac
Src
Integrin
αvβ3
(active)
RAP1
Vitronectin
RhoA
(inactive)
Ras
PDGF
receptor
SPRY2
(inactive)
PDGF
Cell
movement
CDC42
Rac
Cell
proliferation
Reorganization
of the actin
cytoskeleton
CDC42
Rac
c Formation of nectin-based cell–cell adhesion
d AJ formation
Contact inhibition
Endocytosis
of NECL-5
CDC42
RAP1
(inactive)
Rac
Reorganization
of the actin
cytoskeleton
Integrin
αvβ3
(inactive)
PI3K
Rac
(inactive)
Cell movement
RhoA
AKT
Cell survival
Src
Ras
(inactive)
P
SPRY2
CDC42
Rac
Cell proliferation
Figure 5 | Contact inhibition of cell movement and proliferation by the downregulation of NECL-5 and the
inactivation of integrin αvβ3 during adherens-junction formation. a | Nectin-like
protein-5
integrin
αvβ3
Nature
Reviews(NECL-5),
| Molecular
Cell Biology
and platelet-derived growth factor (PDGF) receptor form a complex at the leading edge of a moving cell and enhance cell
movement by inducing Src and RAP1 activation, which leads to the activation of Rac together with the inhibition of RhoA.
The ternary complex also enhances cell proliferation by inducing the activation of the Ras-mediated signalling pathway,
through the inhibition of NECL-5-associated sprouty2 (SPRY2). The activation of integrin αvβ3 is induced by its binding to
an extracellular matrix protein, vitronectin. At this stage, nectins and cadherins are sparsely distributed on the cell surface.
b | The initial cell–cell contact is formed by the trans-interaction of nectin‑3 with NECL-5. At this stage, the reorganization
of the actin cytoskeleton starts with the activation of CDC42 and Rac. c | The trans-interaction of nectin‑3 with NECL-5 is
transient, and NECL-5 is subsequently downregulated from the cell surface by endocytosis. Next, trans-interaction of the
nectins occurs. d | Cadherins are recruited to nectin-based cell–cell adhesion sites and form homophilic interactions in
trans to create adherens junctions (AJs). At this stage, integrin αvβ3 is inactivated by the action of trans-interacting
nectins. Owing to the downregulation of NECL-5 from the cell surface, SPRY2 is released from NECL-5, is phosphorylated
on Tyr by Src and becomes active to inhibit the Ras-mediated cell proliferation signals. The intracellular signalling that is
mediated by integrin αvβ3 and PDGF receptor is then suppressed, resulting in the inhibition of cell movement and
proliferation (contact inhibition). Even after the establishment of AJs, cells continue to survive. The activation of cellsurvival signalling molecules, including phosphoinositide 3-kinase (PI3K) and AKT, is regulated by PDGF receptor that is
associated with the nectin–afadin complex.
Loss of contact inhibition in metastasis. Transformed
cells lose contact inhibition of cell movement and proliferation, resulting in abnormal cell proliferation, invasion
and metastasis96,97. NECL-5 is known to be upregulated in
transformed cells14,37,98,99; it is also upregulated in NIH3T3
cells that overexpress oncogenic Ki-Ras (V12Ras) through
the V12Ras–Raf–MEK–ERK–activator protein-1 (AP1)
pathway17. The expression of NECL-5 also increases
during rat liver regeneration16. The overproduction
of NECL-5 in V12Ras-NIH3T3 cells exceeds the rate of
NECL-5 internalization following cell–cell adhesion,
resulting in the loss of contact inhibition in these cells100.
Consistent with this, an in vivo study showed that
V12Ras-NIH3T3 cells gain metastatic ability owing to
612 | AUGUST 2008 | volume 9
www.nature.com/reviews/molcellbio
© 2008 Macmillan Publishers Limited. All rights reserved.
REVIEWS
the upregulation of NECL-5 (Ref. 75). Conversely, the
development of colitis-associated cancer that is induced
by colonic mucosal cell proliferation is inhibited by the
ablation of NECL-5 in mice (T. Fujimori, personal communication). Taken together, these findings suggest that
the upregulation of NECL-5 following transformation
contributes to the loss of contact inhibition in transformed cells by markedly enhancing cell movement and
proliferation.
Transformed cells that abundantly express NECL-5
form numerous metastatic tumour nodules in the lung75.
Overexpression of NECL-5 is also correlated with the
molecular mechanism that underlies the metastasis of
cancer cells101. Upregulated NECL-5 in cancer cells transinteracts with CD226, a counter-receptor of NECL-5 in
platelets, and promotes the attachment of platelets to
cancer cells, resulting in the formation of large aggregates
that contain cancer cells and platelets in blood vessels.
These aggregates cannot pass through microvessels or
capillaries in the peripheral organs, including the lungs.
Cancer cells that are trapped in capillaries in this way consequently extravasate in order to proliferate ecto­pically. By
contrast, the inhibition of the trans-interaction of NECL-5
with CD226 reduces metastasis. These findings provide
a potential new therapeutic option for the prevention of
metastasis.
Conclusions and perspectives
We have described here how nectins have key roles in
the initiation of cell–cell adhesion, whereas NECL-5 is
crucial for cell movement and proliferation. Although
several CAMs are reported to be involved in the contact
inhibition of cell movement and proliferation94,95, nectins
and NECL-5 also have important functions in contact
1.
2.
3.
4.
5.
6.
7.
8.
Takai, Y. & Nakanishi, H. Nectin and afadin: novel
organizers of intercellular junctions. J. Cell Sci. 116,
17–27 (2003).
Takai, Y., Irie, K., Shimizu, K., Sakisaka, T. & Ikeda, W.
Nectins and nectin-like molecules: roles in cell
adhesion, migration, and polarization. Cancer Sci. 94,
655–667 (2003).
Geraghty, R. J., Krummenacher, C., Cohen, G. H.,
Eisenberg, R. J. & Spear, P. G. Entry of αherpesviruses
mediated by poliovirus receptor-related protein 1 and
poliovirus receptor. Science 280, 1618–1620 (1998).
Warner, M. S. et al. A cell surface protein with
herpesvirus entry activity (HveB) confers susceptibility
to infection by mutants of herpes simplex virus type 1,
herpes simplex virus type 2, and pseudorabies virus.
Virology 246, 179–189 (1998).
Takahashi, K. et al. Nectin/PRR: an immunoglobulinlike cell adhesion molecule recruited to cadherinbased adherens junctions through interaction with
afadin, a PDZ domain-containing protein. J. Cell Biol.
145, 539–549 (1999).
Identifies nectin as a CAM that directly binds to
afadin with Ca2+-independent cell adhesion activity
and that colocalizes with cadherin at AJs.
Kakunaga, S. et al. Nectin-like molecule‑1/TSLL1/
SynCAM3: a neural tissue-specific immunoglobulinlike cell–cell adhesion molecule localizing at nonjunctional contact sites of presynaptic nerve terminals,
axons, and glia cell processes. J. Cell Sci. 118, 1267–
1277 (2005).
Maurel, P. et al. Nectin-like proteins mediate axon
Schwann cell interactions along the internode and are
essential for myelination. J. Cell Biol. 178, 861–874
(2007).
Spiegel, I. et al. A central role for Necl4 (SynCAM4) in
Schwann cell–axon interaction and myelination.
Nature Neurosci. 10, 861–869 (2007).
9.
10.
11.
12.
13.
14.
15.
inhibition, at least in model cell lines such as MDCK and
NIH3T3 cells, which are epithelial cells and fibroblasts,
respectively.
In embryonic development, individually moving and
proliferating mesenchymal cells first form primordial
cell–cell contacts through collision of cells. They are transformed into epithelial cells and form specialized cell–cell
junction complexes, such as AJs and tight junctions. This
phenomenon is called mesenchymal–epithelial transition
(MET). By contrast, in embryonic development and cancer progression, epithelial cells lose their connection to
neighbouring cells and become free, which increases cell
migration and proliferation. This opposite phenomenon
is correlated with epithelial–mesenchymal transition
(EMT), which is characterized by a change to a fibro­
blastoid spindle-shaped cell morphology, loss of epithelial
marks (including E‑cadherin), induction of mesenchymal
markers and acquisition of a motility machinery102. The
dynamic regulation of MET and EMT is crucial for multicellular organisms to develop and survive. Overexpression
of NECL-5 and integrin αvβ3 in cell-culture studies might
explain, at least partly, the mechanism of EMT, because
their overexpression causes the disruption of cell–cell
junctions and loss of contact inhibition, which can induce
the downregulation of E‑cadherin and cell morphological
change. Nectins and Necls are involved in the initial
processes of the formation of cell–cell junctions, but the
question of how tight junctions are formed at the apical
side of AJs during MET. This issue needs to be clarified
in order to understand the mechanism of MET remains
unanswered. The relationship between the in vitro results
that have been obtained from cell-culture studies and the
in vivo processes of EMT and MET will undoubtedly be
the subject of future studies.
Kuramochi, M. et al. TSLC1 is a tumor-suppressor
gene in human non‑small‑cell lung cancer. Nature
Genet. 27, 427–430 (2001).
Fujito, T. et al. Inhibition of cell movement and
proliferation by cell–cell contact-induced interaction of
Necl‑5 with nectin‑3. J. Cell Biol. 171, 165–173
(2005).
Proves that the downregulation of NECL-5, which is
initiated by its trans-interaction with nectin‑3
following cell–cell contact is one of the mechanisms
that underlie contact inhibition of cell movement
and proliferation.
Mendelsohn, C. L., Wimmer, E. & Racaniello, V. R.
Cellular receptor for poliovirus: molecular cloning,
nucleotide sequence, and expression of a new member
of the immunoglobulin superfamily. Cell 56, 855–865
(1989).
Shows the cDNA sequence and expression pattern
of PVR in humans and analyses its molecular
characteristics to examine the mode of poliovirus
attachment and replication.
Koike, S. et al. The poliovirus receptor protein is
produced both as membrane-bound and secreted
forms. EMBO J. 9, 3217–3224 (1990).
Chadeneau, C., LeCabellec, M., LeMoullac, B., Meflah, K.
& Denis, M. G. Over-expression of a novel member of
the immunoglobulin superfamily in Min mouse intestinal
adenomas. Int. J. Cancer 68, 817–821 (1996).
Chadeneau, C., LeMoullac, B. & Denis, M. G. A novel
member of the immunoglobulin gene superfamily
expressed in rat carcinoma cell lines. J. Biol. Chem.
269, 15601–15605 (1994).
Lim, Y. P., Fowler, L. C., Hixson, D. C., Wehbe, T. &
Thompson, N. L. TuAg.1 is the liver isoform of the rat
colon tumor-associated antigen pE4 and a member of
the immunoglobulin-like supergene family. Cancer Res.
56, 3934–3940 (1996).
16. Erickson, B. M., Thompson, N. L. & Hixson, D. C.
Tightly regulated induction of the adhesion molecule
necl‑5/CD155 during rat liver regeneration and
acute liver injury. Hepatology 43, 325–334
(2006).
17. Hirota, T., Irie, K., Okamoto, R., Ikeda, W. & Takai, Y.
Transcriptional activation of the mouse Necl‑5/Tage4/
PVR/CD155 gene by fibroblast growth factor or
oncogenic Ras through the Raf–MEK–ERK–AP‑1
pathway. Oncogene 24, 2229–2235 (2005).
18. Kakunaga, S. et al. Enhancement of serum- and
platelet-derived growth factor-induced cell
proliferation by Necl‑5/Tage4/poliovirus receptor/
CD155 through the Ras–Raf–MEK–ERK signaling.
J. Biol. Chem. 279, 36419–36425 (2004).
19. Fisher, H. W. & Yeh, J. Contact inhibition in colony
formation. Science 155, 581–582 (1967).
20. Bell, P. B. Jr. Contact inhibition of movements in
transformed and nontransformed cells. Birth Defects
Orig. Artic. Ser. 14, 177–194 (1978).
21. Yagi, T. & Takeichi, M. Cadherin superfamily
genes: functions, genomic organization, and
neurologic diversity. Genes Dev. 14, 1169–1180
(2000).
22. Takeichi, M. The cadherin superfamily in neuronal
connections and interactions. Nature Rev. Neurosci. 8,
11–20 (2007).
23. van der Flier, A. & Sonnenberg, A. Function and
interactions of integrins. Cell Tissue Res. 305,
285–298 (2001).
24. Comoglio, P. M., Boccaccio, C. & Trusolino, L.
Interactions between growth factor receptors and
adhesion molecules: breaking the rules. Curr. Opin.
Cell Biol. 15, 565–571 (2003).
25. Perez-Moreno, M., Jamora, C. & Fuchs, E. Sticky
business: orchestrating cellular signals at adherens
junctions. Cell 112, 535–548 (2003).
nature reviews | molecular cell biology
volume 9 | AUGUST 2008 | 613
© 2008 Macmillan Publishers Limited. All rights reserved.
REVIEWS
26. Yap, A. S. & Kovacs, E. M. Direct cadherin-activated
cell signaling: a view from the plasma membrane.
J. Cell Biol. 160, 11–16 (2003).
27. Mueller, S., Cao, X., Welker, R. & Wimmer, E.
Interaction of the poliovirus receptor CD155 with the
dynein light chain Tctex‑1 and its implication for
poliovirus pathogenesis. J. Biol. Chem. 277,
7897–7904 (2002).
28. Takekuni, K. et al. Direct binding of cell polarity
protein PAR‑3 to cell–cell adhesion molecule nectin at
neuroepithelial cells of developing mouse. J. Biol.
Chem. 278, 5497–5500 (2003).
29. Yageta, M. et al. Direct association of TSLC1 and
DAL‑1, two distinct tumor suppressor proteins in lung
cancer. Cancer Res. 62, 5129–5133 (2002).
30. Shingai, T. et al. Implications of nectin-like molecule‑2/
IGSF4/RA175/SgIGSF/TSLC1/SynCAM1 in cell–cell
adhesion and transmembrane protein localization in
epithelial cells. J. Biol. Chem. 278, 35421–35427
(2003).
31. Mandai, K. et al. Afadin: a novel actin filament-binding
protein with one PDZ domain localized at cadherinbased cell‑to‑cell adherens junction. J. Cell Biol. 139,
517–528 (1997).
Shows that afadin purified from rat brain has
F‑actin‑binding properties and specifically localizes
at cadherin-based AJs, indicating a role for afadin
in linking AJ structures to the actin cytoskeleton.
32. Prasad, R. et al. Cloning of the ALL‑1 fusion partner,
the AF‑6 gene, involved in acute myeloid leukemias
with the t(6;11) chromosome translocation. Cancer
Res. 53, 5624–5628 (1993).
33. Saito, S. et al. Complete genomic structure DNA
polymorphisms, and alternative splicing of the human
AF‑6 gene. DNA Res. 5, 115–120 (1998).
34. Satoh-Horikawa, K. et al. Nectin‑3, a new member of
immunoglobulin-like cell adhesion molecules that
shows homophilic and heterophilic cell–cell adhesion
activities. J. Biol. Chem. 275, 10291–10299 (2000).
35. Reymond, N. et al. Nectin4/PRR4, a new afadinassociated member of the nectin family that transinteracts with nectin1/PRR1 through V domain
interaction. J. Biol. Chem. 276, 43205–43215
(2001).
36. Aoki, J. et al. Mouse homolog of poliovirus receptorrelated gene 2 product, mPRR2, mediates homophilic
cell aggregation. Exp. Cell Res. 235, 374–384 (1997).
37. Ikeda, W. et al. Tage4/nectin-like molecule‑5
heterophilically trans-interacts with cell adhesion
molecule nectin‑3 and enhances cell migration. J. Biol.
Chem. 278, 28167–28172 (2003).
38. Bottino, C. et al. Identification of PVR (CD155) and
Nectin‑2 (CD112) as cell surface ligands for the human
DNAM‑1 (CD226) activating molecule. J. Exp. Med.
198, 557–567 (2003).
39. Fuchs, A., Cella, M., Giurisato, E., Shaw, A. S. &
Colonna, M. Cutting edge: CD96 (tactile) promotes NK
cell–target cell adhesion by interacting with the
poliovirus receptor (CD155). J. Immunol. 172,
3994–3998 (2004).
40. Boles, K. S., Barchet, W., Diacovo, T., Cella, M. &
Colonna, M. The tumor suppressor TSLC1/NECL‑2
triggers NK‑cell and CD8+ T‑cell responses through the
cell-surface receptor CRTAM. Blood 106, 779–786
(2005).
41. Koch, A. W., Pokutta, S., Lustig, A. & Engel, J. Calcium
binding and homoassociation of E‑cadherin domains.
Biochemistry 36, 7697–7705 (1997).
42. Honda, T. et al. Antagonistic and agonistic effects of
an extracellular fragment of nectin on formation of
E‑cadherin‑based cell–cell adhesion. Genes Cells 8,
51–63 (2003).
43. Anastasiadis, P. Z. & Reynolds, A. B. The p120 catenin
family: complex roles in adhesion, signaling and
cancer. J. Cell Sci. 113, 1319–1334 (2000).
44. Mandai, K. et al. Ponsin/SH3P12: an l‑afadin‑ and
vinculin-binding protein localized at cell–cell and cell–
matrix adherens junctions. J. Cell Biol. 144,
1001–1017 (1999).
45. Tachibana, K. et al. Two cell adhesion molecules,
nectin and cadherin, interact through their
cytoplasmic domain-associated proteins. J. Cell Biol.
150, 1161–1176 (2000).
46. Pokutta, S., Drees, F., Takai, Y., Nelson, W. J. & Weis,
W. I. Biochemical and structural definition of the
l‑afadin‑ and actin-binding sites of α-catenin. J. Biol.
Chem. 277, 18868–18874 (2002).
47. Asada, M. et al. ADIP, a novel afadin- and
α‑actinin‑binding protein localized at cell–cell
adherens junctions. J. Biol. Chem. 278, 4103–4111
(2003).
48. Ooshio, T. et al. Involvement of LMO7 in the association
of two cell–cell adhesion molecules, nectin and
E‑cadherin, through afadin and α-actinin in epithelial
cells. J. Biol. Chem. 279, 31365–31373 (2004).
49. Ikeda, W. et al. Afadin: a key molecule essential for
structural organization of cell–cell junctions of
polarized epithelia during embryogenesis. J. Cell Biol.
146, 1117–1132 (1999).
50. Ooshio, T. et al. Cooperative roles of Par‑3 and afadin
in the formation of adherens and tight junctions.
J. Cell Sci. 120, 2352–2365 (2007).
51. Yamada, A. et al. Requirement of nectin, but not
cadherin, for formation of claudin-based tight
junctions in annexin II‑knockdown MDCK cells.
Oncogene 25, 5085–5102 (2006).
52. Drees, F., Pokutta, S., Yamada, S., Nelson, W. J. &
Weis, W. I. α-Catenin is a molecular switch that binds
E‑cadherin–β-catenin and regulates actin-filament
assembly. Cell 123, 903–915 (2005).
53. Yamada, S., Pokutta, S., Drees, F., Weis, W. I. &
Nelson, W. J. Deconstructing the cadherin–catenin–
actin complex. Cell 123, 889–901 (2005).
54. Weis, W. I. & Nelson, W. J. Re-solving the
cadherin–catenin–actin conundrum. J. Biol. Chem.
281, 35593–35597 (2006).
55. Nagafuchi, A., Ishihara, S. & Tsukita, S. The roles of
catenins in the cadherin-mediated cell adhesion:
functional analysis of E‑cadherin–α catenin fusion
molecules. J. Cell Biol. 127, 235–245 (1994).
56. Imamura, Y., Itoh, M., Maeno, Y., Tsukita, S. &
Nagafuchi, A. Functional domains of α-catenin
required for the strong state of cadherin-based cell
adhesion. J. Cell Biol. 144, 1311–1322 (1999).
57. Fukuhara, T. et al. Activation of Cdc42 by trans
interactions of the cell adhesion molecules nectins
through c‑Src and Cdc42–GEF FRG. J. Cell Biol. 166,
393–405 (2004).
58. Fukuyama, T. et al. Involvement of the c‑Src–Crk–
C3G–Rap1 signaling in the nectin-induced activation
of Cdc42 and formation of adherens junctions.
J. Biol. Chem. 280, 815–825 (2005).
59. Kawakatsu, T. et al. Vav2 as a Rac-GEF responsible for
the nectin-induced, c‑Src‑ and Cdc42-mediated
activation of Rac. J. Biol. Chem. 280, 4940–4947
(2005).
60. Yonemura, S., Itoh, M., Nagafuchi, A. & Tsukita, S.
Cell‑to‑cell adherens junction formation and actin
filament organization: similarities and differences
between non-polarized fibroblasts and polarized
epithelial cells. J. Cell Sci. 108, 127–142 (1995).
61. Ehrlich, J. S., Hansen, M. D. H. & Nelson, W. J.
Spatio-temporal regulation of Rac1 localization and
lamellipodia dynamics during epithelial cell–cell
adhesion. Dev. Cell 3, 259–270 (2002).
62. Vasioukhin, V., Bauer, C., Yin, M. & Fuchs, E. Directed
actin polymerization is the driving force for epithelial
cell–cell adhesion. Cell 100, 209–219 (2000).
63. Pignatelli, M. Integrins, cadherins, and catenins:
molecular cross-talk in cancer cells. J. Pathol. 186,
1–2 (1998).
64. Siu, M. K. & Cheng, C. Y. Dynamic cross-talk between
cells and the extracellular matrix in the testis.
Bioessays 26, 978–992 (2004).
65. Monier-Gavelle, F. & Duband, J. L. Cross talk between
adhesion molecules: control of N‑cadherin activity by
intracellular signals elicited by β1 and β3 integrins in
migrating neural crest cells. J. Cell Biol. 137,
1663–1681 (1997).
66. Schreider, C., Peignon, G., Thenet, S., Chambaz, J. &
Pincon-Raymond, M. Integrin-mediated functional
polarization of Caco‑2 cells through E‑cadherin–actin
complexes. J. Cell Sci. 115, 543–552 (2002).
67. Sakamoto, Y. et al. Interaction of integrin αvβ3 with
nectin: implication in cross-talk between cell–matrix
and cell–cell junctions. J. Biol. Chem. 281,
19631–19644 (2006).
Shows the importance of crosstalk between the
cell–cell adhesion molecule nectin and the cell–
matrix adhesion molecule integrin αvβ3 for the
formation of AJs.
68. Ozaki, M., Ogita, H. & Takai, Y. Involvement of
integrin-induced activation of protein kinase C in the
formation of adherens junctions. Genes Cells 12,
651–662 (2007).
69. Fukuyama, T., Ogita, H., Kawakatsu, T., Inagaki, M. &
Takai, Y. Activation of Rac by cadherin through the
c‑Src–Rap1–phosphatidylinositol 3‑kinase–Vav2
pathway. Oncogene 25, 8–19 (2006).
70. Hood, J. D. & Cheresh, D. A. Role of integrins in cell
invasion and migration. Nature Rev. Cancer 2,
91–100 (2002).
71. Kanzaki, K. et al. Involvement of the nectin–afadin
complex in platelet-derived growth factor-induced cell
survival. J. Cell Sci. 121, 2008–2017 (2008).
72. Downward, J. PI 3‑kinase, Akt and cell survival.
Semin. Cell Dev. Biol. 15, 177–182 (2004).
73. Ronnstrand, L. & Heldin, C. H. Mechanisms of
platelet-derived growth factor-induced chemotaxis.
Int. J. Cancer 91, 757–762 (2001).
74. Zaidel-Bar, R., Cohen, M., Addadi, L. & Geiger, B.
Hierarchical assembly of cell-matrix adhesion
complexes. Biochem. Soc. Trans. 32, 416–420
(2004).
75. Ikeda, W. et al. Nectin-like molecule‑5/Tage4 enhances
cell migration in an integrin-dependent,
nectin‑3‑independent manner. J. Biol. Chem. 279,
18015–18025 (2004).
76. Minami, Y. et al. Necl‑5/poliovirus receptor interacts in
cis with integrin αvβ3 and regulates its clustering and
focal complex formation. J. Biol. Chem. 282,
18481–18496 (2007).
77. Amano, H. et al. Interaction and localization of
Necl‑5 and PDGF receptor β at the leading edges of
moving NIH3T3 cells: implications for directional
cell movement. Genes Cells 13, 269–284
(2008).
78. Woodard, A. S. et al. The synergistic activity of αvβ3
integrin and PDGF receptor increases cell migration.
J. Cell Sci. 111, 469–478 (1998).
Clearly shows that integrin αvβ3 and the PDGF
receptor associate with each other and
cooperatively enhance cell migration, indicating
that there is crosstalk between CAMs and growth
factor receptors for cell migration.
79. Takahashi, M. et al. Sequential activation of Rap1 and
Rac1 small G proteins by PDGF locally at leading
edges of NIH3T3 cells. Genes Cells 13, 549–569
(2008).
80. Nagamatsu, Y. et al. Roles of Necl‑5/Poliovirus
receptor and ROCK in the regulation of transformation
of integrin αvβ3-based focal complexes into focal
adhesions. J. Biol. Chem. 283, 14532–14541
(2008).
81. Hall, A. Rho GTPases and the actin cytoskeleton.
Science 279, 509–514 (1998).
82. Rottner, K., Hall, A. & Small, J. V. Interplay between
Rac and Rho in the control of substrate contact
dynamics. Curr. Biol. 9, 640–648 (1999).
83. Ballestrem, C., Hinz, B., Imhof, B. A. & Wehrle-Haller, B.
Marching at the front and dragging behind: differential
αvβ3-integrin turnover regulates focal adhesion
behavior. J. Cell Biol. 155,
1319–1332 (2001).
84. Nakata, S. et al. Regulation of PDGF receptor
activation by afadin through SHP‑2: implications for
cellular morphology. J. Biol. Chem. 282,
37815–37825 (2007).
85. Mimori-Kiyosue, Y. & Tsukita, S. ‘Search‑and‑capture’
of microtubules through plus‑end‑binding proteins
(+TIPs). J. Biochem. 134, 321–326 (2003).
86. Ohka, S. et al. Receptor (CD155)-dependent
endocytosis of poliovirus and retrograde axonal
transport of the endosome. J. Virol. 78, 7186–7198
(2004).
87. Calderwood, D. A. Integrin activation. J. Cell Sci. 117,
657–666 (2004).
88. Martel, V. et al. Conformation, localization, and
integrin binding of talin depend on its interaction with
phosphoinositides. J. Biol. Chem. 276, 21217–21227
(2001).
89. Sakamoto, Y., Ogita, H., Komura, H. & Takai, Y.
Involvement of nectin in inactivation of integrin αvβ3
after the establishment of cell–cell adhesion. J. Biol.
Chem. 283, 496–505 (2008).
90. Christofori, G. Split personalities: the agonistic
antagonist Sprouty. Nature Cell Biol. 5, 377–379
(2003).
91. Kim, H. J. & Bar-Sagi, D. Modulation of signalling by
Sprouty: a developing story. Nature Rev. Mol. Cell
Biol. 5, 441–450 (2004).
92. Hacohen, N., Kramer, S., Sutherland, D., Hiromi, Y. &
Krasnow, M. A. sprouty encodes a novel antagonist of
FGF signaling that patterns apical branching of the
Drosophila airways. Cell 92, 253–263 (1998).
Identifies sprouty as a novel negative regulator in
the FGF-induced signalling pathway that controls
the branching of the airways.
93. Kajita, M., Ikeda, W., Tamaru, Y. & Takai, Y.
Regulation of platelet-derived growth factor-induced
Ras signaling by poliovirus receptor Necl‑5 and
negative growth regulator Sprouty2. Genes Cells 12,
345–357 (2007).
614 | AUGUST 2008 | volume 9
www.nature.com/reviews/molcellbio
© 2008 Macmillan Publishers Limited. All rights reserved.
REVIEWS
94. Perrais, M., Chen, X., Perez-Moreno, M. & Gumbiner,
B. M. E‑cadherin homophilic ligation inhibits cell
growth and epidermal growth factor receptor signaling
independently of other cell interactions. Mol. Biol. Cell
18, 2013–2025 (2007).
95. Morrison, H. et al. The NF2 tumor suppressor gene
product, merlin, mediates contact inhibition of growth
through interactions with CD44. Genes Dev. 15,
968–980 (2001).
96. Abercrombie, M. Contact inhibition and malignancy.
Nature 281, 259–262 (1979).
97. Thiery, J. P. Epithelial–mesenchymal transitions in
tumour progression. Nature Rev. Cancer 2, 442–454
(2002).
98. Masson, D. et al. Overexpression of the CD155 gene
in human colorectal carcinoma. Gut 49, 236–240
(2001).
99. Gromeier, M., Lachmann, S., Rosenfeld, M. R.,
Gutin, P. H. & Wimmer, E. Intergeneric poliovirus
recombinants for the treatment of malignant glioma.
Proc. Natl Acad. Sci. USA 97, 6803–6808 (2000).
100.Minami, Y. et al. Involvement of up-regulated Necl‑5/
Tage4/PVR/CD155 in the loss of contact inhibition in
transformed NIH3T3 cells. Biochem. Biophys. Res.
Commun. 352, 856–860 (2007).
101. Morimoto, K. et al. Interaction of cancer cells with
platelets mediated by Necl‑5/poliovirus receptor
enhances cancer cell metastasis to the lungs.
Oncogene 27, 264–273 (2008).
102.Hay, E. D. An overview of epithelio–mesenchymal
transformation. Acta Anat. 154, 8–20 (1995).
103.Abercrombie, M. & Heaysman, J. E. Observations on
the social behaviour of cells in tissue culture. I. Speed
of movement of chick heart fibroblasts in relation to
their mutual contacts. Exp. Cell Res. 5, 111–131
(1953).
First proposes and then addresses the hypothesis
that the velocity of cell movement is affected by the
cell’s contacts with other cells.
104.Abercrombie, M. & Heaysman, J. E. Observations on
the social behaviour of cells in tissue culture. II.
Monolayering of fibroblasts. Exp. Cell Res. 6,
293–306 (1954).
105.Abercrombie, M. Contact inhibition in tissue culture.
In Vitro 6, 128–142 (1970).
106.Abercrombie, M. & Ambrose, E. J. The surface
properties of cancer cells: a review. Cancer Res. 22,
525–548 (1962).
107. Zegers, M. M. et al. Pak1 and PIX regulate contact
inhibition during epithelial wound healing. EMBO J.
22, 4155–4165 (2003).
108.Huttenlocher, A. et al. Integrin and cadherin
synergy regulates contact inhibition of migration
and motile activity. J. Cell Biol. 141, 515–526
(1998).
109.Dunn, G. A. & Ireland, G. W. New evidence that growth
in 3T3 cell cultures is a diffusion-limited process.
Nature 312, 63–65 (1984).
110. Martz, E. & Steinberg, M. S. The role of cell–cell
contact in ‘contact’ inhibition of cell division: a review
and new evidence. J. Cell Physiol. 79, 189–210
(1972).
111. Stoker, M. G. & Rubin, H. Density dependent
inhibition of cell growth in culture. Nature 215,
171–172 (1967).
Acknowledgements
The work presented in this review began at ERATO (Exploratory
Research for Advanced Technology of Japan; 1994–1999) and
was subsequently performed at the Department of Molecular
Biology and Biochemistry, Osaka University Graduate School
of Medicine and Faculty of Medicine, Suita, Japan, with the
support of grants-in-aid for Scientific Research and for Cancer
Research from the Ministry of Education, Culture, Sports,
Science and Technology, Japan (2000–2008). Many faculty
members, including H. Nakanishi, K. Mandai, T. Matozaki,
K. Shimizu, K. Irie, T. Sakisaka and N. Fujita, and many graduate students, postdoctoral fellows and collaborators have made
great contributions to this work. We thank all of them for their
excellent achievements.
DATABASES
UniProtKB: http://ca.expasy.org/sprot
ADIP | afadin | CD226 | CD96 | CDC42 | CRTAM | E-cadherin |
IQGAP1 | LMO7 | MAGUK | N-cadherin | NECL-1 | NECL-2 |
NECL-3 | NECL-4 | NECL-5 | nectin‑1 | nectin‑2 | nectin‑3 |
nectin‑4 | PAR3 | RAP1 | SHP2 | TCTEX1 | VE-cadherin
FURTHER INFORMATION
Yoshimi Takai’s homepage: http://www.med.kobe-u.ac.jp/
gs/field/basic/mol_cell.html (in Japanese)
All links are active in the online pdf
nature reviews | molecular cell biology
volume 9 | AUGUST 2008 | 615
© 2008 Macmillan Publishers Limited. All rights reserved.