Implications of Nectin-like Molecule-2/IGSF4/RA175/SgIGSF/TSLC1/ SynCAM1 in Cell-Cell Adhesion and Transmembrane Protein Localization in Epithelial Cells*

Nectins are Ca 2 (cid:1) -independent immunoglobulin-like cell-cell adhesion molecules that play roles in organization of a variety of cell-cell junctions in cooperation with or independently of cadherins. Four nectins have been identified. Five nectin-like molecules, which have domain structures similar to those of nectins, have been identified, and we characterized here nectin-like mol-ecule-2 (Necl-2)/IGSF4/RA175/SgIGSF/TSLC1/SynCAM1. Necl-2 showed Ca 2 (cid:1) -independent homophilic cell-cell adhesion activity. It furthermore showed Ca 2 (cid:1) -inde-pendent heterophilic cell-cell adhesion activity with Necl-1/TSLL1/SynCAM3 and nectin-3. Necl-2 was widely expressed in rat tissues examined. Necl-2 localized at the basolateral plasma membrane in epithelial cells of the mouse gall bladder, 2-L and nectin-3-L cells were about 5, 10, and 10% that of the aggregates formed between nectin-1-L and nectin-3-L cells, respectively. These results indicate that Necl-2 has both Ca 2 (cid:1) independent homophilic and heterophilic cell-cell adhesion activities.

junctions (AJs), and desmosomes (DSs) (1). These junctional structures are typically aligned from the apical to basal sides, although DSs are independently distributed in other areas. The formation and maintenance of TJs and DSs depend upon the formation and maintenance of AJs. At TJs, claudins are key cell-cell adhesion molecules that form TJ strands (1). At AJs, E-cadherin is a key Ca 2ϩ -dependent cell-cell adhesion molecule (2,3). TJs and AJs are undercoated with actin filament (F-actin) bundles. At DSs, desmosomal cadherins, desmocollin and desmoglein, are key Ca 2ϩ -dependent cell-cell adhesion molecules (4). DSs are linkers of the intermediate filament cytoskeleton.
Nectins are emerging cell-cell adhesion molecules that play roles in the organization of a variety of cell-cell junctions, such as AJs and TJs in epithelial cells, synaptic junctions in neurons, and heterotypic junctions formed between the Sertoli cells and spermatids in the testis, in cooperation with or independently of cadherins (5). Although cadherins are Ca 2ϩ -dependent cell-cell adhesion molecules, nectins are Ca 2ϩ -independent cellcell adhesion molecules that comprise a family of four members, nectin-1, -2, -3, and -4 (5). All nectins have one extracellular region with three Ig-like loops, one transmembrane region, and one cytoplasmic region (5). Each nectin forms homo-cis-dimers followed by formation of homo-trans-dimers, causing cell-cell adhesion (5). Nectin-3 furthermore forms hetero-trans-dimers with either nectin-1 or -2, and the adhesion activity of each hetero-trans-dimers is stronger than that of each homo-trans-dimers (5). Nectin-4 also forms hetero-transdimers with nectin-1 (5). Nectins except nectin-4 have a Cterminal conserved motif of four amino acid (aa) residues that interacts with the PDZ domain of afadin (5). Nectin-4 does not have this consensus motif but binds afadin. Afadin is an Factin-binding protein with one PDZ domain and three other domains and connects nectins to the actin cytoskeleton (5).
Five molecules with one extracellular region containing three Ig-like loops, one transmembrane region, and one cytoplasmic region have thus far been identified. We have proposed, based on their domain structures which are similar to those of nectins, that these molecules are called nectin-like * The work at Osaka University was supported by grants-in-aid for Scientific Research and that for Cancer Research was supported by grants from the Ministry of Education, Culture, Sports, Science, and Technology, Japan (2001,2002). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank TM /EBI Data Bank with accession number(s) AY351388.
We have studied the properties of Necl-2, including its cellcell adhesion activity, its localization, and its binding protein.
We have found that Necl-2 localizes at the extra-junctional region of the basolateral plasma membrane of epithelial cells and directly binds Pals2. Pals2 was originally isolated as a Lin-7-binding protein (20). Lin-7 is a PDZ domain-containing protein that forms a heterotrimeric complex with Lin-2 and -10. This Lin-2⅐Lin-7⅐Lin-10 protein complex is involved in organization of epithelial and neuronal junctions in Caenorhabditis elegans and mammals (21). Pals2 belongs to the membraneassociated guanylate kinase family and consists of two Lin-2/-7 homology domains, one PDZ domain, one Src-homology 3 domain, and one guanylate kinase domain. Pals2 as well as Pals1 and Lin-2 constitutes a subfamily that binds to Lin-7 (20). Here we describe these novel properties of Necl-2.

EXPERIMENTAL PROCEDURES
Molecular Cloning of Mouse Necl-1 and -2 cDNAs-We performed reverse transcription-PCR from mouse brain total RNA using the Isogen RNA extraction kit (Nippon Gene), Ready-To-Go You-Prime First-Strand Beads and pd(N) 6 (Amersham Biosciences), Pfu turbo DNA polymerase (Stratagene), and the specific primers of mouse Necl-1 or -2. The primers were designed based on Necl-1 (GenBank TM /EMBL/DDBJ accession number AF195662) and Necl-2 (GenBank TM /EMBL/DDBJ accession number AB052293). The forward and reverse primers used were; Necl-1 cDNA, 5Ј-gcggaattcaccatgggggccccttccgccc-3Ј and 5Ј-gcggtcgacctagatgaaatattccttcttgtc-3Ј; Necl-2 cDNA, 5Ј-gcggaattcaccatggcgagtgctgtgctgcc-3Ј and 5Ј-gcggtcgacctagatgaagtactctttcttttc-3Ј, respectively. The reverse transcription-PCR products were cloned using Zero Blunt TOPO PCR cloning kit (Invitrogen). DNA sequencing was performed by the dideoxy nucleotide termination method using a DNA sequencer (ABI Prism 3100 Genetic Analyzer, PE Biosystems). The Necl-1 cDNA clone that we isolated was identical to AF195662 (data not shown). The Necl-2 cDNA clone that we isolated was identical to AB052293 except for the deletion of 336 -363 aa at the extracellular region (data not shown, GenBank TM /EMBL/DDBJ accession number AY351388). This deletion has been reported as one of the splicing variants (8).
Other Procedures-Immunofluorescence microscopy of cultured cells and co-immunoprecipitation assay were done as described (28). The cell aggregation assay, chemical cross-linking, and affinity chromatography were done as described (29). Immunoelectron microscopy of mouse tissues was done using the silver enhancement technique as described (31). SDS-PAGE was done as described (32). Protein concentrations were determined with bovine serum albumin as a reference protein as described (33).

Necl-2 and Pals2
sults by measuring the aggregation activity of Necl-2-L cells (cadherin-deficient L cells stably expressing Necl-2). Wild-type L cells endogenously expressed nectin-1 and -2 (22,26), but expression of Necl-2 was undetectable by Western blotting (see Fig. 5A). Wild-type L cells did not form visible cell aggregates (Fig. 1, Aa), but Necl-2-L cells formed aggregates (Fig. 1, Ab). The sizes of the aggregates were not significantly affected by the presence of Ca 2ϩ or EDTA (data not shown). These results are consistent with the earlier observation (8,17) and indicate that Necl-2 has Ca 2ϩ -independent homophilic cell-cell adhesion activity. This cell-cell adhesion activity of Necl-2 was furthermore confirmed by immunofluorescence microscopy. When Necl-2-L cells were cultured, the immunofluorescence signal for Necl-2 was concentrated at cell-cell contact sites (Fig. 1B). We have previously shown that each nectin forms cis-dimers followed by formation of trans-dimers, eventually causing cellcell adhesion (22). Similarly, Necl-2 formed cis-dimers (Fig.  1C). These results are consistent with the earlier observation using HEK293 cells expressing TSLC1 (17). It is likely by analogy with the mode of action of nectins that Necl-2 forms first cis-dimers followed by formation of trans-dimers, eventually causing cell-cell adhesion.
Tissue Distribution and Subcellular Localization of Necl-2-TSLC1 has been shown by Northern blotting to be expressed ubiquitously, except the skeletal muscle, in which the expression of TSLC1 was not detected (11,19). We confirmed these earlier results by Western blotting using the anti-Necl-2 mAb. Western blotting showed that an immunoreactive band at a molecular mass of 92 kDa was detected in various tissues thus far examined, including the brain, the lung, and the kidney (Fig. 3). In the testis, a larger band at 105 kDa was detected and might be one of the alternative splicing variants as described (8). After long exposure, the immunoreactive band of Necl-2 was detected in other tissues including the heart, the spleen, and the liver but was not detected in the skeletal muscle (data not shown). Immunofluorescence microscopy revealed that the signal for Necl-2 was highly concentrated at the basolateral plasma membrane of the epithelial cells of the mouse gall bladder, liver, and pancreas (Fig. 4A, a1, b1, and  c1). It may be noted that the signal for Necl-2 was not overlapped with the signal for afadin, which is known to be confined to AJs undercoated with the F-actin bundles (Fig. 4A, a1-a3), nor the signal for ZO-1, which is known to be confined to TJs (Fig. 4A, b1-b3 and c1-c3). Consistently, immunoelectron microscopy showed that the immunogold particles for Necl-2 were indeed concentrated at the basolateral plasma membrane of the epithelial cells of gall bladder, but was undetectable at the areas of TJs, AJs, and DSs (Fig. 4B, a-c). These findings about Necl-2 showed sharp contrast to the localization of nectins and afadin that are strictly confined to AJs, which are undercoated with F-actin bundles (5).
Expression of Necl-2 in an Epithelial Cell Line, but Not in Fibroblast Cell Lines-We next examined whether Necl-2 is differentially expressed in cells in culture. Western blotting revealed that Necl-2 was detected in mouse MTD-1A epithelial cells but not in mouse fibroblastic L, NIH3T3, or Swiss3T3 cells (Fig. 5A). The larger protein of 105 kDa detected in MTD-1A cells appeared to be an alternative splicing variant of Necl-2 as detected in the testis. In MTD-1A cells the immunofluorescence signal for Necl-2 was concentrated at the basolateral plasma membrane of cell-cell contact sites (Fig. 5B, a1-a3 and b1-b3). In contrast, the signal for Necl-2 was not observed in L, NIH3T3, or Swiss3T3 cells (data not shown). These results suggest that Necl-2 is dominantly expressed in epithelial cells but not in fibroblasts.
Assembly of Necl-2 to Cell-Cell Junctions at the Initial Stage of Their Formation-We next studied how Necl-2 localizes to the basolateral plasma membrane. The nectin-afadin unit plays a key role in the formation of the junctional complex of AJs and TJs in epithelial cells (5). Wound healing assay using MTD-1A cells have revealed that nectin, afadin, ZO-1, E-cadherin, and ␣and ␤-catenins are first assembled at the primordial, spot-like adhesion sites including AJs (34,35). These primordial adhesion sites fuse with each other to form short line-like adhesion sites, which develop into more matured AJs. During and/or after the formation of AJs, junctional adhesion molecule (JAM) is first assembled at these adhesion sites (36). Finally, occludin and claudin are recruited at the apical side of AJs, resulting in the formation of TJs (37,38). We examined at which stage Necl-2 is recruited to the adhesion sites. The immunofluorescence signal for Necl-2 was detected at the spotlike adhesion sites (Fig. 6, Aa-Ac). The signal was also observed at the line-like adhesion sites (Fig. 6, Ba-Bc), but the sites of the signal for Necl-2 were different from those for ZO-1 and afadin (Fig. 6, Ca-Cc and Da-Dc). These results suggest that Necl-2 is assembled to the cell-cell adhesion sites at the very early stage together with the components of the nectinafadin and E-cadherin-catenin units, and thereafter, is translocated to the other sites, presumably the extra-junctional region of the basolateral plasma membrane.
Direct Binding of Pals2 to Necl-2-Nectins except nectin-4 have a C-terminal conserved motif of four aa residues that interacts with the PDZ domain of afadin (5). Although Necl-2 has this motif, Necl-2 did not bind afadin, as estimated by the yeast two-hybrid assay and the affinity chromatography assay (Fig. 7, A and D). Therefore, in the last set of experiments we attempted to isolate a Necl-2-binding protein(s). By use of the transmembrane and cytoplasmic region of Necl-2 (Necl-2-⌬EC) as bait, we searched a Necl-2-binding protein(s) by the yeast two-hybrid screening and isolated one positive clone from a mouse testis library (Fig. 7A). It encoded Pals2 (lacking the N-terminal 1-31 aa). We then examined whether Necl-2 directly binds Pals2 in vitro and in vivo using the constructs of full-length Pals2. FLAG-tagged Necl-2 and HA-tagged Pals2 were coexpressed in HEK293 cells, and FLAG-tagged Necl-2 was immunoprecipitated with the anti-FLAG mAb. HA-tagged Pals2 was co-immunoprecipitated with FLAG-tagged Necl-2 (Fig. 7B). When HA-tagged Pals2 was overexpressed in Necl-2-L cells, the signal for Pals2 was co-localized with that for Necl-2 at the cell-cell contact sites (Fig. 7C, a1-a3). However, when HA-tagged Pals2 was overexpressed in Necl-2-⌬C-L cells (L cells stably expressing the C-terminal four aa-deleted Necl-2), the signal for Necl-2-⌬C was concentrated at the cell-cell contact sites, but the signal for Pals2 was not concentrated there (Fig. 7C, b1-b3). The pure recombinant protein of the cytoplasmic region of Necl-2 (GST-Necl-2-CP) bound the pure recombinant protein of full-length Pals2 (MBP-Pals2) (Fig. 7D) and the PDZ domain of Pals2 (MBP-Pals2-PDZ) (data not shown). However, the pure recombinant protein of the cytoplasmic region of Necl-2, of which the C-terminal four aa were deleted (GST-Necl-2-CP⌬C), did not bind MBP-Pals2 (Fig. 7D) or MBP-Pals2-PDZ (data not shown). These results indicate that Necl-2 directly binds Pals2 and that this binding is mediated through the C-terminal four aa of Necl-2 and the PDZ domain of Pals2.

DISCUSSION
Studies on Necl-2-mediated cell aggregation using MDCKcells expressing GFP-tagged TSLC1 and Drosophila S2 cells expressing SynCAM1 have shown that Necl-2 has Ca 2ϩ -independent homophilic cell-cell adhesion activity (8,17). We have confirmed here these earlier observations by the aggregation assay using L cells expressing each cell-cell adhesion molecule (17). Then we have shown that Necl-2 has furthermore Ca 2ϩindependent heterophilic cell-cell adhesion activity with Necl-1/TSLL1/SynCAM3 and nectin-3 but not with Necl-5/Tage4/ CD155/PVR, nectin-1, or nectin-2, suggesting that Necl-2 is capable of organizing cell-cell adhesion by interactions with these molecules.  TSLC1 and SgIGSF have been shown to be expressed ubiquitously as estimated by Northern blotting (11,19), but SynCAM1 has been shown to be specifically expressed in the mouse brain, as analyzed by Western blotting (8). Our present result indicates that Necl-2 is expressed in a wide variety of mouse tissues thus far examined and is consistent with the results of TSLC1 and SgIGSF but not with that of SynCAM1 (8). The exact reason for this inconsistency between the result of SynCAM1 and those of TSLC1, SgIGSF, and Necl-2 is not known, but it could be attributed to the specificity of the Ab used for SynCAM1.
Immunofluorescence microscopic analysis indicates that Necl-2 localizes at the basolateral plasma membrane of many epithelial cell types, and this result is consistent with the earlier observation that TSLC1 localizes at the basolateral plasma membrane of MDCK cells expressing GFP-tagged TSLC1 (17). Detailed analysis by immunoelectron microscopy indicates that Necl-2 localizes at the basolateral plasma membrane except for specialized cell-cell junctions, such as AJs, TJs, and DSs. This unique localization pattern of Necl-2 is quite different from those of any other known cell-cell adhesion molecules: claudins, occludin, and JAM at TJs, nectins at AJs, E-cadherin at AJs and the lateral plasma membrane, and desmocollin and desmoglein at DSs (1,4,5,39,40).
Nectin-3 has been shown to be involved in the formation of AJs in epithelial cells (5). Therefore, the ability of Necl-2 to interact with nectin-3 suggests that Necl-2 may be recruited to the nectin-3-based cell-cell adhesion in the process of forming AJs. Consistently, wound healing assay analysis using MTD-1A cells indicates that Necl-2 is assembled to the cell-cell adhesion sites at the very early stage together with the components of the nectin-afadin and E-cadherin-catenin units. After Necl-2 is assembled to the primordial cell-cell adhesion sites, it may be translocated from there to the extra-junctional region of the basolateral plasma membrane. Nectins are confined to AJs undercoated with F-actin bundles and are absent from the areas where Necl-2 localizes (5). Therefore, Necl-2 is likely to form homodimers at these areas lacking nectins. The mechanism of segregation of Necl-2 from nectin-3 at the plasma membrane is currently unknown, but it is of crucial importance for our understanding of how the membrane domains of epithelial cells are organized.
It has been shown that TSLC1 binds DAL-1 through a band 4.1-binding motif at the juxtamembrane region (18). We have shown here that Necl-2 does not bind afadin but directly binds Pals2. This binding is mediated through the C-terminal consensus motif of four aa of Necl-2 and the PDZ domain of Pals2. Thus, Necl-2 appears to bind both DAL-1 and Pals2. DAL-1 belongs to the band 4.1 family, connects TSLC1/Necl-2 to the actin cytoskeleton (18), and is known as a tumor suppressor (41). In contrast, Pals2 is a membrane-associated guanylate kinase family member and binds to Lin-7, of which the C. elegans homologue is implicated in the proper localization of the Let-23 protein, the homologue of mammalian epidermal growth factor receptor (42). Taken together, Necl-2 directly binds to Necl-1 and nectin-3 extracellularly, mediating cell adhesions, and also binds Pals2 intracellularly, mediating localizations of transmembrane proteins. Further studies would be necessary for our understanding of the physiological role of Necl-2, which regulates cell-cell adhesion and localization of transmembrane proteins in mammals. FIG. 7. Direct binding of Pals2 to Necl-2. A, yeast two-hybrid analysis of binding of Necl-2-⌬EC to Pals2. Yeast transformants with the indicated plasmids were streaked on synthetic complete medium lacking adenine to score ADE2 reporter activity and incubated at 30°C for 3 days. pACT2-Pals2 (lacking the N-terminal 1-31 aa) was a positive clone that was isolated by yeast two-hybrid screening of pGBD-C1-Necl-2-⌬EC from a mouse testis library. B, co-immunoprecipitation of Pals2 with Necl-2. pFLAG-CMV1-Necl-2 and pCMV-HA-Pals2 (full-length) were transfected to HEK293 cells, and the cell extract (500 g of protein each) was subjected to immunoprecipitation with the anti-FLAG mAb. The immunoprecipitates were then subjected to SDS-PAGE (10% polyacrylamide gel) followed by Western blotting with the anti-HA mAb or the anti-FLAG pAb. C, Necl-2-dependent recruitment of Pals2 to cellcell adhesion sites. Necl