Implications of Nectin-like Molecule-2/IGSF4/RA175/SgIGSF/TSLC1/SynCAM1 in Cell-Cell Adhesion and Transmembrane Protein Localization in Epithelial Cells
Nectins are Ca2+-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 molecule-2 (Necl-2)/IGSF4/RA175/SgIGSF/TSLC1/SynCAM1. Necl-2 showed Ca2+-independent homophilic cell-cell adhesion activity. It furthermore showed Ca2+-independent 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, but not at specialized cell-cell junctions, such as tight junctions, adherens junctions, and desmosomes. Nectins bind afadin, whereas Necl-2 did not bind afadin but bound Pals2, a membrane-associated guanylate kinase family member known to bind Lin-7, implicated in the proper localization of the Let-23 protein in Caenorhabditis elegans, the homologue of mammalian epidermal growth factor receptor. These results indicate the unique localization of Necl-2 and its possible involvement in localization of a transmembrane protein(s) through Pals2. Nectins are Ca2+-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 molecule-2 (Necl-2)/IGSF4/RA175/SgIGSF/TSLC1/SynCAM1. Necl-2 showed Ca2+-independent homophilic cell-cell adhesion activity. It furthermore showed Ca2+-independent 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, but not at specialized cell-cell junctions, such as tight junctions, adherens junctions, and desmosomes. Nectins bind afadin, whereas Necl-2 did not bind afadin but bound Pals2, a membrane-associated guanylate kinase family member known to bind Lin-7, implicated in the proper localization of the Let-23 protein in Caenorhabditis elegans, the homologue of mammalian epidermal growth factor receptor. These results indicate the unique localization of Necl-2 and its possible involvement in localization of a transmembrane protein(s) through Pals2. Cell-cell adhesion is critical for tissue patterning and morphogenesis as well as for maintenance of normal tissues. In polarized epithelial cells, intercellular adhesion is mediated through a junctional complex comprised of tight junctions (TJs), 1The abbreviations used are: TJs, tight junctions; AJs, adherens junctions; DSs, desmosomes; F-actin, actin filament; aa, amino acid(s); Necl, nectin-like molecule; GST, glutathione S-transferase; MBP, maltose-binding protein; nectin-1-L cells, L cells stably expressing human nectin-1α; nectin-2-L cells, L cells stably expressing mouse nectin-2α; nectin-3-L cells, L cells stably expressing mouse nectin-3α; Necl-5-L cells, L cells stably expressing Necl-5; Necl-1-L cells, L cells stably expressing FLAG-Necl-1; non-tagged Necl-2-L cells, L cells stably expressing full-length Necl-2; Necl-2-L cells, L cells stably expressing FLAG-Necl-2; Necl-2-ΔC-L cells, L cells stably expressing the C-terminal four aa-deleted Necl-2; GST-Necl-2-CP, the cytoplasmic region of Necl-2 fused to GST; Ab, antibody; mAb, monoclonal Ab; pAb, polyclonal Ab; MDCK, Madin-Darby canine kidney; GFP, green fluorescent protein; JAM, junctional adhesion molecule; Necl-2-ΔEC, the transmembrane and cytoplasmic region of Necl-2; MBP-Pals2, full-length Pals2 fused to MBP; MBP-Pals2-PDZ, the PDZ domain of Pals2 fused to MBP; GST-Necl-2-CPΔC, the C-terminal four-aa-deleted cytoplasmic region of Necl-2 fused to GST; HA, hemagglutinin. adherens junctions (AJs), and desmosomes (DSs) (1Tsukita S. Furuse M. Itoh M. Nat. Rev. Mol. Cell Biol. 2001; 2: 285-293Crossref PubMed Scopus (2091) Google Scholar). 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 (1Tsukita S. Furuse M. Itoh M. Nat. Rev. Mol. Cell Biol. 2001; 2: 285-293Crossref PubMed Scopus (2091) Google Scholar). At AJs, E-cadherin is a key Ca2+-dependent cell-cell adhesion molecule (2Takeichi M. Development. 1988; 102: 639-655Crossref PubMed Google Scholar, 3Gumbiner B.M. J. Cell Biol. 2000; 148: 399-404Crossref PubMed Scopus (690) Google Scholar). TJs and AJs are undercoated with actin filament (F-actin) bundles. At DSs, desmosomal cadherins, desmocollin and desmoglein, are key Ca2+-dependent cell-cell adhesion molecules (4Garrod D.R. Merritt A.J. Nie Z. Curr. Opin. Cell Biol. 2002; 14: 537-545Crossref PubMed Scopus (193) Google Scholar). 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 (5Takai Y. Nakanishi H. J. Cell Sci. 2003; 116: 17-27Crossref PubMed Scopus (493) Google Scholar). Although cadherins are Ca2+-dependent cell-cell adhesion molecules, nectins are Ca2+-independent cell-cell adhesion molecules that comprise a family of four members, nectin-1, -2, -3, and -4 (5Takai Y. Nakanishi H. J. Cell Sci. 2003; 116: 17-27Crossref PubMed Scopus (493) Google Scholar). All nectins have one extracellular region with three Ig-like loops, one transmembrane region, and one cytoplasmic region (5Takai Y. Nakanishi H. J. Cell Sci. 2003; 116: 17-27Crossref PubMed Scopus (493) Google Scholar). Each nectin forms homo-cis-dimers followed by formation of homo-trans-dimers, causing cell-cell adhesion (5Takai Y. Nakanishi H. J. Cell Sci. 2003; 116: 17-27Crossref PubMed Scopus (493) Google Scholar). 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 (5Takai Y. Nakanishi H. J. Cell Sci. 2003; 116: 17-27Crossref PubMed Scopus (493) Google Scholar). Nectin-4 also forms hetero-trans-dimers with nectin-1 (5Takai Y. Nakanishi H. J. Cell Sci. 2003; 116: 17-27Crossref PubMed Scopus (493) Google Scholar). Nectins except nectin-4 have a C-terminal conserved motif of four amino acid (aa) residues that interacts with the PDZ domain of afadin (5Takai Y. Nakanishi H. J. Cell Sci. 2003; 116: 17-27Crossref PubMed Scopus (493) Google Scholar). Nectin-4 does not have this consensus motif but binds afadin. Afadin is an F-actin-binding protein with one PDZ domain and three other domains and connects nectins to the actin cytoskeleton (5Takai Y. Nakanishi H. J. Cell Sci. 2003; 116: 17-27Crossref PubMed Scopus (493) Google Scholar). 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 molecules (Necls) (6Ikeda W. Kakunaga S. Itoh S. Shingai T. Takekuni K. Satoh K. Inoue Y. Hamaguchi A. Morimoto K. Takeuchi M. Imai T. Takai Y. J. Biol. Chem. 2003; 278: 28167-28172Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). These include Necl-1/TSLL1/SynCAM3 (7Fukuhara H. Kuramochi M. Nobukuni T. Fukami T. Saino M. Maruyama T. Nomura S. Sekiya T. Murakami Y. 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Kuramochi M. Maruyama T. A. Murakami Y. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). It is by with the of of nectins that Necl-2 forms followed by formation of causing cell-cell cell-cell adhesion activity of activity of was for L Necl-2-L localization of Necl-2 at cell-cell adhesion of Necl-2-L The localization of Necl-2 in Necl-2-L cells was by with the formation of homo-cis-dimers of of Necl-2-L cells was in the or of Each of protein was to followed by with the by the The results are of three of homophilic and heterophilic cell-cell adhesion (5Takai Y. Nakanishi H. J. Cell Sci. 2003; 116: 17-27Crossref PubMed Scopus (493) Google Scholar). We by the Necl-2 heterophilic cell-cell adhesion activity with other nectins and Necl-2-L cells formed heterophilic with L cells stably expressing or and nectin-3-L cells, but not with L cells stably expressing nectin-1, or or cells, Necl-1-L cells formed homophilic The of the not by the of or not The of Necl-5-L cells did not form homophilic as (6Ikeda W. Kakunaga S. Itoh S. Shingai T. Takekuni K. Satoh K. Inoue Y. Hamaguchi A. Morimoto K. Takeuchi M. Imai T. Takai Y. J. Biol. Chem. 2003; 278: 28167-28172Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). and cells formed homophilic as K. Nakanishi H. M. K. Satoh K. Satoh A. H. J. A. A. Takai Y. J. Cell Biol. 1999; PubMed Scopus Google Scholar, K. Nakanishi H. K. M. M. K. A. Takai Y. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). The of the formed between nectin-1-L and nectin-3-L cells was the (5Takai Y. Nakanishi H. J. 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Science. 2002; 297: 1525-1531Crossref PubMed Scopus (651) Google Scholar). the of Necl-2 was in other tissues the the and the but was not in the not that the for Necl-2 was at the basolateral plasma membrane of the epithelial cells of the mouse gall bladder, and and It that the for Necl-2 was not with the for afadin, which is known to to AJs undercoated with the the for which is known to to TJs and showed that the for Necl-2 at the basolateral plasma membrane of the epithelial cells of gall bladder, but was at the of TJs, AJs, and DSs These Necl-2 showed to the localization of nectins and afadin that are to AJs, which are undercoated with (5Takai Y. Nakanishi H. J. Cell Sci. 2003; 116: 17-27Crossref PubMed Scopus (493) Google localization of of mouse tissues. The of mouse gall bladder, and with of the and gall and Necl-2; and and the cell-cell junctions of of mouse gall epithelial The with the followed by with the a and junctional complex region at the apical of gall epithelial basolateral plasma membrane of gall epithelial The results are of three of Necl-2 in an Cell but in Cell Necl-2 is expressed in cells in that Necl-2 was in mouse epithelial cells but not in mouse or cells The protein of in cells to an of Necl-2 as in the In cells the for Necl-2 was at the basolateral plasma membrane of cell-cell and In the for Necl-2 was not in or cells not These results that Necl-2 is expressed in epithelial cells but not in of Necl-2 to at the of Necl-2 to the basolateral plasma The a key in the formation of the junctional complex of AJs and TJs in epithelial cells (5Takai Y. Nakanishi H. J. Cell Sci. 2003; 116: 17-27Crossref PubMed Scopus (493) Google Scholar). cells have that afadin, and and are at the adhesion AJs T. Nakanishi H. T. K. K. M. T. Takai Y. 1999; PubMed Scopus Google Scholar, S. Itoh M. A. S. J. Cell Sci. PubMed Google Scholar). These adhesion with each other to form adhesion which AJs. the formation of AJs, junctional adhesion molecule is at these adhesion K. A. Y. T. S. D. J. 2001; 20: PubMed Scopus Google Scholar). and are at the apical of AJs, in the formation of TJs A. K. Shimizu M. K. S. J. Cell Sci. 2002; PubMed Scopus Google Scholar, Y. S. Itoh M. Furuse M. S. J. Cell 1999; PubMed Scopus Google Scholar). We at which Necl-2 is to the adhesion The for Necl-2 was at the adhesion The was also at the adhesion but the of the for Necl-2 from those for and afadin and These results that Necl-2 is to the cell-cell adhesion at the with the of the and and is to the other the region of the basolateral plasma of Pals2 to except nectin-4 have a C-terminal conserved motif of four residues that interacts with the PDZ domain of afadin (5Takai Y. Nakanishi H. J. Cell Sci. 2003; 116: 17-27Crossref PubMed Scopus (493) Google Scholar). Although Necl-2 this Necl-2 did not bind afadin, as by the and the and in the of we to a of the transmembrane and cytoplasmic region of Necl-2 as we a protein(s) by the and one from a mouse It Pals2 the We Necl-2 binds Pals2 in and in the of full-length Pals2. Necl-2 and Pals2 in cells, and Necl-2 was with the Pals2 was with Necl-2 Pals2 was in Necl-2-L cells, the for Pals2 was with that for Necl-2 at the cell-cell Pals2 was in Necl-2-ΔC-L cells cells stably expressing the C-terminal four aa-deleted the for was at the cell-cell but the for Pals2 was not The protein of the cytoplasmic region of Necl-2 bound the protein of full-length Pals2 and the PDZ domain of Pals2 not the protein of the cytoplasmic region of of which the C-terminal four did not bind or not These results indicate that Necl-2 binds Pals2 and that this is mediated through the C-terminal four of Necl-2 and the PDZ domain of Pals2. on expressing and cells expressing have that Necl-2 Ca2+-independent homophilic cell-cell adhesion activity (8Biederer T. Sara Y. Mozhayeva M. Atasoy D. Liu X. Kavalali E.T. Sudhof T.C. Science. 2002; 297: 1525-1531Crossref PubMed Scopus (651) Google Scholar, M. M. H. Kuramochi M. Maruyama T. A. Murakami Y. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). We have here these by the L cells expressing each cell-cell adhesion molecule M. M. H. Kuramochi M. Maruyama T. A. Murakami Y. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). we have that Necl-2 furthermore Ca2+-independent heterophilic cell-cell adhesion activity with Necl-1/TSLL1/SynCAM3 and but not with nectin-1, or that Necl-2 is of cell-cell adhesion by with these and have been to expressed as by T. Ohashi K. Mizuno K. Iseki S. Mol. Reprod. Dev. 2001; 60: 158-164Crossref PubMed Scopus (86) Google Scholar, T. Satoh H. Fujita E. Maruyama T. H. Kuramochi M. S. Momoi T. Murakami Y. 2002; PubMed Scopus Google Scholar), but been to expressed in the mouse as by (8Biederer T. Sara Y. Mozhayeva M. Atasoy D. Liu X. Kavalali E.T. Sudhof T.C. Science. 2002; 297: 1525-1531Crossref PubMed Scopus (651) Google Scholar). that Necl-2 is expressed in a variety of mouse tissues thus far and is with the results of and but not with that of (8Biederer T. Sara Y. Mozhayeva M. Atasoy D. Liu X. Kavalali E.T. Sudhof T.C. Science. 2002; 297: 1525-1531Crossref PubMed Scopus (651) Google Scholar). The for this between the of and those of and Necl-2 is not but to the of the used for that Necl-2 at the basolateral plasma membrane of epithelial and this is with the that at the basolateral plasma membrane of cells expressing M. M. H. Kuramochi M. Maruyama T. A. Murakami Y. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). by that Necl-2 at the basolateral plasma membrane except for specialized cell-cell junctions, such as AJs, TJs, and unique localization of Necl-2 is from those of other known cell-cell adhesion and at TJs, nectins at AJs, E-cadherin at AJs and the plasma and desmocollin and at DSs (1Tsukita S. Furuse M. Itoh M. Nat. Rev. Mol. Cell Biol. 2001; 2: 285-293Crossref PubMed Scopus (2091) Google Scholar, D.R. Merritt A.J. Nie Z. Curr. Opin. Cell Biol. 2002; 14: 537-545Crossref PubMed Scopus (193) Google Scholar, Y. Nakanishi H. J. Cell Sci. 2003; 116: 17-27Crossref PubMed Scopus (493) Google Scholar, S. A. S. Curr. Opin. Cell Biol. PubMed Scopus Google Scholar, B.M. Full Text Full Text PDF PubMed Scopus Google Scholar). Nectin-3 been to in the formation of AJs in epithelial cells (5Takai Y. Nakanishi H. J. Cell Sci. 2003; 116: 17-27Crossref PubMed Scopus (493) Google Scholar). the of Necl-2 to with that Necl-2 to the cell-cell adhesion in the of AJs. cells that Necl-2 is to the cell-cell adhesion at the with the of the and Necl-2 is to the cell-cell adhesion from to the region of the basolateral plasma Nectins are to AJs undercoated with and are from the Necl-2 (5Takai Y. Nakanishi H. J. Cell Sci. 2003; 116: 17-27Crossref PubMed Scopus (493) Google Scholar). 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Necl-2 binds to and and also binds Pals2 of transmembrane for of the of which cell-cell adhesion and localization of transmembrane in We S. for with L and cells and of for with of Pals2. We Inoue and Hamaguchi for