Direct Binding of Cell Polarity Protein PAR-3 to Cell-Cell Adhesion Molecule Nectin at Neuroepithelial Cells of Developing Mouse*

PAR-3 is a cell polarity protein that localizes at tight junctions (TJs) by direct binding to an immunoglobulin (Ig)-like cell-cell adhesion molecule JAM-1 in mammalian epithelial cells. Another Ig-like cell-cell adhesion molecule nectin plays a role in the localization of JAM-1 at TJs in epithelial cells. Nectin furthermore plays a role in the organization of adherens junctions (AJs) and TJs. Nectin comprises a family of four members, nectin-1, -2, -3, and -4. Nectins are associated with the actin cytoskeleton through afadin, of which the PDZ domain binds to nectins through their C-terminal four amino acids. We show here that PAR-3 binds to nectin-1 and -3 in neuroepithelial cells of the embryonic telencephalon, which are equipped with AJs, but not with typical TJs. Nec-tin-1, -2, -3, and afadin, but not JAM-1, were concentrated at AJs in neuroepithelial cells of the embryonic telencephalon at E13.5 and PAR-3 co-localized with nectins. PAR-3 was

Cell polarity is fundamental not only for cell functions but also for development and tissue maintenance (1,2). Studies in mammalian epithelial cells have revealed that establishment of cell polarity depends upon cell adhesion, intracellular signaling, cytoskeletal organization, and protein sorting. Mechanisms of establishment of cell polarity have been studied most extensively in Caenorhabditis elegans and Drosophila (3). PAR proteins, PAR-1 to -6, were first identified in C. elegans as indispensable proteins that are involved in establishment of the anterior-posterior cell polarity of the one-cell embryo (4). Thereafter, homologous proteins have been identified in Drospophila and mammals (5). In mammals, PAR-3, a mammalian homologue of the par-3 gene product, was first identified as an atypical protein kinase C (aPKC) 1 -binding protein (6).
PAR-3 localizes at tight junctions (TJs) and forms a ternary complex with PAR-6 and aPKC in mammalian epithelial cells (5). These three proteins directly interact with each other and play a critical role in the apico-basal polarization of mammalian epithelial cells (5). This PAR-3-aPKC-PAR-6 complex is an evolutionarily conserved cell polarization machinery that works ubiquitously in a variety of biological context from warm embryos to differentiated mammalian cells (5). PAR-3 has recently been shown to be directly bound to junctional adhesion molecule-1 (JAM-1), a Ca 2ϩ -independent immunoglobulin (Ig)like cell-cell adhesion molecule that localizes at TJs in epithelial cells (7,8). PAR-3 has three PDZ domains and one aPKCbinding domain (6), and this binding to JAM-1 is mediated through the first PDZ domain of PAR-3 and the C-terminal four aa (amino acids) of JAM-1 (7). The PAR-3-aPKC-PAR-6 complex is tethered to TJs through its binding to JAM-1. At TJs, claudin is a key cell-cell adhesion molecule which forms TJ strands (9). Claudin is associated with the actin cytoskeleton through ZO-1. ZO-1 furthermore binds both to the C-terminal four aa of JAM-1 through the third PDZ domain of ZO-1 (8) and to the cytoplasmic tail of claudin through the first PDZ domain of ZO-1 (10). Through these interactions, JAM-1 might be recruited and tethered to TJ strands.
In epithelial cells, TJs constitute a junctional complex with adherens junctions (AJs) and desmosomes (9). These junctional structures are typically aligned from the apical to the basal side, although desmosomes are independently distributed in other areas. The formation and maintenance of TJs are dependent on the cell-cell adhesion activity of E-cadherin. E-Cadherin is a key Ca 2ϩ -dependent cell-cell adhesion molecule at AJs (11,12). E-Cadherin is associated with the actin cytoskeleton through peripheral membrane proteins, including ␣-, ␤-, and ␥-catenins, ␣-actinin, and vinculin (11,12). Nectin and afadin constitute another cell-cell adhesion unit at cell-cell AJs (13). Nectin is a Ca 2ϩ -independent Ig-like cell-cell adhesion molecule which comprises a family of four members, nectin-1, -2, -3, and -4. Most of the nectin family members have a C-terminal conserved motif of four aa residues (E/A-X-Y-V), which interacts with the PDZ domain of afadin. Afadin is an actin filament-binding protein with one PDZ domain and four other * The work at Osaka University was supported by grants-in-aid for Scientific Research and for Cancer Research from the Ministry of Education, Culture, Sports, Science, and Technology, Japan (2001,2002). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
domains and connects nectin to the actin cytoskeleton. The nectin-afadin unit has roles in the organization of E-cadherinbased AJs and claudin-based TJs in epithelial cells (14 -17), in the organization of AJs in fibroblasts (14,15,17), in the formation of synapses in neurons (18), and in the formation of Sertoli cell-spermatids junctions in the testis (19), in cooperation with or independently of cadherin.
In addition to the localization of the PAR-3-aPKC-PAR-6 complex at TJs in epithelial cells, this complex localizes at AJs in fibroblasts (6) and in the neuroepithelial cells of the embryonic telencephalon during E10.5 to E14.5 (20). Neuroepithelial cells at these stages contain only AJs and gap junctions, but no typical TJs (21,22), and the mechanism of the localization of the PAR-3-aPKC-PAR-6 complex remains unknown in this cell type. We show here that nectin-1 and -3, but not JAM-1, are involved in the localization of PAR-3 at AJs in the neuroepithelial cells of the embryonic telencephalon.
Immunoprecipitation-The whole brain was dissected from mouse embryo at E13.5, and the sample was incubated in 0.75 mM dithiobis (succinimidylpropionate) in PBS at 4°C for 2 h. The cross-linking reaction was stopped by adding 1 M glycine at pH 7.5 in PBS. The sample was homogenized in a homogenization buffer (20 mM Tris-HCl at pH 8.0, 150 mM NaCl, 10% glycerol, 5 mM CaCl 2 , 5 mM MgCl 2 , 10 g/ml leupepitin, 1.5 g/ml aprotinin, and 10 M p-amidinophenylmethanesulfonyl fluoride). An equal volume of the homogenization buffer containing 2% Nonidet P-40 was added to the homogenate, and the sample was subjected to centrifugation at 20,000 ϫ g for 20 min. The extract (2 mg of protein) was incubated with protein G-Sepharose beads (Amersham Biosciences) coated with the anti-PAR-3 pAb or control IgG at 4°C for 16 h. After the beads were extensively washed with the homogenization buffer containing 1% Nonidet P-40, the bound proteins were eluted by boiling the beads in the SDS sample buffer (60 mM Tris-HCl at pH 6.7, 3% SDS, 2% 2-mercaptoethanol, and 5% glycerol) for 5 min and subjected to SDS-PAGE, followed by Western blotting.
Affinity Chromatography-To determine the binding domains of Other Procedures-Immunofluorescence microscopy of frozen sections of mouse embryonic neuroepithelial cells and cultured cells was done as described (23,29). Immunoelectron microscopy of mouse embryonic neuroepithelial cells was done using the silver enhancement technique as described (30). SDS-PAGE was done as described (31). Protein concentrations were determined with bovine serum albumin as a reference protein as described (32).

RESULTS
In neuroepithelial cells of the embryonic telencephalon at E13.5, the immunofluorescence signals for nectin-1, -2, and -3 and afadin were highly concentrated at the lumenal surface of neuroepithelial cells (Fig. 1, A-C). All of these signals apparently co-localized. The signal for PAR-3 colocalized with the signal for nectin-3 (Fig. 1D). The signals for ZO-1 and ␤-catenin colocalized with the signal for afadin as described (20) (data not shown). However, the signal for occludin or JAM-1 was not detected (Fig. 1, E and F).
We then examined by the immunoprecipitation assay whether PAR-3 binds to nectins in the neuroepithelial cells of the embryonic telencephalon at E13.5. When PAR-3 was immunoprecipitated by its Ab from the embryonic mouse whole brain at E13.5, including telencephalon, nectin-1 and -3, but not nectin-2 or JAM-1, were co-immunoprecipitated with PAR-3 ( Fig. 2A). These results suggest that PAR-3 binds to nectin-1 and -3 in the neuroepithelial cells of the embryonic telencephalon.
It has been shown that AJs, but not typical TJs, are observed in neuroepithelial cells (21,22). Consistently, AJs, which are undercoated with actin filament bundles, but not typical TJs, Direct Binding of PAR-3 to Nectin 5498 were observed (Fig. 3). Immunogolds of nectin-3, PAR-3, and afadin localized at AJs. Immunogolds of nectin-1 also localized at AJs (data not shown). These results, together with the result obtained by the co-immunprecipitation experiment, suggest that PAR-3 binds to nectin-1 and -3 and co-localize with them at AJs of neuroepothelial cells of the embryonic telencephalon.
Thus, both afadin and PAR-3 bind to the C-terminal four aa of nectin-1 and -3. We therefore compared the affinities of afadin and PAR-3 for nectin-1 and -3. Various amounts of either afadin or PAR-3 alone or both were incubated with nectin-3-CP. When the same amounts of afadin and PAR-3 were mixed with nectin-3-CP, apparently the same amounts of afadin and PAR-3 bound to it, and 1 mol of each maximally bound to 2 mol of nectin-3-CP (Fig. 2D). The essentially same results were obtained for nectin-1 (data not shown). These results indicate that the affinities of PAR-3 and afadin for nectin-1 and -3 were apparently similar.

FIG. 2. Direct binding of PAR-3 to nectin-3.
A, co-immunoprecipitation of nectin-3 with PAR-3. A tissue extract from the whole brain of mouse embryo at E13.5, including telencephalon, was subjected to immunoprecipitation with the anti-PAR-3 pAb or the control IgG. The immunoprecipitate was then subjected to SDS-PAGE (10% polyacrylamide gel), followed by Western blotting with the anti-PAR-3 pAb, the anti-nectin-1␣ pAb, the anti-nectin-2 mAb, the anti-nectin-3␣ pAb, or the anti-JAM-1 pAb. Lane 1, the anti-PAR-3 pAb; lane 2, the control IgG. B, direct binding of PAR-3 to nectin-3 through the C-terminal four aa of nectin-3␣. GST-nectin-3␣-CP or GST-nectin-3␣-CP⌬C was applied to the MBP-PAR-3-PDZ1-3-immobilized beads. After the beads were extensively washed, the bound proteins were subjected to SDS-PAGE (10% polyacrylamide gel), followed by protein staining with Coomassie Brilliant Blue. Direct Binding of PAR-3 to Nectin 5499 trated there, either (data not shown). Nectin-1-L cells and nectin-1-L cells formed cell-cell junctions, and PAR-3 as well as afadin was concentrated there (data not shown). Nectin-1⌬C-L cells and nectin-1⌬C-L cells formed cell-cell junctions, but PAR-3 or afadin was not concentrated there (data not shown). Nectin-3 forms not only homo-trans-dimers with nectin-3 but also hetero-trans-dimers with nectin-1 and -2 (13). PAR-3 as well as afadin was concentrated at the junctions formed between nectin-3-L cells and L cells stably expressing nectin-1 or -2 (data not shown). These results are consistent with the above biochemical results and indicate that PAR-3 binds to nectin-1 and -3 in intact cells. DISCUSSION We have shown here that PAR-3 directly binds to nectin-1 and -3 and localizes with them at AJs of the neuroepithelial cells of the embryonic telencephalon. It has been shown that PAR-3 binds to JAM-1 and localizes with it at TJs in epithelial cells (7,8). In the neuroepithelial cells of the embryonic telencephalon, TJs are not well developed, and we could not significantly observe the immunofluorescence signal for occludin or JAM-1 at the cell-cell junction area. Furthermore, JAM-1 was not co-immunoprecipitated with PAR-3 under the conditions where nectin-1 and -3 were co-immunoprecipitated with PAR-3 from the embryonic mouse whole brain at E13.5 including telencephalon. It has been shown that PAR-3 forms a ternary complex with aPKC and PAR-6 (5) and that this complex localizes at AJs in the neuroepithelial cells of the embryonic telencephalon (20). Taken together, it is likely that the PAR-3-aPKC-PAR-6 complex localizes at AJs in the neuroepithelial cells by directly binding of PAR-3 to nectin-1 and -3. The physiological function of this direct binding remains unknown, but it may recruit aPKC and PAR-6 and play a role in the formation and maintenance of polarity of the neuroepithelial cells.
We have shown here that all nectin-1, -2, and -3 localize at AJs of the neuroepithelial cells of the embryonic telencephalon, but that the first PDZ domain of PAR-3 directly binds to the C-terminal four aa of nectin-1 and -3, but not to that of nectin-2. All nectin-1, -2, and -3 have a C-terminal consensus motif with four aa for the binding to PDZ domains and bind afadin. The aa sequences of the motif of nectin-1 and -3 are the same, whereas that of nectin-2 is different from them (24). This difference may determine the specific binding of PAR-3 to nectin-1 and -3.
All nectins directly bind afadin, and this binding is mediated through the PDZ domain of afadin and the C-terminal four aa of nectins (13). This paper demonstrates for the first time that nectin-1 and -3 directly bind not only afadin but also PAR-3. We have shown here that the affinities of PAR-3 and afadin for nectin-1 and -3 are roughly similar and that 1 mol of each afadin and PAR-3 binds to 2 mol of nectin-1 and -3. All nectins first form cis-dimers, followed by the formation of trans-dimers, eventually inducing cell-cell adhesion (13). It remains unknown whether both afadin and PAR-3 bind to the same cisdimer or either afadin or PAR-3 alone binds to the different cis-dimer.
Nectin-1 and -3 are expressed in many cell types in addition to the neuroepithelial cells (13). They may be involved in the localization of the PAR-3-aPKC-PAR-6 complex in cells where JAM-1 is not expressed. We have previously shown that the nectin-afadin unit plays a role in the localization of JAM-1 at TJs (16), where the PAR-3-aPKC-PAR-6 complex is associated with JAM-1. Another possible function of nectin-1 and -3 is that PAR-3 first binds to nectin-1 or -3 and then transferred to JAM-1 during the organization of the junctional complex in the epithelial cells equipped with TJs. Further studies are necessary for our understanding of the physiological role of the direct binding of PAR-3 to nectin-1 and -3 in the neuroepithelial cells and other cell types.