Correspondence to: Yoshimi Takai, Department 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.
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Abstract |
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We have found a new cellcell adhesion system at cadherin-based cellcell adherens junctions (AJs) consisting of at least nectin and l-afadin. Nectin is a Ca2+-independent homophilic immunoglobulin-like adhesion molecule, and l-afadin is an actin filament-binding protein that connects the cytoplasmic region of nectin to the actin cytoskeleton. Both the trans-interaction of nectin and the interaction of nectin with l-afadin are necessary for their colocalization with E-cadherin and catenins at AJs. Here, we examined the mechanism of interaction between these two cellcell adhesion systems at AJs by the use of -catenindeficient F9 cell lines and cadherin-deficient L cell lines stably expressing their various components. We showed here that nectin and E-cadherin were colocalized through l-afadin and the COOH-terminal half of
-catenin at AJs. Nectin trans-interacted independently of E-cadherin, and the complex of E-cadherin and
- and ß-catenins was recruited to nectin-based cellcell adhesion sites through l-afadin without the trans-interaction of E-cadherin. Our results indicate that nectin and cadherin interact through their cytoplasmic domainassociated proteins and suggest that these two cellcell adhesion systems cooperatively organize cellcell AJs.
Key Words: immunoglobulin superfamily, afadin, ponsin, catenin, cellcell adherens junctions
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Introduction |
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We have recently identified a novel cellcell adhesion system at cadherin-based cellcell adherens junctions (AJs)1 consisting of at least nectin and l-afadin (-herpes virus entry and cellcell spread mediator (
and -1ß/HIgR (
and -2
(
, -3ß, and -3
(
, -2
, -2
, -3
, and -3ß, but not nectin-1ß/HIgR and -3
, have a COOH-terminal conserved motif of four amino acid (aa) residues (E/A-X-Y-V) that interacts with the PDZ domain of l-afadin (
We have isolated another l-afadinbinding protein, named ponsin, which is colocalized with nectin and l-afadin at cadherin-based AJs (-catenin (
The cadherin/catenin system plays essential roles not only in the formation and maintenance of cellcell AJs, but also in the organization of tight junctions (TJs; -catenin (
-Catenin interacts with F-actin (
-catenin interacts with other F-actinbinding proteins,
-actinin and vinculin, through the NH2-terminal half (
By analogy with cadherin, we have shown that nectin exhibits cis-dimerization, which may be a key regulatory step for its trans-interaction ( with l-afadin and their colocalization with the cadherin/catenin system are necessary for efficient cellcell spread of herpes simplex virus type 1 (HSV1; Sakisaka, T., T. Taniguchi, H. Nakanishi, K. Takahashi, M. Miyahara, W. Ikeda, S. Yokoyama, Y.-F. Peng, K. Yamanishi, and Y. Takai, manuscript submitted for publication). However, it has not been understood which component(s) of the cadherin/catenin system is required for the colocalization with the nectin/afadin system. It has not been determined how these two cell adhesion systems are functionally correlated to each other. In this study, we have examined the mechanism of interaction between the nectin/afadin and cadherin/catenin systems by the use of
-catenindeficient F9 cell lines and cadherin-deficient L cell lines stably expressing their various components.
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Materials and Methods |
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Construction, Expression, and Purification
Mammalian expression vectors were constructed with pPGKIH (
Expression vectors for human nectin-1 and mouse nectin-2
(shown in Fig 1) contained the following aa: pPGKIH-nectin-1
, aa 1518 (full-length); pPGKIH-nectin-1
-
C, aa 1514 (deletion of the COOH-terminal four aa residues); pPGKIH-FLAG-nectin-1
, aa 27518; pPGKIH-FLAG-nectin-1
-
C, aa 27514; pPGKIH-nectin-2
, aa 1467 (full-length); and pPGKIH-nectin-2
-
C, aa 1463 (deletion of the COOH-terminal four aa residues). An expression vector for mouse ponsin-2 (pEGFP-ponsin) contained aa 1724 (full-length; see Fig 1). An expression vector for l-afadin (pFLAG-CMV2-l-afadin) contained aa 11,829 (full-length; see Fig 1). Expression vectors for mouse E-cadherin and
-catenin contained the following aa: pEF-tEC, aa 562728 (cytoplasmic region and partial transmembrane segment of E-cadherin); pPGKIZ-HA-
-catenin, aa 1906 (full-length); and pPGKIZ-HA-
-catenin-C, aa 509906 (COOH-terminal half; see Fig 1). pEF-tEC was designed to express the NH2-terminal T7 peptide (M-A-S-M-T-G-G-Q-Q-M-G)tagged protein.
|
Baculovirus transfer vectors were constructed with pFastBac1-Myc-His6 and -Msp-Fc. pFastBac1-Myc-His6 was constructed to express a COOH-terminal Myc- and His6-tagged protein by inserting a cDNA fragment encoding the Myc epitope and His6 (hexahistidine) tags of pcDNA3.1(-)/Myc-His (Invitrogen) into pFastBac1 (GIBCO BRL). pFastBac1-Msp-Fc was constructed to express a chimeric protein fused with the NH2-terminal honeybee melittin signal peptide and the COOH-terminal human IgG Fc by inserting cDNA fragments encoding the signal peptide (5'-ATG AAA TTC TTA GTC AAC GTT GCC CTT GTT TTT ATG GTC GTG TAC ATT TCT TAC ATC TAT GCG-3';
Glutathione S-transferase (GST) fusion vectors for -catenin contained the following aa: GST-
-catenin, aa 1906 (full-length;
-catenin-C, aa 509906 (COOH-terminal half; see Fig 1). A GST fusion vector for ZO-1 (GST-ZO-1-N) contained aa 1862 (NH2-terminal half;
Cell Culture and DNA Transfection
F9, L, EL, and COS7 cell lines were maintained in Dulbecco's modified Eagle's medium supplemented with 10% FCS. F9 cells were cultured in gelatin-coated (0.1%) culture dishes. -Catenindeficient F9 cells (F9D
(-/-) cells) and F9D
(-/-) cells reexpressing
-catenin (
F9D
(-/-) cells) were obtained as previously described (
-L cells, the fusion molecule consisting of nonfunctional E-cadherin lacking its catenin-binding domain (aa 1657) and full-length
-catenin (aa 1906;
N-L cells, the fusion molecule consisting of nonfunctional E-cadherin lacking its catenin-binding domain and the NH2-terminal half of
-catenin (aa 1508;
C-L cells, the fusion molecule consisting of nonfunctional E-cadherin lacking its catenin-binding domain and the COOH-terminal half of
-catenin (aa 509906;
-L, nE
N-L, and nE
C-L cells were obtained as previously described (
The following L and EL cell lines were cloned by the introduction of the pPGKIH and pPGKIH-FLAG vectors, respectively, as described previously (-L cells, pPGKIH-nectin-1
(aa 1518); nectin-1
-
C-L cells, pPGKIH-nectin-1
-
C (aa 1514); nectin-2
-L cells, pPGKIH-nectin-2
(aa 1467); nectin-2
-
C-L cells; pPGKIH-nectin-2
-
C (aa 1463); nectin-1
-EL cells, pPGKIH-FLAG-nectin-1
(aa 27518); and nectin-1
-
C-EL cells, pPGKIH-FLAG-nectin-1
-
C (aa 27514). In brief, L and EL cells were transfected with the vectors described above using Lipofectamine reagent (GIBCO BRL) according to the manufacturer's protocol. The cells were cultured for 1 d, replated, and selected by culturing in the presence of 500 µg/ml of hygromycin (GIBCO BRL). To prepare nectin-1
-L and -2
-L cells, both of which transiently expressed
- and/or ß-catenins, the cells were transfected with pPGKIZ-HA-
-catenin or pEF-tEC using Lipofectamine reagent. The cells were cultured for 1 d, replated, and cultured for 3 d.
Antibodies
One rabbit polyclonal antinectin-1 antibody (Ab) was raised against GST-nectin-1
-CPN (aa 379438) as previously described (
Ab1. Another rabbit polyclonal antinectin-1
Ab was raised against the 19-mer synthetic peptide (corresponding to aa 450468 of nectin-1
) as previously described (
Ab2. A rabbit polyclonal antinectin-2
Ab was prepared as described previously (
and -2
, was prepared as described previously (
Yeast Two-Hybrid Interaction and ß-Galactosidase Assay
The bait vectors, pBTM116-HA--catenin (full-length, aa 1906), -HA-
-catenin-N (NH2-terminal half, aa 1508), -HA-
-catenin-C (COOH-terminal half, aa 509906), and -HA-nectin-2
-CP (cytoplasmic region, aa 387467) were constructed by inserting cDNA fragments encoding the respective aa residues of
-catenin and nectin-2
into pBTM116-HA (see Fig 1;
Affinity Chromatography and Immunoprecipitation
Myc-His6-l-afadin (20 µg of protein) was immobilized on TALON metal affinity beads (CLONTECH Laboratories, Inc.) (100 µl of wet volume). GST--catenin and -
-catenin-C (100 µg of protein each) were separately applied to the Myc-His6-l-afadinimmobilized beads equilibrated with buffer A (20 mM Tris-Cl, pH 7.5, 150 mM NaCl, 0.1% Triton X-100, 10 µg/ml leupeptin, 1 mM PMSF, and 1 µg/ml pepstatin A). After the beads were extensively washed with buffer A, elution was performed with buffer A containing 100 mM imidazole chloride, pH 7.5. Each fraction was subjected to SDS-PAGE (10% polyacrylamide gel), followed by protein staining with Coomassie brilliant blue. Where indicated, GST-ZO-1-N and -
-catenin-C (100 µg of protein each) were mixed and subjected to affinity chromatography.
Immunoprecipitation was performed as described previously (-catenin or -
-catenin-C were prepared with transfection of pFLAG-CMV2-l-afadin and pPGKIZ-HA-
-catenin or -HA-
-catenin-C, respectively, using Lipofectamine reagent. The cells were sonicated in buffer B (20 mM Tris-Cl, pH 7.5, 150 mM NaCl, 1% Triton X-100, 1 mM EDTA, 10 µg/ml leupeptin, 1 mM PMSF, and 1 µg/ml pepstatin A), followed by centrifugation at 100,000 g for 30 min. The supernatant was incubated with the anti-FLAG or anti-HA mAb at 4°C for 2 h. Protein GSepharose beads (Amersham Pharmacia Biotech) were added to this sample, and incubation was further performed at 4°C for 1 h. After the beads were extensively washed with buffer B, the bound proteins were eluted by boiling the beads in an SDS sample buffer (60 mM Tris-Cl, pH 6.7, 3% SDS, 2% [vol/vol] 2-mercaptoethanol, and 5% glycerol), and subjected to SDS-PAGE, followed by Western blot analysis.
Coculture of L and EL Cells Stably Expressing Nectin
Nectin-1-L, -1
-EL, and -1
-
C-EL cells were washed with PBS, incubated with 0.2% trypsin and 1 mM EDTA at 37°C for 5 min, and dispersed by gentle pipetting to obtain single cell suspensions. The cell number of each cell line was counted, and nectin-1
-EL or -1
-
C-EL cells were mixed with a 10-fold number of nectin-1
-L cells. These mixed cells were plated at 2 x 105 cells on a 35-mm culture dish and cultured for 2 d. Where indicated, gD was added to the mixed cell suspension to give a final concentration of 0.3 µM. The cells were further cultured with the same concentration of gD for 2 d.
Other Procedures
Immunofluorescence microscopy of cultured cells was done as described previously (
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Results |
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-Catenindependent Colocalization of Nectin and l-Afadin with E-Cadherin
We first examined whether -catenin is involved in the colocalization of nectin and l-afadin with E-cadherin. For this purpose, we used an
-catenindeficient F9 cell line, F9D
(-/-) cells (
(-/-) cells were generated by a disruption of both
-catenin alleles with a targeting vector. As a control, we used
F9D
(-/-) cells, which were generated by the introduction of an expression vector encoding full-length
-catenin into F9D
(-/-) cells (
F9D
(-/-) cells reexpress
-catenin in an amount similar to that of wild-type F9 cells (
F9D
(-/-) cells, E-cadherin was localized at beltlike cellcell adhesion sites, where nectin-2 and l-afadin were colocalized (Fig 2 A). In F9D
(-/-) cells, E-cadherin showed the similar staining pattern, but nectin-2 and l-afadin were hardly concentrated at cellcell adhesion sites between two cells, where E-cadherin was concentrated (Fig 2 B). Nectin-2 and l-afadin were colocalized with E-cadherin only at spotlike cellcell adhesion sites where >2 cells adhered to each other. Western blot analysis indicated that the protein levels of nectin-2
and l-afadin were similar between
F9D
(-/-) and F9D
(-/-) cells (data not shown). It has been shown that the protein levels of E-cadherin and ß-catenin in
F9D
(-/-) cells are comparable to those in F9D
(-/-) cells (
-catenin, but not on the cytoplasmic region of E-cadherin or ß-catenin, in F9 cells.
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ß-Cateninindependent Colocalization of Nectin and l-Afadin with E-Cadherin
We have previously shown that nectin-2 and l-afadin are colocalized with E-cadherin at cellcell AJs in EL cells () of catenin-binding domain-deleted E-cadherin fused with full-length
-catenin (nE
-L cells). In the junctional level of nE
-L cells, nectin-2 and l-afadin were colocalized at cellcell adhesion sites where the nE
molecule was localized (Fig 3 A). Ponsin and vinculin were also colocalized with nE
there. In the basal level, nE
, nectin-2, or l-afadin was not concentrated at focal contacts (data not shown), whereas ponsin and vinculin were colocalized there (Fig 3 B). These results are consistent with those obtained with the two F9 cell lines described above, and indicate that ß-catenin is not essential for the colocalization of nectin, l-afadin, ponsin, and vinculin with E-cadherin at cellcell adhesion sites.
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Ponsin- and Vinculin-independent Colocalization of Nectin and l-Afadin with E-Cadherin
We next examined whether ponsin and vinculin are necessary for the colocalization of nectin and l-afadin with E-cadherin. For this purpose, we took advantage of two L cell lines. One cell line (nEN-L) is an L cell clone stably expressing a chimeric protein (nE
N) of catenin-binding domaindeleted E-cadherin fused with the NH2-terminal half of
-catenin, which is capable of binding vinculin and
-actinin but not ZO-1 (
C-L) is an L cell clone stably expressing a chimeric protein (nE
C) of catenin-binding domaindeleted E-cadherin fused with the COOH-terminal half of
-catenin, which is capable of binding ZO-1, but not vinculin or
-actinin (
N-L cells, the nE
N molecule was localized at cellcell adhesion sites where neither nectin-2 nor l-afadin was concentrated (Fig 4 A). However, vinculin was colocalized with nE
N there. Ponsin appeared to be also colocalized with nE
N there, but the staining was not clear (data not shown). When a GFP-tagged protein of full-length ponsin (GFP-ponsin) was transiently expressed, it was clearly colocalized with nE
N at cellcell adhesion sites (Fig 4 A). In the basal level of nE
N-L cells, ponsin and vinculin were colocalized at focal contacts (Fig 4 B).
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In the junctional level of nEC-L cells, the nE
C molecule was localized at cellcell adhesion sites, where nectin-2 and l-afadin were colocalized (Fig 5 A). However, neither ponsin nor vinculin was concentrated with nE
C there (Fig 5 A), although they were colocalized at focal contacts (Fig 5 B). We have previously shown that the protein levels of nectin-1
and l-afadin are similar among nE
-L, nE
N-L, and nE
C-L cells (Sakisaka, T., T. Taniguchi, H. Nakanishi, K. Takahashi, M. Miyahara, W. Ikeda, S. Yokoyama, Y.-F. Peng, K. Yamanishi, and Y. Takai, manuscript submitted for publication). In addition, Western blot analysis indicated that the protein levels of nectin-2
, ponsin, and vinculin were also similar among these three L cell lines (data not shown). These results indicate that neither ponsin nor vinculin is necessary for the colocalization of nectin and l-afadin with E-cadherin at cellcell adhesion sites, and that their colocalization is dependent on the COOH-terminal half of
-catenin, but not on the cytoplasmic region of E-cadherin or the NH2-terminal half of
-catenin.
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Molecular Linkage between l-Afadin and -Catenin
We have previously shown that l-afadin does not directly interact with -catenin by affinity chromatography using the purified samples (
-catenin is homologous to vinculin (
-catenin. The yeast two-hybrid analysis revealed that l-afadin (pGAD424-HA-l-afadin) interacted with the COOH-terminal half (pBTM-116-HA-
-catenin-C), but not with the full-length (pBTM-116-HA-
-catenin) or the NH2-terminal half (pBTM-116-HA-
-catenin-N; Table 1). This yeast two-hybrid interaction of l-afadin with the COOH-terminal half of
-catenin was comparable to that of l-afadin with the cytoplasmic region of nectin-2
(pBTM-116-HA-nectin-2
-CP). We further confirmed the in vitro direct interaction of l-afadin with
-catenin by affinity chromatography. A GST fusion protein of the COOH-terminal half of
-catenin (GST-
-catenin-C) bound to a Myc- and His6-tagged protein of full-length l-afadin (Myc-His6-l-afadin) immobilized on metal affinity beads (Fig 6), whereas a GST fusion protein of the full-length (GST-
-catenin) hardly bound to the beads (data not shown). However, the stoichiometry of the interaction of GST-
-catenin-C with Myc-His6-l-afadin was
0.1:1. It has been shown that the NH2-terminal half of ZO-1 directly interacts with the AF-6 protein/s-afadin (
-catenin (
-catenin with l-afadin, a GST fusion protein of the NH2-terminal half of ZO-1 (GST-ZO-1-N) was mixed with GST-
-catenin-C, and the mixture was subjected to the Myc-His6-l-afadinimmobilized affinity chromatography. However, GST-ZO-1-N did not enhance the stoichiometry of the interaction of GST-
-catenin-C with Myc-His6-l-afadin (data not shown). To further confirm the interaction of l-afadin with the COOH-terminal half of
-catenin in intact cells, a FLAG-tagged protein of full-length l-afadin (FLAG-l-afadin) and an HA-tagged protein of the COOH-terminal half of
-catenin (HA-
-catenin-C) were coexpressed in COS7 cells and immunoprecipitated by either the anti-FLAG or anti-HA Ab, but they were not coimmunoprecipitated to a significant extent (data not shown). However, negative results obtained from immunoprecipitation experiments do not necessarily reflect in vivo negative proteinprotein interactions. Therefore, these results suggest that l-afadin directly interacts with
-catenin under appropriate conditions.
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l-Afadindependent Recruitment of -Catenin to Nectin-based CellCell Adhesion Sites in the Absence of E-Cadherin
In the next series of experiments, we examined whether -catenin and ß-catenin are recruited through l-afadin to nectin-based cellcell adhesion sites in the absence of E-cadherin. For this purpose, we used L cells stably expressing full-length nectin-2
(nectin-2
-L cells) or the COOH-terminal four aa-deleted nectin-2
(nectin-2
-
C-L cells). We have previously shown that nectin-2
-L and -2
-
C-L cells show similar cell adhesion activity, as estimated by a cell aggregation assay (
and l-afadin in nectin-2
-L cells are similar to those in nectin-2
-
C-L cells, respectively. However, in nectin-2
-L cells, l-afadin is concentrated at nectin-2
based cell adhesion sites, whereas in nectin-2
-
C-L cells, l-afadin is not recruited to nectin-2
-
Cbased cell adhesion sites (
-catenin (HA-
-catenin) was transiently expressed in nectin-2
-L cells, HA-
-catenin was concentrated at nectin-2
-based cellcell adhesion sites (Fig 8, top). However, when HA-
-catenin was transiently expressed in nectin-2
-
C-L cells, HA-
-catenin was not concentrated at nectin-2
-
Cbased cellcell adhesion sites (Fig 8, bottom). Similar results were obtained with L cells stably expressing full-length nectin-1
(nectin-1
-L cells) or the COOH-terminal four aa-deleted nectin-1
(nectin-1
-
C-L cells; data not shown). These results indicate that
-catenin is recruited through l-afadin to nectin-based cellcell adhesion sites in the absence of E-cadherin.
|
|
l-Afadindependent Recruitment of ß-Catenin to Nectin-based CellCell Adhesion Sites without the trans-Interaction of E-Cadherin
In L cells, endogenous ß-catenin was reduced to a practically undetectable level, but the amount of ß-catenin as well as -catenin was increased upon expression of a T7 peptidetagged protein of the cytoplasmic region and partial transmembrane segment of E-cadherin (tEC; data not shown). In nectin-2
-L cells transiently expressing tEC, ß-catenin was recruited to nectin-2
based cellcell adhesion sites (Fig 9, top). In contrast, in nectin-2
-
C-L cells transiently expressing tEC, ß-catenin was not concentrated at nectin-2
-
Cbased cellcell adhesion sites (Fig 9, bottom). The amounts of increased endogenous ß-catenin were similar between these two types of cells (data not shown). These results indicate that ß-catenin is recruited through l-afadin to nectin-based cellcell adhesion sites without the trans-interaction of E-cadherin.
|
l-Afadindependent Recruitment of E-Cadherin to Nectin-based CellCell Adhesion Sites without the trans-Interaction of E-Cadherin
In the last set of experiments, we examined whether E-cadherin is recruited to nectin-based cellcell adhesion sites without the trans-interaction of E-cadherin. For this purpose, we cocultured nectin-1-L cells with EL cells stably expressing a FLAG-tagged protein of full-length nectin-1
(nectin-1
-EL cells) or a FLAG-tagged protein of the COOH-terminal four aa-deleted nectin-1
(nectin-1
-
C-EL cells). When nectin-1
-L cells were cocultured with nectin-1
-EL cells, nectin-1
was concentrated at adhesion sites between the same type of cells and between two different types of cells (Fig 10, top). E-Cadherin was concentrated at adhesion sites between two nectin-1
-EL cells. In addition, E-cadherin was concentrated at adhesion sites between nectin-1
-L and -1
-EL cells. When nectin-1
-L cells were cocultured with nectin-1
-
C-EL cells, nectin-1
and -1
-
C were also concentrated at adhesion sites between the same type of cells and between the two different types of cells (Fig 10, middle). Consistent with our previous report that the interaction of nectin with l-afadin is necessary for its clustering (
and -1
-
C at adhesion sites between nectin-1
-L and -1
-
C-EL cells was weaker than that of nectin-1
at adhesion sites between nectin-1
-L and -1
-EL cells. Moreover, the recruitment of E-cadherin to adhesion sites between nectin-1
-L and -1
-
C-EL cells was significantly reduced when compared with that between nectin-1
-L and -1
-EL cells. Western blot analysis indicated that the protein levels of nectin-1
or -1
-
C among these three cell lines were similar (data not shown). The polyclonal antinectin-1
Ab2 recognized two or three protein bands. Their relationship is not clear, but this may be due to different levels of the posttranslational modifications such as glycosylation.
|
We have previously shown that gD, an envelope component of HSV1, specifically inhibits nectin-1mediated cell adhesion activity as estimated by a cell aggregation assay (Sakisaka, T., T. Taniguchi, H. Nakanishi, K. Takahashi, M. Miyahara, W. Ikeda, S. Yokoyama, Y.-F. Peng, K. Yamanishi, and Y. Takai, manuscript submitted for publication). When gD was added to the coculture system of nectin-1
-L and -1
-EL cells, nectin-1
was hardly concentrated at adhesion sites between the same type of cells or between two different types of cells (Fig 10, bottom). The recruitment of E-cadherin to adhesion sites between nectin-1
-L and -1
-EL cells was remarkably reduced when compared with that in the absence of gD. However, the concentration of E-cadherin at adhesion sites between two nectin-1
-EL cells was not affected. These results indicate that E-cadherin is recruited through l-afadin to nectin-based cellcell adhesion sites without the trans-interaction of E-cadherin, and that nectin, of which trans-interaction is inhibited, is not recruited to cadherin-based cellcell adhesion sites.
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Discussion |
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Our previous series of studies have shown that l-afadin is essential for the colocalization and compact clustering of nectin with E-cadherin at cellcell AJs and proper organization of E-cadherin-based cellcell AJs (-catenin is, furthermore, essential for this colocalization. This colocalization is not mediated through the cytoplasmic region of E-cadherin or ß-catenin.
-Catenin directly interacts with vinculin and
-actinin through the NH2-terminal half (
-cateninbinding proteins directly interact with F-actin (
-catenin is not essential for the colocalization of nectin and l-afadin with E-cadherin, indicating that neither vinculin,
-actinin, nor ponsin is necessary for this colocalization. This result is consistent with our previous observation that ponsin forms a binary complex with either l-afadin or vinculin, and does not form a ternary complex (
It is of crucial importance to clarify the molecular linkage between l-afadin and the COOH-terminal half of -catenin. We have previously shown that l-afadin does not directly interact with
-catenin as estimated by affinity chromatography using the purified samples (
-catenin and connects them. On the basis of this assumption, we have been attempting to isolate an l-afadinbinding protein in the presence of
-catenin or an
-cateninbinding protein in the presence of l-afadin by using various methods currently available, including yeast two-hybrid, affinity chromatography, immunoprecipitation, and blot overlay. We have not yet obtained any candidate proteins, but we have found, by the yeast two-hybrid method, that full-length l-afadin directly interacts with the COOH-terminal half of
-catenin. We have confirmed this interaction by affinity chromatography, although the stoichiometry of this interaction is small. We have attempted to increase this small stoichiometry by the use of the NH2-terminal or COOH-terminal half of l-afadin. The COOH-terminal half directly interacted with the COOH-terminal half of
-catenin, but the stoichiometry did not increase (data not shown). We have added the NH2-terminal half of ZO-1 because it has been shown to directly interact with the AF-6 protein/s-afadin (
-catenin (
-catenin overexpressed in COS7 cells are not coimmunoprecipitated to a significant extent. Thus, our results concerning the interaction of l-afadin with the COOH-terminal half of
-catenin are apparently inconsistent depending on the methods used: one positive, one semi-positive, and one negative. However, the negative results obtained from the immunoprecipitation experiments do not necessarily reflect in vivo negative proteinprotein interactions, because coimmunoprecipitation of two proteins is sometimes affected by an extraction buffer used in experiments, and sometimes is not observed when they form a very complicated multicomplex. Taken together, l-afadin may directly interact with
-catenin under appropriate conditions, but the molecular linkage between these two proteins may not be so simple and another factor and/or posttranslational modifications of l-afadin and
-catenin may be necessary for this linkage.
Several other possible mechanisms of the linkage between the nectin/afadin and cadherin/catenin systems are conceivable. One possibility is that l-afadin indirectly interacts with -catenin through ZO-1, because it has been shown that ZO-1 directly interacts in vitro with the AF-6 protein/s-afadin (
-catenin (
-catenin. Therefore, this possibility is unlikely. The second possibility is that l-afadin indirectly interacts with
-catenin through vinculin, because it has been shown that the COOH-terminal half of
-catenin directly interacts with vinculin in vitro (
C-L and nE
N-L cells that vinculin is colocalized with the NH2-terminal half of
-catenin (nE
N), but not with the COOH-terminal half (nE
C) or l-afadin. Therefore, this possibility is unlikely either. The third possibility is that F-actin is involved in the linkage between l-afadin and
-catenin, because l-afadin (
-catenin (
-catenin with F-actin remains unknown.
It may be noted that nectin has a potency to recruit - and ß-catenins and E-cadherin through l-afadin at nectin-based cellcell adhesion sites in the absence of E-cadherin or without its trans-interaction. It is likely that
-catenin is recruited there by the direct or indirect interaction with l-afadin, and that ß-catenin is recruited there by the direct interaction with
-catenin. E-cadherin may be recruited there by the direct interaction with the
- and ß-catenin complex through l-afadin. However, it remains unknown whether E-cadherin, which is concentrated at adhesion sites between L and EL cells stably expressing nectin, forms a cis-dimer or not.
In contrast to the recruitment of the cadherin-catenin complex to nectin-based cellcell adhesion sites without the trans-interaction of E-cadherin, we have shown that nectin-1, of which trans-interaction is inhibited by gD, is not recruited to E-cadherinbased cellcell adhesion sites between two nectin-1
-EL cells. Thus, nectin trans-interacts independently of the trans-interaction of E-cadherin, and nectin, of which trans-interaction is inhibited or which lacks the ability to interact with l-afadin, does not appear to be recruited to E-cadherinbased cellcell adhesion sites. However, it remains unresolved whether the trans-interaction of E-cadherin is dependent on the trans-interaction of nectin, because nectin has three isoforms, and cell adhesion activities of all the isoforms cannot be inhibited simultaneously at this time (
On the basis of these present and previous observations, we propose here at least two models for the formation of cellcell AJs. One model is that nectin and E-cadherin independently form the respective trans-interactions and the nectin/afadin and cadherin/catenin systems recruit each other to form compact cellcell AJs. The other model is that nectin first forms a trans-interaction that recruits, through l-afadin, the cadherin-catenin system in which E-cadherin does not trans-interact, followed by the trans-interaction of E-cadherin at nectin-based cellcell adhesion sites, finally leading to the formation of compact cellcell AJs. It is currently unknown which is the case, but it is likely that the nectin/afadin system plays a key role in the organization of cellcell AJs in cooperation with the cadherin-catenin system.
We have previously shown by the use of epithelial cells in afadin (-/-) mice and (-/-) embryoid bodies that not only cadherin-based AJs, but also claudin/occludin-based TJs are impaired in these mutant cells (
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Footnotes |
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1 Abbreviations used in this paper: aa, amino acid(s); Ab, antibody; AJ, adherens junction; F-actin, actin filament; gD, glycoprotein D; GFP, green fluorescent protein; GST, glutathione-S-transferase; HA, hemagglutinin; His6, hexa-histidine; HSV1, herpes simplex virus type 1; Ig, immunoglobulin; TJ, tight junction.
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Acknowledgements |
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We thank Dr. M. Takeichi (Kyoto University, Kyoto, Japan) for helpful discussions and for providing us with the antiE-cadherin mAb. We also thank Drs. J. Miyazaki and H. Niwa (Osaka University, Osaka, Japan) for providing us with the pPCAGIZ vector, and Dr. T. Nakano for pEF-MC1neo (Osaka University, Osaka, Japan).
The work at Osaka University was supported by grants-in-aid for scientific research and for cancer research from the Ministry of Education, Science, Sports and Culture, Japan (1999, 2000).
Submitted: 28 March 2000
Revised: 21 June 2000
Accepted: 10 July 2000
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References |
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