From Istituto di Ricerche Farmacologiche Mario Negri,
Via Eritrea 62, 20157 Milano, Italy, ¶ Commissariat à
l'Energie Atomique, Laboratoire de Transgénèse et
Différenciation Cellulaire, Département de Biologie
Moléculaire et Structurale, 17 rue des Martyrs, 38054 Grenoble,
France, and
Facoltà di Farmacologia, Università
degli Studi di Milano, Via Vanvitelli 32, 20129 Milano, Italy
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ABSTRACT |
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Endothelial cells express two major cadherins, VE- and N-cadherins, but only the former consistently participates in adherens junction organization. In heart microvascular endothelial cells, we identified a new member of the cadherin superfamily using polymerase chain reaction. The entire putative coding sequence was determined. Similarly to protocadherins, while the extracellular domain presented homology with other members of the cadherin superfamily, the intracellular region was unrelated either to cadherins or to any other known protein. We propose for this new protein the name of vascular endothelial cadherin-2. By Northern blot analysis, the mRNA was present only in cultured endothelial cell lines but not in other cell types such as NIH 3T3, Chinese hamster ovary, or L cells. In addition, mRNA was particularly abundant in highly vascularized organs such as lung or kidney. In endothelial cells and transfectants, this cadherin was unable to bind catenins and presented a weak association with the cytoskeleton. This new molecule shares some functional properties with VE-cadherin and other members of the cadherin family. In Chinese hamster ovary transfectants it promoted homotypic Ca2+ dependent aggregation and adhesion and clustered at intercellular junctions. However, in contrast to VE-cadherin, it did not modify paracellular permeability, cell migration, and density-dependent cell growth. These observations suggest that different cadherins may promote homophilic cell-to-cell adhesion but that the functional consequences of this interaction depend on their binding to specific intracellular signaling/cytoskeletal proteins.
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INTRODUCTION |
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Endothelial permeability to plasma proteins and circulating cells is controlled in part by intercellular junctions. Besides their role in promoting homotypic cell adhesion, emerging evidence suggests that intercellular junctions can transfer cell-cell signals and be responsible for complex cellular responses such as contact inhibition of cell growth and cell polarity.
The molecular organization of intercellular junctions in the endothelium has been only partially elucidated in the last few years. At least three types of complex structures have been described: tight junctions, adherens junctions, and complexus adhaerentes. All of these structures are formed by specific transmembrane proteins, which through their extracellular region promote homotypic cell-to-cell adhesion and through the cytoplasmic tail bind to a complex network of cytoskeletal and signaling proteins (1-3). Outside of these junctional structures, other adhesive proteins such as PECAM1 (4) or S-Endo-1/Muc-18 (5) have been found to be clustered at intercellular contacts. The intracellular molecules that associate with adherens junctions are different from those that associate with tight junctions and from those that link other junctional adhesion proteins such as PECAM, suggesting that a certain specificity in signaling should exist (3).
In adherens junctions, the transmembrane proteins responsible for
cell-to-cell adhesion belong to the cadherin superfamily of adhesive
proteins. In endothelial cells, one of the major cadherins is
VE-cadherin or cadherin-5 (VE-cad), which is consistently present at
adherens junctions and is cell-specific (2, 3, 6). Similarly to the
other members of the family, the short intracellular tail of VE-cad is
linked to three cytoplasmic proteins called catenins: -catenin,
plakoglobin, and p120.
-Catenin and plakoglobin bind
-catenin,
which in turn promotes the anchorage of the complex to the actin
cytoskeleton. As the other known cadherins, the extracellular domain of
VE-cad promotes homotypic, calcium-dependent adhesion.
Endothelial cells also express N-cadherin, which in human endothelium does not colocalize with VE-cad at cell contacts but remains diffuse on the cell membrane (7, 8).
Besides these well characterized cadherins, some indirect evidence suggests that other cadherin-like structures may be present in the endothelium (9). Therefore, we started an investigation to test this possibility. Using a polymerase chain reaction method previously introduced by Suzuki et al. (6) and Sano et al. (10), we identified a new protein that, on the extracellular domain, presents homology with cadherins. This protein is concentrated at intercellular junctions and expresses adhesive properties, but, in contrast to VE-cad, does not bind to catenins and does not modify paracellular permeability, cell migration, or growth. This indicates that several proteins may participate in the molecular organization of interendothelial junctions, but each molecule may play a specific functional role and possibly transfer different intracellular signals. For its localization in endothelial cells and for its homology with the cadherin and protocadherin family, we propose for this new protein the name of vascular endothelial cadherin-2 (VE-cad-2).
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MATERIALS AND METHODS |
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All reagents were purchased from Sigma unless indicated otherwise.
PCR-- PCR was performed as described previously by Suzuki et al. (6) and Sano et al. (10). Template cDNA was synthesized using mouse heart microvascular endothelial cell (H5V) (11) total RNA according to the protocol of the GeneAmp RNA PCR kit (Perkin-Elmer). Two different sets of degenerated oligonucleotide primers were used in this study. One set corresponds to two well conserved sequences in the cytoplasmic domain of cadherin; the upstream and downstream primers were 5'-GAATTCAC(A/C/G/T)GC(A/C/G/T)CC(A/C/G/T)TA(C/T)GA-3' and 5'-GAATTCTC(A/C/G/T)GC(A/C/G/T)A(A/G)(C/T)TT(C/T)TT(A/G)AA-3', respectively. The other set corresponds to two conserved regions in the third (EC3) and in the fourth extracellular domain (EC4) as described previously by Sano et al. (10). The upstream and downstream primers were 5'-AA(A/G)(C/G)(C/G)(A/C/G/T)(A/C/G/T)T(A/C/G/T)GA(C/T)T(A/ G)(C/T)GA-3' and 5'-(A/C/G/T)(A/C/G/T)(A/C/G/T)GG(A/C/G/T)GC(A/ G)TT(A/G)TC(A/G)TT-3', respectively. The PCR reaction consisted of 33 cycles at 95 °C for 1 min, 45 °C for 2 min, and 72 °C for 3 min using Taq DNA polymerase obtained from Boehringer Mannheim. The PCR products were subcloned into the pCR II plasmid by use of the TA cloning kit (Invitrogen Co., San Diego, CA) and sequenced according to the dideoxynucleotide chain termination method (12).
Screening of a cDNA Library and Sequence Analysis--
About
8 × 105 plaques of a gt10 cDNA library from
postnatal day 4-8 mouse brain microvasculature (13) were screened for clone 1 and clone 14 by a plaque hybridization method, as described previously (14), using as probes the cDNAs obtained from PCR experiments. cDNAs were radiolabeled with
[32P]
-dCTP (Amersham International, Buckinghamshire,
UK), using a random primer DNA labeling kit obtained from Boehringer
Mannheim. Plaques showing a strong positive hybridization signal were
screened four times to obtain a single phage clone.
Plasmid Construction and Transfection-- The full-length open reading frame for VE-cad-2 was cut with EcoRI, and the insert was subcloned into the pECE eukaryotic expression vector (16) to yield the pECE-VE-cad-2 construct. CHO cells were plated at 5-6 × 105 cells/10-mm Petri dish in Dulbecco's modified Eagle's medium with 10% fetal calf serum. After about 24 h, the cells were transfected by calcium phosphate precipitation with 20 µg of pECE-VE-cad-2 and 2 µg of pCMVneo plasmid. Cells were washed 24 h later with PBS and cultured for another 24-36 h in Dulbecco's modified Eagle's medium with 10% fetal calf serum. They were then cultured in presence of 600 µg/ml G418 (Geneticin; Life Technologies, Inc.). After about 10 days in selective medium, the surviving colonies were ring-cloned. G418-resistant clones were screened for expression by Northern blot analysis and indirect immunofluorescence microscopy. Control cells, CHO cells transfected with pCMVneo were selected, cloned, and cultured in the same way.
RNA Expression and Northern Blot Analysis--
Total RNA was
extracted and purified by use of the Rapid Total RNA isolation kit (5 Prime 3 Prime, Inc., Boulder, CO) and 20 µg were run in a
standard formaldehyde/agarose gel, blotted onto Hybond N membrane
(Amersham International), fixed at 80 °C for 2 h, and
hybridized at 65 °C in a buffer containing 10% dextran sulfate, 3×
SSC, 5× Denhardt's solution, 10% SDS, 100 µg/ml denatured salmon
sperm DNA. The membranes were then washed twice with 2× SSC and 0.1%
SDS at room temperature for 10 min, twice with 0.5× SSC and 0.1% SDS
at 65 °C for 15 min, and once with 0.1× SSC and 0.1% SDS at
65 °C for 10 min and then subjected to autoradiography.
Cells-- CHO, NIH 3T3, and L929 cells were from American Tissue Culture Collection (Rockville, MD). Mouse epithelial cells (PDV), kindly provided by A. Cano (Instituto de Investigaciones Biomedicas, Madrid, Spain) were skin keratinocytes isolated and cultured as described previously (17). Mouse endothelioma cell lines H5V, T-end, E-end, and B-end were obtained and cultured as described (11, 18, 19). CHO-VE-cad transfectants were obtained as described previously (15). Cells were cultured in Dulbecco's modified Eagle's medium with 10% fetal calf serum at 37 °C in a 5% CO2 atmosphere.
Sterile plasticware was from Falcon (Becton Dickinson, Lincoln Park, NJ); both culture medium and serum were from Life Technologies, Inc.Antibodies--
A rabbit antiserum was raised against a
recombinant fragment spanning the extracellular domain of VE-cad-2
(74-335 amino acids). The fragment was generated by PCR. The primers
were designed to create at the 5'-end a BamHI site and at
the 3'-end a HindIII restriction site. The cDNA fragment
was then subcloned into the BamHI-HindIII site
of the expression vector pQE30 (Qiaexpressionist kit; Qiagen,
Chatsworth, CA) in the correct reading frame and sequenced to verify
that no mutation had arisen during PCR. The resulting pQE30-VE-cad-2
vector was then introduced into M15 (pREP4) cells by a single step
transformation method. The fusion protein was induced by the addition
of isopropyl--D-thiogalactopyranoside and was purified
from the extract by nickel-nitrilotriacetic acid resin affinity
chromatography, as described by the manufacturer (Qiaexpressionist kit;
Qiagen). Polyclonal antibody against VE-cad-2 was produced in rabbit by
injecting 0.5 mg of the fusion protein in Freund's complete adjuvant
at three subcutaneous sites. Subsequent injections were in Freund's
incomplete adjuvant with 0.5 mg of the fusion protein. The fragment
antiserum was affinity-purified by affinity chromatography on the
corresponding fragment affinity column CN-Br-Sepharose 4B (Pharmacia
LKB Biotechnology, Uppsala, Sweden).
Immunofluorescence Microscopy-- Cells were grown on glass coverslips, rinsed in PBS, and fixed in methanol. The cells were then rinsed and incubated for 45 min at 37 °C with the relevant primary antibodies, washed three times with PBS, and incubated for 30 min with the fluorophore-conjugated secondary antibodies. Coverslips were then mounted in Mowiol 4-88 (Calbiochem) and examined with a Zeiss Axiophot microscope. Photographs were taken using T-Max P3200 films.
EGTA Treatment-- EGTA was used for chelating calcium ions in the culture medium as described previously (21). A buffer stock solution of 100 mM EGTA was used to obtain a final concentration of 5 mM. Cells grown to confluence on glass coverslips were incubated with 5 mM EGTA at 37 °C for 30 min, fixed, and processed for indirect immunofluorescence as described above.
Cytochalasin Treatment-- Cells were cultured to confluence on glass coverslips and treated with 1 µg/ml cytochalasin D in culture medium. 30 min later, cells were fixed with 3% paraformaldehyde, permeabilized with 0.5% Triton X-100, and processed for indirect immunofluorescence as described above.
Cell Surface Biotinylation-- Biotinylation of cell surface proteins was performed as described elsewhere (22) using sulfonitrohydroxysuccinimido-biotin (Pierce). Samples were analyzed by electrophoresis on a 7.5% SDS-polyacrylamide gel and transferred to nitrocellulose membrane. The membranes were blocked with 10% low fat milk and then incubated in fresh blocking solution with horseradish peroxidase-conjugated streptavidin (Biospa Division, Milano, Italy) for 1 h at room temperature. After three washes with PBS containing 0.1% Tween 20, peroxidase-conjugated streptavidin was visualized using the ECL kit as described under "Blot and Immunoprecipitation."
Western Blot and Immunoprecipitation-- Whole cell extracts were obtained from confluent cells as described previously (23). Detergent solubilization was carried out essentially as reported previously in detail (24). Different cell extracts were adjusted to 1× Laemmli sample buffer and fractionated under reducing conditions on 7.5% SDS-polyacrylamide gels (25).
Western blot analyses of the various cell extracts were carried out essentially as described (24). After blocking with 10% low fat milk, the proteins of interest were detected by specific monoclonal or polyclonal antibodies at the optimal dilution in blocking buffer. This was sequentially followed by incubation with goat anti-mouse IgG peroxidase-conjugated antibody (1 mg/ml) for monoclonal antibodies or protein A peroxidase-conjugated antibody (1 mg/ml) (Pierce) for polyclonal antibody and further development of peroxidase activity using an ECL kit (Amersham Biotech Pharmacia International) and autoradiography. Immunoprecipitation of the cadherin-catenin complex was performed using the nonionic detergent-soluble fraction of cells, as previously reported (24) with some modifications. Briefly, cell extracts were precleared by incubation with uncoupled protein G- or protein A-Sepharose CL-4B (Amersham Biotech Pharmacia) for 2 h. After centrifugation, the precleared supernatants were incubated with protein G- or protein A-Sepharose coupled to mAb TEA 1.31 or polyclonal antibody against VE-cad-2 during 1 h. Immunocomplexes were collected by centrifugation; washed five times in a buffer containing 0.5% Triton X-100, 0.1% bovine serum albumin, 50 mM Tris-HCl, pH 7.4, 0.1 M NaCl, and 2 mM CaCl2; and finally resuspended in 30 µl of 1× Laemmli sample buffer and boiled for 5 min. Samples were analyzed by electrophoresis, transferred to nitrocellulose membranes, and immunoblotted sequentially with polyclonal antibody to VE-cad-2 or mAb TEA 1.31 to VE-cad or mAbs toCell Aggregation-- Calcium-dependent cell aggregation was done under conditions that preserve, by flow cytometry analysis, VE-cad-2 or VE-cad expression as described previously (15). When indicated, cytochalasin D was added at 1 µg/ml after the first centrifugation, and the cells were incubated at 37 °C for 30 min. In some experiments, EGTA (5 mM final concentration) was added to the medium. For heterotypic aggregation assays, CHO-VE-cad-2/VE-cad cells were labeled with 2 mM 2',5'-bis(2-carboxyethyl)-5-carboxyfluorescein acetoxymethyl ester and 2',5'-bis(2-carboxyethyl)-6-carboxyfluorescein acetoxymethyl ester (Molecular Probes, Eugene, OR) in Hanks' balanced salt solution for 10 min at 37 °C and processed as described (26).
Cell Adhesion--
CHO, CHO-VE-cad-2, or CHO-VE-cad cells were
cultured in 96-well plates and grown for 5 days to confluency. All
three types of cells were labeled with
[125I]iododeoxyuridine (1 mCi/ml) overnight prior to the
cell adhesion experiment. 12 h later, the cells were detached as
described above and resuspended at 3 × 105 cells/ml
in Dulbecco's modified Eagle's medium with 10% fetal calf serum. 100 µl of labeled cell suspension were added to different adherent cell
monolayers (CHO, CHO-VE-cad-2, and CHO-VE-cad) and incubated for 1 h at 37 °C. After three washes with PBS containing 10% fetal calf
serum, the cells were solubilized with 0.5 M NaOH, 0.1%
SDS and counted in a -counter.
Permeability Assay-- Permeability across cell monolayers was measured in Transwell units (with polycarbonate filter, 0.4-µm pore; Corning Costar Corp., Cambridge, MA) as described previously (15). Briefly, CHO transfectants were cultured to confluency for 5 days. Then culture medium was replaced with serum-free medium, and horseradish peroxidase conjugated to goat immunoglobulins (8 mg/ml initial concentration in the upper chamber; minimal calculated molecular mass, 200 kDa; specific activity, 28 units/mg) was added to the upper chamber. At 2 h, 100-µl aliquots were collected from the lower compartment and assayed photometrically for the presence of enzymatic activity. In some experiments, EGTA (5 mM, final concentration) was added both to the lower and upper compartments for 2 h at the same time as immunoglobulins.
Cell Migration-- Cell migration was estimated as described previously (15). Briefly, the cell monolayer was wounded with a plastic tip. Four diameters, regularly distanced by about 45°, were removed. The remaining cells were washed twice with culture medium to remove cell debris and incubated at 37 °C in culture medium. At the indicated time intervals, cells were fixed with Fast Green FCF (0.02% in methanol) and stained with crystal violet (0.5% in a 20:80 mixture of methanol/water). The distance migrated by the cells was measured using a micrograduate scale (Nikon) adapted in the ocular of a Nikon inverted microscope under phase contrasts (magnification × 100).
Cell Growth Assay-- Cell growth was evaluated as described previously (27). Cells were plated at 1 × 104/ml (1 ml/well) in 24-well plates (2 cm2/well). Culture medium was not changed for the duration of the experiment (96 h). Cell number was evaluated after trypsinization of the cells and counting (four replicates) in a Bürker chamber.
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RESULTS |
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Cloning of a New Member of the Cadherin Superfamily-- A PCR method was applied to identify new members of the cadherin superfamily. As primers we first used two degenerated oligonucleotides corresponding to two highly conserved sequences in the cytoplasmic domain of cadherins as previously described by Suzuki et al. (6). PCR was carried out using cDNA obtained from mouse heart microvascular endothelial cells (H5V). The resulting 160-bp products were then subcloned in the pCRII plasmid and sequenced. Of 35 clones sequenced, three clones encoded the amino acid sequence of N-cadherin, and two clones encoded the amino acid sequence of VE-cad. The other cDNA clones encoded amino acid sequences that did not present homology with the cytoplasmic domain of cadherins.
In the second part of the research, we used as primers degenerated oligonucleotides corresponding to two conserved sequences of the extracellular domain of cadherins, as described previously by Sano et al. (10). PCR from the cDNA of the cell line H5V yielded four major bands of 450, 370, 300, and 130 bp in size. The 450- and 130-bp bands correspond to the distance between the two primer sites in classic cadherins and the two primer sites in protocadherins, respectively (10). The 370- and 300-bp bands would not be predicted from any of the known members of the cadherin superfamily and were therefore discarded. The 130-bp product was subcloned into the pCR II vector, and 30 clones were isolated and sequenced. Two cDNAs (clones 1 and 14) presented a novel sequence and were considered good candidates to be new putative members of the cadherin superfamily. A cDNA library of postnatal day 4-8 mouse brain capillaries (13) was screened by using clones 1 and 14 as probes. Four clones of 5.8, 4.0, 2.4, and 1.8 kb were obtained by screening with clone 1, while only a clone of 700 bp was obtained using clone 14 as probe. The sequence analysis of the 700-bp clone revealed a partial sequence that did not correspond to any previously identified sequence (data not shown). The sequences of the two cDNA clones of 5.8 and of 4.0 kb obtained by screening with clone 1 overlap and appear to contain the full-length open reading frame of a novel member of the cadherin superfamily. The nucleotide and deduced amino acid sequences of the 4.0-kb clone are shown in Fig. 1. The 3868-bp sequence contains 298 bp of putative 5'-untranslated region, an open reading frame of 3540 nucleotides encoding 1180 amino acids, and a short 3'-untranslated region of 29 bp. At position 299, the cDNA sequence contains a translation initiation site that matches the Kozak criteria (28). The polyadenylation signal was not identified.
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VE-cad-2 Is Expressed in Different Mouse Tissues and Endothelial Cell Lines-- We examined the expression of VE-cad-2 in different mouse tissues and cell lines by Northern blot analysis and immunofluorescence staining. As hybridization probe, we used a cDNA stretch (from nucleotide 2100 to 3868) corresponding to the cytoplasmic tail of VE-cad-2 to avoid aspecific hybridization with other cadherin mRNAs. A single band of about 7-kb mRNA was detected in highly vascularized organs such as lung, heart, liver, and kidney. VE-cad-2 mRNA was barely detectable in brain and thymus (Fig. 2A). VE-cad-2 mRNA was found in four endothelial cell lines of different origin but not in other cell types such as L929 and 3T3 fibroblasts or in cultured epithelial cells (PDV) (Fig. 2B). We then performed immunofluorescence staining of cultured endothelial cells using an affinity-purified rabbit polyclonal antibody directed to a recombinant fragment of VE-cad-2, corresponding to the extracellular region between amino acid residues 74-335. In preliminary experiments, we found that this antibody did not recognize mouse VE-, N-, or E-cadherin transfectant cells by flow cytometry, thus excluding the possibility of cross-reactivity with other cadherins potentially expressed in endothelial cells (1). The antiserum recognized a band of 160 kDa in H5V cells and in VE-cad-2 transfectants (Fig. 3B).
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Characterization of VE-cad-2 Transfectants-- In order to investigate the functional properties of VE-cad-2, its cDNA was subcloned into the pECE expression vector and transfected in CHO cells. Successful transfection and selection were determined by Northern blot and Western blot analysis of VE-cad-2 transfectants (Fig. 3, A and B).
As shown in Fig. 3A, a major band of 4.5 kb was evident in three VE-cad-2 transfectant clones but was absent in CHO control cells (CHO cells transfected with the neomycin resistance gene and the empty pECE plasmid). The size of mRNA observed corresponds to the coding cDNA sequence introduced in the pECE expression vector. Expression of VE-cad-2 protein was analyzed by cell surface biotinylation. As reported in Fig. 3B, a band of 160 kDa was detected in transfectants and in endothelial cells, while no detectable VE-cad-2 expression was found in control transfectants. This size is larger than the molecular weight predictable from the deduced amino acid sequence. This discrepancy is commonly found in members of the cadherin superfamily and is probably due to the state of glycosylation and specific structural properties of cadherins. To investigate the possibility that, like other members of the cadherin family, VE-cad-2 could associate with catenins through the cytoplasmic tail, cell extracts of VE-cad-2 transfectants were immunoprecipitated with the VE-cad-2 antibody and then blotted with anti-VE-cad-2 and anti-
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Functional Properties of VE-cad-2: Comparison with VE-cad-- VE-cad-2 has five cadherin-specific motifs in its extracellular region with putative internal Ca2+ binding sequences but, as described above, lacks the capacity to bind catenins. Since binding to catenins may influence cadherin functional behavior, we studied the adhesive properties of VE-cad-2 transfectants in comparison with VE-cad.
Transfection of CHO cells with VE-cad-2 confers calcium-dependent aggregating activity (Fig. 6, A and B) in a way comparable with, or slightly more effective than, transfection with VE-cad. Aggregation was blocked by the addition of EGTA, while cell treatment with cytochalasin D (in order to disrupt actin cytoskeleton) did not affect both VE-cad-2 and VE-cad-induced aggregation. Similar data were obtained when cells were transfected with the truncated VE-cad mutant lacking the cytoplasmic domain responsible for catenin binding (26).
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DISCUSSION |
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In this paper, we describe the cloning and expression of a new member of the cadherin superfamily. Because of its expression in endothelial cells and its homology with the previously characterized cadherins, we propose for this protein the name VE (vascular endothelial) cadherin-2 (VE-cad-2).
The extracellular domain of VE-cad-2 contains five extracellular repeats and five copies of the four and five amino acid residues LDRE and DXNDN, which are typically repeated three or four times in cadherin (33, 34). The spacing of these sequences in this new cadherin is similar to that observed in other cadherins (34).
However, the cytoplasmic domain is not homologous to cadherins or to any other sequence in the available data bases. Within the cadherin superfamily, members of the protocadherin group have similar characteristics. All protocadherins have an extracellular region homologous to cadherins but a variable intracellular domain (35). In VE-cad-2, this region is not homologous to any known protocadherin.
We have been unable to detect VE-cad-2 association with catenins, which are typically bound to the cytoplasmic domain of classic cadherins (36-38). These proteins are responsible for cadherin binding to the actin cytoskeleton and possibly for intracellular signaling (39). Consistent with this observation, VE-cad-2 is in large part associated with the detergent-soluble fraction of cell extracts. However, we cannot exclude the possibility that catenins might interact weakly with VE-cad-2, even if such association might be undetectable in our experimental system.
VE-cad-2 displays functional properties similar to those described in VE-cad and other cadherins. It clusters at intercellular junctions in endothelial cells and transfectants and promotes homotypic aggregation and adhesion. These properties are Ca2+-dependent and, as expected (26), do not require an intact actin cytoskeleton, since cytochalasin D is ineffective.
In contrast, VE-cad-2 is unable to promote other activities previously ascribed to VE-cadherin, such as reduction of paracellular permeability (15), inhibition of cell migration from a confluent monolayer (15), and contact inhibition of cell growth (15).
Interestingly, the functional behavior of VE-cad-2 is similar to that of a truncated VE-cad mutant lacking the cytoplasmic region responsible for binding to catenins (26). Like VE-cad-2, the truncated VE-cadherin promotes homotypic clustering and aggregation but cannot significantly affect cell migration, permeability, and growth (26).
These data are consistent with the idea that the cadherin-like extracellular region has the structural requirements for promoting homophilic cell recognition but that the functional consequences of this interaction are related to the association with cytoplasmic partners. Cadherin association with catenins and actin is required for strengthening adhesion (40, 41) and for transferring signals that limit cell growth and motility (26). Different cytoplasmic structures may be responsible for different cellular responses.
We still do not have direct information about the intracellular partners of VE-cad-2. Immunoprecipitation analysis of metabolically labeled cells showed that the VE-cad-2 antiserum immunoprecipitates two major bands of about 100 and 200 kDa.2 We are currently trying to identify these structures.
The other members of the protocadherin group have variable cytoplasmic tails that do not bind catenins and do not associate with the actin cytoskeleton. Another protocadherin (protocadherin 2, Pcdh-2) was found to coprecipitate two bands of different molecular mass (180 and 50 kDa) than those found in association with VE-cad-2 (32). This suggests that different protocadherins may link different cytoplasmic proteins.
The expanding list of members of the cadherin superfamily shows that several of them display an homologous extracellular domain but divergent intracellular tails. For instance, LI-cadherin has a short tail (42, 43), and T-cadherin lacks the cytoplasmic region and is linked to the membrane by a glycosyl phosphatidylinositol anchor (44). Desmogleins and desmocollins have an intracellular tail different from that of classic cadherins; they do not bind catenins, and they associate with intermediate filaments rather than actin (45).
The presence of this new cadherin in endothelial cells indicates that the organization of intercellular junctions is complex and that several proteins may cooperate to promote homotypic interaction and signaling (3).
It is possible that some of these proteins play a relevant role in the control of vascular permeability, while others are more important for intercellular signaling or transport functions. Future studies will help us to understand their reciprocal interaction and specific biological relevance.
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ACKNOWLEDGEMENT |
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We thank Dr. G. Bazzoni for helpful comments on the manuscript.
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FOOTNOTES |
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* This work was supported in part by Associazione Italiana per la Ricerca sul Cancro; European Community Projects BI04 CT 960036, BMH4 CT960669, and BMH4 CT 950875; and Human Frontier Science Program Project GR 0006/1997-M).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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) Y08715.
§ To whom correspondence should be addressed: Istituto di Ricerche Farmacologiche Mario Negri, Via Eritrea 62, 20157 Milano, Italy. Tel.: 39-2-390141; Fax: 39-2-3546277; E-mail: telo{at}irfmn.mnegri.it.
1 The abbreviations used are: PECAM, platelet/endothelial cell adhesion molecule; CHO, Chinese hamster ovary; EC1-EC6 domains, extracellular domains 1-6; PCR, polymerase chain reaction; VE-cad, vascular endothelial cadherin; VE-cad-2, vascular endothelial cadherin 2; PBS, phosphate-buffered saline; mAb, monoclonal antibody; bp, base pair(s); kb, kilobase pair(s).
2 P. Telo', unpublished observations.
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REFERENCES |
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