Nectins and nectin-like molecules (Necls) are immu-noglobulin (Ig)-like cell adhesion molecules (CAMs) that have recently been shown to be essential contribu-tors 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 mem-bers 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), respectively3,4. 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 move-ment 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 under-lying 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

*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: ytakai@med.kobe‑u.ac.jpdoi:10.1038/nrm2457

Adherens junctionThis 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.

Nectins and nectin-like molecules: roles in contact inhibition of cell movement and proliferationYoshimi 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.

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Tight junctionThe 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 complexA 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 adhesionA mature cell–extracellular matrix junction that associates with integrin signalling factors, filamentous-actin-binding proteins and actin stress fibres.

factor receptors. Cadherins are single-membrane-spanning 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, respec-tively. Integrins are the CAMs that are involved in cell–extracellular matrix (ECM) junctions, and are com-posed 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 move-ment 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 NeclsThe 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 membrane-associated 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 medi-ated by the conserved C-terminal motif of nectins and the PDZ domain of afadin1. Afadin was originally identi-fied 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 con-tacts by forming homophilic or heterophilic trans-dimers. Heterophilic interactions have been detected between nectin-1 and nectin-3, between nectin-2 and nectin-3,

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 mossy-fibre 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.

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Nature Reviews | Molecular Cell Biology

Loss of contact inhibition

Contact inhibition

Transformed cells

Normal cells

Cell movement,cell proliferation

Cell movement,cell proliferation

Disruptedcell–cell adhesion

Cell–cell adhesion

PDZ domainA protein–protein interaction domain that was first found in postsynaptic density protein-95 (PsD95), Discs-large (DLG) and zona occludens-1 (ZO1).

and between nectin-1 and nectin-4 (fIG. 1c). It is note-worthy 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, includ-ing CD226 (also known as DNAX accessory molecule-1; DNAM1), CD96 (also known as Tactile) and class-I-MHC-restricted 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 cell-mediated lysis of target cells38. CD96 that is expressed on

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.

Role of nectins in cell–cell adhesionTwo types of CAMs — nectins and cadherins — localize at AJs and have essential cooperative roles in the forma-tion 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 sys-tem. However, whether nectins and Necls have cooperative roles in cell–cell adhesions remains unknown.

Formation of AJs by nectins and cadherins. Nectins local-ize 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 adhe-sions, 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 activ-ity 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 lat-eral 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 implica-tion of this differential distribution of these molecules in epithelial cells remains elusive, but all of these molecules colocalize at AJs in fibroblasts.

Box 1 | Contact inhibition of cell movement and proliferation

The concepts of ‘contact inhibition of cell movement’ (REf. 20) and ‘contact inhibition 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.

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Plasmamembrane

Nature Reviews | Molecular Cell Biology

TMNecls

TMNectins S–S

S–S S–S S–S

S–S S–SE/A-X-Y-V

AfadinPR1 PR2 PR3RA1 FHA DIL PDZRA2

F-actin

a

Monomer cis-Dimer

cis-Dimerization

trans-Interaction

FirstSecondThird

Ig-likeloop

trans-Dimer

NectinorNecl

b

c

Necl-5CD96/Tactile

?

CD226/DNAM1?

Nectin-4

Nectin-3Nectin-1 Nectin-2Necl-4

Necl-2Necl-1 CRTAM

?

Necl-3

?

PAR3

NECL-1NECL-2

NECL-5

MAGUK family (PALS2, DLG3, CASK)Band4.1 family (DAL1)

TCTEX1

Nectin-1Nectin-3

Afadin, α-catenin and their binding proteins are involved in the association between nectins and cad-herins 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 afadin- and 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 com-plex and F actin is mutually exclusive52–54. This model seems to be in conflict with results from previous stud-ies 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 under-estimate 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 trans-interaction 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 associa-tion that is essential for the nectin-induced activation

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 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.

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Vinculin

Nature Reviews | Molecular Cell Biology

a

Nectin

Afadin

Afadin

AfadinPonsin

b

NectinCadherin

Ponsin

Rac

Rac

CDC42

p120 ctnp120ct

n

β-Catenin

α-CateninF-actinbundles

Plasmamembrane

c

NectinCadherin

Ponsin

ADIPLMO7

p120 ctnp120ct

n

α-Actinin

IQGAP1

Vinculin

Afadin

d

NectinCadherin

Ponsin

ADIP

LMO7

p120 ctnp120ct

n

α-Actinin

AJ

Small G proteinA 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.

FilopodiumA thin cellular protrusion that  is formed by bundle-type reorganization of filamentous actin through the activation of CDC42.

LamellipodiumA broad and flat cellular protrusion that is formed by meshwork-type reorganization of filamentous actin through the activation of Rac.

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 activa-tion 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 inter-act 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

Figure 2 | Dynamic reorganization of the actin cytoskeleton in the formation of adherens junctions. a | The first step of 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).

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β-Catenin

α-CateninNectin-basedcell–cell adhesion

Nectin

Nectin

Afadin

Integrinαvβ3(active)

Talin

PKC FAK

SrcPIPKIγ90 Crk C3G

VAV2

Phosphorylation

FRG

RAP1

Rac CDC42

P

P

P P

P

PtdIns4P

PtdIns(4,5)P2

Reorganization of theactin cytoskeleton

Plasma membraneCadherin

Cadherin

Extracellular matrix

Extracellular matrix

P

PIPKIγ90

PTPµ

PDGFPDGFreceptor

Integrinαvβ3(inactive)

Talin

PI3K

AKT

Apoptosis

Nectin-mediated inactivationof integrin αvβ3 after the establishment of AJs

AJ

Nature Reviews | Molecular Cell Biology

Apical surfaceb

a

β-Catenin

α-Catenin

Afadin

PtdIns4P

Figure 3 | Nectin-induced intracellular signalling during and after the formation of adherens junctions. a | During the 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 phos-phatidylinositol 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.

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Peripheral ruffleA 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.

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 strength-ens their adhesion activity. Both ‘outside-in’ signalling (which is initiated by the trans-interactions of the extra-cellular 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 associa-tions 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 preven-tion 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.

NECL-5 and cell movementPrior 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 con-centrations 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 inter-action 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 com-plex 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 dis-sociated from integrin αvβ3 during the transformation of focal complexes to focal adhesions80. Focal adhesions formed in this way are necessary to generate the suf-ficient 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

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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 signal-ling 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 Src-homology-2 (sH2) domain of the protein Tyr phosphatase SHP2 (REf. 84). The binding of afadin increases the phos-phatase 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 inacti-vation 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 leading- edge 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 inhibitionNECL-5 is a notable member of the Necl family because it promotes cell movement and cell proliferation18,75. Importantly, the downregulation of NECL-5 from the cell surface contributes to the induction of contact inhibition. Nectins and NECL-5 concertedly modu-late the sequential events of cell–cell contact, and thus have roles in contact inhibition of cell movement and proliferation.

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 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.

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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 proteinA common designation for the proteins that accumulate at the plus end of microtubules.

ClathrinThe 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 dis-ruption 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 initi-ated by PDGF receptor and integrin αvβ3, as described above. It cannot be completely excluded that the trans-interaction 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 downregula-tion 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 trans-interaction 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 cadherin-based 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, limit-ing cell movement and proliferation for the long-term maintenance of cell–cell junctions in normal cells.

Inactivation of integrin αvβ3 by nectins. After the estab-lishment 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-4,5-bisphosphate (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 poten-tially interact with PTPµ through their extracellular regions, and the trans-interactions of nectins enhance its phosphatase activity, leading to a decrease in the phos-phorylation 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 path-way 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 fac-tors 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 phos-phorylated 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-inter-action 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 con-tact inhibition are complex and other CAMs, such as E-cadherin and CD44, might be involved94,95.

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Loss of contact inhibition in metastasis. Transformed cells lose contact inhibition of cell movement and prolif-eration, 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

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 (NECL-5), integrin αvβ3 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 cell-survival signalling molecules, including phosphoinositide 3-kinase (PI3K) and AKT, is regulated by PDGF receptor that is associated with the nectin–afadin complex.

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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 com-munication). Taken together, these findings suggest that the upregulation of NECL-5 following transformation contributes to the loss of contact inhibition in trans-formed 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 trans-interacts 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 con-sequently 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 perspectivesWe 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

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 trans-formed 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 can-cer 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 multi-cellular 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.

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AcknowledgementsThe 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 gradu-ate students, postdoctoral fellows and collaborators have made great contributions to this work. We thank all of them for their excellent achievements.

DATABASESUniProtKB: http://ca.expasy.org/sprotADIP | 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 INFORMATIONYoshimi Takai’s homepage: http://www.med.kobe-u.ac.jp/gs/field/basic/mol_cell.html (in Japanese)

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