Advertisement
JBC

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M611330200 on April 19, 2007

J. Biol. Chem., Vol. 282, Issue 25, 18481-18496, June 22, 2007
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemental Data
Right arrow All Versions of this Article:
282/25/18481    most recent
M611330200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Minami, Y.
Right arrow Articles by Takai, Y.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Minami, Y.
Right arrow Articles by Takai, Y.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

Necl-5/Poliovirus Receptor Interacts in cis with Integrin {alpha}Vbeta3 and Regulates Its Clustering and Focal Complex Formation*Formula

Yukiko Minami{ddagger}§, Wataru Ikeda{ddagger}, Mihoko Kajita{ddagger}, Tsutomu Fujito{ddagger}§, Hisayuki Amano{ddagger}, Yoshiyuki Tamaru{ddagger}, Kaori Kuramitsu{ddagger}, Yasuhisa Sakamoto{ddagger}, Morito Monden§, and Yoshimi Takai{ddagger}1

From the Departments of {ddagger}Molecular Biology and Biochemistry and §Surgery and Clinical Oncology, Osaka University Graduate School of Medicine/Faculty of Medicine, Suita 565-0871, Osaka, Japan

Received for publication, December 11, 2006 , and in revised form, March 20, 2007.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Integrin {alpha}vbeta3, 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 {alpha}vbeta3 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 {alpha}vbeta3, and enhances integrin {alpha}vbeta3 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 {alpha}vbeta3; however, both regions are necessary for its action. An interaction between integrin {alpha}vbeta3 and vitronectin and PDGF-induced activation of Rac are also necessary for integrin {alpha}vbeta3 clustering. The interaction between Necl-5 and integrin {alpha}vbeta3 enhances PDGF-induced Rac activation, facilitating integrin {alpha}vbeta3 clustering presumably in a feedback amplification manner. Thus, Necl-5 has a critical role in integrin {alpha}vbeta3 clustering and focal complex formation.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Integrins are key molecules for adhesion between cells and extracellular matrix (ECM)2 proteins (1). They are heterodimers of {alpha} and beta 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 {alpha}-actinin, and intracellular signaling molecules, such as FAK and c-Src (1, 2). Integrins have at least two forms: conformations with either low or high affinity for their ECM binding partners (1, 3, 4). When talin binds to the low affinity form, this is converted to the high affinity form (4). 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 (4). 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 (5, 6). 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 (5, 7). 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 (8). Of the many integrins, integrin {alpha}Vbeta3 has a particularly important role in focal complex formation (9). This integrin is expressed in many cell types, such as fibroblasts and vascular endothelial cells (10, 11), and is often up-regulated in many cancer cells, such as colon carcinoma, melanoma, and glioblastoma cells (12-14).

We found that an Ig-like molecule Necl-5/poliovirus receptor (PVR)/CD155/Tage4 co-localizes with integrin {alpha}Vbeta3 at leading edges in moving cells and enhances growth factor-induced cell movement (15). Human PVR/CD155 was originally identified as a poliovirus receptor (16, 17), whereas rodent Tage4 was originally identified as the product of a gene that is overexpressed in rodent colon carcinoma (18). PVR/CD155 is also overexpressed in many human cancer cells, such as colon carcinoma, melanoma, and glioblastoma cells, in which integrin {alpha}Vbeta3 is up-regulated (19-21). This molecule, with four nomenclatures, is also named nectin-like molecule-5, Necl-5 (22). Necl-5 is expressed in many cell types, such as fibroblasts and vascular endothelial cells, in which integrin {alpha}Vbeta3 is expressed (23, 24). 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 (22, 25). When cells come into contact with other cells, Necl-5 is down-regulated from the cell surface by trans-interacting with nectin-3 (26); however, when there is no contact with other cells, Necl-5 is up-regulated at the transcriptional level by growth factor-induced signaling (27). Up-regulation of Necl-5 enhances growth factor-induced cell movement, whereas down-regulation of Necl-5 reduces it (26). 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 (28, 29). We have proposed that Necl-5 has a role, at least in part, in this contact inhibition of cell movement (26). 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 {alpha}Vbeta3-based focal complex formation. We attempt to address these issues here using NIH3T3 cells as a model cell type, which express both integrin {alpha}Vbeta3 and Necl-5.


Figure 1
Figure 1
View larger version (156K):
[in this window]
[in a new window]

  		FIGURE1.
Co-localizationofNecl-5withintegrin{alpha}Vbeta3at 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-integrinbeta3 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 beta3 and generation of the intensity ratio images were performed by ImageJ 1.34S software with RG2B colocalization plugin. B, quantitative analysis of various structures in NIH3T3 and Necl-5-NIH3T3 cells. a, quantification of peripheral ruffles at leading edges. To quantify peripheral ruffles, the signal for F-actin, which is a major component of peripheral ruffles, was observed. Peripheral ruffles at leading edges were measured and classified into two categories: small ruffles (less than 10 µm in width) and large ruffles (10 µm or more in width). The results are the means ± S.E. of the three independent experiments. b, strength of peripheral ruffles at leading edges. To analyze the strength of peripheral ruffles, the maximum intensity of F-actin signal at the peripheral ruffles was measured using ImageJ 1.34S software and averaged (n = 27). *, p < 0.0002. The results are the means ± S.E. of the twenty-seven cells. c, quantification of focal complexes at leading edges. The patterns of the signal for integrinbeta3 at focal complexes of leading edges were measured and classified into three categories: scattered focal complexes (Scattered FX), line-like distributed focal complexes (Line-like FX), and belt-like distributed focal complexes, which were assembled more than two line-like FX (Belt-like FX). The results are the means ± S.E. of the three independent experiments. Immunofluorescence images are the staining of integrinbeta3 and examples of three categories of focal complexes. d, quantification of focal adhesions per cell. The signal for integrin beta3 at focal adhesions was counted and averaged per cell (n = 27). **, p < 0.0001. The results are the means ± S.E. of the 27 cells.

 

   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Construction—pCAGIPuro-FLAG-Necl-5, pCAGIZeo-FLAG-Necl-5-{Delta}CP, and pCAGIZeo-FLAG-Necl-5-{Delta}EC were prepared as described (15, 25). 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 (26). To obtain siRNA-resistant mutants, FLAG-Necl-5R and FLAG-Necl-5-{Delta}CPR 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-{Delta}CP (15, 25) and cloned into pCMV5 vector (pCMV5-FLAG-Necl-5R and pCMV5-FLAG-Necl-5-{Delta}CPR, respectively). Necl-5-{Delta}CP fused to a Myc tag at the C terminus was inserted into the vector pCAGIPuro (pCAGI-Puro-Necl-5-{Delta}CP-myc). Expression vectors were kindly provided as follows: wild-type human integrin {alpha}V and human integrin beta3 (pcDNA3-{alpha}V and -beta3) were from Dr. J. C. Norman (University of Leicester, Leicester, UK), a dominant-negative mutant of human integrin beta3 (pcDNA3.1-Myc-His(+)-beta3T329C/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 (30).


Figure 2
View larger version (65K):
[in this window]
[in a new window]

  		FIGURE 2.
Physical interaction of Necl-5 with integrin {alpha}Vbeta3. A, co-immunoprecipitation of integrin {alpha}Vbeta3 with Necl-5. a and b, HEK293 cells were transfected with various combinations of the indicated vectors and cultured in suspension under the indicated conditions. FLAG-tagged Necl-5, Necl-5-{Delta}CP, or Necl-5-{Delta}EC was immunoprecipitated using the anti-FLAG mAb and samples were assessed by Western blotting using the anti-FLAG mAb, the anti-integrin {alpha}V mAb, the anti-integrin beta3 mAb, the anti-integrin {alpha}5 mAb, and the anti-integrin beta1 mAb. a, integrin {alpha}Vbeta3; b, integrin {alpha}5beta1. c, NIH3T3 cells were cultured on vitronectin-coated dishes, starved of serum, and cultured in the presence or absence of PDGF. Cells were treated with DTSSP and then subjected to the co-immunoprecipitation assay using the anti-integrin {alpha}V pAb. Samples were assessed by Western blotting using the anti-Necl-5 mAb, the anti-integrin {alpha}V mAb, and the anti-integrin beta3 pAb. The results shown are representative of three independent experiments. B, in vitro binding of Necl-5 to integrin {alpha}Vbeta3. Necl-5 EC (60 pmol) or buffer alone was incubated with integrin {alpha}Vbeta3 EC (6 pmol)-immobilized Ni-Sepharose beads or Ni-Sepharose beads alone. After the beads were extensively washed, bound proteins were eluted with Elution buffer. The samples were assessed by Western blotting using the anti-Necl-5 mAb, the anti-integrin {alpha}V mAb, and the anti-integrin beta3 mAb. The results shown are representative of three independent experiments.

 
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-{Delta}CP-NIH3T3 cells were prepared as described (15). For transient expression experiments, cells were transfected with various expression vectors using Lipofectamine 2000 reagent. Knockdown of Necl-5 was performed as described (26). 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 (25). Hamster anti-integrin {alpha}V and beta3 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 {alpha}V, anti-integrin beta3, anti-integrin {alpha}5, and anti-integrin beta1 (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 {alpha}V (for immunoprecipitation), and anti-integrin beta3 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 (31). 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 x 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 (26). 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 {alpha}Vbeta3, 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 {alpha}Vbeta3, 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 x 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.


Figure 3
View larger version (51K):
[in this window]
[in a new window]

  		FIGURE 3.
Co-localization of Necl-5 with integrin {alpha}Vbeta3 at contact sites between vitronectin-coated beads and cells. A, immunofluorescence images of the contact sites between the vitronectin- or ConA-coated beads and NIH3T3 cells. Cells were double- or triple-stained with various combinations of the anti-Necl-5 mAb, the anti-integrinbeta3 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; a1, b1, c1, and c2, vitronectin-coated beads; a2 and b2, ConA-coated beads. N, nuclei; DIC, differential interference contrast; asterisks, positions of the beads; scale bars, 10 µm. The results shown are representative of three independent experiments. B, quantification of the accumulation of Necl-5 and integrin beta3 at the bead-cell contact sites in A. The ordinate indicates the percentage of beads positive for immunofluorescence signals for Necl-5 and integrin beta3. a, wild-type NIH3T3 and Necl-5-NIH3T3 cells; b, Necl-5-knockdown-NIH3T3 and control-NIH3T3 cells. *, p < 0.001. The results shown are the means ± S.E. of three independent experiments.

 
To examine the interaction between endogenous Necl-5 and endogenous integrin {alpha}Vbeta3, a co-immunoprecipitation assay was performed using NIH3T3 cells. Cells were plated at a density of 5 x 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 x 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 {alpha}V 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 {alpha}Vbeta3—Necl-5 EC was prepared as described (25). To obtain integrin {alpha}Vbeta3 EC, CHO-lec 3.2.8.1 [EC] cells expressing integrin {alpha}Vbeta3 EC were cultured, and the culture supernatant containing soluble integrin {alpha}Vbeta3 EC was collected (3). 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 {alpha}Vbeta3 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 {alpha}Vbeta3 EC was determined with BSA as a reference protein by SDS-PAGE.

To examine the binding of Necl-5 EC to integrin {alpha}Vbeta3 EC, integrin {alpha}Vbeta3 EC (6 pmol) was immobilized on Ni-Sepharose beads and soluble Necl-5 EC (60 pmol) was incubated with integrin {alpha}Vbeta3 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 x 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 (32) 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 x 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 (26).

Assay for Rac Activation—The assay for Rac activation using GST-PAK CRIB was performed as described (33). Briefly, NIH3T3, Necl-5-NIH3T3, or Necl-5-{Delta}CP-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.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Co-localization of Necl-5 with Integrin {alpha}Vbeta3 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 {alpha}Vbeta3 (1), 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 beta3 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 (15). In the basal section of the cells, the signal for integrin beta3 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 beta3. In addition, the signal for integrin beta3, 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 {alpha}V was stained instead of integrin beta3.3 Indeed, unless otherwise specified, essentially the same results were obtained for both integrin {alpha}V and integrin beta3 in the experiments that follow. The dot-like structures under the peripheral ruffles (which were immunopositive for Necl-5, integrin {alpha}V, and integrin beta3) were smaller in size than those at sites to the rear of the leading edges (which were immunopositive for integrin {alpha}V and integrin beta3, but not for Necl-5). The signal for talin co-localized with both types of dot-like structure, consistent with earlier observations (6). 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 {alpha}V, integrin beta3, and talin and locate under the peripheral ruffles, correspond to focal complexes, whereas the dot-like structures, which are immunopositive for integrin {alpha}V, integrin beta3, and talin, but not for Necl-5, correspond to focal adhesions. These results indicate that Necl-5 co-localizes with integrin {alpha}Vbeta3 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 {alpha}Vbeta3 at these structures includes at least the high affinity form, because talin co-localizes with it (4).

Regulation by Necl-5 of Integrin {alpha}Vbeta3 Clustering at Peripheral Ruffles and Focal Complexes—We previously showed that the amount of cell surface Necl-5 is regulated by cell density (26). Therefore, we next examined the effect of the amount of cell surface Necl-5 on integrin {alpha}Vbeta3 clustering at peripheral ruffles and focal complexes in NIH3T3 cells. When NIH3T3 cells stably overexpressing Necl-5 (Necl-5-NIH3T3 cells) were cultured on µ-slide dishes as described above and directionally stimulated by PDGF, most cells showed morphologies similar to those of wild-type NIH3T3 cells, except that they formed more marked peripheral ruffles than wild-type cells (Fig. 1, Ab, Ba, and Bb, see also Aa). The immunofluorescence signals for Necl-5, integrin beta3, and talin, but not that for N-cadherin, were highly concentrated at peripheral ruffles and focal complexes (Fig. 1Ab and supplemental Fig. S1B). The number of focal complexes in the basal sections of these cells was increased compared with that in wild-type cells, but their diameters were unchanged (Fig. 1, Ab and Bc, see also Aa). In addition, the immunofluorescence signals for integrin beta3 and talin were markedly decreased at focal adhesions in Necl-5-NIH3T3 cells compared with those in wild-type cells (Fig. 1, Ab and Bd, see also Aa). The signal for Necl-5 was observed as belt-like structures at leading edges and partly overlapped with the signals for integrin beta3 and talin at focal complexes. The Necl-5-positive belt-like structures might be due to the increased number of dot-like structures caused by overexpression of Necl-5. When similar experiments were performed using NIH3T3 cells in which Necl-5 was knocked down (Necl-5-knockdown-NIH3T3 cells) by siRNA vector (about 80-90% decrease of Necl-5; supplemental Fig. S2A), most cells did not form definitely leading edges, but randomly formed lamellipodia at various sites; the signal for Necl-5 was negligible (Fig. 1Ac, 1 and 2). The signal for integrin beta3 was not concentrated at any region. The amounts of cell surface integrin {alpha}V and integrin beta3 were indistinguishable among wild-type NIH3T3, Necl-5-NIH3T3, Necl-5-knockdown-NIH3T3, and control-NIH3T3 cells as estimated by FACS analysis (supplemental Fig. S3, A and B). These results indicate that Necl-5 enhances integrin {alpha}Vbeta3 clustering and formation of lamellipodia, peripheral ruffles, and focal complexes at leading edges in NIH3T3 cells, and suggest that Necl-5 physically interacts with integrin {alpha}Vbeta3.


Figure 4
View larger version (64K):
[in this window]
[in a new window]

  		FIGURE 4.
Regulation of integrin {alpha}Vbeta3 clustering by Necl-5-integrin {alpha}Vbeta3 interaction. A, inhibition of Necl-5-integrin {alpha}Vbeta3 interaction by Necl-5-{Delta}CP. HEK293 cells were transfected with various combinations of the indicated vectors and cultured in suspension in the presence of Ca2+ and Mg2+. FLAG-tagged Necl-5 was immunoprecipitated using the anti-FLAG mAb, and samples were assessed by Western blotting using the anti-FLAG mAb, the anti-integrin {alpha}V mAb, the anti-integrin beta3 mAb, and the anti-Myc mAb. B, immunofluorescence images of PDGF-stimulated Necl-5-{Delta}CP-NIH3T3 cells cultured on vitronectin-coated µ-slide dishes. Cells were double-stained with the anti-FLAG mAb and the anti-integrin beta3 mAb. Scale bars, 10 µm. The results shown are representative of three independent experiments.

 
Physical Interaction of Necl-5 with Integrin {alpha}Vbeta3—We therefore examined the physical interaction between Necl-5 and integrin {alpha}Vbeta3 by three methods: immunoprecipitation, in vitro binding, and bead-cell contact assays. In the immunoprecipitation assay, we prepared low and high affinity forms of integrin {alpha}Vbeta3 to determine with which form Necl-5 interacts. To make predominantly high affinity integrin {alpha}Vbeta3, human integrin {alpha}V, human integrin beta3, and FLAG-Necl-5 were co-expressed in HEK293 cells, and the cells were cultured in suspension in the presence of Mn2+ and cyclo-RGDfV (3). Alternatively, to make predominantly low affinity integrin {alpha}Vbeta3, human integrin {alpha}V, human integrin beta3T329C/A347C, and FLAG-Necl-5 were co-expressed in HEK293 cells, and the cells were cultured in suspension in the presence of Ca2+ and Mg2+. Integrin beta3T329C/A347C is stabilized in the low affinity form and cannot convert to the high affinity form with integrin {alpha}V (34). When FLAG-Necl-5 was immunoprecipitated by an anti-FLAG mAb from cell lysates, both human integrin {alpha}V and human integrin beta3 or human integrin beta3T329C/A347C were co-immunoprecipitated (Fig. 2Aa). These results indicate that Necl-5 has the ability to physically interact with both the low and high affinity forms of integrin {alpha}Vbeta3. The interaction of Necl-5 with integrin {alpha}Vbeta3 was noted as being specific, because integrin {alpha}5beta1 was not co-immunoprecipitated with FLAG-Necl-5 under comparable conditions (Fig. 2Ab).

The interaction of endogenous Necl-5 with endogenous integrin {alpha}Vbeta3 was then confirmed by a co-immunoprecipitation assay using lysates of NIH3T3 cells. NIH3T3 cells were cultured on vitronectin-coated dishes in the presence or absence of PDGF, and then treated with or without a chemical cross-linker DTSSP, which is membrane insoluble and has a disulfide bond. When endogenous integrin {alpha}V was immunoprecipitated from lysates of cells cultured in the presence or absence of PDGF and treated with DTSSP, integrin beta3 and Necl-5 were co-immunoprecipitated with it from both cell lysates irrespective of the presence and absence of PDGF, although the amount of co-immunoprecipitated Necl-5 in cells cultured in the presence of PDGF was slightly less than that in cells cultured in the absence of PDGF (Fig. 2Ac). The amount of integrin beta3 co-immunoprecipitated with integrin {alpha}V was not affected by PDGF stimulation. Integrin beta3, but not Necl-5, was co-immunoprecipitated with integrin {alpha}V from lysates of cells which were not pretreated with DTSSP.3 The exact reason why Necl-5 was not co-immunoprecipitated with integrin {alpha}V in the absence of the cross-linker is currently unknown, but may be due to low amounts of these proteins in NIH3T3 cells and low affinities of these proteins.


Figure 5
View larger version (70K):
[in this window]
[in a new window]

  		FIGURE 5.
Necessity of both the extracellular and cytoplasmic regions of Necl-5 for integrin {alpha}Vbeta3 clustering. Wild-type NIH3T3 cells were co-transfected with siRNA vector and FLAG-Necl-5R; with siRNA vector and FLAG-Necl-5-{Delta}CPR; or with siRNA vector and FLAG-Necl-5-{Delta}EC. Cells were cultured on vitronectin-coated µ-slide dishes in the presence of PDGF, and double-stained with the anti-FLAG mAb and the anti-integrin beta3 mAb. A, FLAG-Necl-5R; B, FLAG-Necl-5-{Delta}CPR; C, FLAG-Necl-5-{Delta}EC. Scale bars, 10 µm. The results shown are representative of three independent experiments.

 
We next examined whether the extracellular and/or cytoplasmic regions of Necl-5 are necessary for the interaction with integrin {alpha}Vbeta3. We expressed human integrin {alpha}V, human integrin beta3, or integrin beta3T329C/A347C and FLAG-Necl-5 with the cytoplasmic or extracellular region deleted (FLAG-Necl-5-{Delta}CP or FLAG-Necl-5-{Delta}EC, respectively), in HEK293 cells, and performed similar experiments to those described above. Both human integrin {alpha}V and human integrin beta3 or integrin beta3T329C/A347C were co-immunoprecipitated with FLAG-Necl-5-{Delta}CP, whereas neither of them was co-immunoprecipitated with FLAG-Necl-5-{Delta}EC (Fig. 2Aa). These results indicate that the extracellular region of Necl-5, but not the cytoplasmic region, is essential for the interaction with both low and high affinity forms of integrin {alpha}Vbeta3.

We then confirmed this result by use of purified recombinant protein samples. We prepared recombinant proteins of the extracellular region of Necl-5 fused to human IgG Fc portion (Necl-5 EC) and a heterodimer containing the extracellular regions of human integrin {alpha}V and human integrin beta3 with a hexa-histidine-tag fused to the integrin beta3 COOH terminus (integrin {alpha}Vbeta3 EC) (3). When Necl-5 EC was applied to a column of integrin {alpha}Vbeta3 EC-immobilized Ni-Sepharose beads, Necl-5 EC bound to integrin {alpha}Vbeta3 EC (Fig. 2B).

We furthermore confirmed the physical interaction between Necl-5 and integrin {alpha}Vbeta3 by the bead-cell contact assay. We first prepared microbeads coated with vitronectin and placed them on the surface of NIH3T3 cells that were starved of serum and sparsely plated on glass coverslips precoated with laminin. Consistently, the immunofluorescence signals for integrin beta3 and talin were concentrated at the bead-cell contact sites (Fig. 3Aa1). The signal for Necl-5 was also concentrated there. These signals were markedly enhanced by overexpression of Necl-5, and reduced by its knockdown (Fig. 3A, b1, c1, and c2). These results were statistically significant (Fig. 3B, a and b). When ConA-coated beads were used as a control, none of these proteins were concentrated at the bead-cell contact sites (Fig. 3A, a2 and b2). These results support the cis-interaction between Necl-5 and integrin {alpha}Vbeta3.


Figure 6
Figure 6
View larger version (134K):
[in this window]
[in a new window]

  		FIGURE 6.
Necessity of binding of integrin{alpha}Vbeta3 to specific ECM proteins for its clustering. A, immunofluorescence images of PDGF-stimulated NIH3T3 cells cultured on fibronectin- or laminin-coated dishes. Cells were triple-stained with the anti-Necl-5 mAb, the anti-integrin beta3 mAb, and the anti-talin mAb. a, fibronectin-coated dishes; b, laminin-coated dishes; 1, wild-type NIH3T3 cells; 2, Necl-5-NIH3T3 cells. White arrowheads, leading edges; red arrowheads, fibrillar adhesions; 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 beta3 and generation of the intensity ratio images were performed by ImageJ 1.34S software with RG2B colocalization plugin. B, immunofluorescence images of the contact sites between fibronectin- or laminin-coated beads and NIH3T3 cells. Cells were triple-stained with the anti-Necl-5 mAb, the anti-integrin beta3 mAb, and the anti-talin mAb. a, fibronectin-coated beads; b, laminin-coated beads; 1, wild-type NIH3T3 cells; 2, Necl-5-NIH3T3 cells. N, nuclei; DIC, differential interference contrast; asterisks, positions of the beads; scale bars, 10 µm. The results shown are representative of three independent experiments. c, quantification of the accumulation of Necl-5 and integrin beta3 at bead-cell contact sites in a and b. The ordinate indicates the percentage of beads positive for immunofluorescence signals for Necl-5 and integrin beta3.*, p < 0.001. The results shown are the means ± S.E. of three independent experiments.

 
Regulation of Integrin {alpha}Vbeta3 Clustering by Necl-5-Integrin {alpha}Vbeta3 Interaction—We then examined whether the interaction between Necl-5 and integrin {alpha}Vbeta3 indeed regulates integrin {alpha}Vbeta3 clustering and the formation of lamellipodia, peripheral ruffles, and focal complexes at leading edges in NIH3T3 cells. For this purpose, we took advantage of a Necl-5 construct in which the cytoplasmic region was deleted (Necl-5-{Delta}CP), because we previously showed that this deletion mutant reduces the stimulatory effect of full-length Necl-5 on movement of L cells stably overexpressing Necl-5 and NIH3T3 and V12-Ki-Ras-NIH3T3 cells (15) and showed above that the extracellular region of Necl-5 interacts in cis (i.e. in the same cell membrane) with the extracellular region of integrin {alpha}Vbeta3. Consistently, Necl-5-{Delta}CP inhibited the interaction between Necl-5 and integrin {alpha}Vbeta3 as detected by the immunoprecipitation assay (Fig. 4A). When Necl-5-{Delta}CP was stably expressed in NIH3T3 cells that were cultured on µ-slide dishes as described above and directionally stimulated by PDGF, most cells did not form leading edges but randomly formed lamellipodia at various sites (Fig. 4B). The immunofluorescence signal for Necl-5-{Delta}CP was diffusely distributed. The signal for integrin beta3 was markedly decreased at focal complexes compared with that in wild-type cells. These results indicate that Necl-5-{Delta}CP shows a dominant-negative effect on intact Necl-5 through the inhibition of the interaction between Necl-5 and integrin {alpha}Vbeta3 and that the interaction between Necl-5 and integrin {alpha}Vbeta3 regulates integrin {alpha}Vbeta3 clustering and formation of lamellipodia, peripheral ruffles, and focal complexes at leading edges in NIH3T3 cells.

Necessity of Both the Extracellular and Cytoplasmic Regions of Necl-5 for Integrin {alpha}Vbeta3 Clustering—We examined whether integrin{alpha}Vbeta3 clustering needs both the extracellular and cytoplasmic regions of Necl-5. For this purpose, various Necl-5 mutants were expressed in Necl-5-knockdown-NIH3T3 cells. When full-length FLAG-Necl-5R, which is resistant to Necl-5 siRNA, was expressed in Necl-5-knockdown-NIH3T3 cells (supplemental Fig. S2B, a and b), and the cells were cultured on µ-slide dishes precoated with vitronectin and directionally stimulated by PDGF, they showed morphologies similar to those of Necl-5-NIH3T3 cells (Fig. 5A, see also Fig. 1Ab). They had a polarized structure with lamellipodia and peripheral ruffles and the immunofluorescence signals for Necl-5 and integrin beta3 were concentrated at peripheral ruffles over the lamellipodia and focal complexes under the peripheral ruffles. When FLAG-Necl-5-{Delta}CPR, which is also resistant to Necl-5 siRNA, or FLAG-Necl-5-{Delta}EC was expressed in Necl-5-knockdown-NIH3T3 cells (supplemental Fig. S2B, a and b), the phenotypes of most Necl-5-NIH3T3 cells were not restored (Fig. 5, B and C). Morphologically, most cells did not form leading edges but randomly formed lamellipodia at various sites. The signals for FLAG-Necl-5-{Delta}CPR and FLAG-Necl-5-{Delta}EC were diffusely distributed. The signal for integrin beta3 was not concentrated at any region, although the intensity of its signal was higher in the cells expressing FLAG-Necl-5-{Delta}CPR than in the cells expressing FLAG-Necl-5-{Delta}EC. These results indicate that both the extracellular and cytoplasmic regions of Necl-5 are necessary for integrin {alpha}Vbeta3 clustering and formation of lamellipodia, peripheral ruffles, and focal complexes at leading edges in NIH3T3 cells.

Necessity of Binding of Integrin {alpha}Vbeta3 to Specific ECM Proteins for Necl-5-enhanced Integrin {alpha}Vbeta3 Clustering—It has been shown that the clustering of integrin requires its binding to ECM proteins and that integrin {alpha}Vbeta3 binds both vitronectin and fibronectin, but does not bind laminin (1). We examined whether the binding of integrin {alpha}Vbeta3 to its ECM proteins is necessary for PDGF-induced, Necl-5-enhanced integrin {alpha}Vbeta3 clustering and formation of lamellipodia, peripheral ruffles, and focal complexes at leading edges in NIH3T3 cells. When NIH3T3 cells were sparsely plated on µ-slide dishes precoated with fibronectin, starved of serum, and directionally stimulated by PDGF, essentially the same results were obtained as when dishes were precoated with vitronectin, in regards to the immunofluorescence signals for Necl-5, integrin beta3, and talin at leading edges (Fig. 6Aa1, see also Fig. 1Aa). However, the signal for integrin beta3 was not observed at focal adhesions at sites to the rear of the leading edges, where the signal for integrin beta3 was concentrated when cells were cultured on vitronectin. Instead of focal adhesions, the signal for talin was observed at fibrillar adhesions, which were formed by fibrillogenesis of fibronectin (Fig. 6Aa1). Fibrillar adhesions might be formed by other fibronectin receptors, such as integrin {alpha}5beta1, which is expressed in NIH3T3 cells (1, 35). When similar experiments were performed using Necl-5-NIH3T3 cells, essentially the same results as those obtained for vitronectin were obtained except that the signal for talin was observed at fibrillar adhesions (Fig. 6Aa2, see also Fig. 1Ab). In contrast, when similar experiments were performed using NIH3T3 or Necl-5-NIH3T3 cells that were cultured on dishes precoated with laminin instead of vitronectin, most cells spread but did not form leading edges in response to PDGF (Fig. 6A, b1 and b2). The signal for Necl-5 was diffusely observed on the plasma membrane and the signals for integrin beta3 and talin were negligible at peripheral regions. However, the signal for talin was weakly observed at fibrillar adhesion-like structures. Laminin receptor, integrin {alpha}6beta1, is expressed in NIH3T3 cells, and this result is consistent with earlier observations (36, 37).

Essentially the same results were obtained in the bead-cell contact assay. When fibronectin-coated beads were placed on the surface of NIH3T3 cells that were starved of serum and sparsely plated on the dishes precoated with laminin, the signals, although faint, for Necl-5, integrin beta3, and talin were concentrated at the bead-cell contact sites (Fig. 6Ba1). Furthermore, the concentration of these signals was enhanced at the bead-cell contact sites when Necl-5-NIH3T3 cells were used (Fig. 6Ba2). These results were statistically significant (Fig. 6Bc). In contrast, when laminin-coated beads were used instead of vitronectin-coated ones, the signal for Necl-5, integrin beta3, or talin was not concentrated at the contact sites between the beads and NIH3T3 or Necl-5-NIH3T3 cells (Fig. 6B, b1, b2, and c). Taken together, these results indicate that the binding of integrin {alpha}Vbeta3 to its ECM proteins is necessary for Necl-5-enhanced integrin {alpha}Vbeta3 clustering and formation of lamellipodia, peripheral ruffles, and focal complexes at leading edges in NIH3T3 cells.

Necessity of PDGF Stimulation for Necl-5-enhanced Integrin {alpha}Vbeta3 Clustering—It has been shown that cell movement requires the synergistic activity of PDGF- and integrin-induced signaling (10). We therefore examined the effect of PDGF on Necl-5-enhanced integrin {alpha}Vbeta3 clustering and formation of lamellipodia, peripheral ruffles, and focal complexes at leading edges in NIH3T3 cells. When NIH3T3 cells were cultured on µ-slide dishes precoated with vitronectin and not stimulated by PDGF, most cells failed to form leading edges (Fig. 7A). The immunofluorescence signals for Necl-5, integrin beta3, and talin were negligible at peripheral regions. However, the large dot-like signals for integrin beta3 and talin were concentrated at focal adhesions and the number of focal adhesions was increased compared with those observed in the presence of PDGF (see also Fig. 1Aa). When similar experiments were performed using Necl-5-NIH3T3 cells, most cells did not form leading edges (Fig. 7B). The signal for Necl-5 was diffusely observed on the plasma membrane but the signals for integrin beta3 and talin were again negligible at peripheral regions. However, the signals for integrin beta3 and talin were observed at focal adhesions, although they were not observed in Necl-5-NIH3T3 cells stimulated by PDGF (see also Fig. 1Ab). When similar experiments were performed using Necl-5-knockdown-NIH3T3 cells, most cells did not form leading edges (Fig. 7C). The signal for Necl-5 was essentially nil. The signal for integrin beta3 was not concentrated anywhere, and the phenotype was different to those observed in NIH3T3 and Necl-5-NIH3T3 cells (see also Fig. 7, A and B). These results indicate that PDGF stimulation is necessary for Necl-5-enhanced integrin {alpha}Vbeta3 clustering and formation of lamellipodia, peripheral ruffles, and focal complexes at leading edges in NIH3T3 cells.

Involvement of Rac Activation in PDGF-induced, Necl-5-enhanced Integrin {alpha}Vbeta3 Clustering—It has been shown that Rac, which is activated by the action of PDGF, regulates these morphological changes (6, 8, 38). We previously showed that overexpression of Necl-5 enhances the serum-induced activation of Rac in an integrin {alpha}Vbeta3-dependent manner in L fibroblasts (15). We therefore examined whether Necl-5-integrin {alpha}Vbeta3 interaction enhances PDGF-induced Rac activation in NIH3T3 cells. PDGF-induced Rac activation was estimated by a pull-down assay. This form of Rac activation was enhanced and sustained by overexpression of Necl-5 and reduced by its knockdown (about 70% decrease of Necl-5) (Fig. 8A, a and b). It was also reduced by overexpression of Necl-5-{Delta}CP (Fig. 8Aa). These results indicate that Necl-5-integrin {alpha}Vbeta3 interaction enhances PDGF-induced Rac activation in NIH3T3 cells.

We then examined whether Rac is necessary for PDGF-induced, Necl-5-enhanced integrin {alpha}Vbeta3 clustering in NIH3T3 cells. Transient expression of a dominant-negative mutant of Rac1 (Rac1DN) in NIH3T3 cells or Necl-5-NIH3T3 cells cultured on µ-slide dishes precoated with vitronectin and directionally stimulated by PDGF, inhibited the formation of lamellipodia and ruffles at the peripheral regions of both cell lines (Fig. 8B, a and b). The immunofluorescence signals for Necl-5, integrin beta3, and Rac1DN were not concentrated at peripheral regions, but were concentrated at currently unknown structures on the plasma membrane and inside the cells. These results indicate that Rac is necessary for PDGF-induced, Necl-5-enhanced integrin {alpha}Vbeta3 clustering and formation of lamellipodia, peripheral ruffles, and focal complexes at leading edges in NIH3T3 cells.


Figure 7
View larger version (78K):
[in this window]
[in a new window]

  		FIGURE 7.
Necessity of PDGF stimulation for Necl-5-induced integrin {alpha}Vbeta3 clustering. A-C, immunofluorescence images of NIH3T3 cells cultured on vitronectin-coated µ-slide dishes in the absence of PDGF. Cells were double- or triple-stained with various combinations of the anti-Necl-5 mAb, the anti-integrin beta3 mAb, and the anti-talin mAb. A, wild-type NIH3T3 cells; B, Necl-5-NIH3T3 cells; C, Necl-5-knockdown-NIH3T3 cells. Asterisks, siRNA vector-transfected cells; scale bars, 10 µm. The results shown are representative of three independent experiments.

 
Necessity of Both Rac Activation and Necl-5-Integrin {alpha}Vbeta3 Interaction for PDGF-induced Integrin {alpha}Vbeta3 Clustering—We next examined whether Rac activation alone is sufficient for integrin {alpha}Vbeta3 clustering and focal complex formation in NIH3T3 cells. For this purpose, we expressed a constitutively active mutant of Rac1 (Rac1CA) in the absence of PDGF stimulation. When Rac1CA was transiently expressed in NIH3T3 cells cultured on µ-slide dishes precoated with vitronectin in the absence of PDGF, most cells showed round structures with lamellipodia and peripheral ruffles over all peripheral regions (Fig. 9Aa). The immunofluorescence signals for Necl-5, integrin beta3, and Rac1CA were concentrated at peripheral ruffles and focal complexes. In addition, the signal for integrin beta3, but not that for Necl-5 or Rac1CA, was concentrated at focal adhesions. The number of focal complexes was markedly increased by Rac1CA compared with that in wild-type cells in the absence of PDGF (see also Fig, 7A). The reason why lamellipodia, peripheral ruffles, and focal complexes were formed randomly over all peripheral regions in the cells expressing Rac1CA might be due to the random localization of exogenously expressed Rac1CA to the plasma membrane. When NIH3T3 cells overexpressing Rac1CA were cultured on µ-slide dishes precoated with laminin instead of vitronectin, the cells showed morphologies different from those observed in the wild-type cells cultured on vitronectin, and formed lamellipodia unevenly along the plasma membrane but did not show round shapes (Fig. 9Ab). In these cells, the immunofluorescence signal for Necl-5 or integrin beta3 was not concentrated at any region.

Necl-5-NIH3T3 cells overexpressing Rac1CA showed morphologies and staining patterns of Necl-5, integrin beta3, and Rac1CA similar to those of wild-type cells, except that they formed more marked peripheral ruffles and showed more marked staining of these markers than wild-type cells (Fig. 9Ba, see also Aa). When Necl-5-NIH3T3 cells overexpressing Rac1CA were cultured on µ-slide dishes precoated with laminin instead of vitronectin, the cells showed morphologies and staining patterns of Necl-5, integrin beta3, and Rac1CA similar to those of wild-type cells cultured on laminin (Fig. 9Bb, see also Ab). When NIH3T3 or Necl-5-NIH3T3 cells overexpressing Rac1CA were cultured on µ-slide dishes precoated with vitronectin and directionally stimulated by PDGF, PDGF showed no additional effect.3 In contrast to wild-type and Necl-5-NIH3T3 cells, Necl-5-knockdown-NIH3T3 cells overexpressing Rac1CA showed morphologies different from those observed in wild-type cells: they formed lamellipodia unevenly along the plasma membrane, but did not show round shapes (Fig. 9C, see also Aa). The signal for Necl-5 was negligible. The signal for integrin beta3 was not concentrated at any region, similar to the observation of Necl-5-knockdown-NIH3T3 cells in the absence of PDGF (see also Fig. 7C). Necl-5-{Delta}CP-NIH3T3 cells overexpressing Rac1CA showed phenotypes similar to those of Necl-5-knockdown-NIH3T3 cells overexpressing Rac1CA (Fig. 9D). Taken together, these results indicate that Rac activation alone or Necl-5-integrin {alpha}Vbeta3 interaction alone is not sufficient for integrin {alpha}Vbeta3 clustering and formation of lamellipodia, peripheral ruffles, and focal complexes in NIH3T3 cells. Rather, Rac activation, Necl-5-integrin {alpha}Vbeta3 interaction, and integrin {alpha}Vbeta3-vitronectin interaction are all necessary for integrin {alpha}Vbeta3 clustering and morphological changes; however, Rac activation and integrin {alpha}Vbeta3-vitronectin interaction are sufficient, without Necl-5-integrin {alpha}Vbeta3 interaction, for lamellipodium formation.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
We first showed here that Necl-5 and integrin {alpha}Vbeta3 localized at leading edges in moving NIH3T3 cells predominantly where lamellipodia and peripheral ruffles were formed, and that they co-localized at peripheral ruffles and focal complexes that were formed over and under lamellipodia, respectively. We showed that Necl-5 did not localize at focal adhesions where integrin {alpha}Vbeta3 also localized. The immunofluorescence signal for Necl-5 was observed as fuzzy dot-like structures, which mostly, but not perfectly, overlapped with the dot-like signal for integrin{alpha}Vbeta3 at focal complexes. Because Necl-5 did not localize at focal adhesions and it was reported that focal complexes are transformed into focal adhesions by inactivation of Rac and Cdc42 and activation of RhoA (5, 7), Necl-5 might dissociate from integrin {alpha}Vbeta3 during this transformation, causing the formation of the fuzzy dot-like structures. The mechanism of dissociation of Necl-5 from integrin {alpha}Vbeta3 at focal complexes remains unknown.


Figure 8
View larger version (75K):
[in this window]
[in a new window]

  		FIGURE 8.
Involvement of Rac activation in PDGF-induced, Necl-5-enhanced integrin {alpha}Vbeta3 clustering. A, effect of Necl-5 on PDGF-induced Rac activation. Cells were cultured on vitronectin-coated dishes, starved of serum, stimulated with PDGF, and then subjected to the assay for Rac activation. a, wild-type NIH3T3, Necl-5-NIH3T3, and Necl-5-{Delta}CP-NIH3T3 cells. b, control- and Necl-5-knockdown-NIH3T3 cells. Wild-type NIH3T3 cells were transfected with siRNA oligo. Expression level of endogenous Necl-5 in Necl-5-knockdown-NIH3T3 cells was less than 30% of that in control-NIH3T3 cells. B, immunofluorescence images of PDGF-stimulated NIH3T3 cells expressing Rac1DN, cultured on vitronectin-coated µ-slide dishes. Cells were transfected with Myc-tagged Rac1DN and triple-stained with the anti-Necl-5 mAb, the anti-integrin beta3 mAb, and the anti-Myc mAb. a, wild-type NIH3T3 cells; b, Necl-5-NIH3T3 cells. Scale bars, 10 µm. The results shown are representative of three independent experiments.

 
We also showed that Necl-5 enhanced integrin {alpha}Vbeta3 clustering and formation of lamellipodia, peripheral ruffles, and focal complexes at leading edges in NIH3T3 cells. Studies on the mode of action of Necl-5 revealed that Necl-5 directly interacted with the extracellular regions of integrin {alpha}Vbeta3 to form a binary complex. There are theoretically cis- and trans-interactions between Necl-5 and integrin {alpha}Vbeta3; however, the results of the immunoprecipitation assay using suspended HEK293 cells and the bead-cell contact assay support an interaction in cis. The result that this interaction was not affected by EDTA, which would inhibit the interaction between integrin {alpha}Vbeta3 and a typical ligand, as judged by the co-immunoprecipitation assay using suspended HEK293 cells,3 also supports this conclusion. Necl-5 interacted with both low and high affinity forms of integrin {alpha}Vbeta3. Neither PDGF nor vitronectin were necessary for the Necl-5-integrin {alpha}Vbeta3 interaction as judged by the co-immunoprecipitation assay using suspended HEK293 cells.3 In general, the low affinity integrin does not bind talin and barely clusters, whereas the high affinity form binds talin and interacts with its specific ECM proteins, resulting in the clustering of the integrin owing to both outside-in and inside-out signals. Therefore, the low affinity form of integrin {alpha}Vbeta3, which interacts with Necl-5, might be diffusely present along the plasma membrane, excluding focal adhesions. By contrast, most integrin {alpha}Vbeta3 in clusters is likely to be the high affinity form that interacts with Necl-5 at focal complexes, but not at focal adhesions.

It was previously shown that cell movement requires the synergistic activity of PDGF- and integrin-induced signaling (10). It was also shown that the PDGF receptor and integrin {alpha}Vbeta3 interact with each other, and that PDGF-induced signaling requires integrin {alpha}Vbeta3 activation by binding to its ECM proteins (10). The binding of talin to integrin increases the affinity of integrin for its specific ECM protein, and the binding of integrin to its specific ECM protein transduces signals inside cells leading to the reorganization of the actin cytoskeleton as necessary for integrin clustering (1, 4). These inside-out and outside-in signaling pathways from the growth factor receptor and integrin bring about the formation of focal complexes and focal adhesions. Consistently, we confirmed that both the binding of PDGF to its receptor and the binding of integrin {alpha}Vbeta3 to its ECM proteins, vitronectin and fibronectin, were necessary for integrin {alpha}Vbeta3 clustering and the formation of lamellipodia, peripheral ruffles, and focal complexes at leading edges in NIH3T3 cells. We also showed that Necl-5-integrin {alpha}Vbeta3 interaction is additionally necessary for integrin {alpha}Vbeta3 clustering and morphological changes. Thus, three transmembrane proteins, the PDGF receptor, integrin {alpha}Vbeta3, and Necl-5, physically and functionally associate to regulate integrin {alpha}Vbeta3 clustering and formation of lamellipodia, peripheral ruffles, and focal complexes at leading edges in NIH3T3 cells. We further showed that the cytoplasmic region of Necl-5 was not necessary for Necl-5-integrin {alpha}Vbeta3 interaction, but that both the extracellular and cytoplasmic regions of Necl-5 were necessary for integrin {alpha}Vbeta3 clustering and formation of these structures.


Figure 9
View larger version (73K):
[in this window]
[in a new window]

  		FIGURE 9.
Necessity of both Rac activation and Necl-5-integrin {alpha}Vbeta3 interaction for PDGF-induced integrin {alpha}Vbeta3 clustering. Cells were transfected with Myc-tagged Rac1CA and/or siRNA vector, cultured on vitronectin- or laminin-coated µ-slide dishes in the absence of PDGF, and triple-stained with the anti-Necl-5 mAb, the anti-integrin beta3 mAb, and the anti-Myc mAb. A, wild-type NIH3T3 cells; B, Necl-5-NIH3T3 cells; C, Necl-5-knockdown-NIH3T3 cells; D, Necl-5-{Delta}CP-NIH3T3 cells; Aa, Ba, C, and D, cells cultured on vitronectin; Ab and Bb, cells cultured on laminin. Insets, high magnification of peripheral regions; asterisks, siRNA vector-transfected cells; scale bars, 10 µm. The results shown are representative of three independent experiments.

 
Rac was previously shown to have a critical role in the dynamic reorganization of the actin cytoskeleton necessary for the formation of focal complexes (6, 8, 38). We previously showed that Necl-5 enhances the serum-induced, integrin {alpha}Vbeta3-dependent activation of Rac (15). Consistently, we showed here that PDGF-induced Rac activation was necessary for PDGF-induced integrin {alpha}Vbeta3 clustering and morphological changes in NIH3T3 cells. Rac activation alone or Necl-5-integrin {alpha}Vbeta3 interaction alone was insufficient to induce these events; indeed, Rac activation, Necl-5-integrin {alpha}Vbeta3 interaction, and integrin {alpha}Vbeta3-vitronectin interaction were all necessary for integrin {alpha}Vbeta3 clustering and changes in morphology, although the Rac activation and integrin {alpha}Vbeta3-vitronectin interaction were sufficient, without Necl-5-integrin {alpha}Vbeta3 interaction, for lamellipodium formation. We noted that these structures were randomly formed at peripheral regions when Rac1CA was expressed instead of being directionally stimulated by PDGF. These results indicate that PDGF-induced Rac activation is necessary for cell polarization and specific localization of lamellipodia, peripheral ruffles, and focal complexes in NIH3T3 cells.

Considering all the evidence thus far available, we propose the following mechanism of integrin {alpha}Vbeta3 clustering and formation of lamellipodia, peripheral ruffles, and focal complexes at leading edges in NIH3T3 cells: PDGF reaches and binds to its receptor and induces Rac activation; Rac activated in this way induces reorganization of the actin cytoskeleton, which induces clustering of the Necl-5-integrin {alpha}Vbeta3 complex; the clustered complex then enhances PDGF-induced Rac activation in a positive feedback manner. Such a feedback amplification mechanism might efficiently form leading edges in the direction of higher concentrations of PDGF. We used here only NIH3T3 cells as a model cell, but both Necl-5 and integrin {alpha}Vbeta3 are expressed in the same types of cells, such as other fibroblasts, vascular endothelial cells, colon carcinoma cells, melanoma cells, and glioblastoma cells, this mechanism may be applied to these cell types. Further studies are necessary for generalization of this mechanism.

The exact mechanism of how Necl-5 enhances integrin {alpha}Vbeta3 clustering remains unknown, but one possible explanation is that the Necl-5-integrin {alpha}Vbeta3 interaction induces a conformational change in the cytoplasmic region of integrin {alpha}Vbeta3, making it sensitive to binding talin, which converts the low affinity integrin {alpha}Vbeta3 to the high affinity form. This process and/or the subsequent clustering process might be facilitated by Rac. Another possible explanation is that the cytoplasmic region of Necl-5 makes talin sensitive to binding integrin {alpha}Vbeta3 by an unknown mechanism. It remains unknown how the clustered Necl-5-integrin {alpha}Vbeta3 complex enhances PDGF-induced Rac activation, but signals downstream of the complex might enhance the signaling pathway of the PDGF receptor at a step upstream of Rac. Further studies are necessary to elucidate these unresolved issues.


   FOOTNOTES
 
* This work was supported by grants-in-aid for Scientific Research and for Cancer Research from the Ministry of Education, Culture, Sports, Science, and Technology, Japan (2005, 2006). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

Formula The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1-S3. Back

1 To whom correspondence should be addressed: Dept. of Molecular Biology and Biochemistry, Osaka University Graduate School of Medicine/Faculty of Medicine, Suita, Osaka 565-0871, Japan. Tel.: 81-6-6879-3410; Fax: 81-6-6879-3419; E-mail: ytakai{at}molbio.med.osaka-u.ac.jp.

2 The 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. Back

3 Y. Takai, unpublished data. Back


   ACKNOWLEDGMENTS
 
We thank Dr. M. Takeichi (RIKEN Center for Developmental Biology, Kobe, Japan) for his critical reading of the manuscript, Dr. J. Takagi for his helpful discussion and his generous gifts of reagents, and Drs. J. C. Norman, H. Shibuya, and S. Narumiya for their generous gifts of reagents.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. van der Flier, A., and Sonnenberg, A. (2001) Cell Tissue Res. 305, 285-298[CrossRef][Medline] [Order article via Infotrieve]
  2. Zamir, E., and Geiger, B. (2001) J. Cell Sci. 114, 3577-3579[Medline] [Order article via Infotrieve]
  3. Takagi, J., Petre, B. M., Walz, T., and Springer, T. A. (2002) Cell 110, 599-611[CrossRef][Medline] [Order article via Infotrieve]
  4. Cram, E. J., and Schwarzbauer, J. E. (2004) Trends Cell Biol. 14, 55-57[CrossRef][Medline] [Order article via Infotrieve]
  5. Rottner, K., Hall, A., and Small, J. V. (1999) Curr. Biol. 9, 640-648[CrossRef][Medline] [Order article via Infotrieve]
  6. Zaidel-Bar, R., Cohen, M., Addadi, L., and Geiger, B. (2004) Biochem. Soc. Trans. 32, 416-420[CrossRef][Medline] [Order article via Infotrieve]
  7. Ballestrem, C., Hinz, B., Imhof, B. A., and Wehrle-Haller, B. (2001) J. Cell Biol. 155, 1319-1332[Abstract/Free Full Text]
  8. Hall, A. (1998) Science 279, 509-514[Abstract/Free Full Text]
  9. Guo, W., and Giancotti, F. G. (2004) Nat. Rev. Mol. Cell Biol. 5, 816-826[CrossRef][Medline] [Order article via Infotrieve]
  10. Woodard, A. S., Garcia-Cardena, G., Leong, M., Madri, J. A., Sessa, W. C., and Languino, L. R. (1998) J. Cell Sci. 111, 469-478[Abstract]
  11. Brooks, P. C., Clark, R. A., and Cheresh, D. A. (1994) Science 264, 569-571[Abstract/Free Full Text]
  12. Chiang, H. S., Peng, H. C., and Huang, T. F. (1994) Biochim. Biophys. Acta. 1224, 506-516[Medline] [Order article via Infotrieve]
  13. Gehlsen, K. R., Davis, G. E., and Sriramarao, P. (1992) Clin. Exp. Metastasis. 10, 111-120[Medline] [Order article via Infotrieve]
  14. Chatterjee, S., Matsumura, A., Schradermeier, J., and Gillespie, G. Y. (2000) J. Neurooncol. 46, 135-144[CrossRef][Medline] [Order article via Infotrieve]
  15. Ikeda, W., Kakunaga, S., Takekuni, K., Shingai, T., Satoh, K., Morimoto, K., Takeuchi, M., Imai, T., and Takai, Y. (2004) J. Biol. Chem. 279, 18015-18025[Abstract/Free Full Text]
  16. Mendelsohn, C. L., Wimmer, E., and Racaniello, V. R. (1989) Cell 56, 855-865[CrossRef][Medline] [Order article via Infotrieve]
  17. Koike, S., Horie, H., Ise, I., Okitsu, A., Yoshida, M., Iizuka, N., Takeuchi, K., Takegami, T., and Nomoto, A. (1990) EMBO J. 9, 3217-3224[Medline] [Order article via Infotrieve]
  18. Chadeneau, C., LeMoullac, B., and Denis, M. G. (1994) J. Biol. Chem. 269, 15601-15605[Abstract/Free Full Text]
  19. Masson, D., Jarry, A., Baury, B., Blanchardie, P., Laboisse, C., Lustenberger, P., and Denis, M. G. (2001) Gut 49, 236-240[Abstract/Free Full Text]
  20. Gromeier, M., Lachmann, S., Rosenfeld, M. R., Gutin, P. H., and Wimmer, E. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 6803-6808[Abstract/Free Full Text]
  21. Bottino, 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., and Moretta, A. (2003) J. Exp. Med. 198, 557-567[Abstract/Free Full Text]
  22. Takai, Y., Irie, K., Shimizu, K., Sakisaka, T., and Ikeda, W. (2003) Cancer Sci. 94, 655-667[CrossRef][Medline] [Order article via Infotrieve]
  23. Kakunaga, S., Ikeda, W., Shingai, T., Fujito, T., Yamada, A., Minami, Y., Imai, T., and Takai, Y. (2004) J. Biol. Chem. 279, 36419-36425[Abstract/Free Full Text]
  24. Reymond, N., Imbert, A. M., Devilard, E., Fabre, S., Chabannon, C., Xerri, L., Farnarier, C., Cantoni, C., Bottino, C., Moretta, A., Dubreuil, P., and Lopez, M. (2004) J. Exp. Med. 199, 1331-1341[Abstract/Free Full Text]
  25. Ikeda, W., Kakunaga, S., Itoh, S., Shingai, T., Takekuni, K., Satoh, K., Inoue, Y., Hamaguchi, A., Morimoto, K., Takeuchi, M., Imai, T., and Takai, Y. (2003) J. Biol. Chem. 278, 28167-28172[Abstract/Free Full Text]
  26. Fujito, T., Ikeda, W., Kakunaga, S., Minami, Y., Kajita, M., Sakamoto, Y., Monden, M., and Takai, Y. (2005) J. Cell Biol. 171, 165-173[Abstract/Free Full Text]
  27. Hirota, T., Irie, K., Okamoto, R., Ikeda, W., and Takai, Y. (2005) Oncogene 24, 2229-2235[CrossRef][Medline] [Order article via Infotrieve]
  28. Abercrombie, M., and Heaysman, J. E. (1953) Exp. Cell Res. 5, 111-131[Medline] [Order article via Infotrieve]
  29. Abercrombie, M. (1979) Nature 281, 259-262[CrossRef][Medline] [Order article via Infotrieve]
  30. Komuro, R., Sasaki, T., Takaishi, K., Orita, S., and Takai, Y. (1996) Genes Cells 1, 943-951[Abstract]
  31. Yatohgo, T., Izumi, M., Kashiwagi, H., and Hayashi, M. (1988) Cell Struct. Funct. 13, 281-292[CrossRef][Medline] [Order article via Infotrieve]
  32. Tran, H., Pankov, R., Tran, S. D., Hampton, B., Burgess, W. H., and Yamada, K. M. (2002) J. Cell Sci. 115, 2031-2040[Abstract/Free Full Text]
  33. Kawakatsu, T., Shimizu, K., Honda, T., Fukuhara, T., Hoshino, T., and Takai, Y. (2002) J. Biol. Chem. 277, 50749-50755[Abstract/Free Full Text]
  34. Luo, B. H., Takagi, J., and Springer, T. A. (2004) J. Biol. Chem. 279, 10215-10221[Abstract/Free Full Text]
  35. Cukierman, E., Pankov, R., Stevens, D. R., and Yamada, K. M. (2001) Science 294, 1708-1712[Abstract/Free Full Text]
  36. Lammerding, J., Kazarov, A. R., Huang, H., Lee, R. T., and Hemler, M. E. (2003) Proc. Natl. Acad. Sci. U. S. A. 100, 7616-7621[Abstract/Free Full Text]
  37. Dogic, D., Rousselle, P., and Aumailley, M. (1998) J. Cell Sci. 111, 793-802[Abstract]
  38. Geiger, B., and Bershadsky, A. (2001) Curr. Opin. Cell Biol. 13, 584-592[CrossRef][Medline] [Order article via Infotrieve]

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 J. Cell Sci.Home page
N. Kanzaki, H. Ogita, H. Komura, M. Ozaki, Y. Sakamoto, T. Majima, T. Ijuin, T. Takenawa, and Y. Takai
Involvement of the nectin-afadin complex in PDGF-induced cell survival
J. Cell Sci., June 15, 2008; 121(12): 2008 - 2017.
[Abstract] [Full Text] [PDF]

Home page
 GENES CELLSHome page
M. Takahashi, Y. Rikitake, Y. Nagamatsu, T. Hara, W. Ikeda, K.-i. Hirata, and Y. Takai
Sequential activation of Rap1 and Rac1 small G proteins by PDGF locally at leading edges of NIH3T3 cells.
Genes Cells, June 1, 2008; 13(6): 549 - 569.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
Y. Nagamatsu, Y. Rikitake, M. Takahashi, Y. Deki, W. Ikeda, K.-i. Hirata, and Y. Takai
Roles of Necl-5/Poliovirus Receptor and Rho-associated Kinase (ROCK) in the Regulation of Transformation of Integrin {alpha}V{beta}3-based Focal Complexes into Focal Adhesions
J. Biol. Chem., May 23, 2008; 283(21): 14532 - 14541.
[Abstract] [Full Text] [PDF]

Home page
 GENES CELLSHome page
H. Amano, W. Ikeda, S. Kawano, M. Kajita, Y. Tamaru, N. Inoue, Y. Minami, A. Yamada, and Y. Takai
Interaction and localization of Necl-5 and PDGF receptor beta at the leading edges of moving NIH3T3 cells: Implications for directional cell movement.
Genes Cells, March 1, 2008; 13(3): 269 - 284.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
Y. Sakamoto, H. Ogita, H. Komura, and Y. Takai
Involvement of Nectin in Inactivation of Integrin {alpha}v 3 after the Establishment of Cell-Cell Adhesion
J. Biol. Chem., January 4, 2008; 283(1): 496 - 505.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
S. Nakata, N. Fujita, Y. Kitagawa, R. Okamoto, H. Ogita, and Y. Takai
Regulation of Platelet-derived Growth Factor Receptor Activation by Afadin through SHP-2: IMPLICATIONS FOR CELLULAR MORPHOLOGY
J. Biol. Chem., December 28, 2007; 282(52): 37815 - 37825.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemental Data
Right arrow All Versions of this Article:
282/25/18481    most recent
M611330200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Minami, Y.
Right arrow Articles by Takai, Y.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Minami, Y.
Right arrow Articles by Takai, Y.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
 All ASBMB Journals   Molecular and Cellular Proteomics 
 Journal of Lipid Research   ASBMB Today 
Copyright © 2007 by the American Society for Biochemistry and Molecular Biology.
Advertisement
spacer
Advertisement
Advertisement