Tage4/Nectin-like Molecule-5 Heterophilically trans-Interacts with Cell Adhesion Molecule Nectin-3 and Enhances Cell Migration*

Wataru Ikeda {ddagger}, Shigeki Kakunaga {ddagger}, Shinsuke Itoh {ddagger}, Tatsushi Shingai {ddagger}, Kyoji Takekuni {ddagger}, Keiko Satoh §, Yoko Inoue §, Akiko Hamaguchi §, Koji Morimoto §, Masakazu Takeuchi §, Toshio Imai § and Yoshimi Takai {ddagger} 

From the {ddagger}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
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
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.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
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 {alpha}- and {beta}-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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TABLE I
The proposed nomenclature of nectin-like molecules

 


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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.

 


    EXPERIMENTAL PROCEDURES
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Molecular Cloning of Mouse Necl-5 cDNA—The 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.

 

Construction of Plasmids—Expression 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 1–409 (full-length); pFLAG-CMV1-necl-5, aa 30–409 (deleting the signal peptide); pCAGIPuro-FLAG-necl-5, aa 30–409 (including the preprotrypsin signal peptide); pFLAG-CMV1-necl-5-{Delta}EC, aa 335–409 (deleting the extracellular region); pCAGIZeo-FLAG-necl-5-{Delta}EC, aa 335–409 (including the preprotrypsin signal peptide); pGEX4T-1-necl-5-CP, aa 371–409 (the cytoplasmic region); pGBD-C1-necl-5-{Delta}EC, aa 335–409 (deleting the extracellular region); pFastBac1-Msp-Fc-necl-5-EC, aa 30–347 (the extracellular region lacking the signal peptide); and pDREF-SEAP(His)6-Hyg-necl-5-EC, aa 1–347 (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 1–347), or -nectin-2-EC (aa 1–338) 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 56–400) 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 Transfectants—L 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{alpha}, full-length mouse nectin-2{alpha}, or full-length mouse nectin-3{alpha} (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-{Delta}EC (necl-5-{Delta}EC-L cells) was obtained by transfection with pCAGIZeo-necl-5, pCAGIPuro-FLAG-necl-5, or pCAGIZeo-FLAG-necl-5-{Delta}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).

Antibodies—A rat anti-necl-5 monoclonal antibody (mAb) #1A8-8 was raised against the extracellular fragment of necl-5 (aa 30–347) 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{gamma} fragment specific Ab was purchased from Jackson Laboratory.

Surface Plasmon Resonance Analysis—A 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 (M–1 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 Assay—Intercellular 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 Procedures—The 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).


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Heterophilic trans-Interaction of Necl-5 with Nectin-3—We 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.

 

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, Da–Dc). 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, Ea–Ec and Fa–Fc). 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.

 

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.

 

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.

 

Inability of Necl-5 to Bind Afadin—The extracellular region of necl-5 showed 28–42% 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 Cells—We 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-3—We 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-{Delta}EC-L cells were used as control cells. Necl-5-{Delta}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-{Delta}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-{Delta}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-{Delta}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-{Delta}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.

 


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
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 {alpha}-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 Proof—Mueller 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 {alpha}v{beta}3. Reymond et al. (Reymond, N., Fabre, S., Lecocq, E., Adelaïde, J., Dubreuil, P., and Lopez, M. (2001) J. Biol. Chem. 276, 43205–43215) 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. Back

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{alpha}; nectin-2-L cells, L cells stably expressing full-length mouse nectin-2{alpha}; nectin-3-L cells, L cells stably expressing full-length mouse nectin-3{alpha}; 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-{Delta}EC-L-cells, L cells stably expressing FLAG-necl-5-{Delta}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. Back


    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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