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Necl-5/Poliovirus Receptor Interacts in cis with Integrin αVβ3 and Regulates Its Clustering and Focal Complex Formation

2007; Elsevier BV; Volume: 282; Issue: 25 Linguagem: Inglês

10.1074/jbc.m611330200

1083-351X

Yukiko Minami, Wataru Ikeda, Mihoko Kajita, Tsutomu Fujito, H. Amano, Yoshiyuki Tamaru, Kaori Kuramitsu, Yasuhisa Sakamoto, Morito Monden, Yoshimi Takai,

Cellular Mechanics and Interactions

Integrin αvβ3, which forms focal complexes at leading edges in moving cells, is up-regulated in cancer cells and so is implicated in their invasiveness. Necl-5, originally identified as a poliovirus receptor and also up-regulated in cancer cells, colocalizes with integrin αvβ3 at leading edges in moving cells and enhances growth factor-induced cell movement. Here, we show that Necl-5 interacts directly, in cis, with integrin αvβ3, and enhances integrin αvβ3 clustering and focal complex formation at leading edges in NIH3T3 cells. The extracellular region of Necl-5, but not the cytoplasmic region, is necessary for its interaction with integrin αvβ3; however, both regions are necessary for its action. An interaction between integrin αvβ3 and vitronectin and PDGF-induced activation of Rac are also necessary for integrin αvβ3 clustering. The interaction between Necl-5 and integrin αvβ3 enhances PDGF-induced Rac activation, facilitating integrin αvβ3 clustering presumably in a feedback amplification manner. Thus, Necl-5 has a critical role in integrin αvβ3 clustering and focal complex formation. Integrin αvβ3, which forms focal complexes at leading edges in moving cells, is up-regulated in cancer cells and so is implicated in their invasiveness. Necl-5, originally identified as a poliovirus receptor and also up-regulated in cancer cells, colocalizes with integrin αvβ3 at leading edges in moving cells and enhances growth factor-induced cell movement. Here, we show that Necl-5 interacts directly, in cis, with integrin αvβ3, and enhances integrin αvβ3 clustering and focal complex formation at leading edges in NIH3T3 cells. The extracellular region of Necl-5, but not the cytoplasmic region, is necessary for its interaction with integrin αvβ3; however, both regions are necessary for its action. An interaction between integrin αvβ3 and vitronectin and PDGF-induced activation of Rac are also necessary for integrin αvβ3 clustering. The interaction between Necl-5 and integrin αvβ3 enhances PDGF-induced Rac activation, facilitating integrin αvβ3 clustering presumably in a feedback amplification manner. Thus, Necl-5 has a critical role in integrin αvβ3 clustering and focal complex formation. Integrins are key molecules for adhesion between cells and extracellular matrix (ECM) 2The abbreviations used are: ECM, extracellular matrix; PVR, poliovirus receptor; Necl, nectin-like molecule; DMEM, Dulbecco's modified Eagle's medium; siRNA, small interfering RNA; mAb, monoclonal antibody; pAb, polyclonal antibody; PDGF, platelet-derived growth factor; DTSSP, 3,3′-dithiobis [sulfosuccinimidylpropionate]; DN, dominant-negative; CA, constitutively active; nt, nucleotide; PBS, phosphate-buffered saline; BSA, bovine serum albumin. proteins (1van der Flier A. Sonnenberg A. Cell Tissue Res. 2001; 305: 285-298Crossref PubMed Scopus (818) Google Scholar). They are heterodimers of α and β subunits, both of which have one transmembrane segment. The extracellular region of integrins binds to ECM proteins, whereas the cytoplasmic region is directly and indirectly associated with many F-actin-binding proteins, such as talin, vinculin, and α-actinin, and intracellular signaling molecules, such as FAK and c-Src (1van der Flier A. Sonnenberg A. Cell Tissue Res. 2001; 305: 285-298Crossref PubMed Scopus (818) Google Scholar, 2Zamir E. Geiger B. J. Cell Sci. 2001; 114: 3577-3579Crossref PubMed Google Scholar). Integrins have at least two forms: conformations with either low or high affinity for their ECM binding partners (1van der Flier A. Sonnenberg A. Cell Tissue Res. 2001; 305: 285-298Crossref PubMed Scopus (818) Google Scholar, 3Takagi J. Petre B.M. Walz T. Springer T.A. Cell. 2002; 110: 599-611Abstract Full Text Full Text PDF PubMed Scopus (943) Google Scholar, 4Cram E.J. Schwarzbauer J.E. Trends Cell Biol. 2004; 14: 55-57Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). When talin binds to the low affinity form, this is converted to the high affinity form (4Cram E.J. Schwarzbauer J.E. Trends Cell Biol. 2004; 14: 55-57Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). Upon binding to ECM proteins, integrins transduce signals inside cells that then cause the reorganization of the actin cytoskeleton, eventually resulting in integrin clustering and the formation of focal complexes and focal adhesions (4Cram E.J. Schwarzbauer J.E. Trends Cell Biol. 2004; 14: 55-57Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). Focal complexes are formed at leading edges in moving cells, whereas focal adhesions, also called focal contacts, are formed at sites to the rear of the leading edges (5Rottner K. Hall A. Small J.V. Curr. Biol. 1999; 9: 640-648Abstract Full Text Full Text PDF PubMed Scopus (514) Google Scholar, 6Zaidel-Bar R. Cohen M. Addadi L. Geiger B. Biochem. Soc. Trans. 2004; 32: 416-420Crossref PubMed Scopus (415) Google Scholar). Focal complexes are generally smaller than mature focal adhesions, being less than 0.5 μm in diameter. Protrusions, such as filopodia and lamellipodia, are also formed at leading edges, while ruffles are formed on the dorsal surfaces of lamellipodia. Focal complexes are formed under these protrusions. Focal complexes, protrusions, and ruffles are formed by the actions of the small G proteins Rac and Cdc42; their formation is inhibited by the action of the small G protein RhoA (5Rottner K. Hall A. Small J.V. Curr. Biol. 1999; 9: 640-648Abstract Full Text Full Text PDF PubMed Scopus (514) Google Scholar, 7Ballestrem C. Hinz B. Imhof B.A. Wehrle-Haller B. J. Cell Biol. 2001; 155: 1319-1332Crossref PubMed Scopus (299) Google Scholar). By contrast, focal adhesions are formed by the action of RhoA. Inactivation of Rac and Cdc42 and activation of RhoA lead to the transformation of focal complexes into focal adhesions. The dynamic formation of all of these structures is necessary for efficient cell movement. The dynamic activation and inactivation of Rac, Cdc42, and RhoA are regulated not only by the outside-in signaling of integrins but also by growth factor-induced signaling (8Hall A. Science. 1998; 279: 509-514Crossref PubMed Scopus (5230) Google Scholar). Of the many integrins, integrin αVβ3 has a particularly important role in focal complex formation (9Guo W. Giancotti F.G. Nat. Rev. Mol. Cell Biol. 2004; 5: 816-826Crossref PubMed Scopus (1224) Google Scholar). This integrin is expressed in many cell types, such as fibroblasts and vascular endothelial cells (10Woodard 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, 11Brooks P.C. Clark R.A. Cheresh D.A. Science. 1994; 264: 569-571Crossref PubMed Scopus (2750) Google Scholar), and is often up-regulated in many cancer cells, such as colon carcinoma, melanoma, and glioblastoma cells (12Chiang H.S. Peng H.C. Huang T.F. Biochim. Biophys. Acta. 1994; 1224: 506-516Crossref PubMed Scopus (24) Google Scholar, 13Gehlsen K.R. Davis G.E. Sriramarao P. Clin. Exp. Metastasis. 1992; 10: 111-120Crossref PubMed Scopus (183) Google Scholar, 14Chatterjee S. Matsumura A. Schradermeier J. Gillespie G.Y. J. Neurooncol. 2000; 46: 135-144Crossref PubMed Scopus (79) Google Scholar). We found that an Ig-like molecule Necl-5/poliovirus receptor (PVR)/CD155/Tage4 co-localizes with integrin αVβ3 at leading edges in moving cells and enhances growth factor-induced cell movement (15Ikeda W. Kakunaga S. Takekuni K. Shingai T. Satoh K. Morimoto K. Takeuchi M. Imai T. Takai Y. J. Biol. Chem. 2004; 279: 18015-18025Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). Human PVR/CD155 was originally identified as a poliovirus receptor (16Mendelsohn C.L. Wimmer E. Racaniello V.R. Cell. 1989; 56: 855-865Abstract Full Text PDF PubMed Scopus (824) Google Scholar, 17Koike 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), whereas rodent Tage4 was originally identified as the product of a gene that is overexpressed in rodent colon carcinoma (18Chadeneau C. LeMoullac B. Denis M.G. J. Biol. Chem. 1994; 269: 15601-15605Abstract Full Text PDF PubMed Google Scholar). PVR/CD155 is also overexpressed in many human cancer cells, such as colon carcinoma, melanoma, and glioblastoma cells, in which integrin αVβ3 is up-regulated (19Masson D. Jarry A. Baury B. Blanchardie P. Laboisse C. Lustenberger P. Denis M.G. Gut. 2001; 49: 236-240Crossref PubMed Scopus (209) Google Scholar, 20Gromeier 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, 21Bottino C. Castriconi R. Pende D. Rivera P. Nanni M. Carnemolla B. Cantoni C. Grassi J. Marcenaro S. Reymond N. Vitale M. Moretta L. Lopez M. Moretta A. J. Exp. Med. 2003; 198: 557-567Crossref PubMed Scopus (685) Google Scholar). This molecule, with four nomenclatures, is also named nectin-like molecule-5, Necl-5 (22Takai Y. Irie K. Shimizu K. Sakisaka T. Ikeda W. Cancer Sci. 2003; 94: 655-667Crossref PubMed Scopus (291) Google Scholar). Necl-5 is expressed in many cell types, such as fibroblasts and vascular endothelial cells, in which integrin αVβ3 is expressed (23Kakunaga S. Ikeda W. Shingai T. Fujito T. Yamada A. Minami Y. Imai T. Takai Y. J. Biol. Chem. 2004; 279: 36419-36425Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 24Reymond N. Imbert A.M. Devilard E. Fabre S. Chabannon C. Xerri L. Farnarier C. Cantoni C. Bottino C. Moretta A. Dubreuil P. Lopez M. J. Exp. Med. 2004; 199: 1331-1341Crossref PubMed Scopus (216) Google Scholar). Necl-5 does not show homophilic cell-cell adhesion activity, but it heterophilically interacts in trans (i.e. present on a different cell) with nectin-3, a member of the Ig-like nectin family that is a Ca2+-independent cell-cell adhesion molecule and forms adherens junctions cooperatively with cadherins (22Takai Y. Irie K. Shimizu K. Sakisaka T. Ikeda W. Cancer Sci. 2003; 94: 655-667Crossref PubMed Scopus (291) Google Scholar, 25Ikeda 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). When cells come into contact with other cells, Necl-5 is down-regulated from the cell surface by trans-interacting with nectin-3 (26Fujito T. Ikeda W. Kakunaga S. Minami Y. Kajita M. Sakamoto Y. Monden M. Takai Y. J. Cell Biol. 2005; 171: 165-173Crossref PubMed Scopus (86) Google Scholar); however, when there is no contact with other cells, Necl-5 is up-regulated at the transcriptional level by growth factor-induced signaling (27Hirota T. Irie K. Okamoto R. Ikeda W. Takai Y. Oncogene. 2005; 24: 2229-2235Crossref PubMed Scopus (58) Google Scholar). Up-regulation of Necl-5 enhances growth factor-induced cell movement, whereas down-regulation of Necl-5 reduces it (26Fujito T. Ikeda W. Kakunaga S. Minami Y. Kajita M. Sakamoto Y. Monden M. Takai Y. J. Cell Biol. 2005; 171: 165-173Crossref PubMed Scopus (86) Google Scholar). Cultured cells continue to move until they contact other cells and become confluent. They then undergo cell-cell adhesion and stop moving. This phenomenon has been known for more than fifty years as contact inhibition of cell movement (28Abercrombie M. Heaysman J.E. Exp. Cell Res. 1953; 5: 111-131Crossref PubMed Scopus (279) Google Scholar, 29Abercrombie M. Nature. 1979; 281: 259-262Crossref PubMed Scopus (290) Google Scholar). We have proposed that Necl-5 has a role, at least in part, in this contact inhibition of cell movement (26Fujito T. Ikeda W. Kakunaga S. Minami Y. Kajita M. Sakamoto Y. Monden M. Takai Y. J. Cell Biol. 2005; 171: 165-173Crossref PubMed Scopus (86) Google Scholar). However, we did not previously examine which structure of leading edges Necl-5 localizes to or whether, and if so how, Necl-5 regulates integrin αVβ3-based focal complex formation. We attempt to address these issues here using NIH3T3 cells as a model cell type, which express both integrin αVβ3 and Necl-5. Construction—pCAGIPuro-FLAG-Necl-5, pCAGIZeo-FLAG-Necl-5-ΔCP, and pCAGIZeo-FLAG-Necl-5-ΔEC were prepared as described (15Ikeda W. Kakunaga S. Takekuni K. Shingai T. Satoh K. Morimoto K. Takeuchi M. Imai T. Takai Y. J. Biol. Chem. 2004; 279: 18015-18025Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar, 25Ikeda 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). A small interfering RNA (siRNA) vector against Necl-5 (pBS-H1-Necl-5) and a control siRNA vector against luciferase (pBS-H1-control) were prepared as described (26Fujito T. Ikeda W. Kakunaga S. Minami Y. Kajita M. Sakamoto Y. Monden M. Takai Y. J. Cell Biol. 2005; 171: 165-173Crossref PubMed Scopus (86) Google Scholar). To obtain siRNA-resistant mutants, FLAG-Necl-5R and FLAG-Necl-5-ΔCPR were generated by mutagenesis of 5′-GGTATGTTGGCCTCACTAA-3′ to create 5′-GGTACG-TAGGATTAACGAA-3′ using the QuikChange site-directed mutagenesis kit (Stratagene) from pFLAG-CMV1-Necl-5 and pFLAG-CMV1-Necl-5-ΔCP (15Ikeda W. Kakunaga S. Takekuni K. Shingai T. Satoh K. Morimoto K. Takeuchi M. Imai T. Takai Y. J. Biol. Chem. 2004; 279: 18015-18025Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar, 25Ikeda 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 cloned into pCMV5 vector (pCMV5-FLAG-Necl-5R and pCMV5-FLAG-Necl-5-ΔCPR, respectively). Necl-5-ΔCP fused to a Myc tag at the C terminus was inserted into the vector pCAGIPuro (pCAGI-Puro-Necl-5-ΔCP-myc). Expression vectors were kindly provided as follows: wild-type human integrin αV and human integrin β3 (pcDNA3-αV and -β3) were from Dr. J. C. Norman (University of Leicester, Leicester, UK), a dominant-negative mutant of human integrin β3 (pcDNA3.1-Myc-His(+)-β3T329C/A347C) was from Dr. J. Takagi (Osaka University, Suita, Japan). GST-PAK-Cdc42/Rac-interactive binding region (pGEX-PAK-CRIB) was a kind gift from Dr. S. Narumiya (Kyoto University, Kyoto, Japan). pEFBOS-myc-Rac1CA and -Rac1DN were prepared as described (30Komuro R. Sasaki T. Takaishi K. Orita S. Takai Y. Genes Cells. 1996; 1: 943-951Crossref PubMed Scopus (49) Google Scholar). Cell Culture, Transfection, and siRNA Experiments—NIH3T3 cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% calf serum. HEK293 cells were maintained in DMEM supplemented with 10% fetal calf serum. NIH3T3 cells stably expressing FLAG-tagged Necl-5 (Necl-5-NIH3T3 cells) were obtained by transfection with pCAGIPuro-FLAG-Necl-5 using Lipofectamine 2000 reagent (Invitrogen) and by selection with puromycin. Necl-5-ΔCP-NIH3T3 cells were prepared as described (15Ikeda W. Kakunaga S. Takekuni K. Shingai T. Satoh K. Morimoto K. Takeuchi M. Imai T. Takai Y. J. Biol. Chem. 2004; 279: 18015-18025Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). For transient expression experiments, cells were transfected with various expression vectors using Lipofectamine 2000 reagent. Knockdown of Necl-5 was performed as described (26Fujito T. Ikeda W. Kakunaga S. Minami Y. Kajita M. Sakamoto Y. Monden M. Takai Y. J. Cell Biol. 2005; 171: 165-173Crossref PubMed Scopus (86) Google Scholar). Briefly, NIH3T3 cells were co-transfected with siRNA vector against Necl-5, pBS-H1-Necl-5, and pEGFP-tub (Clontech), because EGFP-tagged tubulin, but not EGFP, is resistant to acetone/methanol fixation. As a control, siRNA vector against luciferase, pBS-H1-control, was used. pBS-H1 vector was a gift from Dr. H. Shibuya (Tokyo Medical and Dental University, Tokyo, Japan). Alternatively, cells were transfected with a double-stranded 19-nt RNA duplex to Necl-5 (5′-GGTATGTTGGCCTCACTAA-3′) or a similar duplex to luciferase (5′-CGTACGCGGAATACTTCGA-3′) as a control using Hyperfect reagent (Qiagen). To confirm knockdown of Necl-5 by FACS, pBS-EGFP-H1-Necl-5 and pBS-EGFP-H1-control were used. These vectors were constructed by subcloning the insert encoding the EGFP expression unit from pEGFP-C1 (Clontech) into a region not related to siRNA expressions of pBS-H1-Necl-5 and pBS-H1-control, respectively. Antibodies and Reagents—A rat monoclonal antibody (mAb) against the extracellular region of Necl-5 (mAb-i, 1A8-8) was prepared as described (25Ikeda 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). Hamster anti-integrin αV and β3 mAbs (H9.2B8 and 2C9.G2, respectively; for immunostaining) were purchased from BD Biosciences. The following mouse mAbs were purchased from commercial sources: anti-integrin αV, anti-integrin β3, anti-integrin α5, and anti-integrin β1 (for Western blotting; BD Biosciences), anti-talin (8D4) and anti-FLAG M2 (for immunoprecipitation, Western blotting and FACS) (Sigma), and anti-actin mAbs (Chemicon). The following rabbit polyclonal antibodies (pAbs) were purchased from commercial sources: anti-N-cadherin (TAKARA), anti-integrin αV (for immunoprecipitation), and anti-integrin β3 pAbs (for Western blotting) (Chemicon). Hybridoma cells (9E10) expressing a mouse anti-Myc mAb were purchased from the American Type Culture Collection. Fatty acid-free BSA, fibronectin, laminin, ConA, and cyclo-RGDfV were purchased from Sigma. Horseradish peroxidase-conjugated secondary Abs was purchased from Amersham Biosciences. Fluorophore (FITC, Cy3, Cy5, and R-PE)-conjugated secondary Abs were purchased from Jackson Immuno Research. Human recombinant platelet-derived growth factor (PDGF)-BB was purchased from PEPROTECH. Vitronectin was purified from human plasma (Kohjinbio) as described (31Yatohgo T. Izumi M. Kashiwagi H. Hayashi M. Cell Struct. Funct. 1988; 13: 281-292Crossref PubMed Scopus (439) Google Scholar). Chemical cross-linker, 3,3′-dithiobis [sulfosuccinimidylpropionate] (DTSSP), was purchased from Pierce. Directional Stimulation by PDGF—To generate a concentration gradient of PDGF, a μ-Slide VI flow (uncoated; Ibidi) was used. The μ-Slide VI flow has six parallel channels, which were coated with 5 μg/ml vitronectin, 25 μg/ml fibronectin, or 80 μg/ml laminin. Cells were seeded at a density of 5 × 103 cells per square centimeter, cultured for 16 h, and starved of serum with DMEM containing 0.5% BSA for 1 h. The concentration gradient of PDGF was made using DMEM containing 0.5% BSA and 30 ng/ml PDGF according to manufacturer's protocol. After 30 min, cells were fixed with acetone/methanol (1:1), incubated with 1% BSA in PBS, and then incubated with 20% BlockAce in PBS, prior to immunofluorescence microscopy (26Fujito T. Ikeda W. Kakunaga S. Minami Y. Kajita M. Sakamoto Y. Monden M. Takai Y. J. Cell Biol. 2005; 171: 165-173Crossref PubMed Scopus (86) Google Scholar). The samples were analyzed by confocal laser-scanning microscope systems, digital eclipse C1si-ready (NIKON), and LSM510 META (Carl Zeiss MicroImaging). Co-immunoprecipitation Assay—HEK293 cells were co-transfected with various combinations of plasmids, cultured for 24 h, detached with 0.05% trypsin and 0.53 mm EDTA, and treated with a trypsin inhibitor. To make predominantly high affinity integrin αVβ3, cells were cultured in suspension with Ca2+-free DMEM (Invitrogen) containing 0.5% BSA, 1 mm MnCl2, and 50 μg/ml cyclo-RGDfV for 1 h, collected by centrifugation, washed with Wash buffer (20 mm Tris-HCl, pH 8.0, 150 mm NaCl, and 1 mm Na3VO4), and lysed with Buffer A (20 mm Tris-HCl, pH 8.0, 150 mm NaCl, 1 mm MnCl2, 10% glycerol, 1% Nonidet P-40, 10 mm NaF, 1 mm Na3VO4, 10 μg/ml leupeptin, 2 μg/ml aprotinin, and 10 μm APMSF). To make predominantly low affinity integrin αVβ3, cells were cultured in suspension with DMEM containing 0.5% BSA for 1 h, collected by centrifugation, washed with Wash buffer and lysed with Buffer B (20 mm Tris-HCl, pH 8.0, 150 mm NaCl, 1 mm CaCl2, 1 mm MgCl2, 10% glycerol, 1% Nonidet P-40, 10 mm NaF, 1 mm Na3VO4, 10 μg/ml leupeptin, 2 μg/ml aprotinin, and 10 μm APMSF). The lysates were rotated for 30 min and subjected to centrifugation at 12,000 × g for 20 min. The supernatant was precleared with protein G-Sepharose 4 Fast Flow beads (Amersham Biosciences) at 4 °C for 1 h, incubated with the anti-FLAG mAb at 4 °C for 4 h, and then incubated with protein G-Sepharose beads at 4 °C for 2 h. After the beads were extensively washed with Buffer A or B, bound proteins were eluted by boiling the beads in SDS Sample Buffer (60 mm Tris-HCl, pH 6.7, 3% SDS, 2% 2-mercaptoethanol, and 5% glycerol) for 5 min and subjected to SDS-PAGE followed by Western blotting. To examine the interaction between endogenous Necl-5 and endogenous integrin αVβ3, a co-immunoprecipitation assay was performed using NIH3T3 cells. Cells were plated at a density of 5 × 103 cells per square centimeter on vitronectin-coated dish, cultured for 16 h, starved of serum with DMEM containing 0.5% BSA for 1 h, and then stimulated with DMEM containing 0.5% BSA and/or 3 ng/ml PDGF. After 30 min, cells were washed with ice-cold PBS and incubated with 2 mm DTSSP in PBS at 4 °C for 2 h. To quench the cross-linking reaction, 1 m Tris-HCl (pH 7.5) was added at a final concentration of 50 mm. Cells were washed with PBS and lysed with Buffer B. The lysates were rotated for 30 min and subjected to centrifugation at 12,000 × g for 20 min. The supernatant was precleared with protein A-Sepharose (Amersham Biosciences) at 4 °C for 1 h, incubated with the anti-integrin αV pAb at 4 °C for 16 h, and then incubated with protein A-Sepharose beads at 4 °C for 4 h. After the beads were extensively washed with Buffer B, bound proteins were eluted by boiling the beads in SDS sample buffer for 5 min and subjected to SDS-PAGE followed by Western blotting. In Vitro Binding of Necl-5 to Integrin αVβ3—Necl-5 EC was prepared as described (25Ikeda 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 obtain integrin αVβ3 EC, CHO-lec 3.2.8.1 cells expressing integrin αVβ3 EC were cultured, and the culture supernatant containing soluble integrin αVβ3 EC was collected (3Takagi J. Petre B.M. Walz T. Springer T.A. Cell. 2002; 110: 599-611Abstract Full Text Full Text PDF PubMed Scopus (943) Google Scholar). This cell line was kindly supplied by Dr. J. Takagi (Osaka University, Suita, Japan). The culture supernatant was applied to Ni-Sepharose 6 Fast Flow beads (Amersham Biosciences) and equilibrated with Buffer C (25 mm Tris-HCl at pH 8.0, 200 mm NaCl, 1 mm CaCl2, 1 mm MgCl2, and 20 mm imidazole at pH 8.0). After the beads were extensively washed with Buffer C and then Buffer C containing 0.6 m NaCl, bound integrin αVβ3 EC was eluted with Elution buffer (25 mm Tris-HCl at pH 8.0, 200 mm NaCl, 1 mm CaCl2, 1 mm MgCl2, and 500 mm imidazole at pH 8.0) and the eluate was dialyzed with Buffer C. The protein concentration of integrin αVβ3 EC was determined with BSA as a reference protein by SDS-PAGE. To examine the binding of Necl-5 EC to integrin αVβ3 EC, integrin αVβ3 EC (6 pmol) was immobilized on Ni-Sepharose beads and soluble Necl-5 EC (60 pmol) was incubated with integrin αVβ3 EC-immobilized Ni-Sepharose beads or Ni-Sepharose beads alone in 0.3 ml of Buffer C containing 1% BSA. After the beads were extensively washed with Buffer C, bound proteins were eluted with Elution buffer. The eluate was then subjected to SDS-PAGE, followed by Western blotting. Assay for Bead-Cell Contact—Latex-sulfate microbeads (4.55 × 106; 10-μm diameter; Polyscience Inc.) were washed, resuspended in 0.2 ml of PBS, and incubated with 10 μg of vitronectin, 50 μg of fibronectin, or 160 μg of laminin with gentle mixing at room temperature. After the incubation, the beads were washed three times with 0.5 ml of PBS and resuspended in 0.1 ml of PBS containing 1% BSA. ConA-coated beads were prepared as described (32Tran H. Pankov R. Tran S.D. Hampton B. Burgess W.H. Yamada K.M. J. Cell Sci. 2002; 115: 2031-2040Crossref PubMed Google Scholar) and used as control beads. Cells were starved of serum with DMEM containing 0.5% BSA for 18 h. After serum starvation, cells were detached with 0.05% trypsin and 0.53 mm EDTA and then treated with a trypsin inhibitor (Sigma). Cells were plated at a density of 1 × 104 cells per square centimeter on laminin-coated coverglass, cultured for 3 h, and incubated with vitronectin-, fibronectin-, laminin-, or ConA-coated beads for 1 h. Cells were fixed with acetone/methanol (1:1), incubated with 1% BSA in PBS, and then incubated with 20% BlockAce in PBS, followed by immunofluorescence microscopy (26Fujito T. Ikeda W. Kakunaga S. Minami Y. Kajita M. Sakamoto Y. Monden M. Takai Y. J. Cell Biol. 2005; 171: 165-173Crossref PubMed Scopus (86) Google Scholar). Assay for Rac Activation—The assay for Rac activation using GST-PAK CRIB was performed as described (33Kawakatsu 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). Briefly, NIH3T3, Necl-5-NIH3T3, or Necl-5-ΔCP-NIH3T3 cells were cultured on vitronectin-coated dishes for 16 h, starved of serum with DMEM containing 0.5% BSA for 1 h, stimulated with 3 ng/ml PDGF, and then subjected to the assay for Rac activation. For analysis of Necl-5-knockdown cells, NIH3T3 cells were twice transfected with siRNA oligo every 24 h, cultured for 24 h, and subjected to the assay. Co-localization of Necl-5 with Integrin αVβ3 at Peripheral Ruffles and Focal Complexes—NIH3T3 cells were sparsely plated on μ-slide VI flow dishes precoated with vitronectin, an ECM protein that binds to integrin αVβ3 (1van der Flier A. Sonnenberg A. Cell Tissue Res. 2001; 305: 285-298Crossref PubMed Scopus (818) Google Scholar), starved of serum, and directionally stimulated by PDGF. Most cells became polarized and formed lamellipodia with peripheral ruffles at leading edges, in the direction of higher concentrations of PDGF. The immunofluorescence signals for Necl-5 and integrin β3 were concentrated and co-localized at the peripheral ruffles of the leading edges in the middle section of the cells (Fig. 1Aa), consistent with earlier observations (15Ikeda W. Kakunaga S. Takekuni K. Shingai T. Satoh K. Morimoto K. Takeuchi M. Imai T. Takai Y. J. Biol. Chem. 2004; 279: 18015-18025Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). In the basal section of the cells, the signal for integrin β3 was observed as dot-like structures under the peripheral ruffles. The signal for Necl-5 was also observed as fuzzy dot-like structures and mostly overlapped with the signal for integrin β3. In addition, the signal for integrin β3, but not that for Necl-5, was observed as dot-like structures at sites to the rear of the leading edges. Essentially the same results were obtained when integrin αV was stained instead of integrin β3. 3Y. Takai, unpublished data. Indeed, unless otherwise specified, essentially the same results were obtained for both integrin αV and integrin β3 in the experiments that follow. The dot-like structures under the peripheral ruffles (which were immunopositive for Necl-5, integrin αV, and integrin β3) were smaller in size than those at sites to the rear of the leading edges (which were immunopositive for integrin αV and integrin β3, but not for Necl-5). The signal for talin co-localized with both types of dot-like structure, consistent with earlier observations (6Zaidel-Bar R. Cohen M. Addadi L. Geiger B. Biochem. Soc. Trans. 2004; 32: 416-420Crossref PubMed Scopus (415) Google Scholar). In contrast to these signals, the signal for N-cadherin was not concentrated at any site on the entire plasma membrane (supplemental Fig. S1A). Here we determine that the dot-like structures, which are immunopositive for Necl-5, integrin αV, integrin β3, and talin and locate under the peripheral ruffles, correspond to focal complexes, whereas the dot-like structures, which are immunopositive for integrin αV, integrin β3, and talin, but not for Necl-5, correspond to focal adhesions. These results indicate that Necl-5 co-localizes with integrin αVβ3 both at peripheral ruffles over the lamellipodia and at focal complexes under the peripheral ruffles of leading edges, but not at focal adhesions, in NIH3T3 cells. Integrin αVβ3 at these structures includes at least the high affinity form, because talin co-localizes with it (4Cram E.J. Schwarzbauer J.E. Trends Cell Biol. 2004; 14: 55-57Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar).FIGURE1Co-localizationofNecl-5withintegrinαVβ3at peripheral ruffles and focal complexes.A, immunofluorescence images of PDGF-stimulated NIH3T3 cells cultured on vitronectin-coatedμ-slide dishes. Cells were double- or triple-stained with various combinations of the anti-Necl-5 mAb, the anti-integrinβ3 mAb, and the anti-talin mAb. a, wild-type NIH3T3 cells; b, Necl-5-NIH3T3 cells; c, wild-type NIH3T3 cells co-transfected with siRNA vector and pEGFP-tub; c1, Necl-5-knockdown-NIH3T3 cells; c2, control-NIH3T3 cells. Arrowheads, leading edges; asterisks, siRNA vector-transfected cells; scale bars, 10 μm. Inset boxes show the area of high magnification images. The high magnification images are indicated as intensity ratio images. Analysis of the co-localization of Necl-5 and integrin β3 and generation of the intensity ratio images were performed b