JBC INTERFERin siRNA transfection reagent

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


     

Originally published In Press as doi:10.1074/jbc.M414447200 on April 27, 2005

J. Biol. Chem., Vol. 280, Issue 25, 24095-24103, June 24, 2005
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemental Data
Right arrow All Versions of this Article:
280/25/24095    most recent
M414447200v1
Right arrow Alert me when this article is cited
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 arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Hoshino, T.
Right arrow Articles by Takai, Y.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Hoshino, T.
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?

Regulation of E-cadherin Endocytosis by Nectin through Afadin, Rap1, and p120ctn*

Takashi Hoshino, Toshiaki Sakisaka{ddagger}, Takeshi Baba, Tomohiro Yamada, Toshihiro Kimura, and Yoshimi Takai§

From the Department of Molecular Biology and Biochemistry, Osaka University Graduate School of Medicine/Faculty of Medicine, Osaka 565-0871, Japan

Received for publication, December 22, 2004 , and in revised form, April 19, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Adherens junctions (AJs) are a major cell-cell adhesion structure in epithelial cells that are formed by two major cell-cell adhesion molecules, E-cadherin and nectin. We have previously shown that nectin first forms cell-cell adhesion and then recruits non-trans-interacting E-cadherin to the nectin-based cell-cell adhesion sites, which gradually trans-interact there, eventually forming AJs. We have examined here the effect of trans-interacting nectin on non-trans-interacting E-cadherin endocytosis. Trans-interacting nectin capable of associating with afadin, but not trans-interacting nectin mutant incapable of associating with afadin, inhibited non-trans-interacting E-cadherin endocytosis in intact cells. Afadin is a nectin- and actin filament-binding protein that connects nectin to the actin cytoskeleton. Studies on the mode of action of the nectin-afadin system using cell-free assay revealed that afadin associated with nectin bound Rap1 activated by trans-interacting nectin, interacted with p120ctn, and strengthened the binding of p120ctn to E-cadherin, eventually reducing non-trans-interacting E-cadherin endocytosis. Afadin, which did not bind Rap1, was inactive in this capacity. These results indicate that trans-interacting nectin inhibits non-trans-interacting E-cadherin endocytosis through afadin, Rap1, and p120ctn and thereby further accumulates non-trans-interacting E-cadherin to the nectin-based cell-cell adhesion sites for the formation of AJs.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Adherens junctions (AJs)1 are the principal mediators of cell-cell adhesion in epithelial cells and highly dynamic structures that turn over rapidly. E-cadherin is the major component of AJs in epithelial cells (1-4). E-cadherin first forms cis-dimers on the cell surface of the same cells, followed by formation of trans-dimers (trans-interactions) on the cell surface of two neighboring cells, through the extracellular region in a Ca2+-dependent manner, eventually causing cell-cell adhesion (5-7). The cytoplasmic region is linked to the actin cytoskeleton through {alpha}- and {beta}-catenins (8), which strengthen the cell-cell adhesion activity of E-cadherin (4, 8, 9). p120ctn directly binds to the juxtamembrane region of E-cadherin (10, 11). The definitive role of p120ctn remains unknown, but several lines of evidence for its role have been reported: Mutations of the p120ctn binding region of E-cadherin inhibit transport of de novo synthesized E-cadherin to the plasma membrane in L fibroblasts (12); knock down of p120ctn reduces cell surface E-cadherin, presumably by enhancing its endocytosis (13); and p120ctn binds to kinesin and promotes cell surface trafficking of cadherins (14, 15).

Nectin is a recently emerged Ca2+-independent immunoglobulin (Ig)-like cell-cell adhesion molecule that forms AJs cooperatively with cadherin (16, 17). Nectin comprises a family of four members, nectin-1, -2, -3, and -4. Each member first forms homo-cis-dimers and then homo- or hetero-trans-dimers (trans-interactions) through the extracellular region in a Ca2+-independent manner, inducing cell-cell adhesion. The cytoplasmic region is associated with the actin cytoskeleton through afadin, a nectin- and actin filament-binding protein. Trans-interaction of nectin first forms cell-cell adhesion and then recruits cadherin to the nectin-based cell-cell adhesion sites, eventually forming AJs. In epithelial cells, after the formation of AJs, tight junctions are formed at the apical side of AJs (18). At tight junctions, claudin is a key cell-cell adhesion molecule (19, 20). In addition, trans-interaction of nectin induces activation of Cdc42 and Rac small G proteins (17, 21). Trans-interaction of nectin first recruits and activates c-Src at the nectin-based cell-cell adhesion sites (22). Activated c-Src then tyrosine phosphorylates FRG, a specific GDP/GTP exchange factor (GEF) for Cdc42, FRG (FGD1-related GEF) and activates C3G, a Rap1-GEF, through Crk, an adaptor of c-Src, at the nectin-based cell-cell adhesion sites. Rap1 then enhances the GEF activity on Cdc42 of tyrosine-phosphorylated FRG (23). On the other hand, Vav2 is recruited and tyrosine phosphorylated by c-Src at the nectin-based cell-cell adhesion sites (24). Cdc42 then enhances the GEF activity on Rac of the tyrosine-phosphorylated Vav2. Cdc42 activated in this way induces the formation of filopodia and increases the cell-cell contact sites, whereas Rac activated in this way induces the formation of lamellipodia, which efficiently expands the cell-cell adhesion between filopodia, acting like a "zipper" and eventually enhancing the formation of AJs (22, 25). Both Rac and Cdc42, but mainly Rac, are activated by trans-interaction of E-cadherin (26, 27), but the physiological role of this activation of Rac or Cdc42 remains unknown.

When epithelial cells migrate in response to many extracellular signals, such as hepatocyte growth factor (HGF)/scatter factor and 12-O-tetradecanoyl-phorbol-13-acetate, the cells first spread, followed by disruption of AJs and tight junctions and cell scattering. The disruption of AJs is always associated with E-cadherin endocytosis, but its molecular mechanism has not been fully understood (28-30). We have recently developed a new biochemical assay that efficiently reconstitutes the E-cadherin endocytosis, using an AJ-enriched plasma membrane fraction from rat liver (27). We have found by use of this cell-free assay that non-trans-interacting E-cadherin is constitutively endocytosed in a clathrin-dependent manner. Rac mainly activated by trans-interacting E-cadherin inhibits the E-cadherin endocytosis through IQGAP1 and actin filaments. Because IQGAP1 is an actin filament-cross-linking protein and a downstream target of Rac (31-33), it is likely that IQGAP1 binds to Rac activated by trans-interacting E-cadherin and reorganizes the actin cytoskeleton, resulting in inhibition of E-cadherin endocytosis and stabilization of E-cadherin on the plasma membrane.



View larger version (39K):
[in this window]
[in a new window]
  		FIG. 1.
Requirement of the C-terminal 4 aa of nectin for full inhibition of non-trans-interacting E-cadherin endocytosis by trans-interacting full-length nectin. A, no inhibition of the constitutive E-cadherin endocytosis by non-trans-interacting nectin in intact cells. EL cells, nectin-1-EL cells, and nectin-1-{Delta}C-EL cells were incubated in the medium with 0.4 µM human IgG (control IgG) for 60 min. The cell surface was biotinylated on ice and cultured at 18 °C for the indicated periods of time to allow E-cadherin endocytosis. Biotinylated proteins on the plasma membrane were then stripped off by glutathione treatment, and biotinylated proteins inside the cells were recovered on streptavidin beads. The bound proteins were analyzed by immunoblotting with the anti-E-cadherin mAb. B, inhibition of constitutive E-cadherin endocytosis by trans-interacting nectin in intact cells. EL cells, nectin-1-EL cells, and nectin-1-{Delta}C-EL cells were incubated in the medium with 0.4 µM Nef-3 for 60 min. The cells were then assayed for E-cadherin endocytosis as described in panel A. In all panels, the relative amounts of endocytosed E-cadherin were expressed as percentage of total biotinylated E-cadherin in the bottom panel. The mean (±S.D.) of duplicate assays is shown. Asterisks indicate statistical significance (Student's t test; *, p < 0.05). The results shown in all panels are representative of at least three independent experiments.

 
On the other hand, we have previously shown that nectin, but not E-cadherin, plays an important role in recruiting IQGAP1 to very primordial nectin-based cell-cell adhesion sites through the actin cytoskeleton (34), in contrast to the previous observation that E-cadherin is required for the accumulation of IQGAP1 at AJs solely by binding to {beta}-catenin (35). We have shown that Rac and Cdc42 activated by trans-interacting nectin also have potency to inhibit the E-cadherin endocytosis (27). Therefore, taken together, IQGAP1 would be first recruited through the actin cytoskeleton to the cell-cell adhesion sites where Rac and Cdc42 are activated by trans-interacting nectin. IQGAP1 bound to Rac and Cdc42 then induces reorganization of the actin cytoskeleton. E-cadherin is then recruited to the nectin-based cell-cell adhesion sites and induces the activation of Rac, which then facilitates the recruitment of IQGAP1 and the subsequent reorganization of the actin cytoskeleton. These sequential reorganizations of the actin cytoskeleton are likely to inhibit E-cadherin endocytosis on one hand and strengthen the cell-cell adhesion activity of E-cadherin on the other hand.

In addition, we have recently found that trans-interacting full-length nectin-1 capable of associating with afadin inhibits the non-trans-interacting E-cadherin endocytosis more potently than trans-interacting nectin mutant incapable of associating with afadin. These results suggest that afadin is additionally involved in E-cadherin endocytosis. We studied here the role and mode of action of afadin in E-cadherin endocytosis.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Construction of Expression Vectors—Expression vectors were constructed in pEGFP-C1 (Clontech) and pFastBac1-maltose-binding protein (MBP) using standard molecular biology methods. The pFastBac1-MBP was constructed with a baculovirus transfer vector, pFastBac1 (Invitrogen), to express an N-terminal MBP fusion protein. Constructs of afadin contained the following amino acids (aa): pEGFP-afadin, aa 1-1829; pEGFP-afadin-{Delta}RA, aa 352-1829; pFastBac1-MBP-afadin, aa 1-1829; and pFastBac1-MBP-afadin-{Delta}RA, aa 352-1829. The pIRM21-FLAG-V12Rap1B was provided by Dr. M. Matsuda (Osaka University, Osaka, Japan).

Antibodies—A rat anti-E-cadherin (extracellular portion) mAb (ECCD-2) was provided by Dr. M. Takeichi (Center for Developmental Biology, RIKEN, Kobe, Japan). A mouse anti-p120ctn mAb, a mouse anti-E-cadherin mAb (BD Transduction Laboratories), a rabbit anti-GFP pAb (MBL), a mouse anti-FLAG mAb (Sigma), and a rabbit anti-MBP pAb (New England BioLabs) were purchased from commercial sources.

Biotinylation Assay for E-cadherin Endocytosis—The assay was performed as described with minor modifications (36). Briefly, after preincubation with IgG or Nef-3, EL cells, nectin-1-EL cells, and nectin-1-{Delta}C-EL cells were incubated with 0.5 mg/ml sulfo-NHS-ss-biotin on ice, and the cells were incubated at 18 °C to allow the E-cadherin endocytosis for the indicated periods of time. The cells were incubated in several washes with a glutathione buffer (60 mM glutathione, 83 mM NaCl, 83 mM NaOH, and 10% bovine serum albumin) on ice to remove bound biotinyl groups from remaining cell surface-biotinylated proteins. The cells were then lysed in radioimmune precipitation assay (RIPA) buffer (20 mM Tris-HCl (pH 7.4) with 150 mM NaCl, 0.1% SDS, 1% Triton X-100, 1% deoxycholate, and 5 mM EDTA). The cell extracts were incubated with streptavidin beads (Sigma) to collect bound biotinylated proteins. Bound proteins were then analyzed by SDS-PAGE and immunoblotting with the anti-E-cadherin mAb. The blots were developed using the ECL kit and quantitated using a densitometer FluorchemTM (Alpha Innotech).



View larger version (38K):
[in this window]
[in a new window]
  		FIG. 2.
Inhibition of non-trans-interacting E-cadherin endocytosis by afadin in a Rap1-dependent manner. A, flow diagram of the steps of the cell-free assay and schematic morphologies of the AJ membrane. B, inhibition of E-cadherin endocytosis by afadin-{Delta}RA, but not by afadin. The AJ membrane fraction was incubated in the presence of MBP-afadin or MBP-afadin-{Delta}RA at the indicated concentrations and assayed for E-cadherin endocytosis as described in panel A. The amount of E-cadherin in the endocytosed vesicles (70% of total SDS-solubilized membrane) was quantitated by immunoblotting with anti-E-cadherin mAb. Quantification of immunoblots is shown as the mean (±S.D.) of duplicate assays in the lower panel. C, inhibition of E-cadherin endocytosis by afadin in a Rap1-dependent manner. The AJ membrane fraction was incubated in the presence of MBP-afadin (60 nM) and Rap1B-GTP{gamma}S or Rap1B-GDP{beta}S at the indicated concentrations and assayed for E-cadherin endocytosis as described in panel A. The amount of E-cadherin in the endocytosed vesicles (70% of total SDS solubilized membrane) was quantitated by immunoblotting with the anti-E-cadherin mAb. Quantification of immunoblots is shown as the mean (±S.D.) of duplicate assays in the lower panel. Asterisks indicate statistical significance (Student's t test; *, p < 0.05). The results shown in all panels are representative of at least three independent experiments.

 
Cell-free Assay for E-cadherin Endocytosis—The assay was performed as described (27). Briefly, the AJ-enriched fraction was prepared from rat livers as described (37), washed with 0.5 M Tris-HCl (pH 7.5), resuspended in Buffer A (20 mM Hepes-KOH (pH 7.4) and 125 mM KOAc), and stored at -80 °C until use. The thawed AJ membrane fraction (20 µg of protein) was incubated at 30 °C in a reaction mixture (36 mM Hepes-KOH (pH 7.4), 0.25 M sorbitol, 70 mM KOAc, 5 mM EGTA, 1.8 mM CaCl2, 2.5 mM Mg(OAc)2, an ATP-regenerating system (1 mM ATP, 5 mM creatine phosphate, and 0.2 IU creatine phosphate kinase), 100 µM GTP, and 2.5 mg/ml rat brain cytosol). The reaction was stopped by chilling the tube on ice. The membrane was collected by centrifugation at 20,000 x g for 10 min. The membrane was resuspended by trituration (20 times pipetting) in 50 µl of 20 mM Hepes-KOH (pH 7.2) and 0.25 M sorbitol and then supplemented with KOAc and Mg(OAc)2 to final concentrations of 150 and 2.5 mM, respectively (final volume of 60.6 µl). Immediately after addition of KOAc and Mg(OAc)2, differential centrifugation was performed at medium speed (16,000 x g) for 2 min. The top 42-µl supernatant fraction was harvested and centrifuged at high speed (100,000 x g) for 20 min. Membrane pellets from the high speed spins were solubilized in an SDS sample buffer at room temperature for 30 min with vigorous shaking, and proteins were separated by 10% SDS-PAGE. The proteins were transferred to nitrocellulose membrane sheets and immunoblotted with the anti-E-cadherin mAb, followed by the horseradish peroxidase-conjugated secondary antibody (Amersham Biosciences). The blots were developed using the ECL kit and quantitated using a densitometer FluorchemTM (Alpha Innotech).

Cell-free Assay for p120ctn Release from the AJ Membrane—The thawed AJ membrane fraction (20 µg of protein) was incubated in a reaction mixture (36 mM Hepes-KOH (pH 7.4), 0.25 M sorbitol, 70 mM KOAc, 5 mM EGTA, 1.8 mM CaCl2, 2.5 mM Mg(OAc)2, an ATP-regenerating system, and 100 µM GTP) with or without MBP-afadin, MBP-afadin-{Delta}RA, and a mixture of MBP-afadin and Rap1B-GTP{gamma}S or Rap1B-GDP{beta}S. After incubation at 30 °C for 30 min, the reaction was stopped by chilling the tube on ice and the membrane was collected by centrifugation at 100,000 x g for 20 min. The supernatant fraction and the membrane pellet fraction were solubilized in an SDS sample buffer at room temperature for 30 min with vigorous shaking, and proteins were separated by 8% SDS-PAGE. The proteins were transferred to nitrocellulose membrane sheets and immunoblotted with the anti-p120ctn mAb and the anti-MBP pAb, followed by horseradish peroxidase-conjugated secondary Ab (Amersham Biosciences). The blots were developed using an ECL kit and quantitated using a densitometer FluorchemTM (Alpha Innotech).



View larger version (49K):
[in this window]
[in a new window]
  		FIG. 3.
Enhancement of the binding of p120ctn to E-cadherin by trans-interacting nectin-1 in an afadin-dependent manner. EL cells, nectin-1-EL cells, and nectin-1-{Delta}C-EL cells were plated on a Nef-3-coated dish for 30 min. The extract of the cells was incubated with anti-p120ctn mAb. The immunoprecipitates were analyzed by immunoblotting with the anti-E-cadherin mAb. Quantification of immunoblots is shown as the mean (±S.D.) of duplicate assays in the lower panel. Asterisk indicates statistical significance (Student's t test; *, p < 0.05). The results shown are representative of three independent experiments.

 
Co-immunoprecipitation Assay for p120ctn and E-cadherin in EL Cell Lines—To determine the amount of E-cadherin bound to p120ctn in EL cell lines, cells treated with Nef-3 were extracted by the addition of Buffer A (20 mM Tris-HCl (at pH 8.0), 1 mM EDTA, 1 mM dithiothreitol, 150 mM NaCl, 1% Triton X-100, 10 µM {alpha}-phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, and 10 µg/ml leupeptin). The cell extract was obtained by centrifugation at 100,000 x g for 60 min. The extract was incubated with the anti-p120ctn mAb (20 µg) at 4 °C for 18 h. The immunocomplexes were then precipitated with protein A-Sepharose CL-4B beads (Amersham Biosciences). After the beads were extensively washed with Buffer A, the bound proteins were eluted by boiling in the SDS sample buffer. The samples were then subjected to SDS-PAGE, followed by immunoblotting with the anti-E-cadherin and anti-p120ctn mAbs.

Co-immunoprecipitation Assay for Afadin and p120ctn in HEK293 Cells—To determine the binding of afadin to p120ctn, HEK293 cells were transfected with pEGFP, pEGFP-afadin, pEGFP-afadin-{Delta}RA, or a mixture of pEGFP-afadin and pIRM21-FLAG-V12Rap1B using Lipofectamine 2000 reagent (Invitrogen). After 48 h of incubation, the cells were extracted by the addition of Buffer A. The cell extract was obtained by centrifugation at 100,000 x g for 15 min. The extract was incubated with the anti-GFP pAb (5 µg) at 4 °C for 18 h. The immunocomplexes were then precipitated with protein A-Sepharose CL-4B beads. After the beads were extensively washed with Buffer A, the bound proteins were eluted by boiling in the SDS sample buffer. The samples were then subjected to SDS-PAGE, followed by immunoblotting with the anti-p120ctn mAb, the anti-GFP pAb, and the anti-FLAG mAb.

Immunofluorescence Microscopic Assay for E-cadherin Endocytosis—MDCK cells (2 x 105 cells/35-mm dish) were cultured in Dulbecco's modified Eagle's medium containing 10% fetal calf serum at 37 °C for 48 h and then transfected with pEGFP, pEGFP-afadin, pEGFP-afadin-{Delta}RA, or a mixture of pEGFP-afadin and pIRM21-FLAG-V12Rap1B using Lipofectamine 2000 reagent. After 24 h of culture, the cells were incubated with 10 ng/ml HGF for 4 h. The cells were fixed, followed by immunostaining with the anti-E-cadherin mAb (ECCD-2). Images were captured using a Carl Zeiss confocal laser scanning microscope using a x63 oil immersion objective lens (model LSM 510-V3.2; Carl Zeiss). The fluorescence intensities of E-cadherin and afadin in MDCK cells were measured using the fluorescence intensity profile function of LSM 510 software. Collected data were exported as 8-bit TIFF files and processed using Adobe Photoshop software. The number of endocytosed E-cadherin-positive vesicular structures inside the cells was compared between the transfected and untransfected cells in the same field.

Other Methods—Protein concentrations were determined with bovine serum albumin as a reference protein (38). SDS-PAGE was done as described by Laemmli (39).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Inhibition of Non-trans-interacting E-cadherin Endocytosis by Trans-interacting Nectin—To elucidate the mechanism of the non-trans-interacting E-cadherin endocytosis by the nectin-afadin system, we first examined the effect of the trans- or non-trans-interacting full-length nectin-1 and the C-terminal 4-aa deletion mutant of nectin-1, which does not associate with afadin (nectin-1-{Delta}C), on non-trans-interacting E-cadherin endocytosis in intact cells. For this purpose, we first used L cells stably expressing nectin-1 and E-cadherin (nectin-1-EL cells), L cells stably expressing nectin-1-{Delta}C and E-cadherin (nectin-1-{Delta}C-EL cells), and L cells stably expressing E-cadherin (EL cells), because E-cadherin is constitutively endocytosed and recycled when cells do not contact other cells and E-cadherin does not trans-interact (27, 40). We assayed at 18 °C to stop this recycling and to measure only the E-cadherin endocytosis in the absence or presence of an extracellular fragment of nectin-3 fused to the Fc portion of IgG (Nef-3), known to potently trans-interact with cellular nectin-1 in these cells. When these cells were cultured sparsely and did not contact each other in the absence of Nef-3, the amounts of endocytosed E-cadherin were similar among EL cells, nectin-1-EL cells, and nectin-1-{Delta}C-EL cells (Fig. 1A). In the presence of Nef-3, the amounts of endocytosed E-cadherin were reduced in nectin-1-EL cells and nectin-1-{Delta}C-EL cells, but not in EL cells (Fig. 1B). The amount of endocytosed E-cadherin in nectin-1-EL cells was less than that in nectin-1-{Delta}C-EL cells. Thus, trans-interacting nectin-1 inhibited the non-trans-interacting E-cadherin endocytosis more potently than trans-interacting nectin-1-{Delta}C. We have previously shown that activation of Rac and Cdc42 by trans-interacting full-length nectin inhibits non-trans-interacting E-cadherin endocytosis through the IQGAP1-dependent reorganization of the actin cytoskeleton in nectin-1-EL cells (27). Association of the C-terminal 4 aa of nectin-1 with afadin is not required for this nectin-induced activation of Rac and Cdc42 in nectin-1-EL cells (41). Taken together, in addition to the Rac- and Cdc42-induced inhibition of the non-trans-interacting E-cadherin endocytosis, these results suggest that association of the C-terminal 4 aa of nectin-1 with afadin is required for full inhibition of the non-trans-interacting E-cadherin endocytosis by trans-interacting full-length nectin-1 in nectin-1-EL cells.

Inhibition of Non-trans-interacting E-cadherin Endocytosis by Afadin in a Rap1-dependent Manner—We have previously developed a cell-free assay using an AJ-enriched fraction from rat liver in which non-trans-interacting E-cadherin endocytosis is induced (27). To gain insight into the regulation of the activity of afadin on E-cadherin endocytosis, we examined whether afadin affects the non-trans-interacting E-cadherin endocytosis in this cell-free assay as schematically shown in Fig. 2A. Pure recombinant afadin did not affect the E-cadherin endocytosis, but an RA domain-deleted mutant of afadin, afadin-{Delta}RA, inhibited the E-cadherin endocytosis, suggesting that the RA domain is the negatively regulatory domain for the activity of afadin on E-cadherin endocytosis (Fig. 2B). Afadin binds the GTP-bound form of Rap1B preferentially to the GDP-bound form of Rap1B (42). The GTP{gamma}S-bound form of Rap1B enhanced the inhibitory activity of afadin on the E-cadherin endocytosis in a dose-dependent manner, similarly to that induced by afadin-{Delta}RA, whereas the GDP{beta}S-bound form of Rap1B was less effective (Fig. 2C). These results indicate that afadin inhibits non-trans-interacting E-cadherin endocytosis in a Rap1-dependent manner.



View larger version (54K):
[in this window]
[in a new window]
  		FIG. 4.
Enhancement of the binding of p120ctn to E-cadherin by afadin in a Rap1-dependent manner. A, flow diagram of the steps of the p120ctn-releasing assay and schematic morphologies of the state of p120ctn in the AJ membrane. B, inhibition of p120ctn release from the membrane by afadin-{Delta}RA, but not by afadin. The AJ membrane fraction was incubated in the presence of MBP-afadin or MBP-afadin-{Delta}RA at the indicated concentrations and assayed for the release of endogenous p120ctn as described in panel A. The amounts of p120ctn in the supernatant (sup) and MBP-afadin or MBP-afadin-{Delta}RA in the pellet (ppt) (50% of total sup fraction and 20% of total ppt fraction, respectively) were quantitated by immunoblotting with the anti-p120ctn mAb and the anti-MBP pAb. Quantification of immunoblots is shown as the mean (±S.D.) of duplicate assays in the lower panel. Arrowheads indicate the N-terminal splicing variants of p120ctn (100 kDa). For quantification of immunoblots, we measured the intensity of both bands collectively. C, inhibition of p120ctn release from the AJ membrane by afadin in a Rap1-dependent manner. The AJ membrane fraction was incubated in the presence of MBP-afadin (60 nM) and Rap1B-GTP{gamma}S or Rap1B-GDP{beta}S at the indicated concentrations and assayed for the release of endogenous p120ctn as described in panel A. The amounts of p120ctn in the sup and MBP-afadin in the ppt (50% of total sup fraction and 20% of total ppt fraction, respectively) were quantitated by immunoblotting with the anti-p120ctn mAb and the anti-MBP pAb. Quantification of immunoblots is shown as the mean (±S.D.) of duplicate assays in the lower panel. Arrowheads indicate the N-terminal splicing variants of p120ctn (100 kDa). For quantification of immunoblots, we measured the intensity of both bands collectively. Asterisks indicate statistical significance (Student's t test; *, p < 0.05). The results shown in all panels are representative of at least three independent experiments.

 
Enhancement of the Binding of p120ctn to E-cadherin by Trans-interacting Nectin in an Afadin-dependent Manner—It is well established that p120ctn directly binds to the juxtamembrane region of E-cadherin, and it has been suggested that p120ctn is involved in the stabilization of non-trans-interacting E-cadherin on the cell surface (13, 43). We therefore examined whether the nectin-afadin system inhibits non-trans-interacting E-cadherin endocytosis through p120ctn in nectin-1-EL cells. When p120ctn was immunoprecipitated, E-cadherin was co-immunoprecipitated with p120ctn in EL, nectin-1-EL, and nectin-1-{Delta}C-EL cells (Fig. 3). However, the amount of E-cadherin co-immunoprecipitated with p120ctn in nectin-1-EL cells was much more than that in EL and nectin-1-{Delta}C-EL cells. The amounts of E-cadherin co-immunoprecipitated with p120ctn were similar between EL and nectin-1-{Delta}C-EL cells. These results indicate that nectin-1 stabilizes the association of p120ctn with E-cadherin through afadin and thereby inhibits non-trans-interacting E-cadherin endocytosis.



View larger version (64K):
[in this window]
[in a new window]
  		FIG. 5.
Binding of afadin to p120ctn in a Rap1-dependent manner. HEK293 cells were transfected with empty control vector (GFP), GFP-afadin, GFP-afadin-{Delta}RA, or a mixture of GFP-afadin and FLAG-Rap1B-CA. The extract of HEK293 cells was incubated with the anti-GFP pAb. Immunoprecipitates were analyzed by immunoblotting with the anti-p120ctn mAb, the anti-GFP pAb, and the anti-FLAG mAb. Quantification of immunoblots is shown as the mean (±S.D.) of duplicate assays in the lower panel. Asterisks indicate statistical significance (Student's t test; *, p < 0.05). The results shown are representative of three independent experiments.

 
Enhancement of the Binding of p120ctn to E-cadherin by Afadin in a Rap1-dependent Manner—We next examined whether afadin affects the binding of p120ctn to E-cadherin in a Rap1-dependent manner using the cell-free assay as schematically shown in Fig. 4A. p120ctn was released from the AJ membrane during the incubation. Pure recombinant afadin did not affect the release of p120ctn from the AJ membrane, but afadin-{Delta}RA inhibited the release of p120ctn from the AJ membrane in a dose-dependent manner, suggesting that afadin-{Delta}RA enhances the stabilization of p120ctn on the AJ membrane (Fig. 4B). Afadin and afadin-{Delta}RA bound to the AJ membrane in a dose-dependent manner. Afadin-{Delta}RA bound to it ~3-fold more than afadin. The GTP{gamma}S-bound form of Rap1B enhanced the inhibitory activity of afadin on the release of p120ctn from the AJ membrane in a dose-dependent manner, similarly to that induced by afadin-{Delta}RA, whereas the GDP{beta}S-bound form of Rap1B was less effective (Fig. 4C). Two immunoblot bands were reacted with the anti-p120ctn mAb in the supernatant fraction of the cell-free assay as shown in Fig. 4, B and C. Two major N-terminal splicing variants of p120ctn (120 and 100 kDa) have been reported to be expressed in various mouse tissues (44). The anti-p120ctn mAb used here was raised against the C-terminal fragment of p120ctn and could react with both 120 and 100 kDa isoforms of p120ctn in the AJ fraction from rat liver and other tissues (supplemental Fig. 1). Thus, we assumed that the two bands were the 120 and 100 kDa isoforms of p120ctn. For quantification of immunoblot, we measured the intensity of both bands collectively. These results indicate that afadin inhibits the release of p120ctn from the AJ membrane in a Rap1-dependent manner.

Binding of Afadin to p120ctn in a Rap1-dependent Manner—To examine the in vivo binding of afadin to p120ctn, a co-immunoprecipitation assay was performed using the HEK293 cell lysate. Afadin, a mixture of afadin and a constitutively active mutant of Rap1B (V12Rap1B: Rap1B-CA), or afadin-{Delta}RA was transiently overexpressed in HEK293 cells. When afadin was immunoprecipitated, endogenous p120ctn was co-immunoprecipitated (Fig. 5). The amount of co-immunoprecipitated p120ctn was increased by co-expression with Rap1B-CA. Rap1B-CA indeed bound to afadin. The amount of co-immunoprecipitated p120ctn with afadin-{Delta}RA was more than that with afadin alone and similar to that with the mixture of afadin and Rap1B-CA. Thus, afadin forms a novel trimeric complex with p120ctn and Rap1. To confirm the direct binding of afadin to p120ctn, we performed affinity chromatography using the pure recombinant proteins of afadin and p120ctn. Afadin did not bind p120ctn directly, and this binding was not affected by binding of Rap1B-CA to afadin (data not shown). A modification or another protein(s) may be required for the efficient binding between afadin and p120ctn. Taken together, these results indicate that afadin binds p120ctn in intact cells and inhibits non-trans-interacting E-cadherin endocytosis through p120ctn in a Rap1-dependent manner.

Inhibition of E-cadherin Endocytosis by Afadin in a Rap1-dependent Manner in MDCK Cells—To further validate the results obtained in our assay systems, we finally examined whether afadin affects E-cadherin endocytosis in intact MDCK cells. Activation of c-Met, the cell surface receptor for HGF, enhances E-cadherin endocytosis (28). We examined the effect of afadin on the HGF-induced E-cadherin endocytosis in MDCK cells. Quantitative analysis showed that overexpression of both afadin and Rap1B-CA or afadin-{Delta}RA inhibited the HGF-induced E-cadherin endocytosis, consistent with our results in the cell-free assay (Fig. 6, A and B). Control GFP or afadin alone did not affect the HGF-induced E-cadherin endocytosis. X-Z optical sectioning showed that afadin co-expressed with Rap1B-CA and afadin-{Delta}RA was more highly concentrated at the cell-cell adhesion sites than afadin alone, consistent with our results in the cell-free assay shown in Fig. 4, B and C (Fig. 6, A and C). These results indicate that afadin inhibits the HGF-induced E-cadherin endocytosis in a Rap1-dependent manner in intact MDCK cells.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
We have previously shown that non-trans-interacting E-cadherin, but not the trans-interacting one, undergoes endocytosis and that Rac activated by the action of trans-interacting E-cadherin inhibits E-cadherin endocytosis through the IQGAP1-dependent reorganization of the actin cytoskeleton, which shifts the equilibrium from the cell-cell dissociation state to the cell-cell adhesion state (27). In addition, we have previously shown that Rac and Cdc42 activated by trans-interacting nectin also have a potency to inhibit non-trans-interacting E-cadherin endocytosis through the IQGAP1-dependent reorganization of the actin cytoskeleton (27). For this nectin-induced activation of Rac and Cdc42, the association of the C-terminal tail of nectin with afadin has been shown not to be required (41). On the other hand, for the efficient recruitment of E-cadherin to the nectin-based cell-cell adhesion sites, the association of the C-terminal tail of nectin with afadin has been shown to be required (45). We have shown here that, in addition to the Rac and Cdc42-induced inhibition of non-trans-interacting E-cadherin endocytosis, the association of the C-terminal tail of nectin with afadin is required for full inhibition of non-trans-interacting E-cadherin endocytosis by trans-interacting full-length nectin. The inhibitory effect of afadin on the endocytosis is Rap1-dependent. Afadin enhances the binding of p120ctn to E-cadherin in a Rap1-dependent manner and thereby inhibits non-trans-interacting E-cadherin endocytosis. Moreover, we have recently shown that trans-interacting nectin induces the activation of Rap1 through c-Src/Crk/C3G signaling pathway (23). Taken together, the trans-interacting nectin inhibits non-trans-interacting E-cadherin endocytosis by two pathways, the Rac and Cdc42-IQGAP1 pathway and the Rap1-afadin pathway.



View larger version (54K):
[in this window]
[in a new window]
  		FIG. 6.
Inhibition of E-cadherin endocytosis by afadin in a Rap1-dependent manner in MDCK cells. MDCK cells were transfected with empty control vector (GFP), GFP-afadin, GFP-afadin-{Delta}RA, or a mixture of GFP-afadin and FLAG-Rap1B-CA and cultured for 24 h. After the culture, the cells were incubated with 10 ng/ml HGF for 4 h. The transfected cells were identified by the expression of GFP (green), and their E-cadherin positive internal vesicular structures were examined by immunostaining of E-cadherin (red). A, optical X-Y and X-Z sections in transfected cells are presented. The fluorescence intensity profile showing afadin (green) and E-cadherin (red) in X-Z section is presented below the images. B, quantification is presented of relative fluorescence intensities of E-cadherin at the cell surface and inside cells. C, quantification is presented of relative fluorescence intensities of afadin at the cell surface and inside cells. Bars, 10 µm. Asterisks indicate statistical significance (Student's t test; *, p < 0.05).

 
It has been shown that the Kd value for trans-interaction of E-cadherin is >100-fold higher than that for trans-interaction of nectin (46, 47). E-cadherin recruited to the nectin-based cell-cell adhesion sites is, therefore, likely to be non-trans-interacting; as the concentration of non-trans-interacting E-cadherin increases, it gradually trans-interacts, eventually establishing AJs. Trans-interaction of nectin induces the activation of Rap1, which binds to afadin. The afadin-Rap1 complex then forms a novel trimeric complex with p120ctn and thereby enhances the binding of p120ctn to E-cadherin (Fig. 7). This binding prevents non-trans-interacting E-cadherin from endocytosis and stabilizes E-cadherin at the nectin-based cell-cell adhesion sites. The binding of Rap1 to afadin does not affect the association of nectin with afadin.2 In cooperation with the Rap1-afadin-p120ctn-dependent regulation, trans-interacting nectin induces the activation of Cdc42 and Rac, which then inhibit non-trans-interacting E-cadherin endocytosis through IQGAP1-dependent reorganization of the actin cytoskeleton and thereby accumulate non-trans-interacting E-cadherin at the nectin-based cell-cell adhesion sites. The recruited non-trans-interacting E-cadherin gradually start trans-interacting each other. Rac activated by the action of trans-interacting E-cadherin inhibits non-trans-interacting E-cadherin endocytosis through the IQGAP1-dependent reorganization of the actin cytoskeleton and thereby enhances the accumulation of non-trans-interacting E-cadherin more and more at the nectin-based cell-cell adhesion sites. In these ways, trans-interacting nectin shifts the equilibrium from the non-trans-interaction state of E-cadherin to the trans-interaction state and thereby induces formation of the E-cadherin-based AJs. Moreover, Cdc42 activated by the action of trans-interacting nectin induces the formation of filopodia and increases the cell-cell contact sites, whereas Rac activated by the action of trans-interacting nectin induces the formation of lamellipodia, which efficiently expands the cell-cell adhesion between filopodia, acting like a "zipper." Even after the formation of AJs by trans-interaction of E-cadherin, trans-interacting E-cadherin may dissociate to produce non-trans-interacting E-cadherin, but Cdc42 and Rac activated in these ways inhibit non-trans-interacting E-cadherin endocytosis. Thus, nectin plays important roles in the stabilization of E-cadherin on the plasma membrane and the formation and maintenance of AJs through afadin, the small G proteins, and p120ctn.



View larger version (35K):
[in this window]
[in a new window]
  		FIG. 7.
A model for an inhibitory effect of trans-interacting nectin on non-trans-interacting E-cadherin endocytosis. Trans-interaction of nectin induces the activation of Rap1, which binds to afadin. The afadin-Rap1 complex then forms a novel trimeric complex with p120ctn and thereby enhances the binding of p120ctn to E-cadherin. This binding prevents non-trans-interacting E-cadherin from endocytosis and stabilizes E-cadherin at the nectin-based cell-cell adhesion sites. The recruited non-trans-interacting E-cadherin gradually starts trans-interacting each other on the opposite side of the membrane. In these ways, trans-interacting nectin shifts the equilibrium from the non-trans-interaction state of E-cadherin to the trans-interaction state and thereby induces formation of the E-cadherin-based AJs. To simplify the model, we did not describe the involvement of Rac and Cdc42-IQGAP1 pathway in the non-trans-interacting E-cadherin endocytosis.

 

   FOOTNOTES
 
* This work was supported in part by grants-in-aid for scientific research and for cancer research from the Ministry of Education, Culture, Sports, Science, and Technology, Japan (2004). 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

The on-line version of this article (available at http://www.jbc.org) contains a supplemental figure. Back

{ddagger} Recipient of a Human Frontier Science Program Career Development Award (2003). Back

§ To whom correspondence should be addressed. Tel.: 81-6-6879-3410; Fax: 81-6-6879-3419; E-mail: ytakai{at}molbio.med.osaka-u.ac.jp.

1 The abbreviations used are: AJs, adherens junctions; HGF, hepatocyte growth factor; EL cell, L fibroblast stably expressing E-cadherin; MDCK cell, Madin-Darby canine kidney cell; Nef-3, the extracellular fragment of nectin-3 fused to the Fc portion of human IgG; MBP, maltose-binding protein; GFP, green fluorescent protein; aa, amino acid; HEK, human embryonic kidney; mAb, monoclonal antibody; pAb, polyclonal antibody. Back

2 W. Ikeda and Y. Takai, unpublished data. Back



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Takeichi, M. (1988) Development 102, 639-655[Abstract/Free Full Text]
  2. Yap, A. S., Brieher, W. M., and Gumbiner, B. M. (1997) Annu. Rev. Cell Dev. Biol. 13, 119-146[CrossRef][Medline] [Order article via Infotrieve]
  3. Adams, C. L., and Nelson, W. J. (1998) Curr. Opin. Cell Biol. 10, 572-577[CrossRef][Medline] [Order article via Infotrieve]
  4. Gumbiner, B. M. (2000) J. Cell Biol. 148, 399-404[Abstract/Free Full Text]
  5. Takeichi, M. (1991) Science 251, 1451-1455[Abstract/Free Full Text]
  6. Gumbiner, B. M. (1996) Cell 84, 345-357[CrossRef][Medline] [Order article via Infotrieve]
  7. Vleminckx, K., and Kemler, R. (1999) BioEssays 21, 211-220[CrossRef][Medline] [Order article via Infotrieve]
  8. Aberle, H., Schwartz, H., and Kemler, R. (1996) J. Cell. Biochem. 61, 514-523[CrossRef][Medline] [Order article via Infotrieve]
  9. Nagafuchi, A. (2001) Curr. Opin. Cell Biol. 13, 600-603[CrossRef][Medline] [Order article via Infotrieve]
  10. Daniel, J. M., and Reynolds, A. B. (1995) Mol. Cell. Biol. 15, 4819-4824[Abstract]
  11. Yap, A. S., Niessen, C. M., and Gumbiner, B. M. (1998) J. Cell Biol. 141, 779-789[Abstract/Free Full Text]
  12. Ohkubo, T., and Ozawa, M. (1999) J. Biol. Chem. 274, 21409-21415[Abstract/Free Full Text]
  13. Davis, M. A., Ireton, R. C., and Reynolds, A. B. (2003) J. Cell Biol. 163, 525-534[Abstract/Free Full Text]
  14. Chen, X., Kojima, S., Borisy, G. G., and Green, K. J. (2003) J. Cell Biol. 163, 547-557[Abstract/Free Full Text]
  15. Yanagisawa, M., Kaverina, I. N., Wang, A., Fujita, Y., Reynolds, A. B., and Anastasiadis, P. Z. (2004) J. Biol. Chem. 579, 9512-9521
  16. Takai, Y., and Nakanishi, H. (2003) J. Cell Sci. 116, 17-27[Abstract/Free Full Text]
  17. Takai, Y., Irie, K., Shimizu, K., Sakisaka, T., and Ikeda, W. (2003) Cancer Sci. 94, 655-667[CrossRef][Medline] [Order article via Infotrieve]
  18. Tsukita, S., and Furuse, M. (1999) Trends Cell Biol. 9, 268-273[CrossRef][Medline] [Order article via Infotrieve]
  19. Tsukita, S., and Furuse, M. (2000) Ann. N. Y. Acad. Sci. 915, 129-135[Abstract/Free Full Text]
  20. Tsukita, S., Furuse, M., and Itoh, M. (2001) Nat. Rev. Mol. Cell. Biol. 2, 285-293[CrossRef][Medline] [Order article via Infotrieve]
  21. Shimizu, K., and Takai, Y. (2003) J. Biochem. (Tokyo) 134, 631-636[Abstract/Free Full Text]
  22. Fukuhara, T., Shimizu, K., Kawakatsu, T., Fukuyama, T., Minami, Y., Honda, T., Hoshino, T., Yamada, T., Ogita, H., Okada, M., and Takai, Y. (2004) J. Cell Biol. 166, 393-405[Abstract/Free Full Text]
  23. Fukuyama, T., Ogita, H., Kawakatsu, T., Fukuhara, T., Yamada, T., Sato, T., Shimizu, K., Nakamura, T., Matsuda, M., and Takai, Y. (2005) J. Biol. Chem. 280, 815-825[Abstract/Free Full Text]
  24. Kawakatsu, T., Ogita, H., Fukuhara, T., Fukuyama, T., Minami, Y., Shimizu, K., and Takai, Y. (2005) J. Biol. Chem. 280, 4940-4947[Abstract/Free Full Text]
  25. Ehrlich, J. S., Hansen, M. D. H., and Nelson, W. J. (2002) Dev. Cell 3, 259-270[CrossRef][Medline] [Order article via Infotrieve]
  26. Yap, A. S., and Kovacs, E. M. (2003) J. Cell Biol. 160, 11-16[Abstract/Free Full Text]
  27. Izumi, G., Sakisaka, T., Baba, T., Tanaka, S., Morimoto, K., and Takai, Y. (2004) J. Cell Biol. 166, 237-248[Abstract/Free Full Text]
  28. Kamei, T., Matozaki, T., Sakisaka, T., Kodama, A., Yokoyama, S., Peng, Y. F., Nakano, K., Takaishi, K., and Takai, Y. (1999) Oncogene 18, 6776-6784[CrossRef][Medline] [Order article via Infotrieve]
  29. Fujita, Y., Krause, G., Scheffner, M., Zechner, D., Leddy, H. E., Behrens, J., Sommer, T., and Birchmeier, W. (2002) Nat. Cell Biol. 4, 222-231[CrossRef][Medline] [Order article via Infotrieve]
  30. Palacios, F., Schweitzer, J. K., Boshans, R. L., and D'Souza-Schorey, C. (2002) Nat. Cell Biol. 4, 929-936[CrossRef][Medline] [Order article via Infotrieve]
  31. Kuroda, S., Fukata, M., Kobayashi, K., Nakafuku, M., Nomura, N., Iwamatsu, A., and Kaibuchi, K. (1996) J. Biol. Chem. 271, 23363-23367[Abstract/Free Full Text]
  32. Bashour, A. M., Fullerton, A. T., Hart, M. J., and Bloom, G. S. (1997) J. Cell Biol. 137, 1555-1566[Abstract/Free Full Text]
  33. Fukata, M., Kuroda, S., Fujii, K., Nakamura, T., Shoji, I., Matsuura, Y., Okawa, K., Iwamatsu, A., Kikuchi, A., and Kaibuchi, K. (1997) J. Biol. Chem. 272, 29579-29583[Abstract/Free Full Text]
  34. Katata, T., Irie, K., Fukuhara, A., Kawakatsu, T., Yamada, A., Shimizu, K., and Takai, Y. (2003) Oncogene 22, 2097-2109[CrossRef][Medline] [Order article via Infotrieve]
  35. Fukata, M., and Kaibuchi, K. (2001) Nat. Rev. Mol. Cell. Biol. 2, 887-897[CrossRef][Medline] [Order article via Infotrieve]
  36. Le, T. L., Yap, A. S., and Stow, J. L. (1999) J. Cell Biol. 146, 219-232[Abstract/Free Full Text]
  37. Tsukita, S., and Tsukita, S. (1989) J. Cell Biol. 108, 31-41[Abstract/Free Full Text]
  38. Bradford, M. M. (1976) Anal. Biochem. 72, 248-254[CrossRef][Medline] [Order article via Infotrieve]
  39. Laemmli, U. K. (1970) Nature 277, 680-685
  40. Paterson, A. D., Parton, R. G., Ferguson, C., Stow, J. L., and Yap, A. S. (2003) J. Biol. Chem. 278, 21050-21057[Abstract/Free Full Text]
  41. Kawakatsu, T., Shimizu, K., Honda, T., Fukuhara, T., Hoshino, T., and Takai, Y. (2002) J. Biol. Chem. 277, 50749-50755[Abstract/Free Full Text]
  42. Linnemann, T., Geyer, M., Jaitner, B. K., Block, C., Kalbitzer, H. R., Witting-hofer, A., and Herrmann, C. (1999) J. Biol. Chem. 274, 13556-13562[Abstract/Free Full Text]
  43. Xiao, K., Allison, D. F., Buckley, K. M., Kottke, M. D., Vincent, P. A., Faundez, V., and Kowalczyk, A. P. (2003) J. Cell Biol. 163, 535-545[Abstract/Free Full Text]
  44. Montonen, O., Aho, M., Uitto, J., and Aho, S. (2001) J. Histochem. Cytochem. 49, 1487-1496[Abstract/Free Full Text]
  45. Tachibana, K., Nakanishi, H., Mandai, K., Ozaki, K., Ikeda, W., Yamamoto, Y., Nagafuchi, A., Tsukita, S., and Takai, Y. (2000) J. Cell Biol. 150, 1161-1175[Abstract/Free Full Text]
  46. Koch, A. W., Pokutta, S., Lustig, A., and Engel, J. (1997) Biochemistry 36, 7697-7705[CrossRef][Medline] [Order article via Infotrieve]
  47. 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]

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
 Pharmacol. Rev.Home page
D. D. Mruk, B. Silvestrini, and C. Y. Cheng
Anchoring Junctions As Drug Targets: Role in Contraceptive Development
Pharmacol. Rev., June 1, 2008; 60(2): 146 - 180.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
R. H. Adamson, J. C. Ly, R. K. Sarai, J. F. Lenz, A. Altangerel, D. Drenckhahn, and F. E. Curry
Epac/Rap1 pathway regulates microvascular hyperpermeability induced by PAF in rat mesentery
Am J Physiol Heart Circ Physiol, March 1, 2008; 294(3): H1188 - H1196.
[Abstract] [Full Text] [PDF]

Home page
 GENES CELLSHome page
H. Komura, H. Ogita, W. Ikeda, A. Mizoguchi, J. Miyoshi, and Y. Takai
Establishment of cell polarity by afadin during the formation of embryoid bodies.
Genes Cells, January 1, 2008; 13(1): 79 - 90.
[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]

Home page
 J. Cell Sci.Home page
T. Ooshio, N. Fujita, A. Yamada, T. Sato, Y. Kitagawa, R. Okamoto, S. Nakata, A. Miki, K. Irie, and Y. Takai
Cooperative roles of Par-3 and afadin in the formation of adherens and tight junctions
J. Cell Sci., July 15, 2007; 120(14): 2352 - 2365.
[Abstract] [Full Text] [PDF]

Home page
 Arterioscler. Thromb. Vasc. Bio.Home page
M. J. Lorenowicz, M. Fernandez-Borja, and P. L. Hordijk
cAMP Signaling in Leukocyte Transendothelial Migration
Arterioscler. Thromb. Vasc. Biol., May 1, 2007; 27(5): 1014 - 1022.
[Abstract] [Full Text] [PDF]

Home page
 J. Cell Sci.Home page
M. R. H. Kooistra, N. Dube, and J. L. Bos
Rap1: a key regulator in cell-cell junction formation
J. Cell Sci., January 1, 2007; 120(1): 17 - 22.
[Abstract] [Full Text] [PDF]

Home page