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J. Biol. Chem., Vol. 278, Issue 33, 31251-31260, August 15, 2003
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v
3 Integrin-containing Membrane Microdomains*
From the Department of Molecular Genetics and Microbiology, Stony Brook University, Stony Brook, New York 11794
Received for publication, April 21, 2003
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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v integrin
microdomains on the surface of transfected mouse fibroblasts and at amniotic
epithelial cell junctions. Our findings demonstrate the possible
trans-interaction between the bona fide cell-cell adherens
type adhesion system (cadherin/nectin) and the cell-matrix adhesion system
(integrin/CD155) by virtue of their nectin-3 and CD155 components,
respectively. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The CD155 gene is expressed in four isoforms. Two of the four
alternatively spliced variants, CD155 and CD155
, are
membrane-bound and serve as poliovirus receptor, whereas the significance of
two secreted versions, CD155
and -
, is not clear
(20). CD155 mRNA is widely
expressed, although in relatively low amounts, in human and CD155tg
mouse tissues, including brain, spinal cord, heart, skeletal muscle, kidney,
spleen, leukocyte, liver, lung, and placenta
(20–23).
In humans, CD155 is prominently expressed by enterocytes and cells of
gastrointestinal lymphatic tissues
(24). The ectodomain of CD155
establishes cell-matrix contacts by interaction with vitronectin
(25). Although the overall
structure of CD155 as an Ig superfamily protein and the similarity to nectins
suggests a possible role in cell-cell adhesion, no binding partners that would
support such a notion have been identified thus far. In fact, in contrast to
nectins, there is no evidence for CD155-mediated trans-homophilic
cell-cell adhesion or for CD155 cis-homodimerization
(8–10).
On the other hand, the short cytoplasmic domains of CD155 and
CD155 interact with Tctex-1, a light chain subunit of the dynein motor
complex (26). This interaction
is thought to mediate retrograde axonal transport of CD155 containing
endocytic vesicles (26). In
addition, the cytoplasmic domain of CD155 binds the mu1B subunit of the
clathrin adaptor complex, an interaction that is responsible for sorting of
the CD155 isotype protein to basolateral membranes in epithelial cells
(27).
Studies of CD155 have been complicated by uncertainty over whether a functional rodent homologue of CD155 exists, a conundrum that has restricted and even misled earlier work. It is now clear that a mouse gene termed MPH, which was long held to be the mouse homologue of CD155, is, in fact, the homologue of human nectin-2 (formerly PRR2 or PVRL2) (4, 5). The rodent Tage4 gene, sharing only 46% amino acid identity with CD155, has recently been proposed as the possible CD155 homologue based on conserved gene structure and syngenic chromosomal localization (19). However, no functional relationship between these two molecules could be thus far established. Mice are not a natural host for poliovirus, and cultured mouse cells cannot be infected with poliovirus. Therefore, the development of transgenic mice expressing the human CD155 gene has greatly facilitated the study of CD155 function (21, 23). CD155tg mice are highly susceptible to poliovirus infection and develop disease symptoms very similar to poliomyelitis in humans, indicating that the human transgene functions similarly in mice as it does in humans.
Here we show that nectin-3 interacts with CD155, leading to unidirectional
recruitment of nectin-3 to cell-cell contacts with neighboring
CD155-expressing cells. This interaction was found to depend on
dimerization/multimerization of CD155, which appears to be promoted by cell
type-specific cofactors. Mouse Tage4 (referred to below as mCD155) showed
similar affinity for nectin-3, providing the first evidence for a functional
relationship between CD155 and its mouse counterpart. In vivo,
nectin-3 and CD155 colocalized at lateral cell-cell junctions in epithelia of
proximal kidney tubules and of the amniotic membrane. We determined that CD155
and v integrin colocalize to membrane microdomains on the
cell surface of mouse fibroblasts and to cell-cell and cell-matrix junctions
in the amniotic membrane. We propose that CD155 and nectin-3 are mediators of
trans-heterophilic cell-cell adhesion and may provide a link between
the classical cell-cell adhesion system (cadherin/nectin) and the cell-matrix
adhesion system (integrin/CD155).
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Fusing the coding sequence of the extracellular domains of CD155, nectin-3,
or nectin-1 upstream of the open reading frame of the secreted, heat-stable
human placental alkaline phosphatase
(SEAP),2 in vector
pAPtag2 (28) (J. Flanagan,
Harvard University) resulted in pCD155-AP
(29), pNectin-3-AP, and
pNectin-1-AP,3
respectively. pAPtag4 (J. Flanagan) was used to express untagged SEAP protein.
pCDNA-CD155(AO) (G. Bernhardt, University of Hannover, Germany) contains the
entire CD155 cDNA in pCDNA3. For eukaryotic cell transfection, the
full-length open reading frames of mouse nectin-1, mouse
nectin-2, and mouse nectin-3 were cloned into pCDNA3.1,
resulting in pCDNA-nectin-1, pCDNA-nectin-2, pCDNA-nectin-3, respectively. Two
I.M.A.G.E. consortium clones, 4188634 and 4953896, containing the full-length
open reading frame of mouse Tage4 in vector pCMV-Sport6 were obtained from
ATTC. These plasmids afford eukaryotic expression and were used directly for
DNA transfection. Plasmids encoding the human v and
3 integrin subunits were kindly provided by Drs. Eric Brown
and Mark Ginsberg.
Stably transfected HEK 293 cell lines secreting CD155-AP, nectin-3-AP, or
nectin-1-AP fusion proteins or untagged SEAP alone were produced by
cotransfection of empty pCDNA3.1 vector (carrying a neo selection
marker) with either pCD155-AP, pNectin-3-AP, pNectin-1-AP, or pAPtag4,
respectively. Cells were subjected to selection with 500 µg/ml G418
(Invitrogen), and cell clones were isolated and screened for AP activity in
the supernatant. HeLa, HEK 293, L(tk–) and Neuro2a, and PC12 cells
stably expressing full-length CD155, nectin-1, nectin-2,
or nectin-3 were derived by DNA transfection with pCDNA-CD155(AO),
pCDNA-nectin-1, pCDNA-nectin-2, or pCDNA-nectin-3, followed by selection with
500–750 µg/ml G418. The cell lines were subcloned in order to obtain
lines with similar expression levels.
Antibodies—The following primary antibodies were used in
this study: mouse mAbs 18 (1:2 hybridoma), D171 (1:10 hybridoma supernatants,
or 10 µg/ml for blocking experiments), P286, P403, P275 (all at 10
µg/ml), all against CD155
(24,
30,
31); rat mAbs 17B10 and 6B3
(1:5 diluted hybridoma supernatant) specific for mouse nectin-2
(8); rat mAb 103-A1 (1:5
diluted hybridoma supernatant) specific for mouse nectin-3
(1); mouse mAb 8B6 against
human placental alkaline phosphatase PLAP-1 (1:500; Sigma); rabbit polyclonal
antisera raised against commercially available (Chemicon) purified
v3 integrin (1:2000; M. Roivainen, National
Public Health Institute, Helsinki, Finland) and purified hexahistidine-tagged
CD155 (NAEZ-8; 1:1000; produced in collaboration with M. Roivainen)
(32). The secondary antibodies
were anti-rabbit IgG (1:500), anti-mouse IgG (1:1000, both
Alexa488-conjugated; Molecular Probes, Inc., Eugene, OR), anti-mouse IgG
(1:500), anti-rat IgG (1:1000), anti-rabbit IgG (1:500; all Cy3-conjugated;
Jackson ImmunoResearch), anti-mouse IgG (1:2000; AP-conjugated; Santa Cruz
Biotechnology, Inc., Santa Cruz, CA), and anti-rabbit IgG (1: 10000,
horseradish peroxidase-conjugated; Jackson ImmunoResearch).
Ligand Binding Assays—Receptor-AP in situ ligand binding was performed as previously described (33, 34), with some modifications. Briefly, HEK 293 cell supernatants containing AP fusion protein probes were diluted with growth medium to obtain a uniform AP activity of 2500 A405/ml*h for all probes. COS-1 cells transiently transfected with expression vectors for individual target proteins were incubated with AP fusion protein probes for 1 h at 37 °C. The probes were then aspirated, and cells were washed five times with warm Hanks' balanced salt solution (HBSS), containing 0.5 mg/ml bovine serum albumin. The cells were then fixed with 4% formaldehyde and 60% acetone in PBS for 2 min and washed twice with HBSS/bovine serum albumin, followed by heating of the culture dishes at 65 °C for 4 h in order to inactivate cell endogenous, heat-labile AP. The monolayers were washed twice with double-distilled H2O, and bound AP fusion probes were detected by incubation with 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium colorimetric AP substrate (Sigma). Alternatively, stable HEK 293, HeLa, L(tk–), and PC12 cell lines expressing individual target proteins were produced and processed as described above. To test the effect of cross-linked CD155 binding to nectin-3, CD155-AP supernatant was preincubated with anti-AP mAb 8B6 (1:500) for 1 h at room temperature before being added to the target cells. Alternatively, a fusion protein between the CD155 ectodomain and the Fc portion of mouse IgG2a (CD155-Fc) was used instead of CD155-AP (35). This protein is expressed as a dimer due to the biological activity of the Fc region (35). Binding of CD155-Fc was assessed by subsequent incubation with anti-mouse IgG secondary antibodies conjugated to AP. For antibody blocking of nectin-3-AP binding to CD155-expressing cells, the cells were preincubated with 10 µg/ml of an unrelated mouse mAb or mAb D171, P286, P403, or P275 for 30 min at 37 °C before the addition of nectin-3-AP.
In order to quantitate cell-bound AP fusion proteins, confluent 6-well plates of HeLa, HeLa-CD155, L(tk–), and L(tk–)-CD155 cells were incubated with AP fusion probes as above. However, instead of fixing the cells after the washing step, cells were lysed in 10 mM Tris, pH 7.4, 1% Triton, and the lysate was incubated at 68 °C for 45 min. The amount of AP activity of each binding reaction was determined spectrophotometrically at 405 nm using 9 mg/ml p-nitrophenylphosphate (in 1 M diethanolamine, pH 9.8, 0.5 mM MgCl2) as a colorimetric, water-soluble substrate. In parallel, 105 cell equivalents of total lysate were separated on 7.5% SDS-PAGE followed by Western blot analysis with CD155-specific antiserum NAEZ-8 (1:5000)/anti-rabbit horseradish peroxidase (1:10,000). Immune reactive bands were detected using Lumi-Light chemiluminescence substrate (Roche Molecular Biosciences).
Coculture Assays, Tissue Sections, and Coimmunofluorescence
Analysis—HeLa, HeLa-CD155, and HeLa-nectin-3 (or HeLa-nectin-2)
cell lines were seeded on glass coverslips at a ratio of 3:1:1. Alternatively,
HeLa and HeLa-nectin-3 cells were seeded 2 days prior to superseeding with
Neuro2a-CD155 cells. Cells were incubated for an additional 2 days and then
fixed with either –20 °C cold methanol/acetone (1:1) for 30 min or
4% paraformaldehyde for 10 min, followed by permeabilization with 0.1% Triton
X-100. Adult kidneys and amniotic membranes (embryonic day 15.5) were
recovered from pregnant, CD155tg female mice (ICR-PVRTg21)
(21). Human amniotic membranes
used in this study were discarded clinical samples from term deliveries
(kindly provided by Dr. Diana Roth, Maimonides Medical Center, Brooklyn, NY).
Specimen were fixed with 4% paraformaldehyde in PBS for 4 h, followed by
perfusion with 25% sucrose in PBS. Tissues were then frozen in OCT
cryo-protectant (Tissue-TekTM), and 10-µm-thick cryo-sections were
collected on poly(L)-lysine-coated slides. Sections were air-dried and
postfixed in cold 1:1 methanol/acetone for 90 min at –20 °C before
processing. For immunofluorescence detection, sections were blocked with PBS
containing 5% normal horse and 2% normal goat serum (PBS/HG) for 2 h at room
temperature, followed by overnight incubation at 4 °C with primary
antibodies in PBS/HG at the dilutions stated above. After washing five times
for 10 min with PBS, the sections were incubated with secondary antibody in
PBS/HG for 1 h at 37 °C. The primary/secondary antibody combinations were
either NAEZ-8/mAb 103-A1 with anti-rabbit (Alexa488)/anti-rat (Cy3), mAb
D171/mAb 103-A1 with anti-mouse (Alexa488)/anti-rat (Cy3), or mAb
18/anti-v3 with anti-mouse(Cy3)/anti-rabbit
(Alexa488). The sections were washed five times for 20 min with PBS and
mounted with Immu-Mount (Shandon, Pittsburgh, PA). Images were obtained with a
Bio-Rad Radiance laser-scanning confocal microscope using the LaserSharp 2000
software package and processed with Adobe Photoshop software.
Bis-(sulfosuccinimidyl)suberate (BS3) Surface Cross-linking of CD155—10-cm dishes with confluent monolayers of HeLa-CD155 and L(tk–)-CD155 cells were washed three times with ice-cold HBSS, followed by incubation with 1 mM of the chemical cross-linker BS3 (Pierce) in HBSS under gentle agitation for 30 min at 4 °C. The monolayers were then washed twice with HBSS, and the cross-linking reaction was quenched with HBSS containing 50 mM Tris, pH 7.4. Cells were scraped off the plates and collected by centrifugation at 500 x g for 5 min. The cell pellets were lysed by boiling in gel loading buffer (50 mM Tris, pH 6.8, 2% SDS, 100 mM dithiothreitol, 10% glycerol, 0.1% bromphenol blue), and 105 cell equivalents of lysate were subjected to CD155 Western blot as described above.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Given the recent reports of heterophilic interactions of nectin-3 with both nectin-1 and nectin-2 (1, 11, 12), we asked whether CD155, the closest known relative of nectin-2, would be capable of a similar interaction with nectin-3. For this purpose, COS-1 cells, transiently transfected with individual nectin/CD155 family members, were incubated with AP fusion proteins as indicated in Fig. 2. First, we tested whether our experimental system can reproduce the previously established interactions between nectin family members. Indeed, intense staining, indicating a strong interaction, was observed between cell surface-expressed nectin-3 and the nectin-1-AP probe (Fig. 2A) or the nectin-2-AP probe (Fig. 2B) as well as between nectin-1 and nectin-3-AP (Fig. 2D). Under the conditions of the assay, virtually no homophilic binding resulted from incubation of COS-nectin-3 cells with nectin-3-AP (Fig. 2C). These results support previous results of Satoh-Horikawa and colleagues, who have shown that nectin-3 and nectin-1 or nectin-3 and nectin-2 predominantly undergo trans-heterophilic rather than trans-homophilic interactions (1). When COS-1 cells were transfected with either human CD155 or mouse CD155/Tage4, significant binding of nectin-3-AP resulted from both proteins (Fig. 2, E and F). Judging from the intensity of the staining, the intraspecies interaction between mCD155/Tage4 and nectin-3-AP (containing the mouse nectin-3 ectodomain) appeared to be slightly more efficient (Fig. 2, compare F and E). To rule out the possibility that the observed interactions might be mediated by the AP moiety of our fusion proteins, we incubated CD155-transfected cells with untagged AP protein. As expected, no binding was observed (Fig. 2G). Likewise, nectin-3-AP did not bind to COS-1 cells transfected with an empty expression vector (Fig. 2H), ruling out binding to COS-1 endogenous proteins. It is worth noting that in the reverse binding assay, in which CD155-AP was used to detect nectin-3 expression, we did not observe any significant interaction (Fig. 2I). The reason for this seemingly contradictory result is based on the failure of the soluble CD155-AP protein to form homodimers, as explained below.
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In order to confirm the specificity of the interaction between CD155 and nectin-3, PC12-CD155 cells were preincubated with a panel of CD155-specific mAbs, followed by a nectin-3-AP binding assay (Fig. 3). Three mAbs, D171, P286, and P403, reduced binding of nectin-3-AP binding by 54, 69, and 68%, respectively, whereas mAb 275 had no effect. Interestingly, all three inhibitory antibodies are known to bind to the N-terminal Ig-like domain and also block binding of poliovirus to CD155, an interaction that is solely mediated by this domain (30, 31, 37). This result suggests that, similarly to the interaction between other nectins (1–3), the binding site for nectin-3 lies within the N-terminal Ig-like domain of CD155.
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The above findings present the first evidence for an interaction of CD155 with a bona fide cell-cell adhesion molecule, nectin-3, and for a functional homology between human CD155 and its mouse counterpart. We therefore propose that Tage4 be referred to as mCD155.
Interaction of Nectin-3 and CD155 Requires Dimerized CD155—To our surprise, we noticed that the reverse ligand binding assay, using soluble CD155-AP to detect nectin-3 expressed on the cell surface, did not result in significant binding (Figs. 2I and 4F) on any of the cell lines tested (HeLa, HEK 293, COS-1, L(tk–), PC12). During a parallel analysis of the interaction between nectin-2 and nectin-3, we made the curious observation that preincubation of HEK 293-nectin-2 cells with nectin-2 rat mAbs 17B10 and 6B3 (8), instead of blocking, immensely enhanced the binding of nectin-3-AP to nectin-2 (Fig. 4, compare A and C). The same phenomenon was observed on L(tk–)-nectin-2 cells (data not shown). This effect was specific for the interaction of nectin-2 with nectin-3-AP, since CD155-AP showed no binding to nectin-2 before or after antibody preincubation with 17B10/6B3 (Fig. 4, compare D and B). Furthermore, no cross-reactivity of mAbs 17B10 and 6B3 with nectin-3 was detected (data not shown). Since nectin-2 is already present in form of a dimer on the cell surface (9, 10), our data indicate that clustering of nectin-2 may be necessary for efficient trans-interaction with nectin-3.
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The CD155-specific antibodies available to us did not cause a similar stimulation of nectin-3-AP binding to CD155-expressing cells; all of these antibodies were found to be inhibitory to this interaction or had no effect (Fig. 3). Since soluble CD155 does not form homodimers as nectins do, we speculated that the reason for the failure of CD155-AP binding to nectin-3 (Figs. 2I and 4F) is due to the monomeric nature of CD155. To address this issue, we cross-linked our CD155-AP probe by preincubation with AP-specific mAb 8B6 before adding the fusion protein to HEK 293-nectin-3 cells. We found that antibody-cross-linked CD155-AP now efficiently bound to nectin-3 (Fig. 4, compare F and G). To confirm this observation, we next tested the binding of a recombinant CD155 to which the Fc portion of mouse IgG2a was fused (CD155-Fc) (35). Due to the properties of the Fc region, this fusion protein spontaneously forms dimers. Binding of CD155-Fc to HEK 293-nectin-3 cells was detected with a secondary anti-mouse IgG antibody conjugated to AP. As can be seen in Fig. 4H, CD155-Fc bound well to nectin-3 expressed on the cell surface of HEK 293 cells, similar to the binding of the antibody-cross-linked CD155-AP (Fig. 4G). Our results indicate that CD155 dimerization/oligomerization is essential for efficient binding of nectin-3.
Cell Type-specific Cofactors Are Required for the Proper Display of
CD155 on the Cell Surface and for the Interaction with
Nectin-3—When the binding of nectin-3-AP to different
CD155-expressing cell lines was compared, a considerable and reproducible
difference was noticed. This was particularly striking in the case of
CD155-expressing mouse L(tk–) versus human HeLa cells
(Fig. 5). Although
L(tk–)-CD155 and HeLa-CD155 cells both expressed high levels of CD155
(Fig. 5B,
third and fourth lanes; compare with HeLa cell endogenous
CD155 in first lane), very little nectin-3-AP binding above
background (Fig. 5A,
second bar) was observed on L(tk–)-CD155 cells (compare
Fig. 5A,
third and fourth bars). We do not believe this phenomenon to
be a species-specific effect of mouse versus human cells, since CD155
expressed in other rodent cells, like mouse Neuro2a and rat PC12 cells, was
efficiently recognized by nectin-3-AP (Fig.
3 and data not shown). We deemed it more likely that CD155
expressed on L(tk–) cells is not presented to nectin-3 in a proper
higher order structure (dimer, oligomer, or complex with other proteins). To
address this point, we carried out surface cross-linking of L(tk–)-CD155
and HeLa-CD155 cells with the chemical cross-linker BS3
(Fig. 6). Four high molecular
weight complexes containing CD155 were obtained from HeLa-CD155 cells
(Fig. 6, third lane).
The smallest, and most prominent, of these complexes was 155–160 kDa in
size, which corresponds to the expected size of a CD155 dimer
(Fig. 6, third lane,
asterisk). Three additional higher molecular mass CD155 containing
complexes ranging from 250 to 
400–450 kDa were observed
(Fig. 6, third lane,
arrowheads). The components of these complexes, besides CD155, are not
known at present. In contrast, CD155 expressed on L(tk–) cells appeared
to be exclusively present as the typical 75–80-kDa monomer
(Fig. 6, fourth lane).
We suggest that a correlation exists between the absence of these high
molecular weight CD155 complexes on L(tk–) cells and the inability of
nectin-3-AP to recognize CD155. This is most likely due to missing, as yet
unidentified, cofactors on L(tk–) cells.
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Nectin-3 Is Recruited to Cell-Cell Contacts through Interaction with CD155—The members of the nectin family have been shown to localize to homotypic cadherin-based cell-cell junctions both in cultured cells and in vivo (1, 2, 8, 9). Furthermore, we and others have shown that nectin-3 is also present at specialized heterotypic cell-cell junctions such as Sertoli cell-spermatid junctions in testis (11)1 or at synaptic puncta adherentia (12). Therefore, we asked whether CD155 and nectin-3 colocalize at sites of cell-cell contact. For this purpose, we devised an in vitro coculture system that allowed us to assay for trans-interactions between cells. When nectin-3 expressing HeLa cells (HeLa-nectin-3) were cocultured with parental HeLa cells until confluence (Fig. 7, A–D), nectin-3 distributed evenly to all junctions between neighboring cells (Fig. 7, B and D). There was no noticeable preference of nectin-3 localization to junctions between HeLa-nectin-3 cells over junctions between HeLa-nectin-3 and parental HeLa cells (Fig. 7, B and D). This distribution is similar to previously reported nectin-3 expression in L cells (1). We furthermore detected significant nectin-3 immunoreactivity on the cell membranes not involved in cell-cell contacts (Fig. 7B). To test for interaction of nectin-3 with CD155, HeLa-nectin-3 cells were cocultured together with HeLa-CD155 cells and parental HeLa cells (as an internal control) (Fig. 7, E–H). The cells were then stained simultaneously for nectin-3 and CD155 by indirect immunofluorescence with rat mAb 103-A1 and mouse mAb D171 and analyzed by fluorescence microscopy (see "Materials and Methods"). We found that wherever HeLa-nectin-3 cells came into contact with HeLa-CD155 cells, virtually all nectin-3 was unidirectionally attracted to cell contacts with the CD155-expressing neighboring cells (Fig. 7F). Fig. 7, E–H, shows a typical example of four HeLa-nectin-3 cells (labeled 3 in Fig. 7G) adjacent to three HeLa-CD155 cells (labeled 5 in Fig. 7G). CD155 protein remained evenly distributed over the whole surface of the cell regardless of the identity of its neighbor. In contrast, nectin-3 protein was redistributed to the contact sites with the HeLa-CD155 cells and excluded from the junction with other HeLa-nectin-3 cells or the junction with parental nontransfected HeLa cells (Fig. 7, F and H). Furthermore, any nectin-3 previously found associated with the free cell surface was now incorporated into junctions with HeLa-CD155 cells. We conclude that the efficient recruitment of nectin-3 is a direct result of the interaction with CD155 on HeLa-CD155 cells. An opposite picture emerged when HeLa-CD155 cells were cocultured with HeLa-nectin-2 and parental HeLa cells (Fig. 7, I–L). Here, nectin-2, which does not interact with CD155 (data not shown), was found to be excluded from junctions with HeLa-CD155 or parental HeLa cells, in favor of homophilic interaction with nectin-2 on neighboring HeLa-nectin-2 cells (Fig. 7, J and L). Together, these data indicate that nectin-3 preferentially undergoes trans-heterophilic interactions with CD155 over trans-homophilic interactions, whereas nectin-2, under the experimental conditions of the assay, engages only in the trans-homophilic mode of interaction.
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We extended our coculture system to the analysis of heterotypic cell-cell junctions by using a mouse neuroblastoma cell line, Neuro2a, stably transfected with CD155 (Neuro2a-CD155) together with HeLa-nectin-3 and parental HeLa cells. In contrast to the "HeLa-only" coculture described above, here HeLa-nectin-3 and HeLa cells were seeded first and allowed to grow for 2 days to a 70% cell confluence, during which time nectin-3 distributed evenly to all cell-cell junctions (similarly to Fig. 7B). Neuro2a-CD155 cells were then superseeded, followed by an additional 2 days of coculture. Cells were processed as above and analyzed by laser-scanning confocal microscopy (Fig. 8). Under the given culture conditions, a portion of Neuro2a-CD155 cells were found to extend long neurites. Like the rest of the cell surface, these neurites were richly decorated with CD155 protein (Fig. 8A, a1 and a2). Wherever CD155-positive neurites extended over a HeLa-nectin-3 cell, almost the entire nectin-3 pool of that cell was found to be associated with the crossing section of the CD155-bearing neurite (Fig. 8, B (b1 and b2) and D (d1 and d2)). Fig. 8C (c1 and c2) depicts two CD155-expressing neurites that traverse the middle of several HeLa-nectin-3 cells and in some cases cross over the region of the underlying cell's nucleus (arrowheads). As an interesting implication of this result, we conclude that the junction assembled by CD155 and nectin-3 can form anywhere on the cell surface. Our coculture data suggest to us that nectin-3 is being actively recruited away from HeLa-nectin-3 homotypic cell junction and incorporated into HeLa-nectin-3/Neuro2a-CD155 heterotypic cell junctions. Thus, nectin-3 protein at HeLa-HeLa cell junctions undergoes only weak and transient interactions, and upon encountering CD155 on Neuro2a-CD155 cells, it will be incorporated into a more stable adhesion complex. This leads to a concentration of nectin-3 along CD155-expressing surfaces until all nectin-3 has been has "mopped up" through interaction with CD155.
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Nectin-3 and CD155 Colocalize at Lateral Cell-Cell Junctions of Kidney and Amniotic Epithelia—In order to confirm the physiological significance of the interaction between nectin-3 and CD155, we searched for sites of colocalization of both proteins in mouse tissues. Cryosections of CD155tg mouse kidney and amniotic membranes were stained simultaneously with antibodies 103-A1 and NAEZ-8 and analyzed by laser-scanning confocal microscopy (Fig. 9). Basolateral expression of CD155 was seen in epithelial cells lining the proximal tubules of the kidney, decreasing in intensity toward the most apical portions of the lateral cell-cell junctions (Fig. 9A, a1). Nectin-3 was most concentrated in the anterior aspects of lateral cell-cell junctions (Fig. 9B, b1), consistent with its localization at cadherin-based adherens junctions that has been described in other epithelia (1). The expression level of nectin-3 decreased toward the basal aspect of the lateral membranes (Fig. 9B, b1). Significant colocalization of CD155 staining and nectin-3 staining could be detected in the medial region of the lateral cell contacts (Fig. 9C, c1). A very similar observation was made in the epithelium of the amniotic membrane, except that here CD155 was found mainly along the lateral cell-cell junctions and less at the basal side of the cell (Fig. 9, D–F, d1–f1, and d2–f2). We consider it possible that nectin-3 and CD155 may be involved in maintaining the distinct compartmentalization and polarity of the epithelial cell membrane in the transition zone between the apical and basolateral membrane region of epithelial cells.
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cis-Colocalization of CD155 with
v3
Integrin—The basolateral distribution of CD155 in epithelia
(24,
27,
38), reminiscent of an
integrin expression pattern, and the fact that both CD155 and
v integrins serve as vitronectin receptor led us to
speculate on a functional connection between CD155 and integrins. We therefore
set out to analyze their subcellular expression and possible colocalization in
cultured cells. A mouse mAb specific for the
v3 integrin heterodimer (clone LM609;
Chemicon) and a rabbit polyclonal antibody against
v3 were found to intensely and specifically
stain L(tk–) cells cotransfected with human v and
3 integrin expression vectors (data not shown). When the
cells were cotransfected to express v, 3,
and CD155, the observed punctate surface staining for
v3 integrin
(Fig. 10, A and
D) coincided almost completely with that of CD155
(Fig. 10, B and
E). Although we presently do not know whether they
physically interact, the fact that both CD155 and
v3 are targeted to identical subcellular
membrane microdomains suggests a functional relationship, possibly their
presence in a common supermolecular adhesion complex. Moreover, no significant
colocalization was observed between v3 and
nectin-1, nectin-2, or nectin-3, attesting to the specificity of the observed
colocalization with CD155 (data not shown).
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Finally, we determined that CD155 and v integrin
colocalize at the basolateral membranes in human amniotic epithelium
(Fig. 10, G–I
and insets). Unlike the colocalization with nectin-3 that only
occurred at lateral cell-cell contacts
(Fig. 9F), CD155 and
v colocalized in the entire basolateral region. Adequate
staining in human amniotic epithelium was only obtained with a polyclonal
antibody against v3 and not with the
v3-specific mouse mAb LM609. Thus, it is
not clear whether the observed staining reflects expression of
v3 or that of another
v-containing heterodimer cross-reacting with this polyclonal
antibody. For instance, this polyclonal antibody very efficiently stained
focal contacts in HeLa (data not shown), a cell line that reportedly does not
express v3 but does express
v5
(39,
40). Unfortunately, we were
unable to test colocalization of nectin-3 and CD155 in human amnion due to the
unavailability of suitable nectin-3 antibodies specific for the human
protein.
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DISCUSSION |
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v3
integrin-containing complex, may function as a bona fide cell-cell
adhesion molecule by interacting with nectin-3, an adhesion receptor present
at cadherin-based adherens junctions. We propose that this interaction may
play a role in mediating heterophilic adhesion between the integrin and the
cadherin/nectin adhesive systems. Direct interactions such as between the
21 or E7
integrins and E-cadherin have been described previously
(41,
42). Our results provide a
first indication that an interaction between the cadherin and integrin system
may also occur through their associated proteins.
It has been shown recently that CD155 exhibits affinity for the
extracellular matrix protein vitronectin, an interaction that may be involved
in establishing cell-matrix contacts
(25). The major receptors for
vitronectin are members of the v integrin family, in
particular v3 and
v5. Integrins are considered the
quintessential mediators of adhering epithelial cells to the underlying
extracellular matrix (43).
Their presence has been shown in at least two very distinct adhesion
complexes. These are focal adhesions, in which they bridge the extracellular
matrix and the actin cytoskeleton
(44), and hemidesmosomes
(45,
46) that tie into the
intermediate filament network of the epithelial cell (see Ref.
47 and references therein).
Our finding of cis colocalization of CD155 and
v3 integrin on transfected mouse
fibroblasts together with the fact that both proteins serve as vitronectin
receptors seems to indicate their close association, possibly in a
multiprotein adhesion complex. In addition, the CD155-specific antibody NAEZ-8
blocks the infection of rhabdomyosarcoma cells by Coxsackie virus
A9,4 a virus that uses
v3 as its cognate cellular receptor
(48). Such a blocking effect
could best be explained by steric hindrance, due to the close association of
CD155 and v3 integrin. However, it is
presently unknown whether CD155 and v3
integrin interact physically. Interestingly,
v3, v5,
CD155, and their mutual ligand vitronectin are all up-regulated in glioma
cells, and a positive correlation between metastatic/invasive properties of
gliomas and v3 expression is well
documented
(49–53).
In the future, it will be interesting to address the possible role of CD155 in
such malignancies.
We have also begun to determine the mechanisms of the interaction that we have identified between nectin-3 and CD155. This heterophilic interaction is not a simple bimolecular reaction but rather requires the presence of CD155 in a higher order complex that facilitates dimer formation and proper display of CD155 on the cell surface. The correct presentation of CD155 needed for interaction with nectin-3 appears to be cell type-specific (Figs. 4, 5, 6). L(tk–) cells were found to be unable to assemble such a CD155 complex, possibly due to the lack of an as yet unidentified cellular cofactor(s). As a result, no functional CD155-mediated nectin-3 binding sites were formed on L(tk–)-CD155 cells (Figs. 4, 5, 6). Dimerization of binding partners alone, however, may not explain the complex interactions between nectin/CD155 family members. Although nectin-2 spontaneously forms homodimers in all cell lines that were tested (9, 10), cross-linking with anti-nectin-2 greatly facilitated binding of nectin-3-AP to HEK 293-nectin-2 cells (Fig. 4). Thus, cis-dimer formation may not be the sole requirement for efficient binding. Rather, the formation of higher order structures, such as nectin clusters or "lattices," may be necessary. For example, the requirement of integrins to form supermolecular clusters at focal contacts has been well documented (see Refs. 44 and 54 and references therein). We suggest that this may also be the case for trans-interactions mediated by nectin/CD155 family members.
It is attractive to speculate that cofactors associated in cis
with CD155 such as v3 integrin may aid in
the formation of clusters of CD155 binding sites on the cell surface.
Furthermore, the identification of CD155 as a vitronectin receptor
(25) and its colocalization
with v3 that we report here point toward
the possibility of a trimolecular vitronectin receptor
(v3/CD155).
Our preliminary results indicate that nectin-3-AP binding to HeLa-CD155 cells does not block poliovirus binding or infection (data not shown). Poliovirus virions avidly bind monomeric CD155 in vitro (29), an observation suggesting that monomeric CD155 is an efficient cellular receptor for the virus. This is supported by the fact that L(tk–)-CD155 cells, which only express CD155 monomers (Fig. 6), present an excellent substrate for poliovirus binding and infection (17)5 while not supporting nectin-3-AP binding (Fig. 5). Our data indicate that several distinct CD155 populations exist on HeLa cells (Fig. 6). The majority of CD155 is present as monomers that may serve as poliovirus receptors, whereas CD155 pools that contain dimerized CD155, possibly in a complex with other proteins, may constitute the binding sites for nectin-3.
Finally, we have shown that mouse CD155/Tage4 interacts with nectin-3 much in the same manner as human CD155 (Fig. 2). This result provides the first functional evidence that mouse CD155/Tage4 is, indeed, the authentic rodent homologue of human CD155. In this context, it is interesting to note that both CD155 and Tage4 are overexpressed in colon carcinoma (13, 18).
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grant
AI39485 (to E. W.). The costs of publication of this article were defrayed in
part by the payment of page charges. This article must therefore be hereby
marked "advertisement" in accordance with 18 U.S.C.
Section 1734 solely to indicate this fact. 
Supported by a doctoral fellowship of the Boehringer Ingelheim Fonds,
Heidesheim, Germany.
To whom correspondence should be addressed: Dept. of Molecular Genetics and
Microbiology, Stony Brook University, Stony Brook, NY 11794. Tel.:
631-632-8787; Fax: 631-632-8891; E-mail:
ewimmer{at}ms.cc.sunysb.edu.
1 Mueller, S., Rosenquist, T. A., Takai, Y., Bronson, R. A., and Wimmer, E.
(2003) Biol. Reprod., in press.
2 The abbreviations used are: SEAP, secreted, heat-stable human placental
alkaline phosphatase; AP, alkaline phosphatase; mAb, monoclonal antibody;
HBSS, Hanks' balanced salt solution; PBS, phosphate-buffered saline; BS3,
bis-(sulfosuccinimidyl)suberate.
3 J. Zhan and E. Wimmer, unpublished results.
4 M. Roivainen, personal communication.
5 S. Mueller and E. Wimmer, unpublished results.
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ACKNOWLEDGMENTS |
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