Tage4/Nectin-like Molecule-5 Heterophilically trans-Interacts with Cell Adhesion Molecule Nectin-3 and Enhances Cell Migration
2003; Elsevier BV; Volume: 278; Issue: 30 Linguagem: Inglês
10.1074/jbc.m303586200
ISSN1083-351X
AutoresWataru Ikeda, Shigeki Kakunaga, Shinsuke Itoh, Tatsushi Shingai, Kyoji Takekuni, Keiko Satoh, Yoko Inoue, Akiko Hamaguchi, Koji Morimoto, Masakazu Takeuchi, Toshio Imai, Yoshimi Takai,
Tópico(s)Skin and Cellular Biology Research
ResumoMalignant transformation of cells causes disruption of cell-cell adhesion, enhancement of cell motility, and invasion into surrounding tissues. Nectins have both homophilic and heterophilic cell-cell adhesion activities and organize adherens junctions in cooperation with cadherins. We examined here whether Tage4, which was originally identified to be a gene overexpressed in colon carcinoma and has a domain structure similar to those of nectins, is involved in cell adhesion and/or migration. Tage4 heterophilically trans-interacted with nectin-3, but not homophilically with Tage4. Expression of Tage4 was markedly elevated in NIH3T3 cells transformed by an oncogenic Ki-Ras (V12Ras-NIH3T3 cells) as compared with that of wild-type NIH3T3 cells. trans-Interaction of Tage4 with nectin-3 enhanced motility of V12Ras-NIH3T3 cells. Tage4 did not bind afadin, a nectin- and actin filament-binding protein that connects nectins to the actin cytoskeleton and cadherins through catenins. Thus, Tage4 heterophilically trans-interacts with nectin-3 and regulates cell migration. Tage4 is tentatively re-named here nectin-like molecule-5 (necl-5) on the basis of its function and domain structure similar to those of nectins. Malignant transformation of cells causes disruption of cell-cell adhesion, enhancement of cell motility, and invasion into surrounding tissues. Nectins have both homophilic and heterophilic cell-cell adhesion activities and organize adherens junctions in cooperation with cadherins. We examined here whether Tage4, which was originally identified to be a gene overexpressed in colon carcinoma and has a domain structure similar to those of nectins, is involved in cell adhesion and/or migration. Tage4 heterophilically trans-interacted with nectin-3, but not homophilically with Tage4. Expression of Tage4 was markedly elevated in NIH3T3 cells transformed by an oncogenic Ki-Ras (V12Ras-NIH3T3 cells) as compared with that of wild-type NIH3T3 cells. trans-Interaction of Tage4 with nectin-3 enhanced motility of V12Ras-NIH3T3 cells. Tage4 did not bind afadin, a nectin- and actin filament-binding protein that connects nectins to the actin cytoskeleton and cadherins through catenins. Thus, Tage4 heterophilically trans-interacts with nectin-3 and regulates cell migration. Tage4 is tentatively re-named here nectin-like molecule-5 (necl-5) on the basis of its function and domain structure similar to those of nectins. In multicellular organisms, cell adhesion and migration are critical for many events, including tissue patterning, morphogenesis, and maintenance of normal tissues (1Takeichi M. Curr. Opin. Cell Biol. 1995; 7: 619-627Crossref PubMed Scopus (1272) Google Scholar, 2Gumbiner B.M. Cell. 1996; 84: 345-357Abstract Full Text Full Text PDF PubMed Scopus (2979) Google Scholar, 3Lauffenburger D.A. Horwitz A.F. Cell. 1996; 84: 359-369Abstract Full Text Full Text PDF PubMed Scopus (3319) Google Scholar). They also play roles in malignant transformation of cells (4Thiery J.P. Nat. Rev. Cancer. 2002; 2: 442-454Crossref PubMed Scopus (5603) Google Scholar). Adhesion and migration of non-transformed normal cells are dynamic and well regulated (2Gumbiner B.M. Cell. 1996; 84: 345-357Abstract Full Text Full Text PDF PubMed Scopus (2979) Google Scholar). Cells disrupt cell-cell adhesion and start to migrate in response to extracellular cues, such as growth factors, cytokines, and extracellular matrix molecules (4Thiery J.P. Nat. Rev. Cancer. 2002; 2: 442-454Crossref PubMed Scopus (5603) Google Scholar). When migrating cells contact other cells, they stop migration and proliferation and adhere to each other to become confluent (5Abercrombie M. In Vitro. 1970; 6: 128-142Crossref PubMed Scopus (262) Google Scholar, 6Martz E. Steinberg M.S. J. Cell. Physiol. 1972; 79: 189-210Crossref PubMed Scopus (112) Google Scholar). This phenomenon is known for a long time as contact inhibition of cell movement and proliferation. Transformation of cells causes disruption of cell-cell adhesion, increase of cell motility, and loss of contact inhibition of cell movement and proliferation, eventually leading the transformed cells to invasion into surrounding tissues and metastasis to other organs (4Thiery J.P. Nat. Rev. Cancer. 2002; 2: 442-454Crossref PubMed Scopus (5603) Google Scholar, 7Abercrombie M. Nature. 1979; 281: 259-262Crossref PubMed Scopus (295) Google Scholar). However, molecular mechanisms underlying these physiological or pathological processes are not fully understood. Cell-cell adherens junctions (AJs) 1The abbreviations used are: AJ, adherens junction; aa, amino acid(s); necl, nectin-like molecule; V12Ras-NIH3T3 cells, NIH3T3 cells stably expressing V12Ki-Ras; SEAP, secreted alkaline phosphatase; Neap, the extracellular fragment of nectin fused to SEAP; Nef, the extracellular fragment of nectin fused to the human IgG Fc; nectin-1-L cells, L cells stably expressing full-length human nectin-1α; nectin-2-L cells, L cells stably expressing full-length mouse nectin-2α; nectin-3-L cells, L cells stably expressing full-length mouse nectin-3α; non-tagged-necl-5-L-cells, L cells stably expressing full-length necl-5; necl-5-L-cells, L cells stably expressing FLAG-necl-5; necl-5-ΔEC-L-cells, L cells stably expressing FLAG-necl-5-ΔEC; Ab, antibody; Lef, the extracellular fragment of necl fused to the human IgG Fc; GST, glutathione S-transferase; GST-necl-5-CP, the cytoplasmic tail of necl-5 fused to GST; Leap, the extracellular fragment of necl fused to SEAP; mAb, monoclonal Ab; pAb, polyclonal antibody; DiI, 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate; EL cells, L cells stably expressing full-length E-cadherin. play major roles in cell-cell adhesion in fibroblasts and epithelial cells (1Takeichi M. Curr. Opin. Cell Biol. 1995; 7: 619-627Crossref PubMed Scopus (1272) Google Scholar, 2Gumbiner B.M. Cell. 1996; 84: 345-357Abstract Full Text Full Text PDF PubMed Scopus (2979) Google Scholar). Cadherins are key Ca2+-dependent cell-cell adhesion molecules at AJs (1Takeichi M. Curr. Opin. Cell Biol. 1995; 7: 619-627Crossref PubMed Scopus (1272) Google Scholar, 2Gumbiner B.M. Cell. 1996; 84: 345-357Abstract Full Text Full Text PDF PubMed Scopus (2979) Google Scholar). Cadherins are associated with the actin cytoskeleton through peripheral membrane proteins, including α- and β-catenins, in fibroblasts and epithelial cells (1Takeichi M. Curr. Opin. Cell Biol. 1995; 7: 619-627Crossref PubMed Scopus (1272) Google Scholar). This association strengthens the cell-cell adhesion activity of cadherins (1Takeichi M. Curr. Opin. Cell Biol. 1995; 7: 619-627Crossref PubMed Scopus (1272) Google Scholar). Nectins and afadin constitute another cell-cell adhesion unit that localizes at cell-cell AJs and regulates organization of AJs in cooperation with cadherins in fibroblasts and epithelial cells (8Takai Y. Nakanishi H. J. Cell Sci. 2003; 116: 17-27Crossref PubMed Scopus (493) Google Scholar). Nectins are Ca2+-independent Ig-like cell-cell adhesion molecules. Afadin is a nectin- and actin filament-binding protein that connects nectins to the actin cytoskeleton. Nectins comprise a family of four members, nectin-1, -2, -3, and -4, each of which has two or three splicing variants. Nectins have one extracellular region with three Ig-like loops, one transmembrane region, and one cytoplasmic region. All nectins except nectin-4 have a C-terminal conserved motif of four amino acids (aa) residues, which interacts with the PDZ domain of afadin. Nectin-4 does not have this motif but binds afadin. Each nectin forms homo-cis-dimers, followed by the formation of homo-trans-dimers, causing cell-cell adhesion. Nectin-3 furthermore heterophilically trans-interacts with nectin-1 or -2 and the adhesion activity of these heterophilic trans-interactions is stronger than that of the homophilic trans-interactions. Nectin-4 also heterophilically trans-interacts with nectin-1. Five or six molecules having one extracellular region with three Ig-like loops, one transmembrane region, and one cytoplasmic region have thus far been identified (Table I) (9Fukuhara H. Kuramochi M. Nobukuni T. Fukami T. Saino M. Maruyama T. Nomura S. Sekiya T. Murakami Y. Oncogene. 2001; 20: 5401-5407Crossref PubMed Scopus (70) Google Scholar, 10Biederer T. Sara Y. Mozhayeva M. Atasoy D. Liu X. Kavalali E.T. Sudhof T.C. Science. 2002; 297: 1525-1531Crossref PubMed Scopus (652) Google Scholar, 11Urase K. Soyama A. Fujita E. Momoi T. Neuroreport. 2001; 12: 3217-3221Crossref PubMed Scopus (57) Google Scholar, 12Gomyo H. Arai Y. Tanigami A. Murakami Y. Hattori M. Hosoda F. Arai K. Aikawa Y. Tsuda H. Hirohashi S. Asakawa S. Shimizu N. Soeda E. Sakaki Y. Ohki M. Genomics. 1999; 62: 139-146Crossref PubMed Scopus (103) Google Scholar, 13Wakayama T. Ohashi K. Mizuno K. Iseki S. Mol. Reprod. Dev. 2001; 60: 158-164Crossref PubMed Scopus (87) Google Scholar, 14Kuramochi M. Fukuhara H. Nobukuni T. Kanbe T. Maruyama T. Ghosh H.P. Pletcher M. Isomura M. Onizuka M. Kitamura T. Sekiya T. Reeves R.H. Murakami Y. Nat. Genet. 2001; 27: 427-430Crossref PubMed Scopus (403) Google Scholar, 15Fukami T. Satoh H. Fujita E. Maruyama T. Fukuhara H. Kuramochi M. Takamoto S. Momoi T. Murakami Y. Gene (Amst.). 2002; 295: 7-12Crossref PubMed Scopus (34) Google Scholar, 16Chadeneau C. LeMoullac B. Denis M.G. J. Biol. Chem. 1994; 269: 15601-15605Abstract Full Text PDF PubMed Google Scholar, 17Chadeneau C. LeCabellec M. LeMoullac B. Meflah K. Denis M.G. Int. J. Cancer. 1996; 68: 817-821Crossref PubMed Scopus (44) Google Scholar, 18Mendelsohn C.L. Wimmer E. Racaniello V.R. Cell. 1989; 56: 855-865Abstract Full Text PDF PubMed Scopus (862) Google Scholar, 19Koike S. Horie H. Ise I. Okitsu A. Yoshida M. Iizuka N. Takeuchi K. Takegami T. Nomoto A. EMBO J. 1990; 9: 3217-3224Crossref PubMed Scopus (274) Google Scholar). We tentatively name here these molecules nectin-like molecules (necls) on the basis of their domain structures similar to those of nectins (see "Discussion"). Of these necls, Tage4 was originally identified to be a gene overexpressed in rat and mouse colon carcinoma (16Chadeneau C. LeMoullac B. Denis M.G. J. Biol. Chem. 1994; 269: 15601-15605Abstract Full Text PDF PubMed Google Scholar, 17Chadeneau C. LeCabellec M. LeMoullac B. Meflah K. Denis M.G. Int. J. Cancer. 1996; 68: 817-821Crossref PubMed Scopus (44) Google Scholar). Northern blot analysis has revealed that Tage4 is expressed in normal adult rat and mouse tissues to small extents (16Chadeneau C. LeMoullac B. Denis M.G. J. Biol. Chem. 1994; 269: 15601-15605Abstract Full Text PDF PubMed Google Scholar, 17Chadeneau C. LeCabellec M. LeMoullac B. Meflah K. Denis M.G. Int. J. Cancer. 1996; 68: 817-821Crossref PubMed Scopus (44) Google Scholar), but its function remains unknown, except that it mediates entry of porcine pseudorabies virus and bovine herpesvirus 1 (20Baury B. Geraghty R.J. Masson D. Lustenberger P. Spear P.G. Denis M.G. Gene (Amst.). 2001; 265: 185-194Crossref PubMed Scopus (22) Google Scholar). We have studied here the function of Tage4 and revealed that Tage4 heterophilically trans-interacts with nectin-3 and regulates cell migration. Tage4 is tentatively re-named here necl-5 on the basis of its function and phylogenetic tree of nectins and necls (Fig. 1) (see "Discussion").Table IThe proposed nomenclature of nectin-like moleculesProposed nomenclatureOld nomenclatureAccession No.Submitted yearRef.Necl-1Human NECL1AF0627331998Direct submission onlyMouse NECL1AF1956621999Direct submission onlyHuman TSLL1AF36336720019Fukuhara H. Kuramochi M. Nobukuni T. Fukami T. Saino M. Maruyama T. Nomura S. Sekiya T. Murakami Y. Oncogene. 2001; 20: 5401-5407Crossref PubMed Scopus (70) Google ScholarMouse TSLL1AY0593932001Direct submission onlyMouse SynCAM310Biederer T. Sara Y. Mozhayeva M. Atasoy D. Liu X. Kavalali E.T. Sudhof T.C. Science. 2002; 297: 1525-1531Crossref PubMed Scopus (652) Google ScholarNecl-2Mouse NECL2AF0612601998Direct submission onlyMouse RA175AB021964-AB021966199811Urase K. Soyama A. Fujita E. Momoi T. Neuroreport. 2001; 12: 3217-3221Crossref PubMed Scopus (57) Google ScholarHuman NECL2AF1328111999Direct submission onlyHuman IGSF4NM_018770199912Gomyo H. Arai Y. Tanigami A. Murakami Y. Hattori M. Hosoda F. Arai K. Aikawa Y. Tsuda H. Hirohashi S. Asakawa S. Shimizu N. Soeda E. Sakaki Y. Ohki M. Genomics. 1999; 62: 139-146Crossref PubMed Scopus (103) Google ScholarMouse SgIGSFAB052293200013Wakayama T. Ohashi K. Mizuno K. Iseki S. Mol. Reprod. Dev. 2001; 60: 158-164Crossref PubMed Scopus (87) Google ScholarHuman TSLC114Kuramochi M. Fukuhara H. Nobukuni T. Kanbe T. Maruyama T. Ghosh H.P. Pletcher M. Isomura M. Onizuka M. Kitamura T. Sekiya T. Reeves R.H. Murakami Y. Nat. Genet. 2001; 27: 427-430Crossref PubMed Scopus (403) Google ScholarMouse TSLC1AF434663200115Fukami T. Satoh H. Fujita E. Maruyama T. Fukuhara H. Kuramochi M. Takamoto S. Momoi T. Murakami Y. Gene (Amst.). 2002; 295: 7-12Crossref PubMed Scopus (34) Google ScholarMouse SynCAM1AF539424200210Biederer T. Sara Y. Mozhayeva M. Atasoy D. Liu X. Kavalali E.T. Sudhof T.C. Science. 2002; 297: 1525-1531Crossref PubMed Scopus (652) Google ScholarNecl-3Human NECL3AF5389732002Direct submission onlyMouse similar to NECL3XM_1396892002Direct submission onlyMouse SynCAM210Biederer T. Sara Y. Mozhayeva M. Atasoy D. Liu X. Kavalali E.T. Sudhof T.C. Science. 2002; 297: 1525-1531Crossref PubMed Scopus (652) Google ScholarNecl-4Human TSLL2AF36336820019Fukuhara H. Kuramochi M. Nobukuni T. Fukami T. Saino M. Maruyama T. Nomura S. Sekiya T. Murakami Y. Oncogene. 2001; 20: 5401-5407Crossref PubMed Scopus (70) Google ScholarMouse TSLL2AY0593942001Direct submission onlyMouse SynCAM410Biederer T. Sara Y. Mozhayeva M. Atasoy D. Liu X. Kavalali E.T. Sudhof T.C. Science. 2002; 297: 1525-1531Crossref PubMed Scopus (652) Google ScholarNecl-5Rat Tage4L12025199316Chadeneau C. LeMoullac B. Denis M.G. J. Biol. Chem. 1994; 269: 15601-15605Abstract Full Text PDF PubMed Google ScholarMouse Tage4MMU35836199517Chadeneau C. LeCabellec M. LeMoullac B. Meflah K. Denis M.G. Int. J. Cancer. 1996; 68: 817-821Crossref PubMed Scopus (44) Google ScholarMouse similar to Tage4BC0136732001Direct submission onlyNecl-6Human PVR/CD155M24407, M24406198918Mendelsohn C.L. Wimmer E. Racaniello V.R. Cell. 1989; 56: 855-865Abstract Full Text PDF PubMed Scopus (862) Google ScholarX64116-X64123 (exons 1-8)199019Koike S. Horie H. Ise I. Okitsu A. Yoshida M. Iizuka N. Takeuchi K. Takegami T. Nomoto A. EMBO J. 1990; 9: 3217-3224Crossref PubMed Scopus (274) Google Scholar Open table in a new tab Molecular Cloning of Mouse Necl-5 cDNA—The cDNA of mouse Tage4/necl-5 was originally isolated by reverse transcriptase-PCR from the C26 mouse colon carcinoma cell line (DDBJ/GenBank™/EBI accession number MMU35836) (17Chadeneau C. LeCabellec M. LeMoullac B. Meflah K. Denis M.G. Int. J. Cancer. 1996; 68: 817-821Crossref PubMed Scopus (44) Google Scholar). This cell line was derived from BALB/c mice. Because we generally use nectins derived from C57BL/6 mice, we re-cloned the Tage4/necl-5 cDNA derived from C57BL/6 mice. We searched in the DNA data base and found one sequence similar to that of Tage4/necl-5 (DDBJ/GenBank™/EBI accession number BC013673). We performed reverse transcriptase-PCR from mouse brain total RNA of C57BL/6 mice on the basis of BC013673. The new sequence of Tage4/necl-5 showed 93% nucleotide identity to that of the original one. The C-terminal half was identical, but the N-terminal half was slightly different. The new sequence was identical to BC013673 except for the exchange of a single nucleotide from cytosine to adenine, at position 854 (open reading frame). The reason for this difference is not known, but may be due to the different strains of mice. We confirmed that the isolated cDNA encodes the full-length protein: the protein was expressed in L cells and the molecular mass of the expressed protein was compared with that of the endogenous protein, which was expressed in NIH3T3 cells stably expressing V12Ki-Ras, an oncogenic Ki-Ras, (V12Ras-NIH3T3 cells). The molecular masses of the two proteins were apparently similar as estimated by SDS-PAGE, followed by Western blotting (see Fig. 5). Construction of Plasmids—Expression vectors were constructed in pFLAG-CMV1 (Sigma), pCAGIPuro (21Miyahara M. Nakanishi H. Takahashi K. Satoh-Horikawa K. Tachibana K. Takai Y. J. Biol. Chem. 2000; 275: 613-618Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar), pCAGIZeo (22Niwa H. Burdon T. Chambers I. Smith A. Genes Dev. 1998; 12: 2048-2060Crossref PubMed Scopus (1268) Google Scholar), pGEX4T-1 (Amersham Biosciences), pGBD-C1 (23James P. Halladay J. Craig E.A. Genetics. 1996; 144: 1425-1436Crossref PubMed Google Scholar), pFastBac1-Msp-Fc (24Sakisaka T. Taniguchi T. Nakanishi H. Takahashi K. Miyahara M. Ikeda W. Yokoyama S. Peng Y.F. Yamanishi K. Takai Y. J. Virol. 2001; 75: 4734-4743Crossref PubMed Scopus (83) Google Scholar), and pDREF-SEAP(His)6-Hyg (25Imai T. Baba M. Nishimura M. Kakizaki M. Takagi S. Yoshie O. J. Biol. Chem. 1997; 272: 15036-15042Abstract Full Text Full Text PDF PubMed Scopus (476) Google Scholar). Constructs of necl-5 contained the following aa: pCAGIZeo-necl-5, aa 1–409 (full-length); pFLAG-CMV1-necl-5, aa 30–409 (deleting the signal peptide); pCAGIPuro-FLAG-necl-5, aa 30–409 (including the preprotrypsin signal peptide); pFLAG-CMV1-necl-5-ΔEC, aa 335–409 (deleting the extracellular region); pCAGIZeo-FLAG-necl-5-ΔEC, aa 335–409 (including the preprotrypsin signal peptide); pGEX4T-1-necl-5-CP, aa 371–409 (the cytoplasmic region); pGBD-C1-necl-5-ΔEC, aa 335–409 (deleting the extracellular region); pFastBac1-Msp-Fc-necl-5-EC, aa 30–347 (the extracellular region lacking the signal peptide); and pDREF-SEAP(His)6-Hyg-necl-5-EC, aa 1–347 (the extracellular region). To express the extracellular fragment of nectin-1 or -2 fused to secreted alkaline phosphatase (SEAP) (Neap-1 or -2), pDREF-SEAP(His)6-Hyg-nectin-1-EC (aa 1–347), or -nectin-2-EC (aa 1–338) was constructed into pDREF-SEAP(His)6-Hyg. The SEAP fusion proteins were expressed in 293/EBNA-1 cells (Invitrogen) and purified as described previously (25Imai T. Baba M. Nishimura M. Kakizaki M. Takagi S. Yoshie O. J. Biol. Chem. 1997; 272: 15036-15042Abstract Full Text Full Text PDF PubMed Scopus (476) Google Scholar). To express the extracellular fragment of nectin-3 fused to the human IgG Fc (Nef-3), pFastBac1-Msp-Fc-nectin-3-EC (aa 56–400) was prepared as described previously (26Satoh-Horikawa K. Nakanishi H. Takahashi K. Miyahara M. Nishimura M. Tachibana K. Mizoguchi A. Takai Y. J. Biol. Chem. 2000; 275: 10291-10299Abstract Full Text Full Text PDF PubMed Scopus (244) Google Scholar). The IgG Fc fusion proteins were prepared as a secreted protein from the baculovirus transfer system (Invitrogen) and purified by use of protein A-Sepharose beads (Amersham Biosciences) as described previously (24Sakisaka T. Taniguchi T. Nakanishi H. Takahashi K. Miyahara M. Ikeda W. Yokoyama S. Peng Y.F. Yamanishi K. Takai Y. J. Virol. 2001; 75: 4734-4743Crossref PubMed Scopus (83) Google Scholar). The GST fusion proteins were purified by use of glutathione-Sepharose beads (Amersham Biosciences). Cell Culture and Establishment of Transfectants—L and MTD-1A cells were kindly supplied by Dr. S. Tsukita (Kyoto University, Kyoto, Japan). MDCK cells were kindly supplied by Dr. W. Birchmeier (Max-Delbruck-Center for Molecular Medicine, Berlin, Germany). V12Ras-NIH3T3 cells were prepared as described (27Fujioka H. Kaibuchi K. Kishi K. Yamamoto T. Kawamura M. Sakoda T. Mizuno T. Takai Y. J. Biol. Chem. 1992; 267: 926-930Abstract Full Text PDF PubMed Google Scholar). L, MTD-1A, and MDCK cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% FCS. NIH3T3 and V12Ras-NIH3T3 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% calf serum. L cell lines stably expressing full-length human nectin-1α, full-length mouse nectin-2α, or full-length mouse nectin-3α (nectin-1-L, -2-L, or -3-L cells, respectively) were prepared as described previously (21Miyahara M. Nakanishi H. Takahashi K. Satoh-Horikawa K. Tachibana K. Takai Y. J. Biol. Chem. 2000; 275: 613-618Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar, 26Satoh-Horikawa K. Nakanishi H. Takahashi K. Miyahara M. Nishimura M. Tachibana K. Mizoguchi A. Takai Y. J. Biol. Chem. 2000; 275: 10291-10299Abstract Full Text Full Text PDF PubMed Scopus (244) Google Scholar, 28Takahashi K. Nakanishi H. Miyahara M. Mandai K. Satoh K. Satoh A. Nishioka H. Aoki J. Nomoto A. Mizoguchi A. Takai Y. J. Cell Biol. 1999; 145: 539-549Crossref PubMed Scopus (449) Google Scholar). An L cell line stably expressing full-length necl-5 (non-tagged-necl-5-L cells), FLAG-necl-5 (necl-5-L cells), or FLAG-necl-5-ΔEC (necl-5-ΔEC-L cells) was obtained by transfection with pCAGIZeo-necl-5, pCAGIPuro-FLAG-necl-5, or pCAGIZeo-FLAG-necl-5-ΔEC, respectively, using LipofectAMINE PLUS reagent (Invitrogen). We mostly used necl-5-L cells (FLAG-tagged necl-5) in the present study, but the essentially similar results were obtained with non-tagged-necl-5-L cells (data not shown). Antibodies—A rat anti-necl-5 monoclonal antibody (mAb) #1A8-8 was raised against the extracellular fragment of necl-5 (aa 30–347) fused to the human IgG Fc (Lef-5). A rabbit antisera against necl-5 was raised against the cytoplasmic tail of necl-5 fused to GST (GST-necl-5-CP). An affinity-purified F(ab′)2 fragment goat anti-human IgG, Fcγ fragment specific Ab was purchased from Jackson Laboratory. Surface Plasmon Resonance Analysis—A BIAcore X surface plasmon resonance-based biosensor (BIAcore Inc., Piscataway, NJ) was used to measure kinetic parameters for the interaction between Neap-1, Neap-2, or the extracellular fragment of necl-5 fused to SEAP (Leap-5) and immobilized Nef-3 or Lef-5. The F(ab′)2 fragment goat anti-human IgG Fc polyclonal Ab (pAb) was immobilized at a concentration of about 4800 resonance units (4.8 ng/mm2) to the sensor chip surface by the amine-coupling method. Nef-3 or Lef-5 was immobilized at a concentration of about 500 resonance units to the sensor chip via the anti-human IgG Fc Ab. Neap-1, Neap-2, or Leap-5 was then diluted in HBS-EP buffer (10 mm HEPES, pH7.4, 150 mm NaCl, 3 mm EDTA, 0.005% Tween 20; BIAcore) to 40 nm and injected at a flow rate of 20 μl/min at 25 °C for 210 s. Both an association rate constant k a (m–1 s–1) and a dissociation rate constant k d (s–1) were obtained using the BIAevaluation software version 3.2 (BIAcore), and the dissociation constant (K D = k d/k a) was derived from the two deduced rate constants. Intercellular Motility Assay—Intercellular motility assay was done as described previously (29Nagafuchi A. Ishihara S. Tsukita S. J. Cell Biol. 1994; 127: 235-245Crossref PubMed Scopus (363) Google Scholar). Briefly, the cells were labeled with 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI), and the 1 × 102 labeled cells were seeded on a confluent culture of 2 × 105 unlabeled nectin-3-L cells in a 24-well dish. After 36 or 48 h of culture, four sister cells that seemed to be derived from one seeded cell were examined by fluorescence microscopy. In the experiments using the anti-necl-5 mAb, this mAb was added at a final concentration of 50 μg/ml in the medium. When a cell line A was seeded on a confluent culture of a cell line B, we designated the experiment as A/B analysis. For quantification of intercellular motility, intercellular distances of all combinations between four sister cells were measured and summed as Dc. As a control experiment, the labeled cells were seeded on dishes in the absence of a cell layer. In this case, the intercellular distances were summed as Dd. The degree of intercellular motility was represented as Dc/Dd. At least 24 independent samples were picked up to determine Dc or Dd for each cell line. Other Procedures—The cell aggregation assay, chemical cross-linking, and SDS-PAGE were done as described previously (21Miyahara M. Nakanishi H. Takahashi K. Satoh-Horikawa K. Tachibana K. Takai Y. J. Biol. Chem. 2000; 275: 613-618Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar, 26Satoh-Horikawa K. Nakanishi H. Takahashi K. Miyahara M. Nishimura M. Tachibana K. Mizoguchi A. Takai Y. J. Biol. Chem. 2000; 275: 10291-10299Abstract Full Text Full Text PDF PubMed Scopus (244) Google Scholar, 28Takahashi K. Nakanishi H. Miyahara M. Mandai K. Satoh K. Satoh A. Nishioka H. Aoki J. Nomoto A. Mizoguchi A. Takai Y. J. Cell Biol. 1999; 145: 539-549Crossref PubMed Scopus (449) Google Scholar, 30Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (212360) Google Scholar). The inability of necl-5 to bind afadin was confirmed by yeast two-hybrid assay, co-immunoprecipitation assay, and affinity chromatography as described previously (23James P. Halladay J. Craig E.A. Genetics. 1996; 144: 1425-1436Crossref PubMed Google Scholar, 26Satoh-Horikawa K. Nakanishi H. Takahashi K. Miyahara M. Nishimura M. Tachibana K. Mizoguchi A. Takai Y. J. Biol. Chem. 2000; 275: 10291-10299Abstract Full Text Full Text PDF PubMed Scopus (244) Google Scholar, 28Takahashi K. Nakanishi H. Miyahara M. Mandai K. Satoh K. Satoh A. Nishioka H. Aoki J. Nomoto A. Mizoguchi A. Takai Y. J. Cell Biol. 1999; 145: 539-549Crossref PubMed Scopus (449) Google Scholar), under the conditions where nectin-2 and -3 bound afadin. Protein concentrations were determined with bovine serum albumin as a reference protein as described previously (31Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (222135) Google Scholar). Heterophilic trans-Interaction of Necl-5 with Nectin-3—We first examined by the aggregation assay using necl-5-L cells whether necl-5 has cell-cell adhesion activity. In wild-type L cells, nectin-1 and -2, but not cadherin or nectin-3, are expressed as estimated by Western blotting (21Miyahara M. Nakanishi H. Takahashi K. Satoh-Horikawa K. Tachibana K. Takai Y. J. Biol. Chem. 2000; 275: 613-618Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar, 24Sakisaka T. Taniguchi T. Nakanishi H. Takahashi K. Miyahara M. Ikeda W. Yokoyama S. Peng Y.F. Yamanishi K. Takai Y. J. Virol. 2001; 75: 4734-4743Crossref PubMed Scopus (83) Google Scholar, 26Satoh-Horikawa K. Nakanishi H. Takahashi K. Miyahara M. Nishimura M. Tachibana K. Mizoguchi A. Takai Y. J. Biol. Chem. 2000; 275: 10291-10299Abstract Full Text Full Text PDF PubMed Scopus (244) Google Scholar). Expression of necl-5 was not detected in wild-type L cells by Western blotting using any Abs, which recognized exogenously expressed necl-5 (see Fig. 5). Wild-type L cells did not form visible cell aggregates as described previously (28Takahashi K. Nakanishi H. Miyahara M. Mandai K. Satoh K. Satoh A. Nishioka H. Aoki J. Nomoto A. Mizoguchi A. Takai Y. J. Cell Biol. 1999; 145: 539-549Crossref PubMed Scopus (449) Google Scholar) (Fig. 2A). Necl-5-L cells did not form visible cell aggregates, either (Fig. 2B). These results indicate that necl-5 does not homophilically trans-interact with necl-5, causing no homophilic cell-cell adhesion. We then examined whether necl-5 has heterophilic cell-cell adhesion activity with other nectins. Necl-5-L cells were mixed with nectin-1-L, -2-L, or -3-L cells followed by the aggregation assay. Nectin-3-L cells formed small aggregates in the absence of necl-5-L cells as described previously (26Satoh-Horikawa K. Nakanishi H. Takahashi K. Miyahara M. Nishimura M. Tachibana K. Mizoguchi A. Takai Y. J. Biol. Chem. 2000; 275: 10291-10299Abstract Full Text Full Text PDF PubMed Scopus (244) Google Scholar) (Fig. 2C), but formed relatively big aggregates with necl-5-L cells (Fig. 2D, Da–Dc). This aggregate was similarly formed even in the absence of Ca2+ (data not shown). The size of the aggregates formed between necl-5-L and nectin-3-L cells were about 20% that of the aggregates formed between nectin-1-L and -3-L cells, which formed the biggest aggregates among various combinations of nectins thus far examined (32Honda T. Shimizu K. Kawakatsu T. Yasumi M. Shingai T. Fukuhara A. Ozaki-Kuroda K. Irie K. Nakanishi H. Takai Y. Genes Cells. 2003; 8: 51-63Crossref PubMed Scopus (80) Google Scholar) (see Fig. 7, Aa and Ba). Necl-5-L cells did not form mixed aggregates with nectin-1-L or -2-L cells (Fig. 2, Ea–Ec and Fa–Fc). Small aggregates observed were formed by nectin-1-L or nectin-2-L cells by themselves as described (28Takahashi K. Nakanishi H. Miyahara M. Mandai K. Satoh K. Satoh A. Nishioka H. Aoki J. Nomoto A. Mizoguchi A. Takai Y. J. Cell Biol. 1999; 145: 539-549Crossref PubMed Scopus (449) Google Scholar) (Fig. 2, Ea, Ec, Fa, and Fc). These results indicate that necl-5 heterophilically trans-interacts selectively with nectin-3 in a Ca2+-independent manner, causing heterophilic cell-cell adhesion selectively with nectin-3. To confirm that necl-5 directly interacts with nectin-3, we performed surface plasmon resonance analysis using Nef-3 and Leap-5. Neap-1 and -2 were used as controls. Nef-3 is the extracellular fragment of nectin-3 fused to the human IgG Fc; Leap-5 is the extracellular fragment of necl-5 fused to SEAP; Neap-1 is the extracellular fragment of nectin-1 fused to SEAP; and Neap-2 is the extracellular fragment of nectin-2 fused to SEAP. Nef-3 bound all of these molecules, and the K d value of Nef-3 for Leap-5 was about 17 nm, whereas the K d values of Nef-3 for Neap-1 and -2 were about 2.3 and 360 nm, respectively (Fig. 3). Lef-5 did not bind Leap-5 (data not shown). Lef-5 is the extracellular fragment of necl-5 fused to the human IgG Fc. We have previously shown that nectin-2 forms homo-cis-dimers, followed by the formation of homo- or hetero-trans-dimers, eventually causing cell-cell adhesion (21Miyahara M. Nakanishi H. Takahashi K. Satoh-Horikawa K. Tachibana K. Takai Y. J. Biol. Chem. 2000; 275: 613-618Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar). Similarly, necl-5 formed homo-cis-dimers (Fig. 4). In addition, necl-5 furthermore formed a multimer. Taken together, it is likely by analogy with the mode of action of nectin-2 that necl-5 forms first homo-cis-dimers, followed by the heterophilic trans-interaction with nectin-3, eventually causing cell-cell adhesion. Inability of Necl-5 to Bind Afadin—The extracellular region of necl-5 showed 28–42% aa identity to that of nectins, but necl-5 does not have a C-terminal consensus motif with four aa for binding to the PDZ domain (data not shown). We examined whether necl-5 binds afadin. Necl-5 did not bind afadin as estimated by yeast two-hybrid assay, co-immunoprecipitation assay, and affinity chromatography under the conditions where nectin-2 or -3 bound it (data not shown). Elevated Expression of Necl-5 in V12Ras-NIH3T3 Cells—We then examined the tissue distribution of necl-5 in mouse by Western blotting, but the significant immunoreactive band was not detected in any normal tissue examined, including heart, brain, spleen, lung, liver, kidney, skeletal muscle, and testis (data not shown), consistent with the earlier observation (17Chadeneau C. LeCabellec M. LeMoullac B. Meflah K. Denis M.G. Int. J. Cancer. 1996; 68: 817-821Crossref PubMed Scopus (44) Google Scholar). Any band was not detected in cultured cell lines, including L (Fig. 5), MTD-1A (data not shown), and MDCK cells (data not shown), but two faint bands were detected in NIH3T3 cells (Fig. 5). Tage4 was originally isolated from rat and mouse colon carcinoma (16Chadeneau C. LeMoullac B. Denis M.G. J. Biol. Chem. 1994; 269: 15601-15605Abstract Full Text PDF PubMed Google Scholar, 17Chadeneau C. LeCabellec M. LeMoullac B. Meflah K. Denis M.G. Int. J. Cancer. 1996; 68: 817-821Crossref PubMed Scopus (44) Google Scholar). We therefore examined the expression of necl-5 in V12Ras-NIH3T3 cells. Expression of necl-5 was markedly elevated in the transformed cells as compared with that of the wild-type cells (Fig. 5). Enhancement of Motility of V12Ras-NIH3T3 and Necl-5-L Cells by trans-Interaction of Necl-5 with Nectin-3—We finally studied the role of the trans-interaction of necl-5 with nectin-3 on motility of V12Ras-NIH3T3 cells by using intercellular motility assay, because transformed cells show generally enhanced migration activity (4Thiery J.P. Nat. Rev. Cancer. 2002; 2: 442-454Crossref PubMed Scopus (5603) Google Scholar). Necl-5-L and necl-5-ΔEC-L cells were used as control cells. Necl-5-ΔEC-L cells were L cells expressing necl-5, of which extracellular region except the juxtamembrane 13 aa were deleted. In this assay, cell motility in a confluent cell sheet, which is influenced by dynamic cell-cell adhesion, could be measured. V12Ras-NIH3T3 cells labeled with DiI, a fluorescence dye, were seeded on a confluent culture of non-labeled nectin-3-L cells, and after 36 h (twice the doubling time), the cell scatter property was analyzed by measuring the mass distance among four sister labeled cells. As a control experiment, labeled V12Ras-NIH3T3 cells were seeded on the dish in the absence of nectin-3-L cells. V12Ras-NIH3T3 cells scattered on a confluent culture of nectin-3-L cells more actively than on the dish (Fig. 6, Aa1, Aa2, and B). The scattering of V12Ras-NIH3T3 cells on nectin-3-L cells was inhibited by the anti-necl-5 mAb, whereas the scattering on the dish was not affected by this mAb (Fig. 6, Aa3, Aa4, and B). The anti-necl-5 mAb inhibited the interaction of necl-5 with nectin-3 as estimated by the aggregation assay using necl-5-L and nectin-3-L cells (Fig. 7, Aa and Ab). This mAb did not affect the interaction of nectin-1 with nectin-3 (Fig. 7, Ba and Bb). Necl-5-L cells labeled with DiI were seeded on a confluent culture of non-labeled nectin-3-L cells, and after 48 h (twice the doubling time), the cell scatter property was similarly analyzed. Necl-5-L cells scattered on a confluent culture of nectin-3-L cells more actively than on the dish (Fig. 6, Ab1, Ab2, and B). The scattering of necl-5-L cells on nectin-3-L cells was inhibited by the anti-necl-5 mAb, whereas the scattering of necl-5-L cells on the dish was not affected by this mAb (Fig. 6, Ab3, Ab4, and B). Necl-5-ΔEC-L cells labeled with DiI scattered on a confluent culture of nectin-3-L cells less actively than on the dish (Fig. 6, Ac1, Ac2, and B). Necl-5-ΔEC-L cells did not adhere to nectin-3-L cells as estimated by the aggregation assay (data not shown). The scattering of necl-5-ΔEC-L cells in the presence or absence of nectin-3-L cells was not affected by the anti-necl-5 mAb (Fig. 6, Ac3, Ac4, and B). These results indicate that the trans-interaction of necl-5 with nectin-3 enhances motility of V12Ras-NIH3T3 and necl-5-L cells. We have shown here that necl-5 does not homophilically trans-interact with necl-5, but heterophilically trans-interacts selectively with nectin-3, causing cell-cell adhesion. This property of necl-5 is quite different from that of nectins which both homophilically and heterophilically trans-interact (8Takai Y. Nakanishi H. J. Cell Sci. 2003; 116: 17-27Crossref PubMed Scopus (493) Google Scholar). We have previously proposed that nectins are involved in the formation of AJs in cooperation with E-cadherin, on the basis of the observations that the trans-interaction of nectins recruits E-cadherin to the nectin-based cell-cell adhesion sites, resulting in formation of AJs, and that the disruption of this trans-interaction of nectins by their antagonists impairs the formation of E-cadherin-based AJs (8Takai Y. Nakanishi H. J. Cell Sci. 2003; 116: 17-27Crossref PubMed Scopus (493) Google Scholar). The association of nectins and E-cadherin at AJs is mediated through afadin and α-catenin (8Takai Y. Nakanishi H. J. Cell Sci. 2003; 116: 17-27Crossref PubMed Scopus (493) Google Scholar). We have shown here that necl-5 does not bind afadin. The inability of necl-5 to bind afadin suggests that necl-5 has no potency to recruit cadherins to the cell-cell adhesion site formed by the trans-interaction of necl-5 with nectin-3 and is not involved in the formation of AJs. We have shown here that the heterophilic trans-interaction of necl-5 with nectin-3 rather enhances motility of V12Ras-NIH3T3 and necl-5-L cells. It has previously been reported that L cells stably expressing full-length E-cadherin (EL cells) shows inter-EL-cellular EL cell motility (29Nagafuchi A. Ishihara S. Tsukita S. J. Cell Biol. 1994; 127: 235-245Crossref PubMed Scopus (363) Google Scholar, 33Itoh M. Nagafuchi A. Moroi S. Tsukita S. J. Cell Biol. 1997; 138: 181-192Crossref PubMed Scopus (581) Google Scholar). The mechanism of this intercellular motility of EL cells is not clear, but it has been suggested that dynamic attachment of EL cells to neighboring EL cells and dynamic detachment of EL cells from neighboring EL cells are necessary for the motility of EL cells (29Nagafuchi A. Ishihara S. Tsukita S. J. Cell Biol. 1994; 127: 235-245Crossref PubMed Scopus (363) Google Scholar, 33Itoh M. Nagafuchi A. Moroi S. Tsukita S. J. Cell Biol. 1997; 138: 181-192Crossref PubMed Scopus (581) Google Scholar). The mechanism of intercellular motility of V12Ras-NIH3T3 and necl-5-L cells is not known, either, but may be analogous to that of EL cells. Transformation of cells increases cell motility, causing invasion into surrounding tissues. Since expression of necl-5 is elevated by transformation as shown here and described previously (16Chadeneau C. LeMoullac B. Denis M.G. J. Biol. Chem. 1994; 269: 15601-15605Abstract Full Text PDF PubMed Google Scholar, 17Chadeneau C. LeCabellec M. LeMoullac B. Meflah K. Denis M.G. Int. J. Cancer. 1996; 68: 817-821Crossref PubMed Scopus (44) Google Scholar), this elevation of necl-5 may be at least partly responsible for the enhanced cell motility and invasion of transformed cells. Intercellular motility is observed in vivo in the process of morphogenetic rearrangement of cells in embryonic tissues (2Gumbiner B.M. Cell. 1996; 84: 345-357Abstract Full Text Full Text PDF PubMed Scopus (2979) Google Scholar, 3Lauffenburger D.A. Horwitz A.F. Cell. 1996; 84: 359-369Abstract Full Text Full Text PDF PubMed Scopus (3319) Google Scholar). It remains unknown what kind of the cells express necl-5 in embryonic tissues, but if necl-5 is expressed in rapidly migrating cells, such as mesenchymal cells, the dynamic trans-interaction of necl-5 with nectin-3 may also play a role in their intercellular motility. Further studies are necessary for establishing the physiological and pathological roles of necl-5 in these processes. We lastly discuss about other necls which have thus far been identified in addition to necl-5/Tage4 (Table I). Five or six necls including necl-5 have been identified but have many nomenclatures. We propose here that a group of proteins with structures similar to those of nectins but without ability to directly bind afadin are called nectin-like molecules (necls). NECL1/TSLL1/SynCAM3, NECL2/IGSF4/RA175/SgIGSF/TSLC1/SynCAM1, NECL3/similar to NECL3/SynCAM2, and TSLL2/SynCAM4 have a C-terminal consensus motif with four aa for binding to PDZ domains, and these cytoplasmic regions show high similarity. Necl-5/Tage4 or PVR/CD155 does not have this motif. Our analysis has revealed that NECL1/TSLL1/SynCAM3 or NECL2/IGSF4/RA175/SgIGSF/TSLC1/SynCAM1 as well as necl-5/Tage4 does not bind afadin as estimated by yeast two-hybrid assay, co-immunoprecipitation assay, and affinity chromatography (data not shown). It remains to be examined whether NECL3/similar to NECL3/SynCAM2, TSLL2/SynCAM4, or PVR/CD155 binds afadin. On the assumption that these three proteins do not directly bind afadin, we propose that all of these molecules are called necls. Then, we propose the following nomenclatures according to the phylogenetic tree shown in Fig. 1: necl-1 for NECL1/TSLL1/SynCAM3, necl-2 for NECL2/IGSF4/RA175/SgIGSF/TSLC1/SynCAM1, necl-3 for NECL3/similar to NECL3/SynCAM2, necl-4 for TSLL2/SynCAM4, necl-5 for Tage4, and necl-6 for PVR/CD155. The necl-6/PVR/CD155 gene has thus far been found only in the primates (34Koike S. Ise I. Sato Y. Yonekawa H. Gotoh, O. Nomoto A. J. Virol. 1992; 66: 7059-7066Crossref PubMed Google Scholar), and necl-5/Tage4 has thus far been found only in the rodent. The sequence of necl-5/Tage4 shows 42% aa identity to that of necl-6/PVR/CD155. The necl-5/Tage4 gene may be an ortholog of the necl-6/PVR/CD155 gene (20Baury B. Geraghty R.J. Masson D. Lustenberger P. Spear P.G. Denis M.G. Gene (Amst.). 2001; 265: 185-194Crossref PubMed Scopus (22) Google Scholar), but the phylogenetic tree of nectins and necls can not clearly conclude that necl-5/Tage4 and necl-6/PVR/CD155 are derived from the same or different ancestor gene. Therefore, we reserve the conclusion for the classification of these two genes. We thank Dr. S. Tsukita (Kyoto University, Kyoto, Japan) for providing us with L and MTD-1A cells and Dr. W. Birchmeier (Max-Delbruck-Center for Molecular Medicine, Berlin, Germany) for providing us with MDCK cells. We are also grateful to Dr. A. Nagafuchi (Kumamoto University, Kumamoto, Japan) for his helpful discussion.
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