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Originally published In Press as doi:10.1074/jbc.M209846200 on October 11, 2002

J. Biol. Chem., Vol. 277, Issue 52, 50749-50755, December 27, 2002
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trans-Interactions of Nectins Induce Formation of Filopodia and Lamellipodia through the Respective Activation of Cdc42 and Rac Small G Proteins*

Tomomi Kawakatsu, Kazuya Shimizu, Tomoyuki Honda, Tatsuro Fukuhara, Takashi Hoshino, and Yoshimi TakaiDagger

From the Department of Molecular Biology and Biochemistry, Osaka University Graduate School of Medicine/Faculty of Medicine, Suita 565-0871, Japan

Received for publication, September 25, 2002

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Nectins and afadin constitute a novel cell-cell adhesion system that plays a cooperative role with cadherins in the organization of adherens junctions (AJs). Nectins are Ca2+-independent immunoglobulin-like cell-cell adhesion molecules, and afadin is a nectin- and actin filament-binding protein that connects nectins to the actin cytoskeleton. Rac and Cdc42 small G proteins have been implicated in the organization of AJs, but their modes of action remain unknown. The trans-interaction of E-cadherin has recently been shown to induce the activation of Rac, but not that of Cdc42. We show here that the trans-interactions of nectins induce the formation of filopodia and lamellipodia through the respective activation of Cdc42 and Rac. The Cdc42 activation is necessary, but not sufficient, for the Rac-induced formation of lamellipodia, whereas the Rac activation is not necessary for the Cdc42-induced formation of filopodia. These effects of nectins require their cytoplasmic tail but not their association with afadin. We propose here the functional relationship between nectins and the small G proteins in the organization of AJs.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Cadherins are Ca2+-dependent cell-cell adhesion molecules at adherens junctions (AJs)1 (for reviews, see Refs. 1-3). Cadherins form cis-dimers and then trans-dimers (trans-interactions) through the extracellular region, causing cell-cell adhesion, whereas the cytoplasmic tail is linked to the actin cytoskeleton through many peripheral membrane proteins, including alpha -catenin, beta -catenin, vinculin, and alpha -actinin, which strengthen the cell-cell adhesion activity of cadherins (1-3). Nectins are Ca2+-independent immunoglobulin-like cell-cell adhesion molecules that play roles in the organization of AJs in epithelial cells and fibroblasts in cooperation with E-cadherin (4-8), the organization of synapses in neurons in cooperation with N-cadherin (9, 10), and the organization of Sertoli cell-spermatid junctions in the testis independently of cadherins (11). Nectins comprise a family consisting of four members, nectin-1, nectin-2, nectin-3, and nectin-4 (7, 12-16). Nectin-1 was originally isolated as one of the poliovirus receptor-related proteins and named PRR-1 (17). Nectin-2 was originally isolated as the murine homolog of the human poliovirus receptor (18), but it turned out to be another poliovirus receptor-related protein and was named PRR-2 (19). Neither PRR-1 nor PRR-2 has thus far been shown to serve as a poliovirus receptor. PRR-1 and PRR-2 were later shown to serve as receptors for alpha -herpes viruses, facilitating their entry and intercellular spreading, and they were renamed HveC and HveB, respectively (20-24). It remains unknown whether nectin-3 and nectin-4 serve as receptors for viruses. All nectins form homo-cis-dimers and then homo-trans-dimers (trans-interactions), causing cell-cell adhesion (13-15, 24, 25). Furthermore, nectin-3 forms hetero-trans-dimers with nectin-1 and nectin-2 (15). Nectin-4 also forms hetero-trans-dimers with nectin-1 (16). All nectins are associated with the actin cytoskeleton through afadin, which is a nectin- and actin filament (F-actin)-binding protein (4, 7, 15, 16). The C-terminal conserved motif of four amino acid (aa) residues of nectins interacts with the PDZ domain of afadin (7, 15, 16).

It has been shown that the extracellular fragment of E-cadherin fused to the Fc portion of IgG coated on microbeads trans-interacts with cellular E-cadherin and recruits it to the bead-cell contact sites (26). By analogy, we have recently shown that the extracellular fragment of nectin-3 fused to the Fc portion of IgG (Nef-3) coated on microbeads trans-interacts with cellular nectin-1 and recruits cellular nectin-1 and then E-cadherin to the bead-cell contact sites.2 We have furthermore shown that soluble Nef-3 trans-interacts with cellular nectin-1 and thereby diminishes the formation of the nectin-1-based cell-cell adhesion, resulting in a reduction of the formation of the E-cadherin-based cell-cell adhesion.2 These results have provided an additional line of evidence to show that nectins are involved in the formation of AJs in cooperation with E-cadherin.2

The formation of AJs has been shown to be enhanced by Rac and Cdc42 small G proteins (27-31), but their modes of action remain unknown. The trans-interaction of E-cadherin has recently been shown to induce the activation of Rac but not that of Cdc42 (26, 32-34). One group has shown that the trans-interaction of E-cadherin induces the activation of Cdc42 (35), but this result has not been reproduced by another group (32). We show here by use of Nef-3 together with Nef-1 and Nef-2 (the extracellular fragments of nectin-1 and nectin-2 fused to the Fc portion of IgG, respectively) that the trans-interactions of nectins induce the activation of Cdc42 and Rac.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Construction and Purification-- An eukaryotic expression vector, pFLAG-CMV1, and baculovirus expression vectors, pFastBac1-Msp-Fc and pFastBac1-Fc, were prepared as described (14, 15, 24). The construct of pFLAG-CMV1-nectin-2alpha /TEM5 contained the cDNA fragment encoding the extracellular and transmembrane domains of mouse nectin-2alpha (aa 1-394) fused to the cytoplasmic domain of TEM5 (GenBankTM accession number AF378755) (aa 1062-1331). The construct of pEGFP-NWASP-Cdc42 and Rac interactive binding domain (CRIB) contained the cDNA fragment encoding the CRIB domain of NWASP (36).

Nef-1, Nef-2, and Nef-3 were prepared as described previously2 (15). Briefly, pFastBac1-Msp-Fc-nectin-3-EX was prepared as described (15). pFastBac1-Msp-Fc-nectin-1-EX and pFastBac1-Fc-nectin-2-EX were similarly prepared by constructing the cDNA fragments encoding the extracellular fragments of nectin-1alpha (GenBankTM accession number AF270977) (aa 27-347) and nectin-2alpha (GenBankTM accession number M80206) (aa 1-338) in pFastBac1-Msp-Fc and pFastBac1-Fc, respectively. Baculoviruses bearing the cDNAs encoding Nef-1, Nef-2, and Nef-3 were prepared with pFastBac1-Msp-Fc-nectin-1-EX, pFastBac1-Msp-Fc-nectin-2-EX, and pFastBac1-Msp-Fc-nectin-3-EX, respectively, according to the manufacturer's protocol (Invitrogen). The Nef-1, Nef-2, and Nef-3 proteins were expressed in High Five insect cells (Invitrogen) infected with the baculoviruses and bound to protein A-Sepharose beads (Amersham Biosciences). The bound proteins were eluted with 0.1 M glycine/HCl, pH 2.5, and immediately neutralized to pH 7.5 with 1.5 M Tris followed by dialysis against PBS. The purified proteins were frozen and stored at -80 °C until use.

pGEX-PAK-CRIB was a gift of Dr. S. Narumiya (Kyoto University, Kyoto, Japan). The glutathione-S-transferase (GST)·PAK·CRIB fusion protein was purified from overexpressing Escherichia coli according to the manufacturer's protocol (Amersham Biosciences).

Cell Culture and DNA Transfection-- L, EL, and rat 3Y1 cells were supplied from Dr. Sh. Tsukita (Kyoto University, Kyoto, Japan). EL cells were cloned by introduction of the exogenous E-cadherin cDNA to cadherin-deficient L cells (37). L cells stably expressing exogenous full-length nectin-1alpha , nectin-2alpha , nectin-3alpha , and C-terminal four aa-deleted nectin-1alpha (nectin-1-L, nectin-2-L, nectin-3-L, and nectin-1-Delta C-L cells, respectively) were prepared as described (8, 14, 15). EL cells stably expressing exogenous full-length nectin-1alpha (nectin-1-EL cells) were prepared as described (8). MDCK cells were kindly supplied from Dr. W. Birchmeier (Max-Delbruck-Center for Molecular Medicine, Berlin, Germany). MDCK cells stably expressing exogenous full-length nectin-1alpha (nectin-1-MDCK cells) were prepared as described (7). Transfection was done as described (7) with pEGFP, pEGFP-Rac1, pEGFP-NWASP-CRIB, pEGFP-N17Rac1, pEGFP-Cdc42, and pFLAG-CMV1-nectin-2alpha /TEM5 plasmids.

Assay for Formation of Filopodia and Lamellipodia-- For the assay by scanning electron microscopy, Nef-3- or human IgG-coated microbeads were prepared2 (38). Briefly, latex-sulfate micro-beads (3 × 108; 5.2-µm diameter; Interfacial Dynamics Corporation, Portland, OR) were washed, resuspended in 0.2 ml of 0.1 M borate buffer, pH 8.0, and incubated with 0.1 mg of a goat anti-human IgG (Fc-specific) polyclonal antibody (pAb) (Sigma) with gentle mixing at room temperature for 18 h. The beads were then centrifuged at 16,000 × g at 4 °C for 10 min and washed with 1 ml of PBS three times. The beads were incubated with 1 ml of PBS containing 0.5% bovine serum albumin (BSA) at room temperature for 3 h. The bead suspension (0.2 ml, 6 × 107 beads) was then incubated with 30 µg of Nef-3 or 50 µg of human IgG at room temperature for 3 h. After the incubation, the beads were washed with 1 ml of PBS containing 0.5% BSA three times and re-suspended in 0.2 ml of PBS containing 0.5% BSA. The human IgG-coated beads were used as control beads. Nectin-1-EL cells were cultured on glass coverslips in Dulbecco's modified Eagle's medium (DMEM) containing 10% FCS for 12 h, and then Nef-3- or human IgG-coated beads were added. The cells were further cultured for 60 min and then fixed in 2.5% glutaraldehyde in PBS at 4 °C for 1 h. The samples were rinsed with PBS and fixed with 1% osmium tetroxide in PBS at 4 °C for 1 h. The samples were dehydrated in graded ethanol and dried with liquid CO2. The samples were then coated with platinum (EIKO, IB-5) and analyzed at an accelerating voltage of 10 kV by a Hitachi S-800 scanning electron microscope.

For the assay by confocal laser scanning microscopy, coverslips were coated with Nef-1, Nef-2, Nef-3, or human IgG (50 µg/ml) for 15 h and blocked with 1% BSA in Hanks' balanced salt solution for 1 h. Each cell line except the nectin-1-EL cell line was washed with PBS, incubated with 0.2% trypsin and 1 mM EDTA at 37 °C for 5 min, and dispersed gently by pipetting. Nectin-1-EL cells were washed with PBS, incubated with 0.01% trypsin and 1 mM CaCl2 at 37 °C for 15 min, and dispersed by pipetting. The dispersed cells were then suspended in DMEM containing 10% FCS. The cells (1.0 × 104) were placed on the coverslip pre-coated with each Nef or human IgG as a control in a 24-well dish and cultured for indicated periods of time. The cells were then fixed, followed by immunostaining. Immunofluorescence microscopy of cultured cells was done as described (4, 7).

Pull-down Assay-- The pull-down assay was performed as described (39). Briefly, 25 µg of Nef-3 or human IgG (Sigma) was preclustered using 10 µg of the anti-human IgG pAb in 50 µl of PBS at room temperature for 1 h. Nectin-1-MDCK cells (3 × 106) were cultured in DMEM without FCS for 24 h. The cells were further cultured with Nef-3 or human IgG preclustered by the anti-human IgG pAb in 1 ml of DMEM for indicated periods of time. After being washed with ice-cold PBS, the cells were lysed with 0.5 ml of the lysis buffer (50 mM Tris/HCl, pH 7.4, 100 mM NaCl, 10 mM MgCl2, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM dithiothreitol, 1% Triton X-100, 10 µg/ml leupeptin, 10 µg/ml aprotinin, 10 µM (p-amidinophenyl) methanesulfonyl fluoride hydrochloride) containing 20 µg of recombinant GST·PAK·CRIB and incubated at 4 °C for 1 h. After incubation, the lysates were cleared by centrifugation. The cleared lysates were then incubated with glutathione-agarose beads (Amersham Biosciences) at 4 °C for 1 h. After centrifugation, the beads were washed with 0.5 ml of the lysis buffer three times. Cdc42 and Rac bound to the beads were eluted with Laemmli buffer and analyzed by Western blotting using anti-Cdc42 (Transduction Laboratories) and anti-Rac (Upstate Biotechnology) monoclonal Abs, respectively. The whole cell lysates were also analyzed for the presence of Cdc42 and Rac for normalization.

Other Procedures-- SDS-polyacrylamide gel electrophoresis was done as described (40). The amounts of recombinant proteins were determined with BSA as a standard protein by densitometric tracing of protein bands stained with Coomassie Brilliant Blue on an SDS-polyacrylamide gel (41).

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Formation of Filopodia and Lamellipodia by trans-Interaction of Nectin-1 with Nef-3-- We have shown previously that when the microbeads coated with Nef-3 are put on the surface of nectin-1-EL cells (L cells stably expressing exogenous nectin-1alpha and E-cadherin) and the cells are cultured, Nef-3 trans-interacts with cellular nectin-1 and recruits cellular nectin-1 and then E-cadherin to the bead-cell contact sites.2 When these bead-cell contact sites were analyzed by scanning electron microscopy, filopodia and lamellipodia were observed over the surface of the beads (Fig. 1a). Such structures were not observed with the microbeads coated with human IgG as a control (Fig. 1b). When nectin-1-EL cells were cultured on the coverslips coated with Nef-3 in the dishes, the cells first formed filopodia and then lamellipodia (Fig. 2, a1, b1, and c1). In the control experiments using IgG instead of Nef-3, the cells formed filopodia and lamellipodia to a much lesser extent (Fig. 2, a2, b2, and c2). Nef-3 formed filopodia and lamellipodia in wild-type EL cells but less than in nectin-1-EL cells (Fig. 3a1, and see also Fig. 2c1), whereas IgG formed filopodia and lamellipodia to a lesser extent in wild-type EL cells (Fig. 3a2). When nectin-1-L cells (L cells stably expressing exogenous nectin-1alpha alone) were used instead of nectin-1-EL cells, Nef-3 showed similar effects to those obtained with nectin-1-EL cells (Fig. 3b1, and see also Fig. 2c1), whereas IgG formed filopodia and lamellipodia to a much lesser extent (Fig. 3b2). When wild-type L cells were used, Nef-3 showed similar effects to those obtained with wild-type EL cells (Fig. 3c1, and see also Fig. 3a1), whereas IgG formed filopodia and lamellipodia to a lesser extent (Fig. 3c2). These results indicate that the trans-interaction of nectin-1 and nectin-3 forms filopodia and lamellipodia in a manner independent of E-cadherin.


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  		Fig. 1.   Scanning electron microscopy analysis on the formation of filopodia and lamellipodia by the trans-interaction of nectin-1 with Nef-3. Nectin-1-EL cells were incubated with beads coated with Nef-3 or human IgG for 60 min. The cells were fixed and observed by the scanning electron microscope. a, Nef-3. b, IgG. Bars, 1 µm. The results shown are representative of three independent experiments.


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  		Fig. 2.   Confocal laser scanning microscopy analysis on the formation of filopodia and lamellipodia by the trans-interaction of nectin-1 with Nef-3. Nectin-1-EL cells were cultured on the coverslips coated with Nef-3 or human IgG for indicated periods of time. The cells were fixed and then stained with rhodamine-phalloidin. a, 10 min. b, 30 min. c, 60 min. 1, Nef-3. 2, IgG. Bars, 10 µm. The results shown are representative of three independent experiments.


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  		Fig. 3.   Formation of filopodia and lamellipodia by the trans-interaction of nectin-1 with Nef-3 in wild-type EL, nectin-1-L, and wild-type L cells. Wild-type EL, nectin-1-L, and wild-type L cells were cultured on the coverslips coated with Nef-3 or human IgG for 60 min. The cells were fixed and then stained with rhodamine-phalloidin. a, wild-type EL cells. b, nectin-1-L cells. c, wild-type L cells. 1, Nef-3. 2, IgG. Bars, 10 µm. The results shown are representative of three independent experiments.

Nef-3 further formed filopodia and lamellipodia in wild-type 3Y1 cells (Fig. 4A, a1, b1, and c1), whereas IgG formed filopodia and lamellipodia to a much lesser extent (Fig. 4A, a2, b2, and c2). Wild-type 3Y1 cells expressed the endogenous nectin-1 protein (data not shown). Nef-3 furthermore formed filopodia and lamellipodia in nectin-1-MDCK cells (MDCK cells stably expressing exogenous nectin-1alpha ) (Fig. 4B, a1, b1, and c1), whereas IgG formed filopodia and lamellipodia to a negligible extent (Fig. 4B, a2, b2, and c2). The time courses of the formation of filopodia and lamellipodia in nectin-1-MDCK cells were not significantly different. These results indicate that the trans-interaction of nectin-1 and nectin-3 forms filopodia and lamellipodia in a variety of cell lines.


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  		Fig. 4.   Formation of filopodia and lamellipodia by the trans-interaction of nectin-1 with Nef-3 in wild-type 3Y1 and nectin-1-MDCK cells. Wild-type 3Y1 and nectin-1-MDCK cells were cultured on the coverslips coated with Nef-3 or human IgG for indicated periods of time. The cells were fixed and then stained with rhodamine-phalloidin. A, wild-type 3Y1 cells. B, nectin-1-MDCK cells. a, 15 min. b, 30 min. c, 60 min. 1, Nef-3. 2, IgG. Bars, 10 µm. The results shown are representative of three independent experiments.

Formation of Filopodia and Lamellipodia by trans-Interactions of Various Combinations of Nectins and Nefs-- When nectin-3-L cells and Nef-1 were used, essentially the same results as those observed with nectin-1-L cells and Nef-3 (Fig. 5a3) were obtained (Fig. 5c1). When nectin-1-L cells and Nef-3 were used, essentially the same results were obtained, although the Nef-3 effects were less than those on nectin-1-L cells (Fig. 5b3, and see also Fig. 5a3). Essentially the same results were obtained with other combinations such as nectin-1-L cells and Nef-1 (Fig. 5a1), nectin-2-L cells and Nef-2 (Fig. 5b2), nectin-3-L cells and Nef-2 (Fig. 5c2), and nectin-3-L cells and Nef-3 (Fig. 5c3), although their effects and the time courses varied depending on nectins and Nefs. Nectin-1 does not trans-interact with nectin-2 (15). Nef-1 and Nef-2 were inactive on nectin-2-L and nectin-1-L cells, respectively (Fig. 5, b1 and a2). IgG as a control formed filopodia and lamellipodia to a much lesser extent in nectin-1-L, nectin-2-L, and nectin-3-L cells (Fig. 5, a4, b4, and c4). These effects of Nefs were apparently parallel to the activities of homo- and hetero-trans-interactions of nectins to induce the aggregation of L cells2 (15). These results indicate that the homo- and hetero-trans-interactions of nectins transduce signals bidirectionally and form filopodia and lamellipodia.


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  		Fig. 5.   Formation of filopodia and lamellipodia by the trans-interactions of various combinations of nectins and Nefs. Nectin-1-L, nectin-2-L, or nectin-3-L cells were cultured on the coverslips coated with Nef-1, Nef-2, Nef-3, or human IgG for 60 min. The cells were fixed and then stained with rhodamine-phalloidin. a, nectin-1-L cells. b, nectin-2-L cells. c, nectin-3-L cells. 1, Nef-1. 2, Nef-2. 3, Nef-3. 4, IgG. Bars, 10 µm. The result shown in a3 was the reproduction of that shown in Fig. 3b1. The results shown are representative of three independent experiments.

Involvement of Cdc42 and Rac in the Nef-3 Effects-- Cdc42 and Rac are involved in the formation of filopodia and lamellipodia, respectively (for a review, see Ref. 42). We next examined with the use of nectin-1-L cells and Nef-3 whether these small G proteins are involved in the Nef-3 effects. NWASP-CRIB specifically binds to GTP-Cdc42 and suppresses the Cdc42 action (for a review, see Ref. 43). Overexpression of green fluorescent protein (GFP)-NWASP-CRIB in nectin-1-L cells cultured on the Nef-3-coated coverslips reduced the formation of filopodia and lamellipodia (Fig. 6, a1-a4). N17Rac1, a dominant negative mutant of Rac1, inhibits the production of GTP-Rac (44). Overexpression of GFP-N17Rac1 reduced the formation of lamellipodia but not that of filopodia (Fig. 6, b1-b4). In contrast, overexpression of GFP alone inhibited neither the formation of filopodia nor that of lamellipodia (Fig. 6, c1-c4). These results suggest that Nef-3-formed filopodia and lamellipodia are mediated through the activation of Cdc42 and Rac, respectively. Overexpression of C3, which inhibits the Rho actions, resulted in rounding up the cells, and we could not judge whether Rho is involved in the Nef-3 effects (data not shown).


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  		Fig. 6.   Involvement of Cdc42 and Rac in the Nef-3 effects. GFP-NWASP-CRIB, GFP-N17Rac1, or GFP was transiently expressed in nectin-1-L cells. The cells were then cultured on the coverslips coated with Nef-3 for 60 min, followed by immunostaining with rhodamine-phalloidin and the anti-nectin-1alpha pAb (15). a, GFP-NWASP-CRIB. b, GFP-N17Rac1. c, GFP. 1, the F-actin signal. 2, the GFP signal. 3, the nectin-1alpha signal. 4, merge. Bars, 10 µm. The results shown are representative of three independent experiments.

Nef-3-induced Activation of Cdc42 and Rac-- We then examined whether Nef-3 induces the activation of Cdc42 and Rac in nectin-1-L cells. When GFP-Cdc42 or GFP-Rac1 was transiently expressed in nectin-1-L cells, and these cells were cultured with the Nef-3-coated beads, GFP-Cdc42 and GFP-Rac1 were highly concentrated at the bead-cell contact sites (Fig. 7A, a1 and b1). However, neither GFP-Cdc42 nor GFP-Rac1 was concentrated at the IgG-coated bead-cell contact sites (Fig. 7A, a2 and b2). These results suggest that the trans-interaction of nectin-1 with Nef-3 induces the activation of Cdc42 and Rac at the Nef-3-coated bead-cell contact sites.


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  		Fig. 7.   Nef-3-induced activation of Cdc42 and Rac. A, localization of Cdc42 and Rac1. GFP-Cdc42 or GFP-Rac1 was transiently expressed in nectin-1-L cells. The cells were cultured with the Nef-3- or human IgG-coated beads for 60 min. The cells were fixed and observed by the confocal laser scanning microscope. a, GFP-Cdc42. b, GFP-Rac1. 1, Nef-3. 2, IgG. Bars, 2.5 µm. The positions of the beads are marked with asterisks. The arrows indicate the bead-cell contact sites. B, activation of Cdc42 and Rac by Nef-3. Nectin-1-MDCK cells were cultured with Nef-3 or human IgG preclustered by the anti-human IgG pAb for indicated periods of time. The cells were then subjected to the pull-down assay. a, Cdc42. b, Rac. The results shown are representative of three independent experiments.

We next measured the formation of GTP-Cdc42 and GTP-Rac by pull-down assay using GST·PAK·CRIB. The expression levels of the endogenous Cdc42 and Rac proteins in nectin-1-L cells were hardly detectable by Western blotting (data not shown), whereas the expression levels of these proteins in nectin-1-MDCK cells were detectable (Fig. 7B, a and b). Therefore, we used nectin-1-MDCK cells for the pull-down assay. First, we detached nectin-1-MDCK cells from a cell culture dish and then replated them on the Nef-3-coated dish in the same way as that shown in Fig. 4B. This detachment caused elevation of the levels of GTP-Cdc42 and GTP-Rac, and the elevated levels did not decrease to the basal levels for at least 1 h after the replating (data not shown). Therefore, we cultured nectin-1-MDCK cells for 24 h and then stimulated the cells by adding the medium containing soluble Nef-3, which was preclustered by the anti-human IgG pAb. After being stimulated by soluble Nef-3 for 15, 30, and 60 min, nectin-1-MDCK cells were washed with PBS and immediately lysed on the dish with the lysis buffer containing GST·PAK·CRIB. The lysates were subjected to the pull-down assay. The level of GTP-Cdc42 started to increase at 30 min after the addition of soluble Nef-3 (Fig. 7Ba), whereas the level of GTP-Cdc42 did not change by the addition of human IgG as a control. The level of GTP-Rac also started to increase at 30 min after the addition of soluble Nef-3 (Fig. 7Bb), whereas the level of GTP-Rac did not change with the addition of human IgG. These results indicate that Nef-3 indeed induces the activation of Cdc42 and Rac. We could not practically observe different time courses between the activation of Cdc42 and Rac by this assay.

The Cytoplasmic Tail of Nectin-dependent Effect of Nef-3-- We examined whether the Nef-3 effects are dependent on the association of nectins with afadin. In nectin-1-Delta C-L cells stably expressing C-terminal four aa-deleted nectin-1alpha incapable of binding afadin, Nef-3 formed filopodia and lamellipodia to extents similar to those observed with nectin-1-L cells (Fig. 8a1, and see also Fig. 3b1), indicating that the association of nectins with afadin is not essential for the Nef-3 effects. In contrast, nectin-1-Delta C-L cells on the IgG-coated coverslips formed filopodia and lamellipodia to a much lesser extent (Fig. 8a2). We then examined whether the Nef-3 effects are dependent on the cytoplasmic tail of nectins. For this experiment, we used nectin-2-L cells. Nef-3 formed filopodia and lamellipodia in nectin-2-L cells (see Fig. 5b3), whereas IgG formed filopodia and lamellipodia to a much lesser extent (see Fig. 5b4). We attempted to make L cells stably expressing the nectin-2alpha mutant consisting of the extracellular and transmembrane domains and the first six aa of the cytoplasmic tail (aa 1-383). However, this mutant was not sorted into the plasma membrane (data not shown). Therefore, we expressed the nectin-2/TEM5 chimera protein, the fragment of nectin-2alpha containing the extracellular and transmembrane domains, which is fused to the cytoplasmic tail of TEM5 in L cells. This protein was sorted into the plasma membrane (data not shown). TEM5 is a seven-pass transmembrane protein of which function is unknown (45). Nef-3 formed filopodia and lamellipodia to a small extent in L cells transiently expressing the nectin-2/TEM5 chimera protein (Fig. 8b1). The extent of this effect of Nef-3 was less than that obtained with nectin-2-L cells (see Fig. 5b3) and similar to that obtained with wild-type L cells (see Fig. 3c1). The effect of Nef-3 on L cells expressing the nectin-2/TEM5 chimera protein may be due to the endogenously expressed nectins in L cells. These results indicate that the cytoplasmic tail of nectins is essential for the Nef-3 effects.


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  		Fig. 8.   The cytoplasmic tail of nectin-dependent effect of Nef-3. Each L cell line was cultured on the coverslips coated with Nef-3 or human IgG for 60 min. The cells were fixed and then stained with rhodamine-phalloidin. a, nectin-1-Delta C-L cells. b, L cells transiently expressing the nectin-2/TEM5 chimera protein. 1, Nef-3. 2, IgG. Bars, 10 µm. The results shown are representative of three independent experiments.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

We have shown here by the use of Nefs that the homo- and hetero-trans-interactions of nectins form filopodia and lamellipodia in a manner independent of E-cadherin. The formation of filopodia and lamellipodia in this way is reduced by an inhibitor of Cdc42 (NWASP-CRIB), whereas the formation of lamellipodia but not of filopodia is reduced by a dominant negative mutant of Rac1 (N17Rac1). We have moreover shown that the trans-interactions of nectins induce the activation of Cdc42 and Rac. We have previously shown in L cells that a dominant active mutant of Cdc42 (V12Cdc42) induces the formation of filopodia but not of lamellipodia, whereas a dominant active mutant of Rac1 (V12Rac1) induces only the formation of lamellipodia, indicating that Cdc42 alone does not activate Rac in this cell line (36). Taken together, it is likely in L cells that the trans-interactions of nectins induce the formation of filopodia and lamellipodia through the respective activation of Cdc42 and Rac and that the activation of Cdc42 is necessary, but not sufficient, for the activation of Rac, whereas the activation of Rac is not necessary for the activation of Cdc42. We have moreover shown here that the effects of the trans-interactions of nectins require their cytoplasmic tail but not their association with afadin. It remains unknown how Cdc42 is activated by the trans-interactions of nectins, but the trans-interactions of nectins may induce the activation of a GDP/GTP exchange protein (GEP) for Cdc42 by the direct or indirect binding of the GEP to the cytoplasmic tail of nectins. It also remains unknown how Rac1 is activated by the trans-interactions of nectins, but the trans-interactions of nectins may induce the activation of a GEP for Rac1 in cooperation with Cdc42.

Three functions of the nectin-induced activation of Cdc42 and Rac1 are conceivable. 1) the nectin-induced activation of Cdc42 and Rac1 reorganizes the actin cytoskeleton through their F-actin-binding downstream effectors such as NWASP, IQGAP, and IRSp53-WAVE (43) in the process of the AJ formation. When the two migrating cells meet through the protrusions, such as Cdc42-induced filopodia and Rac1-induced lamellipodia, primordial spot-like junctions are formed followed by maturation to AJs. We have previously proposed that nectins first trans-interact to form micro-clusters, which then recruit E-cadherin at the initial cell-cell contact sites, resulting in the formation of the mixture of the nectin- and cadherin-based micro-clusters.2 These primordial junctions fuse with each other to form short line-like junctions, which develop into more maturated AJs. Once nectins trans-interact, they reorganize the actin cytoskeleton through the respective activation of Cdc42 and Rac1, which in turn enhance the formation of maturated AJs. The trans-interaction of E-cadherin further reorganizes the actin cytoskeleton through the activation of Rac1 (26, 32-34). 2) the nectin-induced activation of Cdc42 and Rac1 may be involved in gene expression through the activation of the mitogen-activated protein (MAP) kinase cascades. 3) the nectin-induced activation of Cdc42 and Rac1 may be involved in the assembly of the cell polarity proteins Par6, Par3, and atypical protein kinase C, which form a ternary complex (for a review, see Ref. 46). Par6 is a downstream effector of Cdc42 and Rac (46). Par3 has recently been shown to directly associate with the junctional adhesion molecule (JAM) (46), and we have recently found that nectins are involved in the localization of JAM at tight junctions (38). Further studies are necessary for establishing the physiological role of the nectin-induced activation of Cdc42 and Rac1.

    ACKNOWLEDGEMENTS

We thank Dr. S. Narumiya (Kyoto University, Kyoto, Japan) for his generous gifts of pEGFP-Cdc42, pEGFP-Rac1, pEGFP-N17Rac1, and pGEX-PAK-CRIB constructs, Dr. Sh. Tsukita (Kyoto University, Kyoto, Japan) for his generous gifts of L, EL, and rat 3Y1 cells, and Dr. W. Birchmeier (Max-Delbruck-Center for Molecular Medicine, Berlin, Germany) for his generous gift of MDCK cells.

    FOOTNOTES

* This work 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. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed. Tel.: 81-6-6879-3410; Fax: 81-6-6879-3419; E-mail: ytakai@molbio.med.osaka-u.ac.jp.

Published, JBC Papers in Press, October 11, 2002, DOI 10.1074/jbc.M209846200

2 Honda, T., Shimizu, K., Kawakatsu, T., Yasumi, M., Shingai, T., Fukuhara, A., Ozaki-Kuroda, K., Irie, K., Nakanishi, H., and Takai, Y. (2002) Genes Cells, in press.

    ABBREVIATIONS

The abbreviations used are: AJ, adherens junction; aa, amino acid(s); BSA, bovine serum albumin; CRIB, Cdc42 and Rac interactive binding domain; DMEM, Dulbecco's modified Eagle's medium; F-actin, actin filaments; GEP, GDP/GTP exchange protein; GFP, green fluorescent protein; GST, glutathione-S-transferase; MDCK, Madin-Darby canine kidney cells; Nef, the extracellular fragment of nectin fused to the Fc portion of IgG; pAb, polyclonal antibody; PBS, phosphate-buffered saline.

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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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