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Nectin-like Molecule-5/Tage4 Enhances Cell Migration in an Integrin-dependent, Nectin-3-independent Manner

2004; Elsevier BV; Volume: 279; Issue: 17 Linguagem: Inglês

10.1074/jbc.m312969200

ISSN

1083-351X

Autores

Wataru Ikeda, Shigeki Kakunaga, Kyoji Takekuni, Tatsushi Shingai, Keiko Satoh, Koji Morimoto, Masakazu Takeuchi, Toshio Imai, Yoshimi Takai,

Tópico(s)

Proteoglycans and glycosaminoglycans research

Resumo

Cell migration plays roles in invasion of transformed cells and scattering of embryonic mesenchymal cells into surrounding tissues. We have found that Ig-like Necl-5/Tage4 is up-regulated in NIH3T3 cells transformed by an oncogenic Ras (V12Ras-NIH3T3 cells) and heterophilically trans-interacts with a Ca2+-independent Ig-like cell adhesion molecule nectin-3, eventually enhancing their intercellular motility. We show here that Necl-5 furthermore enhances cell migration in a nectin-3-independent manner. Studies using L fibroblasts expressing various mutants of Necl-5, NIH3T3 cells, and V12Ras-NIH3T3 cells have revealed that Necl-5 enhances serum- and platelet-derived growth factor-induced cell migration. The extracellular region of Necl-5 is necessary for directional cell migration, but not for random cell motility. The cytoplasmic region of Necl-5 is necessary for both directional and random cell movement. Necl-5 colocalizes with integrin αVβ3 at leading edges of migrating cells. Analyses using an inhibitor or an activator of integrin αVβ3 or a dominant negative mutant of Necl-5 have shown the functional association of Necl-5 with integrin αVβ3 in cell motility. Cdc42 and Rac small G proteins are activated by the action of Necl-5 and required for the serum-induced, Necl-5-enhanced cell motility. These results indicate that Necl-5 regulates serum- and platelet-derived growth factor-induced cell migration in an integrin-dependent, nectin-3-independent manner, when cells do not contact other cells. We furthermore show here that enhanced motility and metastasis of V12Ras-NIH3T3 cells are at least partly the result of up-regulated Necl-5. Cell migration plays roles in invasion of transformed cells and scattering of embryonic mesenchymal cells into surrounding tissues. We have found that Ig-like Necl-5/Tage4 is up-regulated in NIH3T3 cells transformed by an oncogenic Ras (V12Ras-NIH3T3 cells) and heterophilically trans-interacts with a Ca2+-independent Ig-like cell adhesion molecule nectin-3, eventually enhancing their intercellular motility. We show here that Necl-5 furthermore enhances cell migration in a nectin-3-independent manner. Studies using L fibroblasts expressing various mutants of Necl-5, NIH3T3 cells, and V12Ras-NIH3T3 cells have revealed that Necl-5 enhances serum- and platelet-derived growth factor-induced cell migration. The extracellular region of Necl-5 is necessary for directional cell migration, but not for random cell motility. The cytoplasmic region of Necl-5 is necessary for both directional and random cell movement. Necl-5 colocalizes with integrin αVβ3 at leading edges of migrating cells. Analyses using an inhibitor or an activator of integrin αVβ3 or a dominant negative mutant of Necl-5 have shown the functional association of Necl-5 with integrin αVβ3 in cell motility. Cdc42 and Rac small G proteins are activated by the action of Necl-5 and required for the serum-induced, Necl-5-enhanced cell motility. These results indicate that Necl-5 regulates serum- and platelet-derived growth factor-induced cell migration in an integrin-dependent, nectin-3-independent manner, when cells do not contact other cells. We furthermore show here that enhanced motility and metastasis of V12Ras-NIH3T3 cells are at least partly the result of up-regulated Necl-5. In multicellular organisms, cell migration is essential for normal development and responses to tissue damages and infection throughout life (1Lauffenburger D.A. Horwitz A.F. Cell. 1996; 84: 359-369Abstract Full Text Full Text PDF PubMed Scopus (3291) Google Scholar, 2Keller R. Science. 2002; 298: 1950-1954Crossref PubMed Scopus (572) Google Scholar). Cell migration is also observed in many diseases, such as cancer and atherosclerosis (3Libby P. Nature. 2002; 420: 868-874Crossref PubMed Scopus (7017) Google Scholar, 4Thiery J.P. Nat. Rev. Cancer. 2002; 2: 442-454Crossref PubMed Scopus (5531) Google Scholar). Cells migrate as individuals or as groups; leukocytes, lymphocytes, and fibroblasts migrate as individuals, whereas epithelial and endothelial cells migrate as groups. Cell migration is divided into at least four mechanistically separate steps: extension of protrusions, formation of new cell-matrix adhesions, contraction of cell body, and tail detachment (1Lauffenburger D.A. Horwitz A.F. Cell. 1996; 84: 359-369Abstract Full Text Full Text PDF PubMed Scopus (3291) Google Scholar, 5Gumbiner B.M. Cell. 1996; 84: 345-357Abstract Full Text Full Text PDF PubMed Scopus (2948) Google Scholar). Cell migration is normally directed and controlled by extracellular cues, such as growth factors, cytokines, and extracellular matrix molecules. These cues stimulate cell surface receptors to initiate intracellular signaling through second messengers, protein kinases, protein phosphatases, and heterotrimeric large and monomeric small G proteins to regulate the multiple steps. When migrating cells contact other cells, they stop migration and proliferation (6Abercrombie M. In Vitro. 1970; 6: 128-142Crossref PubMed Scopus (258) Google Scholar, 7Martz 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 transformed cells to invasion into surrounding tissues and metastasis to other organs (4Thiery J.P. Nat. Rev. Cancer. 2002; 2: 442-454Crossref PubMed Scopus (5531) Google Scholar, 8Abercrombie M. Nature. 1979; 281: 259-262Crossref PubMed Scopus (290) Google Scholar). Cell-cell adhesion is mainly mediated by cell-cell adherens junctions (AJs), 1The abbreviations used are: AJ, adherens junction; aa, amino acid(s); PVR, poliovirus receptor; Necl, nectin-like molecule; V12Ras-NIH3T3 cells, NIH3T3 cells transformed by an oncogenic Ki-Ras; PDGF, platelet-derived growth factor; Nef-3, the extracellular fragment of nectin-3 fused to the human IgG Fc; DMEM, Dulbecco's modified Eagle's medium; non-tagged Necl-5-L cells, L cells stably expressing Necl-5; Necl-5-L cells, L cells stably expressing FLAG-Necl-5; Necl-5-ΔEC-L cells, L cells stably expressing Necl-5 of which extracellular region is deleted; Necl-5-ΔCP-L cells, L cells stably expressing Necl-5 of which cytoplasmic region is deleted; Necl-5-ΔCP-NIH3T3 cells, NIH3T3 cells stably expressing Necl-5 of which cytoplasmic region is deleted; Necl-5-ΔCP-V12Ras-NIH3T3 cells, V12Ras-NIH3T3 cells stably expressing Necl-5 of which cytoplasmic region is deleted; Ab, antibody; mAb, monoclonal antibody; pAb, polyclonal antibody; BSA, bovine serum albumin; FRET, fluorescent resonance energy transfer; PBS, phosphate-buffered saline; YFP, yellow fluorescent protein; CFP, cyan fluorescent protein. where cadherins are key Ca2+-dependent cell-cell adhesion molecules (5Gumbiner B.M. Cell. 1996; 84: 345-357Abstract Full Text Full Text PDF PubMed Scopus (2948) Google Scholar, 9Takeichi M. Curr. Opin. Cell Biol. 1995; 7: 619-627Crossref PubMed Scopus (1262) Google Scholar). Cadherins are associated with the actin cytoskeleton through many peripheral membrane proteins, including α- and β-catenins, α-actinin, and vinculin, which strengthen cell-cell adhesion activity of cadherins. Nectins are Ca2+-independent Ig-like cell-cell adhesion molecules that also localize at cell-cell AJs and regulate organization of AJs in cooperation with cadherins (10Takai Y. Nakanishi H. J. Cell Sci. 2003; 116: 17-27Crossref PubMed Scopus (488) Google Scholar, 11Takai Y. Irie K. Shimizu K. Sakisaka T. Ikeda W. Cancer Sci. 2003; 94: 655-667Crossref PubMed Scopus (291) Google Scholar). Nectins are similarly associated with the actin cytoskeleton through afadin. Nectins comprise a family of four members, nectin-1, -2, -3, and -4. 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 acid (aa) residues that 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 formation of homo-trans-dimers (homo-trans-interaction), causing cell-cell adhesion. Nectin-3 furthermore forms hetero-trans-dimers (hetero-trans-interaction) with nectin-1 or -2, and the adhesion activity of the hetero-trans-dimers is stronger than that of the homo-trans-dimers. Nectin-4 also forms hetero-trans-dimers with nectin-1. In addition to the cell-cell adhesion activity, nectins have an activity to induce activation of Cdc42 and Rac small G proteins, which regulate cell-cell adhesion through reorganization of the actin cytoskeleton and gene expression through activation of c-Jun N-terminal kinase (12Kawakatsu T. Shimizu K. Honda T. Fukuhara T. Hoshino T. Takai Y. J. Biol. Chem. 2002; 277: 50749-50755Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar, 13Honda T. Shimizu K. Kawakatsu T. Fukuhara A. Irie K. Nakamura T. Matsuda M. Takai Y. Genes Cells. 2003; 8: 481-491Crossref PubMed Scopus (43) Google Scholar, 14Fukuhara A. Shimizu K. Kawakatsu T. Fukuhara T. Takai Y. J. Biol. Chem. 2003; 278: 51885-51893Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). Nectins furthermore directly bind PAR-3, a cell polarity protein, and regulate cell polarization (15Takekuni K. Ikeda W. Fujito T. Morimoto K. Takeuchi M. Monden M. Takai Y. J. Biol. Chem. 2003; 278: 5497-5500Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar). Five other molecules with one extracellular region with three Ig-like loops, one transmembrane region, and one cytoplasmic region have thus far been identified. These include Necl-1/TSLL1/SynCAM3 (16Fukuhara H. Kuramochi M. Nobukuni T. Fukami T. Saino M. Maruyama T. Nomura S. Sekiya T. Murakami Y. Oncogene. 2001; 20: 5401-5407Crossref PubMed Scopus (69) Google Scholar, 17Biederer T. Sara Y. Mozhayeva M. Atasoy D. Liu X. Kavalali E.T. Sudhof T.C. Science. 2002; 297: 1525-1531Crossref PubMed Scopus (647) Google Scholar), Necl-2/IGSF4/RA175/SgIGSF/TSLC1/SynCAM1 (17Biederer T. Sara Y. Mozhayeva M. Atasoy D. Liu X. Kavalali E.T. Sudhof T.C. Science. 2002; 297: 1525-1531Crossref PubMed Scopus (647) Google Scholar, 18Gomyo 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, 19Kuramochi 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 (399) Google Scholar, 20Urase K. Soyama A. Fujita E. Momoi T. Neuroreport. 2001; 12: 3217-3221Crossref PubMed Scopus (57) Google Scholar, 21Wakayama T. Ohashi K. Mizuno K. Iseki S. Mol. Reprod. Dev. 2001; 60: 158-164Crossref PubMed Scopus (85) Google Scholar), Necl-3/similar to NECL3/SynCAM2 (17Biederer T. Sara Y. Mozhayeva M. Atasoy D. Liu X. Kavalali E.T. Sudhof T.C. Science. 2002; 297: 1525-1531Crossref PubMed Scopus (647) Google Scholar), Necl-4/TSLL2/SynCAM4 (16Fukuhara H. Kuramochi M. Nobukuni T. Fukami T. Saino M. Maruyama T. Nomura S. Sekiya T. Murakami Y. Oncogene. 2001; 20: 5401-5407Crossref PubMed Scopus (69) Google Scholar, 17Biederer T. Sara Y. Mozhayeva M. Atasoy D. Liu X. Kavalali E.T. Sudhof T.C. Science. 2002; 297: 1525-1531Crossref PubMed Scopus (647) Google Scholar), and Necl-5/Tage4/human poliovirus receptor (PVR)/CD155 (22Mendelsohn C.L. Wimmer E. Racaniello V.R. Cell. 1989; 56: 855-865Abstract Full Text PDF PubMed Scopus (824) Google Scholar, 23Koike 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 (270) Google Scholar, 24Chadeneau C. LeMoullac B. Denis M.G. J. Biol. Chem. 1994; 269: 15601-15605Abstract Full Text PDF PubMed Google Scholar, 25Chadeneau C. LeCabellec M. LeMoullac B. Meflah K. Denis M.G. Int. J. Cancer. 1996; 68: 817-821Crossref PubMed Scopus (43) Google Scholar). The domain structures of this group of molecules are similar to those of nectins, but they do not bind afadin (11Takai Y. Irie K. Shimizu K. Sakisaka T. Ikeda W. Cancer Sci. 2003; 94: 655-667Crossref PubMed Scopus (291) Google Scholar, 26Ikeda 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 (112) Google Scholar). We have proposed that this group of molecules is tentatively called nectin-like molecules (Necls). Of these Necls, we focus here on Necl-5/Tage4/PVR/CD155. Tage4 was originally identified to be the product of a gene overexpressed in rat and mouse colon carcinoma (24Chadeneau C. LeMoullac B. Denis M.G. J. Biol. Chem. 1994; 269: 15601-15605Abstract Full Text PDF PubMed Google Scholar, 25Chadeneau C. LeCabellec M. LeMoullac B. Meflah K. Denis M.G. Int. J. Cancer. 1996; 68: 817-821Crossref PubMed Scopus (43) Google Scholar). The Tage4 gene has been mapped to rat chromosome 1q22 (27Chadeneau C. Liehr T. Rautenstrauss B. Denis M.G. Mamm. Genome. 1997; 8: 157-158Crossref PubMed Scopus (8) Google Scholar) and mouse 7A2-B1 (28Chadeneau C. LeMoullac B. LeCabellec M. Mattei M. Meflah K. Denis M.G. Mamm. Genome. 1996; 7: 636-637Crossref PubMed Scopus (18) Google Scholar). Northern blot analysis has revealed that the Tage4 mRNA is expressed in normal adult rat and mouse tissues to small extents (24Chadeneau C. LeMoullac B. Denis M.G. J. Biol. Chem. 1994; 269: 15601-15605Abstract Full Text PDF PubMed Google Scholar, 25Chadeneau C. LeCabellec M. LeMoullac B. Meflah K. Denis M.G. Int. J. Cancer. 1996; 68: 817-821Crossref PubMed Scopus (43) Google Scholar). Tage4 has been shown to mediate entry of porcine pseudorabies virus and bovine herpesvirus 1 (29Baury 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). PVR/CD155 was originally identified as a human poliovirus receptor (22Mendelsohn C.L. Wimmer E. Racaniello V.R. Cell. 1989; 56: 855-865Abstract Full Text PDF PubMed Scopus (824) Google Scholar, 23Koike 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 (270) Google Scholar). The PVR/CD155 gene has been mapped to the long arm of human chromosome 19, and this region is homologous to the regions of rat and mouse Tage4 genes (23Koike 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 (270) Google Scholar, 29Baury 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). PVR/CD155 has not been thought to be a cell-cell adhesion molecule because it does not homophilically trans-interact (30Aoki J. Koike S. Asou H. Ise I. Suwa H. Tanaka T. Miyasaka M. Nomoto A. Exp. Cell Res. 1997; 235: 374-384Crossref PubMed Scopus (115) Google Scholar). PVR/CD155 has been shown to serve as an entry receptor not only for human poliovirus but also for porcine pseudorabies virus and bovine herpesvirus 1 (29Baury 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, 31Geraghty R.J. Krummenacher C. Cohen G.H. Eisenberg R.J. Spear P.G. Science. 1998; 280: 1618-1620Crossref PubMed Scopus (777) Google Scholar). PVR/CD155 is overexpressed in human colorectal carcinoma and malignant glioma (32Masson D. Jarry A. Baury B. Blanchardie P. Laboisse C. Lustenberger P. Denis M.G. Gut. 2001; 49: 236-240Crossref PubMed Scopus (209) Google Scholar, 33Gromeier M. Lachmann S. Rosenfeld M.R. Gutin P.H. Wimmer E. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 6803-6808Crossref PubMed Scopus (306) Google Scholar). PVR/CD155 has been shown to be physically associated with CD44 on human monocyte cell surfaces (34Freistadt M.S. Eberle K.E. Mol. Immunol. 1997; 34: 1247-1257Crossref PubMed Scopus (30) Google Scholar). CD44 is known to be a transmembrane protein that is involved in cell migration and metastasis of cancer cells (35Ponta H. Sherman L. Herrlich P.A. Nat. Rev. Mol. Cell Biol. 2003; 4: 33-45Crossref PubMed Scopus (1871) Google Scholar). The extracellular region of PVR/CD155 has been reported to bind to the extracellular matrix molecule vitronectin (36Lange R. Peng X. Wimmer E. Lipp M. Bernhardt G. Virology. 2001; 285: 218-227Crossref PubMed Scopus (77) Google Scholar). The cytoplasmic region of PVR/CD155 binds to Tctex-1, a subunit of the dynein motor complex (37Mueller S. Cao X. Welker R. Wimmer E. J. Biol. Chem. 2002; 277: 7897-7904Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar). Thus, the roles of Tage4 and PVR/CD155 as viral receptors have been established, but their physiological function(s) remained unknown. We have recently shown that Tage4 does not homophilically trans-interact, but heterophilically trans-interacts with nectin-3, that its expression is very low in normal adult tissues but up-regulated in NIH3T3 cells transformed by an oncogenic Ki-Ras (V12Ras-NIH3T3 cells) as estimated by Western blotting, and that the heterophilic trans-interaction of Tage4 with nectin-3 enhances intercellular motility of V12Ras-NIH3T3 cells (26Ikeda 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 (112) Google Scholar). Consistently, Mueller and Wimmer (38Mueller S. Wimmer E. J. Biol. Chem. 2003; 278: 31251-31260Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar) have recently shown that PVR/CD155 heterophilically trans-interacts with nectin-3. The phylogenetic analysis of nectins and Necls cannot clearly conclude that the genes of Tage4 and PVR/CD155 are derived from the common or different ancestor gene (26Ikeda 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 (112) Google Scholar), and their exact relationship is currently unknown. However, on the basis of similar properties of Tage4 and PVR/CD155 thus far elucidated (29Baury 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, 39Ravens I. Seth S. Forster R. Bernhardt G. Biochem. Biophys. Res. Commun. 2003; 312: 1364-1371Crossref PubMed Scopus (41) Google Scholar), we tentatively propose here that they are derived from the common ancestor gene and called Necl-5. Extending these earlier observations, we have first examined here whether Necl-5 regulates motility of cells that do not contact other cells expressing nectin-3 and have found that Necl-5 enhances serum- and platelet-derived growth factor (PDGF)-induced cell motility without its trans-interaction with nectin-3. We have then examined whether the serum-induced, Necl-5-enhanced cell motility requires integrins, because integrins have been shown to play a key role in cell motility (40Hood J.D. Cheresh D.A. Nat. Rev. Cancer. 2002; 2: 91-100Crossref PubMed Scopus (1495) Google Scholar), and have found that Necl-5 is functionally associated with integrin αVβ3 in cell motility. Analysis on the mode of action of Necl-5 has revealed that it enhances cell motility through activation of Cdc42 and Rac. We show here that Necl-5 regulates serum- and PDGF-induced cell motility in an integrin-dependent, nectin-3-independent manner. We furthermore show that enhanced motility and metastasis of V12Ras-NIH3T3 cells are at least partly the result of up-regulated Necl-5. Construction—Expression vectors were constructed in pCAGIZeo (41Niwa H. Burdon T. Chambers I. Smith A. Genes Dev. 1998; 12: 2048-2060Crossref PubMed Scopus (1256) Google Scholar), pFLAG-CMV1 (Sigma), and pFastBac1-Msp-Fc (42Sakisaka 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). Constructs of Necl-5 contained the following aa: pCAGIZeo-Necl-5-ΔCP, aa 1-374 (deletion of the cytoplasmic region); pFLAG-CMV1-Necl-5-ΔCP, aa 30-374 (deletion of the signal peptide and the cytoplasmic region); and pFastBac1-Msp-Fc-Necl-5-EC (the extracellular fragment of Necl-5 fused to the human IgG Fc), aa 30-347 (26Ikeda 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 (112) Google Scholar). To express the extracellular fragment of nectin-3 fused to the human IgG Fc (Nef-3), pFast-Bac1-Msp-Fc-nectin-3-EC (aa 56-400) was prepared as described (43Satoh-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 fusion protein with IgG Fc was produced as a secreted protein by the baculovirus transfer system (Invitrogen, Carlsbad, CA) and purified by use of protein A-Sepharose beads (Amersham Biosciences) as described (42Sakisaka 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). pEF-BOS-Myc-NWASP-CRIB and pEF-BOS-Myc-N17Rac1 were prepared as described (44Ono Y. Nakanishi H. Nishimura M. Kakizaki M. Takahashi K. Miyahara M. Satoh-Horikawa K. Mandai K. Takai Y. Oncogene. 2000; 19: 3050-3058Crossref PubMed Scopus (55) Google Scholar, 45Yasuda T. Ohtsuka T. Inoue E. Yokoyama S. Sakisaka T. Kodama A. Takaishi K. Takai Y. Genes Cells. 2000; 5: 583-591Crossref PubMed Scopus (15) Google Scholar). pRaichu-Rac1 and pRaichu-Cdc42 were kind gifts from Drs. M. Matsuda and T. Nakamura (Osaka University, Osaka, Japan). Cell Culture, DNA Transfection, and Establishment of Transformants—L cells were kindly supplied by Dr. S. Tsukita (Kyoto University, Kyoto, Japan). L cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum. L cell lines stably expressing mouse Necl-5 (full-length, aa 1-409), FLAG-Necl-5 (aa 30-409), or Necl-5, of which the extracellular region was deleted (aa 335-409) (non-tagged Necl-5-L, Necl-5-L, or Necl-5-ΔEC-L cells, respectively) were prepared as described (26Ikeda 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 (112) Google Scholar). NIH3T3 and V12Ras-NIH3T3 cells were maintained in DMEM supplemented with 10% calf serum. V12Ras-NIH3T3 cells were prepared as described (46Fujioka 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). An L cell line, an NIH3T3 cell line, or a V12Ras-NIH3T3 cell line stably expressing Necl-5, from which the cytoplasmic region was deleted (Necl-5-ΔCP-L, Necl-5-ΔCP-NIH3T3, or Necl-5-ΔCP-V12Ras-NIH3T3 cells, respectively), was obtained by transfection with pCAGIZeo-Necl-5-ΔCP using LipofectAMINE PLUS reagent (Invitrogen). For transient expression of Myc-NWASP, Myc-N17Rac1, FLAG-Necl-5-ΔCP, or Necl-5-ΔCP, L or NIH3T3 cell lines were transfected with pEF-BOS-Myc-NWASP-CRIB, pEF-BOS-Myc-N17Rac1, or pFLAG-CMV1-Necl-5-ΔCP, respectively, as described above. Antibodies and Reagents—A rat anti-Necl-5 monoclonal antibody (mAb) (1A8-8; mAb-i) was prepared as described (26Ikeda 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 (112) Google Scholar). Another rat anti-Necl-5 mAb (3A4-2; mAb-s) was raised against the fusion protein of the extracellular region of Necl-5 (aa 30-347) with IgG Fc. A mouse anti-integrin αV mAb (clone 21), a rat anti-integrin αV mAb (RMV-7), and an Armenian hamster anti-integrin β3 mAb (2C9.G2) were purchased from BD Biosciences Pharmingen (San Diego, CA). A rabbit anti-integrin αV polyclonal Ab (pAb) and β3 pAb were purchased from Chemicon (Temecula, CA). Hybridoma cells expressing the mouse anti-Myc mAb (9E10) were obtained from American Type Culture Collection (Manassas, VA). A rabbit anti-FLAG pAb, a mouse anti-FLAG mAb, fatty acid-free bovine serum albumin (BSA), and echistatin were purchased from Sigma. A horseradish peroxidase-conjugated goat anti-mouse IgG was purchased from American Qualex (San Clemente, CA). A horseradish peroxidase-conjugated donkey anti-rabbit IgG and a horseradish peroxidase-conjugated goat anti-Armenian hamster IgG were purchased from Jackson Immunoresearch Laboratories (West Grove, PA). Vitronectin was kindly supplied by Dr. K. Sekiguchi (Osaka University, Osaka, Japan). Human recombinant PDGF-BB was purchased from PEPROTECH (Rocky Hill, NJ). Immunofluorescence Microscopy—Immunofluorescence microscopy of various cell lines was done as described (47Takahashi 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 (447) Google Scholar). For the experiments using V12Ras-NIH3T3 cells, the samples were fixed with acetone/methanol (1:1) at -20 °C for 1 min. For the experiments using Necl-5-L cells with various anti-integrin-αv or -β3 Ab, the signal was enhanced using a Tyramide Signal Amplification kit (Molecular Probes, Eugene, OR) according to the protocol from the manufacturer. The samples were analyzed by Radiance 2000 or 2100 confocal laser scanning microscope (Bio-Rad). Phagokinetic Track Motility Assay—The uniform carpet of gold particles was prepared on glass coverslips as described (48Albrecht-Buehler G. Cell. 1977; 11: 395-404Abstract Full Text PDF PubMed Scopus (384) Google Scholar). The colloidal gold-coated coverslips were placed in 35-mm nontreated dishes, and the coverslips were coated with or without various reagents (50 μg/ml of the anti-Necl-5 mAb-s, mAb-i, or Nef-3, or 10 μg/ml of vitronectin) by incubation at room temperature for 1 h. Cells were seeded at a density of 2 × 103 cells/35-mm dish. When the anti-Necl-5 mAb-i or vitronectin was added into the medium, the final concentration of the anti-Necl-5 mAb-i or vitronectin was 50 or 20 μg/ml, respectively. When the cells were incubated in the absence of serum, DMEM supplemented with 0.5% fatty acid-free BSA was used. After incubation for 16 h, the samples were fixed with PBS containing 3.7% formaldehyde and cell motility was analyzed by measuring the areas free of gold particles around a single cell. Five independent experiments were performed, and at least 24 independent samples in each experiment were picked up to determine the areas. The statistical significance was determined by paired t test. Boyden Chamber Assay—The Boyden chamber assay was performed as described (49Woodard A.S. Garcia-Cardena G. Leong M. Madri J.A. Sessa W.C. Languino L.R. J. Cell Sci. 1998; 111: 469-478Crossref PubMed Google Scholar) with some modifications. Falcon™ cell culture inserts (8.0-μm pores, Becton Dickinson Labware, Franklin Lakes, NJ) were coated with 3 μg/ml vitronectin or 1% BSA at 37 °C for 1 h. The inserts were then blocked with 1% BSA at 37 °C for 30 min. NIH3T3 cell lines, which had been serum starved with DMEM supplemented with 0.5% fatty acid-free BSA for 24 h, were detached with 0.05% trypsin and 0.53 mm EDTA and then treated with a trypsin inhibitor (Sigma). The cells were then resuspended in DMEM supplemented with 0.5% fatty acid-free BSA and seeded at a density of 4 × 104 cells/insert. The cells were incubated at 37 °C for 14 h in the presence or absence of 30 ng/ml of PDGF-BB. PDGF-BB was added only to the bottom well to generate a concentration gradient. After incubation, the inserts were washed with PBS, the cells were fixed using 3.7% formaldehyde and subsequently stained with 0.5% Crystal Violet (Sigma). The cells, which had not migrated, were removed by wiping the top of the membrane with a cotton swap. The number of stained cells in five randomly chosen fields per filter were counted by microscopic examination. Fluorescent Resonance Energy Transfer (FRET) Imaging—The FRET imaging was performed as described (13Honda T. Shimizu K. Kawakatsu T. Fukuhara A. Irie K. Nakamura T. Matsuda M. Takai Y. Genes Cells. 2003; 8: 481-491Crossref PubMed Scopus (43) Google Scholar) with some modifications. Necl-5-L, Necl-5-ΔEC-L, or Necl-5-ΔCP-L cells were transfected with pRaichu-Rac1 or pRaichu-Cdc42 using LipofectAMINE PLUS reagent. The FRET probes for wild-type Rac1 and Cdc42 consisted of a CRIB domain of PAK, Rac1 or Cdc42, a pair of green fluorescent protein mutants, and a CAAX box of Ki-Ras (50Itoh R.E. Kurokawa K. Ohba Y. Yoshizaki H. Mochizuki N. Matsuda M. Mol. Cell Biol. 2002; 22: 6582-6591Crossref PubMed Scopus (459) Google Scholar). Twenty-four h after the transfection, the cells were replated on the glass base dishes (Iwaki, Tokyo, Japan). Sixteen h after replating, the cells were then imaged with an Olympus IX71 inverted microscope equipped with a cooled charge-coupled device camera, CoolSNAP HQ (Roper Scientific, Trenton, NJ), controlled by MetaMorph software (Universal Imaging, West Chester, PA) (51Miyawaki A. Llopis J. Heim R. McCaffery J.M. Adams J.A. Ikura M. Tsien R.Y. Nature. 1997; 388: 882-887C

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