Tage4/Nectin-like Molecule-5 Heterophilically trans-Interacts with Cell Adhesion Molecule Nectin-3 and Enhances Cell Migration*
Wataru Ikeda
,
Shigeki Kakunaga
,
Shinsuke Itoh
,
Tatsushi Shingai
,
Kyoji Takekuni
,
Keiko Satoh
,
Yoko Inoue
,
Akiko Hamaguchi
,
Koji Morimoto
,
Masakazu Takeuchi
,
Toshio Imai
and
Yoshimi Takai
¶
From the
Department of Molecular Biology and
Biochemistry, Osaka University Graduate School of Medicine/Faculty of
Medicine, Suita 565-0871, Japan and the
KAN
Research Institute Inc., 93 Chudoji-Awatamachi, Shimogyo-ku, Kyoto 600-8815,
Japan
Received for publication, April 7, 2003
 |
ABSTRACT
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Malignant transformation of cells causes disruption of cell-cell adhesion,
enhancement of cell motility, and invasion into surrounding tissues. Nectins
have both homophilic and heterophilic cell-cell adhesion activities and
organize adherens junctions in cooperation with cadherins. We examined here
whether Tage4, which was originally identified to be a gene overexpressed in
colon carcinoma and has a domain structure similar to those of nectins, is
involved in cell adhesion and/or migration. Tage4 heterophilically
trans-interacted with nectin-3, but not homophilically with Tage4.
Expression of Tage4 was markedly elevated in NIH3T3 cells transformed by an
oncogenic Ki-Ras (V12Ras-NIH3T3 cells) as compared with that of wild-type
NIH3T3 cells. trans-Interaction of Tage4 with nectin-3 enhanced
motility of V12Ras-NIH3T3 cells. Tage4 did not bind afadin, a nectin- and
actin filament-binding protein that connects nectins to the actin cytoskeleton
and cadherins through catenins. Thus, Tage4 heterophilically
trans-interacts with nectin-3 and regulates cell migration. Tage4 is
tentatively re-named here nectin-like molecule-5 (necl-5) on the basis of its
function and domain structure similar to those of nectins.
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INTRODUCTION
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In multicellular organisms, cell adhesion and migration are critical for
many events, including tissue patterning, morphogenesis, and maintenance of
normal tissues
(13).
They also play roles in malignant transformation of cells
(4). Adhesion and migration of
non-transformed normal cells are dynamic and well regulated
(2). Cells disrupt cell-cell
adhesion and start to migrate in response to extracellular cues, such as
growth factors, cytokines, and extracellular matrix molecules
(4). When migrating cells
contact other cells, they stop migration and proliferation and adhere to each
other to become confluent (5,
6). This phenomenon is known
for a long time as contact inhibition of cell movement and proliferation.
Transformation of cells causes disruption of cell-cell adhesion, increase of
cell motility, and loss of contact inhibition of cell movement and
proliferation, eventually leading the transformed cells to invasion into
surrounding tissues and metastasis to other organs
(4,
7). However, molecular
mechanisms underlying these physiological or pathological processes are not
fully understood.
Cell-cell adherens junctions
(AJs)1 play major
roles in cell-cell adhesion in fibroblasts and epithelial cells
(1,
2). Cadherins are key
Ca2+-dependent cell-cell adhesion molecules at AJs
(1,
2). Cadherins are associated
with the actin cytoskeleton through peripheral membrane proteins, including
- and
-catenins, in fibroblasts and epithelial cells
(1). This association
strengthens the cell-cell adhesion activity of cadherins
(1). Nectins and afadin
constitute another cell-cell adhesion unit that localizes at cell-cell AJs and
regulates organization of AJs in cooperation with cadherins in fibroblasts and
epithelial cells (8). Nectins
are Ca2+-independent Ig-like cell-cell adhesion
molecules. Afadin is a nectin- and actin filament-binding protein that
connects nectins to the actin cytoskeleton. Nectins comprise a family of four
members, nectin-1, -2, -3, and -4, each of which has two or three splicing
variants. Nectins have one extracellular region with three Ig-like loops, one
transmembrane region, and one cytoplasmic region. All nectins except nectin-4
have a C-terminal conserved motif of four amino acids (aa) residues, which
interacts with the PDZ domain of afadin. Nectin-4 does not have this motif but
binds afadin. Each nectin forms homo-cis-dimers, followed by the
formation of homo-trans-dimers, causing cell-cell adhesion. Nectin-3
furthermore heterophilically trans-interacts with nectin-1 or -2 and
the adhesion activity of these heterophilic trans-interactions is
stronger than that of the homophilic trans-interactions. Nectin-4
also heterophilically trans-interacts with nectin-1.
Five or six molecules having one extracellular region with three Ig-like
loops, one transmembrane region, and one cytoplasmic region have thus far been
identified (Table I)
(919).
We tentatively name here these molecules nectin-like molecules (necls) on the
basis of their domain structures similar to those of nectins (see
"Discussion"). Of these necls, Tage4 was originally identified to
be a gene overexpressed in rat and mouse colon carcinoma
(16,
17). Northern blot analysis
has revealed that Tage4 is expressed in normal adult rat and mouse tissues to
small extents (16,
17), but its function remains
unknown, except that it mediates entry of porcine pseudorabies virus and
bovine herpesvirus 1 (20). We
have studied here the function of Tage4 and revealed that Tage4
heterophilically trans-interacts with nectin-3 and regulates cell
migration. Tage4 is tentatively re-named here necl-5 on the basis of its
function and phylogenetic tree of nectins and necls
(Fig. 1) (see
"Discussion").

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FIG. 1. Phylogenic analysis of nectin and necl families. The aa sequences
were aligned using the CLASTALW program, and the phylogenic tree was
constructed by tree drawing software TreeViewPPC 1.6.6.
(taxonomy.zoology.gla.ac.uk/rod/rod.html).
Blanch lengths are drawn to scale, with longer branches indicating more
changes. Necl-1, NECL1/TSLL1/SynCAM3; Necl-2,
NECL2/IGSF4/RA175/SgIGSF/TSLC1/SynCAM1; Necl-3, NECL3/similar to
NECL3/SynCAM2; Necl-4, TSLL2/SynCAM4; Necl-5, Tage4; and Necl-6,
PVR/CD155.
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EXPERIMENTAL PROCEDURES
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Molecular Cloning of Mouse Necl-5 cDNAThe cDNA of mouse
Tage4/necl-5 was originally isolated by reverse transcriptase-PCR from the C26
mouse colon carcinoma cell line (DDBJ/GenBankTM/EBI accession number
MMU35836) (17). This cell line
was derived from BALB/c mice. Because we generally use nectins derived from
C57BL/6 mice, we re-cloned the Tage4/necl-5 cDNA derived from C57BL/6 mice. We
searched in the DNA data base and found one sequence similar to that of
Tage4/necl-5 (DDBJ/GenBankTM/EBI accession number BC013673
[GenBank]
). We performed
reverse transcriptase-PCR from mouse brain total RNA of C57BL/6 mice on the
basis of BC013673
[GenBank]
. The new sequence of Tage4/necl-5 showed 93% nucleotide
identity to that of the original one. The C-terminal half was identical, but
the N-terminal half was slightly different. The new sequence was identical to
BC013673
[GenBank]
except for the exchange of a single nucleotide from cytosine to
adenine, at position 854 (open reading frame). The reason for this difference
is not known, but may be due to the different strains of mice. We confirmed
that the isolated cDNA encodes the full-length protein: the protein was
expressed in L cells and the molecular mass of the expressed protein was
compared with that of the endogenous protein, which was expressed in NIH3T3
cells stably expressing V12Ki-Ras, an oncogenic Ki-Ras, (V12Ras-NIH3T3 cells).
The molecular masses of the two proteins were apparently similar as estimated
by SDS-PAGE, followed by Western blotting (see
Fig. 5).

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FIG. 5. Expression levels of necl-5 in various cell lines. The cell lysates
of wild-type L, necl-5-L, NIH3T3, and V12Ras-NIH3T3 cells (20 µg of protein
each) were subjected to SDS-PAGE (13% polyacrylamide gel), followed by Western
blotting using the anti-necl-5 mAb. The results shown are representative of
three independent experiments.
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Construction of PlasmidsExpression vectors were constructed
in pFLAG-CMV1 (Sigma), pCAGIPuro
(21), pCAGIZeo
(22), pGEX4T-1 (Amersham
Biosciences), pGBD-C1 (23),
pFastBac1-Msp-Fc (24), and
pDREF-SEAP(His)6-Hyg
(25). Constructs of necl-5
contained the following aa: pCAGIZeo-necl-5, aa 1409 (full-length);
pFLAG-CMV1-necl-5, aa 30409 (deleting the signal peptide);
pCAGIPuro-FLAG-necl-5, aa 30409 (including the preprotrypsin signal
peptide); pFLAG-CMV1-necl-5-
EC, aa 335409 (deleting the
extracellular region); pCAGIZeo-FLAG-necl-5-
EC, aa 335409
(including the preprotrypsin signal peptide); pGEX4T-1-necl-5-CP, aa
371409 (the cytoplasmic region); pGBD-C1-necl-5-
EC, aa
335409 (deleting the extracellular region); pFastBac1-Msp-Fc-necl-5-EC,
aa 30347 (the extracellular region lacking the signal peptide); and
pDREF-SEAP(His)6-Hyg-necl-5-EC, aa 1347 (the extracellular
region). To express the extracellular fragment of nectin-1 or -2 fused to
secreted alkaline phosphatase (SEAP) (Neap-1 or -2),
pDREF-SEAP(His)6-Hyg-nectin-1-EC (aa 1347), or -nectin-2-EC
(aa 1338) was constructed into pDREF-SEAP(His)6-Hyg. The
SEAP fusion proteins were expressed in 293/EBNA-1 cells (Invitrogen) and
purified as described previously
(25). To express the
extracellular fragment of nectin-3 fused to the human IgG Fc (Nef-3),
pFastBac1-Msp-Fc-nectin-3-EC (aa 56400) was prepared as described
previously (26). The IgG Fc
fusion proteins were prepared as a secreted protein from the baculovirus
transfer system (Invitrogen) and purified by use of protein A-Sepharose beads
(Amersham Biosciences) as described previously
(24). The GST fusion proteins
were purified by use of glutathione-Sepharose beads (Amersham
Biosciences).
Cell Culture and Establishment of TransfectantsL and MTD-1A
cells were kindly supplied by Dr. S. Tsukita (Kyoto University, Kyoto, Japan).
MDCK cells were kindly supplied by Dr. W. Birchmeier (Max-Delbruck-Center for
Molecular Medicine, Berlin, Germany). V12Ras-NIH3T3 cells were prepared as
described (27). L, MTD-1A, and
MDCK cells were maintained in Dulbecco's modified Eagle's medium supplemented
with 10% FCS. NIH3T3 and V12Ras-NIH3T3 cells were maintained in Dulbecco's
modified Eagle's medium supplemented with 10% calf serum. L cell lines stably
expressing full-length human nectin-1
, full-length mouse
nectin-2
, or full-length mouse nectin-3
(nectin-1-L, -2-L, or
-3-L cells, respectively) were prepared as described previously
(21,
26,
28). An L cell line stably
expressing full-length necl-5 (non-tagged-necl-5-L cells), FLAG-necl-5
(necl-5-L cells), or FLAG-necl-5-
EC (necl-5-
EC-L cells) was
obtained by transfection with pCAGIZeo-necl-5, pCAGIPuro-FLAG-necl-5, or
pCAGIZeo-FLAG-necl-5-
EC, respectively, using LipofectAMINE PLUS reagent
(Invitrogen). We mostly used necl-5-L cells (FLAG-tagged necl-5) in the
present study, but the essentially similar results were obtained with
non-tagged-necl-5-L cells (data not shown).
AntibodiesA rat anti-necl-5 monoclonal antibody (mAb)
#1A8-8 was raised against the extracellular fragment of necl-5 (aa
30347) fused to the human IgG Fc (Lef-5). A rabbit antisera against
necl-5 was raised against the cytoplasmic tail of necl-5 fused to GST
(GST-necl-5-CP). An affinity-purified F(ab')2 fragment goat
anti-human IgG, Fc
fragment specific Ab was purchased from Jackson
Laboratory.
Surface Plasmon Resonance AnalysisA BIAcore X surface
plasmon resonance-based biosensor (BIAcore Inc., Piscataway, NJ) was used to
measure kinetic parameters for the interaction between Neap-1, Neap-2, or the
extracellular fragment of necl-5 fused to SEAP (Leap-5) and immobilized Nef-3
or Lef-5. The F(ab')2 fragment goat anti-human IgG Fc
polyclonal Ab (pAb) was immobilized at a concentration of about 4800 resonance
units (4.8 ng/mm2) to the sensor chip surface by the amine-coupling
method. Nef-3 or Lef-5 was immobilized at a concentration of about 500
resonance units to the sensor chip via the anti-human IgG Fc Ab. Neap-1,
Neap-2, or Leap-5 was then diluted in HBS-EP buffer (10 mM HEPES,
pH7.4, 150 mM NaCl, 3 mM EDTA, 0.005% Tween 20; BIAcore)
to 40 nM and injected at a flow rate of 20 µl/min at 25 °C
for 210 s. Both an association rate constant ka
(M1 s1) and a
dissociation rate constant kd
(s1) were obtained using the BIAevaluation
software version 3.2 (BIAcore), and the dissociation constant
(KD =
kd/ka) was derived
from the two deduced rate constants.
Intercellular Motility AssayIntercellular motility assay
was done as described previously
(29). Briefly, the cells were
labeled with
1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine
perchlorate (DiI), and the 1 x 102 labeled cells were seeded
on a confluent culture of 2 x 105 unlabeled nectin-3-L cells
in a 24-well dish. After 36 or 48 h of culture, four sister cells that seemed
to be derived from one seeded cell were examined by fluorescence microscopy.
In the experiments using the anti-necl-5 mAb, this mAb was added at a final
concentration of 50 µg/ml in the medium. When a cell line A was seeded on a
confluent culture of a cell line B, we designated the experiment as A/B
analysis. For quantification of intercellular motility, intercellular
distances of all combinations between four sister cells were measured and
summed as Dc. As a control experiment, the labeled cells were seeded on dishes
in the absence of a cell layer. In this case, the intercellular distances were
summed as Dd. The degree of intercellular motility was represented as Dc/Dd.
At least 24 independent samples were picked up to determine Dc or Dd for each
cell line.
Other ProceduresThe cell aggregation assay, chemical
cross-linking, and SDS-PAGE were done as described previously
(21,
26,
28,
30). The inability of necl-5
to bind afadin was confirmed by yeast two-hybrid assay, co-immunoprecipitation
assay, and affinity chromatography as described previously
(23,
26,
28), under the conditions
where nectin-2 and -3 bound afadin. Protein concentrations were determined
with bovine serum albumin as a reference protein as described previously
(31).
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RESULTS
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Heterophilic trans-Interaction of Necl-5 with Nectin-3We
first examined by the aggregation assay using necl-5-L cells whether necl-5
has cell-cell adhesion activity. In wild-type L cells, nectin-1 and -2, but
not cadherin or nectin-3, are expressed as estimated by Western blotting
(21,
24,
26). Expression of necl-5 was
not detected in wild-type L cells by Western blotting using any Abs, which
recognized exogenously expressed necl-5 (see
Fig. 5). Wild-type L cells did
not form visible cell aggregates as described previously
(28)
(Fig. 2A). Necl-5-L
cells did not form visible cell aggregates, either
(Fig. 2B). These
results indicate that necl-5 does not homophilically trans-interact
with necl-5, causing no homophilic cell-cell adhesion.

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FIG. 2. Heterophilic cell aggregation activity of necl-5. A single-cell
suspension was rotated for 10 min. A, wild-type L cells; B,
necl-5-L cells; C, nectin-3-L cells; D, DiI-labeled necl-5-L
cells and unlabeled nectin-3-L cells; E, DiI-labeled necl-5-L cells
and unlabeled nectin-1-L cells; and F, DiI-labeled necl-5-L cells and
unlabeled nectin-2-L cells. A, B, C, Da, Ea, and Fa, phase
contrast image; Db, Eb, and Fb, fluorescence image; and
Dc, Ec, and Fc, superimposed image. Bars, 100
µm. The results shown are representative of three independent
experiments.
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We then examined whether necl-5 has heterophilic cell-cell adhesion
activity with other nectins. Necl-5-L cells were mixed with nectin-1-L, -2-L,
or -3-L cells followed by the aggregation assay. Nectin-3-L cells formed small
aggregates in the absence of necl-5-L cells as described previously
(26)
(Fig. 2C), but formed
relatively big aggregates with necl-5-L cells
(Fig. 2D,
DaDc). This aggregate was similarly formed even in the
absence of Ca2+ (data not shown). The size of the
aggregates formed between necl-5-L and nectin-3-L cells were about 20% that of
the aggregates formed between nectin-1-L and -3-L cells, which formed the
biggest aggregates among various combinations of nectins thus far examined
(32) (see
Fig. 7, Aa and
Ba). Necl-5-L cells did not form mixed aggregates with
nectin-1-L or -2-L cells (Fig. 2,
EaEc and FaFc). Small aggregates
observed were formed by nectin-1-L or nectin-2-L cells by themselves as
described (28)
(Fig. 2, Ea, Ec, Fa, and
Fc). These results indicate that necl-5 heterophilically
trans-interacts selectively with nectin-3 in a
Ca2+-independent manner, causing heterophilic cell-cell
adhesion selectively with nectin-3.

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FIG. 7. Specific inhibition of the interaction of necl-5 with nectin-3 by the
anti-necl-5 mAb. The inhibitory activity of the anti-necl-5 mAb was
assayed by the cell aggregation assay. A, necl-5-L and nectin-3-L
cells; B, nectin-1-L and nectin-3-L cells. Aa and
Ba, in the absence of the anti-necl-5 mAb; Ab and
Bb, in the presence of the anti-necl-5 mAb. Bars, 100 µm.
The results shown are representative of three independent experiments.
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To confirm that necl-5 directly interacts with nectin-3, we performed
surface plasmon resonance analysis using Nef-3 and Leap-5. Neap-1 and -2 were
used as controls. Nef-3 is the extracellular fragment of nectin-3 fused to the
human IgG Fc; Leap-5 is the extracellular fragment of necl-5 fused to SEAP;
Neap-1 is the extracellular fragment of nectin-1 fused to SEAP; and Neap-2 is
the extracellular fragment of nectin-2 fused to SEAP. Nef-3 bound all of these
molecules, and the Kd value of Nef-3 for Leap-5
was about 17 nM, whereas the Kd values
of Nef-3 for Neap-1 and -2 were about 2.3 and 360 nM, respectively
(Fig. 3). Lef-5 did not bind
Leap-5 (data not shown). Lef-5 is the extracellular fragment of necl-5 fused
to the human IgG Fc.

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FIG. 3. Binding kinetics of recombinant necl-5 to Nef-3. The binding of
Leap-5 (necl-5) to Nef-3 (nectin-3) was measured by surface plasmon resonance.
Neap-1 and -2 (nectin-1 and -2, respectively) were used as controls. The
results shown are representative of three independent experiments.
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We have previously shown that nectin-2 forms homo-cis-dimers,
followed by the formation of homo- or hetero-trans-dimers, eventually
causing cell-cell adhesion
(21). Similarly, necl-5 formed
homo-cis-dimers (Fig.
4). In addition, necl-5 furthermore formed a multimer. Taken
together, it is likely by analogy with the mode of action of nectin-2 that
necl-5 forms first homo-cis-dimers, followed by the heterophilic
trans-interaction with nectin-3, eventually causing cell-cell
adhesion.

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FIG. 4. Formation of homo-cis-dimers of necl-5. A single-cell
suspension of necl-5-L cells was incubated in the presence or absence of
bis(sulfosuccinimidyl) suberate (BS3). Each cell lysate (10 µg of
protein) was subjected to SDS-PAGE (13% polyacrylamide gel), followed by
Western blotting using the anti-necl-5-CP pAb. Arrow, monomer;
arrowhead, dimer; and double-arrowhead, multimer. The
results shown are representative of three independent experiments.
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Inability of Necl-5 to Bind AfadinThe extracellular region
of necl-5 showed 2842% aa identity to that of nectins, but necl-5 does
not have a C-terminal consensus motif with four aa for binding to the PDZ
domain (data not shown). We examined whether necl-5 binds afadin. Necl-5 did
not bind afadin as estimated by yeast two-hybrid assay, co-immunoprecipitation
assay, and affinity chromatography under the conditions where nectin-2 or -3
bound it (data not shown).
Elevated Expression of Necl-5 in V12Ras-NIH3T3 CellsWe then
examined the tissue distribution of necl-5 in mouse by Western blotting, but
the significant immunoreactive band was not detected in any normal tissue
examined, including heart, brain, spleen, lung, liver, kidney, skeletal
muscle, and testis (data not shown), consistent with the earlier observation
(17). Any band was not
detected in cultured cell lines, including L
(Fig. 5), MTD-1A (data not
shown), and MDCK cells (data not shown), but two faint bands were detected in
NIH3T3 cells (Fig. 5). Tage4
was originally isolated from rat and mouse colon carcinoma
(16,
17). We therefore examined the
expression of necl-5 in V12Ras-NIH3T3 cells. Expression of necl-5 was markedly
elevated in the transformed cells as compared with that of the wild-type cells
(Fig. 5).
Enhancement of Motility of V12Ras-NIH3T3 and Necl-5-L Cells by
trans-Interaction of Necl-5 with Nectin-3We finally studied the
role of the trans-interaction of necl-5 with nectin-3 on motility of
V12Ras-NIH3T3 cells by using intercellular motility assay, because transformed
cells show generally enhanced migration activity
(4). Necl-5-L and
necl-5-
EC-L cells were used as control cells. Necl-5-
EC-L cells
were L cells expressing necl-5, of which extracellular region except the
juxtamembrane 13 aa were deleted. In this assay, cell motility in a confluent
cell sheet, which is influenced by dynamic cell-cell adhesion, could be
measured. V12Ras-NIH3T3 cells labeled with DiI, a fluorescence dye, were
seeded on a confluent culture of non-labeled nectin-3-L cells, and after 36 h
(twice the doubling time), the cell scatter property was analyzed by measuring
the mass distance among four sister labeled cells. As a control experiment,
labeled V12Ras-NIH3T3 cells were seeded on the dish in the absence of
nectin-3-L cells. V12Ras-NIH3T3 cells scattered on a confluent culture of
nectin-3-L cells more actively than on the dish
(Fig. 6, Aa1, Aa2, and
B). The scattering of V12Ras-NIH3T3 cells on nectin-3-L
cells was inhibited by the anti-necl-5 mAb, whereas the scattering on the dish
was not affected by this mAb (Fig. 6,
Aa3, Aa4, and B). The anti-necl-5 mAb inhibited
the interaction of necl-5 with nectin-3 as estimated by the aggregation assay
using necl-5-L and nectin-3-L cells (Fig.
7, Aa and Ab). This mAb did not affect the
interaction of nectin-1 with nectin-3 (Fig.
7, Ba and Bb). Necl-5-L cells labeled with DiI
were seeded on a confluent culture of non-labeled nectin-3-L cells, and after
48 h (twice the doubling time), the cell scatter property was similarly
analyzed. Necl-5-L cells scattered on a confluent culture of nectin-3-L cells
more actively than on the dish (Fig. 6,
Ab1, Ab2, and B). The scattering of necl-5-L
cells on nectin-3-L cells was inhibited by the anti-necl-5 mAb, whereas the
scattering of necl-5-L cells on the dish was not affected by this mAb
(Fig. 6, Ab3, Ab4, and
B). Necl-5-
EC-L cells labeled with DiI scattered
on a confluent culture of nectin-3-L cells less actively than on the dish
(Fig. 6, Ac1, Ac2, and
B). Necl-5-
EC-L cells did not adhere to nectin-3-L
cells as estimated by the aggregation assay (data not shown). The scattering
of necl-5-
EC-L cells in the presence or absence of nectin-3-L cells was
not affected by the anti-necl-5 mAb (Fig.
6, Ac3, Ac4, and B). These results indicate that
the trans-interaction of necl-5 with nectin-3 enhances motility of
V12Ras-NIH3T3 and necl-5-L cells.

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FIG. 6. Enhancement of motility of V12Ras-NIH3T3 and necl-5-L cells by
trans-interaction of necl-5 with nectin-3. A,
inter-nectin-3-L-cellular motility. Panels a,
V12Ras-NIH3T3/nectin-3-L analysis; panels b, necl-5-L/nectin-3-L
analysis; and panels c, necl-5- EC-L/nectin-3-L analysis.
Panels a1, b1, and c1, on the cell layer; panels a2,
b2, and c2, on the dish; panels a3, b3, and
c3, on the cell layer in the presence of the anti-necl-5 mAb; and
panels a4, b4, and c4, on the dish in the presence of the
anti-necl-5 mAb. The labeled four sister cells were indicated as red
images by the fluorescence microscopy. In panels a1, a3, b1, b3,
c1, and c3, these images were superimposed on the phase contrast
image of a confluent cell layer, and the labeled four sister cells were
indicated as white arrows. Bars, 100 µm. B, quantitative
analysis of the intercellular motility. The average value of intercellular
motility is shown here. At least 24 independent samples were picked up to
determine the intercellular motility index for each analysis.
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DISCUSSION
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We have shown here that necl-5 does not homophilically
trans-interact with necl-5, but heterophilically
trans-interacts selectively with nectin-3, causing cell-cell
adhesion. This property of necl-5 is quite different from that of nectins
which both homophilically and heterophilically trans-interact
(8). We have previously
proposed that nectins are involved in the formation of AJs in cooperation with
E-cadherin, on the basis of the observations that the
trans-interaction of nectins recruits E-cadherin to the nectin-based
cell-cell adhesion sites, resulting in formation of AJs, and that the
disruption of this trans-interaction of nectins by their antagonists
impairs the formation of E-cadherin-based AJs
(8). The association of nectins
and E-cadherin at AJs is mediated through afadin and
-catenin
(8). We have shown here that
necl-5 does not bind afadin. The inability of necl-5 to bind afadin suggests
that necl-5 has no potency to recruit cadherins to the cell-cell adhesion site
formed by the trans-interaction of necl-5 with nectin-3 and is not
involved in the formation of AJs.
We have shown here that the heterophilic trans-interaction of
necl-5 with nectin-3 rather enhances motility of V12Ras-NIH3T3 and necl-5-L
cells. It has previously been reported that L cells stably expressing
full-length E-cadherin (EL cells) shows inter-EL-cellular EL cell motility
(29,
33). The mechanism of this
intercellular motility of EL cells is not clear, but it has been suggested
that dynamic attachment of EL cells to neighboring EL cells and dynamic
detachment of EL cells from neighboring EL cells are necessary for the
motility of EL cells (29,
33). The mechanism of
intercellular motility of V12Ras-NIH3T3 and necl-5-L cells is not known,
either, but may be analogous to that of EL cells.
Transformation of cells increases cell motility, causing invasion into
surrounding tissues. Since expression of necl-5 is elevated by transformation
as shown here and described previously
(16,
17), this elevation of necl-5
may be at least partly responsible for the enhanced cell motility and invasion
of transformed cells. Intercellular motility is observed in vivo in
the process of morphogenetic rearrangement of cells in embryonic tissues
(2,
3). It remains unknown what
kind of the cells express necl-5 in embryonic tissues, but if necl-5 is
expressed in rapidly migrating cells, such as mesenchymal cells, the dynamic
trans-interaction of necl-5 with nectin-3 may also play a role in
their intercellular motility. Further studies are necessary for establishing
the physiological and pathological roles of necl-5 in these processes.
We lastly discuss about other necls which have thus far been identified in
addition to necl-5/Tage4 (Table
I). Five or six necls including necl-5 have been identified but
have many nomenclatures. We propose here that a group of proteins with
structures similar to those of nectins but without ability to directly bind
afadin are called nectin-like molecules (necls). NECL1/TSLL1/SynCAM3,
NECL2/IGSF4/RA175/SgIGSF/TSLC1/SynCAM1, NECL3/similar to NECL3/SynCAM2, and
TSLL2/SynCAM4 have a C-terminal consensus motif with four aa for binding to
PDZ domains, and these cytoplasmic regions show high similarity. Necl-5/Tage4
or PVR/CD155 does not have this motif. Our analysis has revealed that
NECL1/TSLL1/SynCAM3 or NECL2/IGSF4/RA175/SgIGSF/TSLC1/SynCAM1 as well as
necl-5/Tage4 does not bind afadin as estimated by yeast two-hybrid assay,
co-immunoprecipitation assay, and affinity chromatography (data not shown). It
remains to be examined whether NECL3/similar to NECL3/SynCAM2, TSLL2/SynCAM4,
or PVR/CD155 binds afadin. On the assumption that these three proteins do not
directly bind afadin, we propose that all of these molecules are called necls.
Then, we propose the following nomenclatures according to the phylogenetic
tree shown in Fig. 1: necl-1
for NECL1/TSLL1/SynCAM3, necl-2 for NECL2/IGSF4/RA175/SgIGSF/TSLC1/SynCAM1,
necl-3 for NECL3/similar to NECL3/SynCAM2, necl-4 for TSLL2/SynCAM4, necl-5
for Tage4, and necl-6 for PVR/CD155. The necl-6/PVR/CD155 gene has thus far
been found only in the primates
(34), and necl-5/Tage4 has
thus far been found only in the rodent. The sequence of necl-5/Tage4 shows 42%
aa identity to that of necl-6/PVR/CD155. The necl-5/Tage4 gene may be an
ortholog of the necl-6/PVR/CD155 gene
(20), but the phylogenetic
tree of nectins and necls can not clearly conclude that necl-5/Tage4 and
necl-6/PVR/CD155 are derived from the same or different ancestor gene.
Therefore, we reserve the conclusion for the classification of these two
genes.
 |
FOOTNOTES
|
---|
Note Added in ProofMueller and Wimmer (Mueller, S., and
Wimmer, E. (2003) J. Biol. Chem. 278, in press) demonstrated
the heterophilic trans-interactions of PVR/CD155 and Tage4/necl-5
with nectin-3, and they furthermore showed that PVR/CD155 colocalized with
integrin
v
3. Reymond et al.
(Reymond, N., Fabre, S., Lecocq, E., Adelaïde, J., Dubreuil, P., and
Lopez, M. (2001) J. Biol. Chem. 276, 4320543215)
previously claimed the PVR/CD155 bound nectin-3, although no experimental
evidence for this interaction was presented.
* The work at Osaka University was supported by grants-in-aid for Scientific
Research and for Cancer Research from the Ministry of Education, Culture,
Sports, Science, and Technology, Japan (2001, 2002). The costs of publication
of this article were defrayed in part by the payment of page charges. This
article must therefore be hereby marked "advertisement"
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 
¶
To whom correspondence should be addressed: Dept. of Molecular Biology and
Biochemistry, Osaka University Graduate School of Medicine/Faculty of
Medicine, Suita 565-0871, Osaka, Japan. Tel.: 81-6-6879-3410; Fax:
81-6-6879-3419; E-mail:
ytakai{at}molbio.med.osaka-u.ac.jp.
1 The abbreviations used are: AJ, adherens junction; aa, amino acid(s); necl,
nectin-like molecule; V12Ras-NIH3T3 cells, NIH3T3 cells stably expressing
V12Ki-Ras; SEAP, secreted alkaline phosphatase; Neap, the extracellular
fragment of nectin fused to SEAP; Nef, the extracellular fragment of nectin
fused to the human IgG Fc; nectin-1-L cells, L cells stably expressing
full-length human nectin-1
; nectin-2-L cells, L cells stably expressing
full-length mouse nectin-2
; nectin-3-L cells, L cells stably expressing
full-length mouse nectin-3
; non-tagged-necl-5-L-cells, L cells stably
expressing full-length necl-5; necl-5-L-cells, L cells stably expressing
FLAG-necl-5; necl-5-
EC-L-cells, L cells stably expressing
FLAG-necl-5-
EC; Ab, antibody; Lef, the extracellular fragment of necl
fused to the human IgG Fc; GST, glutathione S-transferase;
GST-necl-5-CP, the cytoplasmic tail of necl-5 fused to GST; Leap, the
extracellular fragment of necl fused to SEAP; mAb, monoclonal Ab; pAb,
polyclonal antibody; DiI,
1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine
perchlorate; EL cells, L cells stably expressing full-length E-cadherin. 
 |
ACKNOWLEDGMENTS
|
---|
We thank Dr. S. Tsukita (Kyoto University, Kyoto, Japan) for providing us
with L and MTD-1A cells and Dr. W. Birchmeier (Max-Delbruck-Center for
Molecular Medicine, Berlin, Germany) for providing us with MDCK cells. We are
also grateful to Dr. A. Nagafuchi (Kumamoto University, Kumamoto, Japan) for
his helpful discussion.
 |
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