ADIP, a Novel Afadin- and alpha -Actinin-Binding Protein Localized at Cell-Cell Adherens Junctions*

Masanori AsadaDagger , Kenji IrieDagger , Koji Morimoto§, Akio YamadaDagger , Wataru IkedaDagger , Masakazu Takeuchi§, and Yoshimi TakaiDagger

From the Dagger  Department of Molecular Biology and Biochemistry, Osaka University Graduate School of Medicine/ Faculty of Medicine, Suita 565-0871, Japan and § KAN Research Institute Inc., 93 Chudoji-Awatamachi, Shimogyo-ku, Kyoto 600-8815, Japan

Received for publication, September 25, 2002, and in revised form, November 8, 2002

    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Afadin is an actin filament (F-actin)-binding protein that is associated with the cytoplasmic tail of nectin, a Ca2+-independent immunoglobulin-like cell-cell adhesion molecule. Nectin and afadin strictly localize at cell-cell adherens junctions (AJs) undercoated with F-actin bundles and are involved in the formation of AJs in cooperation with E-cadherin in epithelial cells. In epithelial cells of afadin (-/-) mice and (-/-) embryoid bodies, the proper organization of AJs is markedly impaired. However, the molecular mechanism of how the nectin-afadin system is associated with the E-cadherin-catenin system or functions in the formation of AJs has not yet been fully understood. Here we identified a novel afadin-binding protein, named ADIP (afadin DIL domain-interacting protein). ADIP consists of 615 amino acids with a calculated Mr of 70,954 and has three coiled-coil domains. Northern and Western blot analyses in mouse tissues indicated that ADIP was widely distributed. Immunofluorescence and immunoelectron microscopy revealed that ADIP strictly localized at cell-cell AJs undercoated with F-actin bundles in small intestine absorptive epithelial cells. This localization pattern was the same as those of afadin and nectin. ADIP was undetectable at cell-matrix AJs. ADIP furthermore bound alpha -actinin, an F-actin-bundling protein known to be indirectly associated with E-cadherin through its direct binding to alpha -catenin. These results indicate that ADIP is an afadin- and alpha -actinin-binding protein that localizes at cell-cell AJs and may have two functions. ADIP may connect the nectin-afadin and E-cadherin-catenin systems through alpha -actinin, and ADIP may be involved in organization of the actin cytoskeleton at AJs through afadin and alpha -actinin.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Cells in multicellular organisms recognize their neighboring cells, adhere to them, and form intercellular junctions. Such junctions have essential roles in various cellular functions, including morphogenesis, differentiation, proliferation, and migration (for reviews, see Refs. 1-6). In polarized epithelial cells, intercellular adhesion is mediated through a junctional complex composed of tight junctions (TJs),1 adherens junctions (AJs), and desmosomes. These junctional structures are typically aligned from the apical to basal sides, although desmosomes are independently distributed in other areas.

AJs were originally defined using ultrastructural analysis as closely apposed plasma membrane domains reinforced by a dense cytoplasmic plaque, where actin filament (F-actin) bundles are undercoated (7). Molecular analysis shows that AJs are cell-cell adhesion sites where classic cadherins function as cell adhesion molecules and where the actin-based cytoskeleton and several cytoplasmic components are assembled (8-10). E-cadherin, like other classical cadherins, is a single-pass transmembrane protein whose extracellular domain mediates homophilic recognition and adhesive binding in a Ca2+-dependent manner (10). E-cadherin associates with the actin cytoskeleton through peripheral membrane proteins, including alpha -, beta -, and gamma -catenins, alpha -actinin, and vinculin (9, 11-14). beta -Catenin directly interacts with the cytoplasmic tail of E-cadherin and connects E-cadherin to alpha -catenin that directly binds to F-actin (15). alpha -Actinin and vinculin are also F-actin-binding proteins that directly bind to alpha -catenin (13, 14, 16). The association of E-cadherin with the actin cytoskeleton through these peripheral membrane proteins strengthens the cell-cell adhesion activity of E-cadherin (1, 17).

We have found that another cell-cell adhesion molecule, nectin, and its associated F-actin-binding protein, afadin, strictly localize at AJs undercoated with F-actin bundles (18-20). In contrast, E-cadherin is concentrated at AJs but is more widely distributed from the apical to basal sides of the lateral plasma membranes (2, 21). Nectin is a Ca2+-independent immunoglobulin-like cell-cell adhesion molecule (19, 22-26). Nectin comprises a family, which, at present, consists of four members, nectin-1, -2, -3, and -4. All nectins have two or three splice variants (19, 25-31). Nectin-1 was originally identified as one of the poliovirus receptor-related proteins (PRR-1) (30). Nectin-2 was originally identified as the murine homolog of human poliovirus receptor protein (27) but turned out to be another poliovirus receptor-related protein (PRR-2) (29, 30). Neither PRR-1 nor PRR-2 has thus far been shown to serve as a poliovirus receptor. PRR-1 and PRR-2 were later shown to serve as the receptors for alpha -herpes virus, facilitating their entry and intercellular spread and so were renamed HveC and HveB, respectively (31-36). It remains unknown whether nectin-3 and nectin-4 serve as receptors for viruses. All of the members have an extracellular domain with three immunoglobulin-like loops, a single transmembrane region, and a cytoplasmic region. Furthermore, all of them, except nectin-1beta , -3gamma , and -4, have a conserved motif of 4 amino acid (aa) residues (Glu/Ala-X-Tyr-Val) at their carboxyl termini, and this motif binds the PDZ domain of afadin (18, 19, 25, 26).

Afadin has at least two splice variants, l- and s-afadin (18). l-Afadin, the larger splice variant, binds nectin and, through its F-actin binding domain, F-actin. l-afadin binds to the side of F-actin but does not cross-link it to form bundles. Afadin also has two Ras-associated domains, a forkhead-associated domain, a dilute (DIL) domain, a PDZ domain, and three proline-rich domains (see Fig. 1A). F-actin binds to the region containing the third proline-rich domain (18). s-afadin, the smaller splice variant, has two Ras-associated domains, a forkhead-associated domain, a DIL, a PDZ, and two proline-rich domains, but lacks the F-actin-binding domain. Human s-afadin is identical to the gene product of AF-6, a gene that has been identified as an ALL-1 fusion partner that is involved in acute myeloid leukemias (37). Unless otherwise specified, afadin refers to l-afadin in this paper.

Nectin has a potency to recruit the E-cadherin-beta -catenin complex to the nectin-based cell-cell adhesion sites through afadin and alpha -catenin in fibroblasts and epithelial cells (38-40). Nectin has furthermore a potency to recruit the components of TJs including ZO-1, claudin, occludin, and junctional adhesion molecule (JAM) to the nectin-based cell-cell adhesion sites through afadin in fibroblasts and epithelial cells (39-42). Claudin is a key cell-cell adhesion molecule that forms TJ strands (43-46), and occludin and JAM are other transmembrane proteins at TJs (45-47). Claudin, occludin, and JAM interact with an F-actin-binding scaffold molecule, ZO-1 (48-59). In epithelial cells of afadin (-/-) mice and (-/-) embryoid bodies, the proper organization of AJs and TJs is impaired (60). Nectin-1 has recently been determined by positional cloning to be responsible for cleft lip/palate-ectodermal dysplasia, which is characterized by cleft lip/palate, syndactyly, mental retardation, and ectodermal dysplasia (61). In addition, we have recently found that the nectin-afadin system is involved in the formation of synapses in cooperation with N-cadherin in neurons (62) and that the nectin-afadin system constitutes an important adhesion system in the organization of Sertoli cell-spermatid junctions in the testis (63). Nectin and afadin are therefore important for the organization of a wide variety of intercellular junctions either with or independently of known cell adhesion molecules. However, the molecular mechanism of how the nectin-afadin system is associated with the E-cadherin-catenin system or functions in the formation of these intercellular junctions has not yet been fully understood.

To gain the insight into these issues, we attempted here to identify an afadin-binding protein using yeast two-hybrid screening. For this purpose, we used the DIL domain of afadin as a bait. The DIL domain has been found in afadin and type V myosins including dilute, Myo2, and Myo4, but its function remains unknown (64). The recent finding that the region of Myo4 containing its DIL domain binds to an adapter protein, She3 (65, 66), implies that the DIL domain is involved in the protein-protein interaction. We have identified and characterized here a novel afadin-binding protein, named ADIP (afadin DIL domain-interacting protein), that binds to the DIL domain of afadin. ADIP furthermore binds alpha -actinin, an F-actin-bundling protein known to be indirectly associated with E-cadherin through its direct binding to alpha -catenin (9, 16). These results indicate that ADIP is an afadin- and alpha -actinin-binding protein that localizes at cell-cell AJs and may have two functions. ADIP may connect the nectin-afadin and E-cadherin-catenin systems through alpha -actinin, and ADIP may be involved in organization of the actin cytoskeleton at AJs through afadin and alpha -actinin.

    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
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Yeast Two-hybrid Screening-- One bait vector, pGBDU-afadin (aa 511-981), was constructed by subcloning the insert encoding the aa residue of afadin into pGBDU-C1 (67). Another bait vector, pGBD-mouse ADIP (mADIP)-M (aa 152-436), was constructed by subcloning the insert encoding the aa residue of mADIP into pGBD-C1 (67). The plasmids, pGBDU-MYO4-DIL and pGAD-SHE3, were constructed by subcloning the fragments of MYO4 (aa 920-1471) and SHE3 (aa 1-425) into pGBDU-C2 and pGAD-C2, respectively. Yeast two-hybrid libraries constructed from mouse 11-day embryo, rat brain, rat lung, and human testis cDNAs were purchased from Clontech. Two-hybrid screening using the yeast strain PJ69-4A (MATa trp1-901 leu2-3, 112 ura3-52 his3-200 gal4Delta gal80Delta GAL2-ADE2 LYS2::GAL1-HIS3 met2:: GAL7-lacZ) was performed as described (67). Standard procedures for yeast manipulations were performed as described (68).

Construction of Expression Vectors-- For full-length cDNAs of mADIP and rat ADIP (rADIP), BLAST searches were conducted against GenBankTM and EMBL databases. A selection of hits obtained by BLAST searches against the human subset of GenBankTM and EMBL sequences was used to assemble the cDNA sequence of the human homologue of KIAA0923 (AB023140; GenBankTM/EMBL/DDBJ). A cDNA of KIAA0923 was kindly supplied by Dr. T. Nagase (Kazusa DNA Research Institute). The full-length cDNAs of mADIP (accession number AF532969) and rADIP (accession number AF532970) were generated from mouse and rat brain cDNAs (Clontech), respectively, by reverse transcription-coupled PCR using the following primer sets: for mADIP cDNA, 5'-CGTAGGAGAGTGACAGGAGCTG-3' and 5'-GGTTATCGAGTTTTTCTACATGAC-3'; for rADIP cDNA, 5'-CGTAGGAGAGTGACAGGAGCTG-3' and 5'-TTCCTGTTTTTGCACTGTAGCTG-3'.

The PCR products were subcloned into pCR4B (Invitrogen), and nucleotide sequence analysis was performed by the dideoxynucleotide termination method using a DNA sequencer (model 3100; Applied Biosystems, Inc.). The mammalian expression vectors, pCMV-FLAG, pCMV-T7, and pCMV-HA, were designed to express N-terminal FLAG-, T7-, and hemagglutinin (HA)-tagged proteins, respectively (69). The mammalian expression vector expressing the DIL domain of afadin (aa 606-983) was constructed with pCMV-T7. The mammalian expression vectors expressing mADIP and rADIP, pCMV-HA-mADIP (aa 1-615), pCMV-HA-mADIP-C (aa 339-615), pCMV-FLAG-mADIP-C (aa 339-615), and pCMV-FLAG-mADIP-M (aa 152-436), were constructed with pCMV-FLAG and pCMV-HA. The mammalian expression vector expressing alpha -actinin-1 (human, BC015766; GenBankTM), pCMV-HA-alpha -actinin-1-C (aa 406-892), was constructed with pCMV-HA. The glutathione S-transferase (GST) fusion or maltose-binding protein (MBP) fusion vectors of mADIP and rADIP, MBP-mADIP (aa 1-615), MBP-rADIP-C (aa 159-613), MBP-mADIP-C (aa 339-615), MBP-mADIP-F (aa 121-436), GST-rADIP-C (aa 159-613), GST-mADIP-N (aa 1-226), and GST-mADIP-C (aa 339-615), were constructed with pGEX-KG (70) and pMal-C2 (New England Biolabs). The GST fusion vector of the DIL domain of afadin (aa 606-983), GST-DIL, was constructed with pGEX4T-1 (Amersham Biosciences). The GST fusion vector of the two EF-hand domains of alpha -actinin-1 (aa 406-892), GST-alpha -actinin-1-C, was constructed with pGEX4T-1. The GST and MBP fusion proteins were purified using glutathione-Sepharose beads (Amersham Biosciences) and amylose resin beads (New England Biolabs), respectively.

Antibodies-- The GST fusion proteins with fragments of rADIP-C (aa 159-613), mADIP-N (aa 1-226), and mADIP-C (aa 339-615) were produced in Escherichia coli, purified, and used as each antigen to raise polyclonal antibodies (pAbs) in rabbits. Two pAbs against rADIP-C (aa 159-613) and mADIP-C (aa 339-615), M05 and M01, were used after affinity purification with MBP-rADIP-C (aa 159-613) and MBP-mADIP-C (aa 339-615), respectively. Another pAb against mADIP-N (aa 1-226), M57, was used after affinity purification with GST-mADIP-N (aa 1-226). A mouse anti-afadin monoclonal Ab (mAb) was prepared as described (71). A rat anti-ZO-1 mAb was purchased from Chemicon. A mouse anti-E-cadherin mAb was purchased from Transduction Laboratories. Mouse anti-alpha -actinin, anti-vinculin, and anti-FLAG-M2 mAbs were purchased from Sigma. A rat anti-HA mAb was purchased from Roche Molecular Biochemicals. A rabbit anti-alpha -actinin pAb and a mouse anti-GST mAb were purchased from Santa Cruz. A mouse anti-T7 mAb was purchased from Novagen.

Cell Culture and Transfection-- MDCK cells were kindly supplied by Dr. W. Birchmeier (Max-Delbruck-Center for Molecular Medicine, Berlin, Germany). HEK293 and MDCK were cultured in Dulbecco's modified Eagle's medium with 10% fetal calf serum. HEK293 cells were transfected using the CalPhos mammalian transfection kit (Clontech).

Assay for Co-immunoprecipitation of ADIP with Afadin and alpha -Actinin-- Co-immunoprecipitation experiments using HEK293 cells were performed as follows. HEK293 cells were transfected with the expression plasmids in various combinations. The cells were suspended in 1 ml of Buffer A (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Triton X-100, 1 mM EDTA, 10 µM alpha -phenylmethanesulfonyl fluoride hydrochloride, and 10 µg/ml aprotinin), sonicated for 10 s three times at 10-s intervals, and incubated on ice for 30 min. The cell extract (1.2 mg of protein) was obtained by centrifugation at 100,000 × g for 15 min and then precleared by incubation with protein G-Sepharose 4 Fast Flow beads (Amersham Biosciences). The cell extract was incubated with 20 µl of anti-FLAG M2 mAb-coated protein G-Sepharose 4 Fast Flow beads at 4 °C for 18 h. After the beads were washed with Buffer A, the bound proteins were eluted by boiling the beads in an SDS sample buffer (60 mM Tris-HCl, pH 6.7, 3% SDS, 2% 2-mercaptoethanol, and 5% glycerol) for 10 min. The samples were then subjected to SDS-PAGE, followed by Western blotting with the anti-T7, anti-FLAG, anti-HA, and anti-alpha -actinin Abs.

Co-immunoprecipitation experiments using MDCK cells were performed as follows. MDCK cells on two 10-cm dishes were sonicated in 2 ml of Buffer A, followed by ultracentrifugation at 100,000 × g for 15 min. The cell extract was precleared by incubation with protein A-Sepharose CL-4B beads (Amersham Biosciences) and then incubated with 20 µl of anti-ADIP pAb (M05)-coated protein A-Sepharose CL-4B beads at 4 °C for 18 h. After the beads were washed with Buffer A, the bound proteins were eluted by boiling the beads in the SDS sample buffer for 10 min. The samples were then subjected to SDS-PAGE, followed by Western blotting with the anti-ADIP, anti-afadin, and anti-alpha -actinin Abs.

Assay for the Binding of ADIP to Afadin and alpha -Actinin-- Affinity chromatography using MDCK cells were done as follows: MDCK cells on two 10-cm dishes were sonicated in 2 ml of Buffer A, followed by ultracentrifugation at 100,000 × g for 15 min. The supernatant was incubated with MBP or MBP-mADIP (200 pmol each) immobilized on 20 µl (wet volume) of amylose resin beads (New England Biolabs) at 4 °C for 18 h. After the beads were extensively washed with Buffer A, the bound proteins were eluted by Buffer A containing 20 mM maltose. The eluates were boiled in the SDS sample buffer. The samples were then subjected to SDS-PAGE, followed by Western blotting with the anti-afadin mAb.

Direct binding of ADIP to the DIL domain of afadin or the C-terminal region of alpha -actinin was done as follows: GST-DIL or GST-alpha -actinin-1-C was incubated with MBP or MBP-mADIP-F (200 pmol each) immobilized on 20 µl (wet volume) of amylose resin beads (New England Biolabs) at 4 °C for 1 h. After the beads were extensively washed with PBS, the bound proteins were eluted with PBS containing 20 mM maltose. The eluates were boiled in the SDS sample buffer. The samples were then subjected to SDS-PAGE, followed by Western blotting with the anti-GST mAb.

Other Procedures-- Northern blotting was performed as described (72). The mADIP cDNA fragment was radiolabeled with alpha -32P by a standard random priming method and used to probe a mouse multiple tissue Northern blot (Clontech). Subcellular fractionation of rat liver was performed as described (73). Immunofluorescence microscopy of cultured cells and frozen sections of mouse tissues was performed as described (18, 19). Immunoelectron microscopy of mouse intestine absorptive epithelial cells using the ultrathin cryosection technique was performed as described (18). Protein concentrations were determined with bovine serum albumin as a reference protein (74). SDS-PAGE was performed as described by Laemmli (75).

    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
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Identification of a Novel Afadin-binding Protein-- To identify an afadin-binding protein, we performed the yeast two-hybrid screening using the region of afadin containing the DIL domain (aa 606-983) as a bait (Fig. 1A). We screened 2 × 105 clones of a mouse embryo library and 2 × 105 clones of a rat brain library and obtained 31 and 25 positive clones, respectively. Four mouse clones and one rat clone encoded proteins similar to the carboxyl-terminal portion of human KIAA0923 (AB023140; GenBankTM/EMBL/DDBJ). The full-length clones of these mouse and rat cDNAs were isolated, and they encoded proteins composed of 615 aa with a calculated Mr of 70,954 and 613 aa with a calculated Mr of 70,684, respectively (Fig. 1B). We named this protein ADIP ((afadin DIL domain-interacting protein)). ADIP has three coiled-coil domains (Figs. 1B and 2A). The aa sequences of mADIP and rADIP were 92% identical to each other and 88 and 87% identical to that of human KIAA0923, respectively (Fig. 1B).


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Fig. 1.   Amino acid sequences of ADIP. A, schematic structure of afadin. RA, Ras-associated domain; FHA, forkhead-associated domain. PDZ, PDZ domain; PR, proline-rich domain. B, deduced aa sequences of rat ADIP, mouse ADIP, and human KIAA0923. Asterisks, identical sequences. Putative coiled-coil domains are underlined. C, comparison of molecular masses of native and recombinant ADIP proteins. pCMV-HA-mADIP was transfected to HEK293 cells, and the cell extract was subjected to SDS-PAGE (10% polyacrylamide gel), followed by Western blotting with the anti-ADIP pAb (M57). The extracts of control HEK293 and MDCK cells were similarly subjected to SDS-PAGE, followed by Western blotting with the anti-ADIP pAb (M57). Lane 1, control HEK293 cells (20 µg of protein); lane 2, pCMV-HA-mADIP-transfected HEK293 cells (20 µg of protein); lane 3, MDCK cells (20 µg of protein).


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Fig. 2.   Binding of ADIP to the DIL domain of afadin. A, schematic structure of mouse ADIP. CC, coiled-coil domain. Yeast two-hybrid analysis of the afadin- or alpha -actinin-binding regions of ADIP is shown. ADIP, afadin (DIL domain), and alpha -actinin were constructed into pGBDU or pGAD and co-transformed into the reporter yeast strains. Binding of ADIP to afadin or alpha -actinin, as shown by expression of the HIS3 and ADE2 reporter genes, was monitored by scoring growth on synthetic complete medium lacking histidine and adenine, respectively. +, interacted; -, not interacted; NT, not tested. B, yeast two-hybrid assay showing the specific binding of ADIP to the DIL domain of afadin. Yeast transformants with the indicated plasmids were streaked on synthetic complete medium lacking histidine to score HIS3 reporter activity and incubated for 3 days at 30 °C. The results are the representative of three independent experiments.

To confirm whether the isolated cDNAs encode full-length ADIP, HEK293 cells were transfected with pCMV-HA-mADIP, which expressed HA-tagged full-length mADIP. The cell extract was subjected to SDS-PAGE, followed by Western blotting with the anti-ADIP pAbs. Three anti-ADIP pAbs, M57 against the N-terminal portion of mADIP (aa 1-226), M01 against the C-terminal portion of mADIP (aa 339-615), and M05 against the C-terminal portion of rADIP (aa 159-613), were generated. A protein with a molecular mass of about 78 kDa was detected in the extracts of HEK293 cells expressing HA-mADIP by these three pAbs (Fig. 1C and data not shown). When the extracts of MDCK and HEK293 cells were subjected to SDS-PAGE, followed by Western blotting with the three anti-ADIP pAbs, a protein with a similar molecular mass to that of HA-tagged ADIP was detected (Fig. 1C and data not shown). Therefore, we concluded that the isolated cDNA encodes full-length ADIP.

In Vitro and in Vivo Binding of ADIP to Afadin-- Two-hybrid analysis revealed that ADIP specifically bound to the DIL domain of afadin but not to the DIL domain of yeast Myo4 (Fig. 2B). The region containing the third coiled-coil domain (aa 339-436) of ADIP was required for the binding to the DIL domain of afadin (Fig. 2A). We further confirmed the in vitro and in vivo binding of ADIP to afadin. First, we performed the immunoprecipitation analysis. HEK293 cells were co-transfected with the T7-tagged DIL domain of afadin (T7-DIL; aa 606-983) and the FLAG-tagged C-terminal portion of ADIP containing the third coiled-coil domain (FLAG-mADIP-C; aa 339-615), which was isolated in the two-hybrid screening. When FLAG-mADIP-C was immunoprecipitated from the cell extract with the anti-FLAG mAb, T7-DIL was co-immunoprecipitated, as detected by Western blotting with the T7 mAb (Fig. 3A). Second, we performed the affinity chromatography using MDCK cells. The extract of MDCK cells expressing endogenous afadin was incubated with an MBP-fusion protein of full-length mADIP (aa 1-615) immobilized on amylose-resin beads. After the beads were washed with the lysis buffer, the bound proteins were eluted, and the eluate was subjected to SDS-PAGE, followed by Western blotting with the anti-afadin mAb. Afadin indeed bound to MBP-mADIP, but not to MBP alone (Fig. 3B). Third, we examined whether ADIP directly interacted with afadin. A GST fusion protein of the DIL domain of afadin (GST-DIL) bound to an MBP-fusion protein of the region of ADIP containing the all three coiled-coil domains (aa 121-436) (MBP-mADIP-F) immobilized on amylose resin beads. GST-DIL bound to MBP-mADIP-F, but not to MBP alone (Fig. 3C). Finally, to confirm that ADIP binds to afadin in vivo, we examined whether endogenous afadin was co-immunoprecipitated with endogenous ADIP from the extract of MDCK cells. When endogenous ADIP was immunoprecipitated from the extract of MDCK cells with anti-ADIP pAb, endogenous afadin was co-immunoprecipitated (Fig. 3D). Afadin was not co-immunoprecipitated with control IgG. These results together with the two-hybrid analysis indicate that ADIP directly binds to afadin both in vitro and in vivo.


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Fig. 3.   In vitro and in vivo binding of ADIP to afadin. A, co-immunoprecipitation of the T7-tagged DIL domain of afadin with the FLAG-tagged mADIP-C. Expression vectors were transfected into HEK293 cells as indicated. The T7-tagged DIL domain of afadin was specifically co-immunoprecipitated with the FLAG-tagged mADIP-C, as is shown by Western blotting with the anti-T7 and anti-FLAG mAbs. B, in vitro binding of afadin to MBP-mADIP. The extract of MDCK cells was incubated with either MBP or MBP-mADIP (full-length) immobilized on amylose resin beads. The beads were then subjected to SDS-PAGE (10% polyacrylamide gel), followed by Western blotting with the anti-afadin mAb. C, in vitro binding of the DIL domain of afadin to mADIP. The purified GST-DIL protein was incubated with either MBP or MBP-mADIP-F (aa 121-436) immobilized on amylose resin beads. The eluates were then subjected to SDS-PAGE (10% polyacrylamide gel), followed by Western blotting with the anti-GST mAb. D, co-immunoprecipitation of endogenous afadin with endogenous ADIP from MDCK cells. The extracts of MDCK cells were immunoprecipitated (IP) with the anti-ADIP pAb (M05) and analyzed by Western blotting (IB) with the anti-ADIP pAb (M05) and the anti-afadin mAb. The results are the representative of three independent experiments.

Tissue and Subcellular Distribution of ADIP-- Northern blot analysis using the fragment of mADIP cDNA (bp 552-3194) as a probe detected an ~4.3-kb mRNA in all the mouse tissues examined, including heart, brain, spleen, lung, liver, skeletal muscle, kidney, and testis (Fig. 4A). The smaller band (~3.0 kb) was also detected in the liver and the testis (Fig. 4A, lanes 5 and 8). Western blot analysis using the anti-ADIP pAb, M57, detected an ~78-kDa protein, which was the same size of ADIP detected in HEK293 and MDCK cells and in mouse spleen, lung, and kidney (Fig. 4B, lanes 3, 4, 7, and 9). The smaller bands (~60 kDa in heart, ~76 and ~40 kDa in testis) were also detected, suggesting that smaller splice variants of ADIP may be expressed (Fig. 4B, lanes 1 and 8). Subcellular fractionation analysis of ADIP in rat liver indicated that it was enriched in the fraction rich in AJs and TJs, where afadin and E-cadherin were also enriched (Fig. 4C, lanes 4 and 5). The reason why the Western blot analysis of tissue distribution did not detect the ~78-kDa protein in the liver (Fig. 4B, lanes 5) was probably its low expression level. These results indicate that ADIP is widely expressed, although its expression levels vary depending on tissues.


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Fig. 4.   Tissue and subcellular distribution of ADIP. A, Northern blotting. Mouse RNA blot membranes (Clontech) were hybridized with the 32P-labeled fragment (bp 552-3194) of the ADIP cDNA, followed by autoradiography. Lane 1, heart; lane 2, brain; lane 3, spleen; lane 4, lung; lane 5, liver; lane 6, skeletal muscle; lane 7, kidney; lane 8, testis (short exposed). B, Western blotting. The homogenates of various mouse tissues (30 µg of protein each) were subjected to SDS-PAGE (10% polyacrylamide gel), followed by Western blotting with the anti-ADIP pAb (M57). Lane 1, heart; lane 2, brain; lane 3, spleen; lane 4, lung; lane 5, liver; lane 6, skeletal muscle; lane 7, kidney; lane 8, testis; lane 9, MDCK cells. C, subcellular distribution of ADIP in rat liver. Subcellular fractionation of rat liver was performed, and each fraction (30 µg of protein each) was subjected to SDS-PAGE (10% polyacrylamide gel), followed by Western blotting with the anti-ADIP pAb (M57), the anti-afadin mAb, or the E-cadherin mAb. Lane 1, the homogenate fraction; lane 2, the soluble fraction; lane 3, the pellet fraction; lane 4, the fraction rich in bile canaliculi; lane 5, the fraction rich in AJs and TJs. The results are representative of three independent experiments.

Co-localization of ADIP with Afadin at AJs in Epithelial Cells-- Since afadin has been shown to strictly localize at AJs undercoated with F-actin bundles (18), we examined by immunofluorescence microscopy whether ADIP co-localized with afadin at AJs in MDCK cells. ADIP and afadin co-localized at the sites of cell-cell contacts (Fig. 5A). The cross-sectional analysis using a confocal microscopy revealed that ADIP co-localized with afadin at the junctional complex region (data not shown). Thus, ADIP and afadin co-localized at the cell-cell junctions. Essentially the same results were obtained with three anti-ADIP pAbs, M57, M01, and M05 (data not shown). ADIP and afadin were also co-stained at the perinuclear regions, most presumably the Golgi complex, as estimated by the co-staining with Golgi 58-kDa protein, a marker for the Golgi complex (Fig. 5A and data not shown), but the physiological significance of this staining is currently unknown.


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Fig. 5.   Localization of ADIP at AJs in MDCK cells and mouse small intestine absorptive epithelial cells. A, MDCK cells were doubly stained with the anti-ADIP (M05) and anti-afadin Abs. B, the frozen sections were triple-stained with the anti-ADIP pAb (M01) and the anti-afadin and anti-ZO-1 mAb. C, left column, the frozen sections were double-stained with the anti-ADIP pAb (M01) and the anti-afadin mAb; right column, the frozen sections were double-stained with the anti-ADIP pAb (M01) and the anti-ZO-1 mAb. Arrowhead, the ADIP signal co-localized with the afadin signal; arrow, the ADIP signal localized at a slightly more basal side than the ZO-1 signal; bars, 10 µm. The results are representative of three independent experiments.

To examine the precise localization of ADIP at the junctional complex region, the frozen sections of small intestine were triple-stained with the anti-ADIP pAb, the anti-afadin mAb, and the anti-ZO-1 mAb, since TJs and AJs are well separated in this cell type (49). ZO-1 is known to be a marker for TJs (48, 49). Immunofluorescence microscopy revealed that ADIP co-localized with afadin but localized at the slightly more basal side than ZO-1 in the absorptive epithelia (Fig. 5, B and C, arrows). A signal for ADIP was also observed in the microvilli, but its significance is not clear. It may be nonspecific staining. Finally, immunoelectron microscopy revealed that ADIP was exclusively localized at AJs undercoated with F-actin bundles (Fig. 6). The signal for ADIP was rarely observed at TJs and desmosomes. This localization pattern of ADIP was the same as those of afadin and nectin but was different from that of E-cadherin, which is concentrated at AJs but is more widely distributed from the apical to basal sides of the lateral plasma membranes (18, 19). These results indicate that ADIP strictly co-localizes with afadin and nectin at cell-cell AJs undercoated with F-actin bundles.


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Fig. 6.   Ultrastructural localization of ADIP in mouse small intestine absorptive epithelial cells. The mouse small intestine absorptive epithelial cells were labeled with the anti-ADIP pAb (M01) using the ultrathin cryosection technique. Upper right inset, the image at a high magnification; DS, desmosome; bar, 0.1 µm.

The ADIP signal was not detected at focal adhesions where the vinculin signal was detected in MDCK cells (Fig. 7A). In the heart, focal adhesions, named costameres, are well developed and periodically located along the lateral borders of cardiac muscle cells (76). Vinculin localizes at costameres, whereas nectin and afadin do not (18, 19). The ADIP signal was not detected at costameres where the vinculin signal was detected (Fig. 7B, arrows). Both the ADIP and vinculin signals were detected at the intercalated discs, corresponding to cell-cell AJs (Fig. 7B, arrowheads). In addition to the signals for ADIP and vinculin at intercalated discs, these signals were also observed in stripes within the cardiac muscle cells, but their significance is not clear. They may be nonspecific staining. These results indicate that ADIP does not localize at cell-matrix junctions.


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Fig. 7.   Absence of ADIP at cell-matrix junctions in MDCK cells and mouse cardiac cells. A, MDCK cells were doubly stained with the anti-ADIP (M05) and anti-vinculin Abs. MDCK cells in this figure are less confluent than those in Fig. 5A. Bars, 10 µm. B, the frozen sections were double-stained with the anti-ADIP pAb (M01) and the anti-vinculin mAb. Arrowheads, intercalated disc; arrows, costamere; bar, 50 µm. The results are the representative of three independent experiments.

Binding of ADIP to alpha -Actinin-- To gain insight into the function of ADIP, we attempted to identify an ADIP-binding protein(s). As described above, ADIP has three coiled-coil domains, and the region containing the third coiled-coil domain (aa 339-436) of ADIP is required for the binding to the DIL domain of afadin (Fig. 2A). We performed the yeast two-hybrid screening using the region of ADIP containing all three coiled-coil domains (aa 152-436) as a bait (Fig. 2A). We screened 7 × 105 clones of a rat lung library and obtained 18 positive clones. Nine clones encoded the C-terminal portion of alpha -actinin-1 (aa 406-892; human, BC015766; GenBankTM). alpha -Actinin is a well characterized protein that shows F-actin-cross-linking activity (77). Four isoforms of human alpha -actinin have been identified: nonmuscle actinin-1 and actinin-4 and muscle actinin-2 and actinin-3 (78-81). ADIP bound alpha -actinin-2 in addition to alpha -actinin-1 as estimated by yeast two-hybrid analysis (data not shown), indicating that the binding of ADIP to alpha -actinin is not specific for alpha -actinin-1. Two-hybrid analysis revealed that the region containing only the first coiled-coil domain (aa 1-226) of ADIP bound alpha -actinin-1 (Fig. 2A) and that the two C-terminal EF-hand motifs of alpha -actinin-1 (aa 740-892) were required for its binding to ADIP (data not shown). These results indicate that the first coiled-coil domain of ADIP binds to the EF-hand motifs of alpha -actinin-1.

To confirm the binding of ADIP to alpha -actinin-1 in vivo, we performed the immunoprecipitation analysis. HEK293 cells were co-transfected with the HA-tagged C-terminal portion of alpha -actinin-1 (HA-alpha -actinin-1-C; aa 406-892) and FLAG-tagged ADIP (FLAG-mADIP-M; aa 152-436). When FLAG-tagged ADIP was immunoprecipitated from the cell extract with the anti-FLAG mAb, HA-alpha -actinin-1-C was co-immunoprecipitated, as detected by Western blotting with the HA mAb (Fig. 8A). When FLAG-mADIP-M was immunoprecipitated with the anti-FLAG Ab from the extract of HEK293 cells transiently expressing the FLAG-tagged mADIP (FLAG-mADIP-M; aa 152-436) alone, endogenous alpha -actinin was co-immunoprecipitated (Fig. 8B). We next examined whether alpha -actinin-1 directly interacted with ADIP in vitro. A GST fusion protein of the C-terminal region of alpha -actinin-1 (GST-alpha -actinin-1-C) bound to an MBP fusion protein of the region of ADIP containing all three coiled-coil domains (aa 121-436) (MBP-mADIP-F) immobilized on amylose resin beads. GST-alpha -actinin-1-C bound to MBP-mADIP-F but not to MBP alone (Fig. 8C). Finally, when endogenous ADIP was immunoprecipitated from the extract of MDCK cells with the anti-ADIP pAb, endogenous alpha -actinin was co-immunoprecipitated (Fig. 8D). alpha -Actinin was not co-immunoprecipitated with control IgG. These results indicate that ADIP directly binds alpha -actinin in vivo and in vitro.


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Fig. 8.   In vivo binding of ADIP to alpha -actinin. A, co-immunoprecipitation (IP) of the HA-tagged alpha -actinin-1-C with the FLAG-tagged mADIP-M. Expression vectors were transfected into HEK293 cells as indicated. The HA-tagged alpha -actinin-1-C was specifically co-immunoprecipitated with the FLAG-tagged mADIP-M, as is shown by Western blotting with the anti-HA and anti-FLAG mAbs. B, co-immunoprecipitation of endogenous alpha -actinin with the FLAG-tagged mADIP-M. Expression vectors were transfected into HEK293 cells as indicated. Endogenous alpha -actinin-1 was specifically immunoprecipitated with FLAG-tagged mADIP-M, as is shown by Western blotting (IB) with the anti-alpha -actinin pAb and the anti-FLAG mAb. C, in vitro binding of alpha -actinin-1 to mADIP. The purified protein of GST-alpha -actinin-1-C (aa 406-892) was incubated with either MBP or MBP-mADIP-F (aa 121-436) immobilized on amylose resin beads. The eluates were then subjected to SDS-PAGE (10% polyacrylamide gel), followed by Western blotting with the anti-GST mAb. D, co-immunoprecipitation of endogenous alpha -actinin and endogenous afadin with endogenous ADIP from MDCK cells. The extracts of MDCK cells were immunoprecipitated with the anti-ADIP pAb (M05) and analyzed by Western blotting with the anti-ADIP (M05), anti-alpha -actinin, and anti-afadin Abs. The results are representative of three independent experiments.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We have isolated here a novel afadin-binding protein and named it ADIP. Several lines of evidence suggest that ADIP binds to afadin in vivo at cell-cell AJs: 1) ADIP binds to afadin, as estimated by the yeast two-hybrid analysis and co-immunoprecipitation from the extracts of cells exogenously expressing the fragments of ADIP; 2) recombinant ADIP directly binds to the DIL domain of recombinant afadin in a cell-free system; 3) endogenous ADIP and afadin are co-immunoprecipitated from the extracts of MDCK cells; and 4) ADIP co-localizes with afadin and nectin at cell-cell AJs. Several proteins, including E-cadherin, alpha -catenin, beta -catenin, and alpha -actinin, are concentrated at AJs but more widely distributed from the apical to basal sides of the lateral plasma membranes (2, 21). Four proteins (vinculin, afadin, nectin, and ponsin) strictly localize at AJs, which are defined using ultrastructural analysis as closely apposed plasma membrane domains reinforced by a dense cytoplasmic plaque to which F-actin bundles attach (7, 18, 19, 82), whereas vinculin and ponsin furthermore localize at cell-matrix junctions (82). Ponsin is the afadin- and vinculin-binding protein containing three Src homology 3 domains (82). ADIP is the fifth protein that strictly localizes at AJs.

We have furthermore shown that ADIP binds alpha -actinin. Our results suggest that ADIP binds to alpha -actinin in vivo. 1) ADIP binds to alpha -actinin as estimated by the yeast two-hybrid analysis and co-immunoprecipitation from the extracts of HEK293 cells exogenously expressing the full-length ADIP or fragments; 2) recombinant ADIP directly binds to recombinant alpha -actinin in a cell-free system; and 3) endogenous ADIP and alpha -actinin are co-immunoprecipitated from the extracts of MDCK cells.

What is the function of ADIP? Our study indicates that ADIP binds two F-actin-binding proteins, afadin and alpha -actinin. Afadin binds along the sides of F-actin but does not have a marked F-actin-cross-linking activity (18). alpha -Actinin has an F-actin-cross-linking activity (77). Thus, ADIP links afadin to alpha -actinin and functions in the formation of the specialized actin structure at cell-cell AJs. Recently, we have shown that the heterotypic trans-interaction between nectin-2 in Sertoli cells and nectin-3 in spermatids is formed at Sertoli cell-spermatid junctions, heterotypic AJs in the testis, and that each nectin-based adhesive membrane domain exhibits one-to-one co-localization with each actin bundle underlying Sertoli cell-spermatid junctions (63). Afadin also co-localizes with nectin at Sertoli cell-spermatid junctions. Our present finding that ADIP binds both afadin and alpha -actinin suggests that afadin, together with ADIP and alpha -actinin, functions in the formation of the actin bundle at the nectin-based cell-cell adhesion sites. Since it has been reported that E-cadherin also associates with alpha -actinin through alpha -catenin (9, 16), two cell-cell adhesion systems at AJs, the nectin-afadin and cadherin-catenin systems, are connected to actin cytoskeleton through alpha -actinin. We have previously shown that nectin has a potency to recruit the E-cadherin-beta -catenin complex to the nectin-based cell-cell adhesion sites through afadin and alpha -catenin in fibroblasts and epithelial cells (38-40). Our finding that ADIP binds both afadin and alpha -actinin suggests that ADIP serves as a linker between the nectin-afadin and cadherin-catenin systems.

We originally isolated afadin as an F-actin-binding protein (18). We subsequently found that afadin binds nectin and ponsin (18, 19) and furthermore isolated here another afadin-binding protein, ADIP, which bound alpha -actinin in this study (Fig. 9). Afadin binds these proteins through different regions; nectin binds to the PDZ domain (19), ponsin binds to the third proline-rich domain (82), and ADIP binds to the DIL domain (Fig. 9). It has furthermore been reported that afadin directly binds alpha -catenin, although its binding is not strong (38, 83). In addition, the splice variant of afadin, AF-6, binds Rap1 and profilin through the Ras-associated domain and the carboxyl-terminal region, respectively (84), suggesting that afadin also binds them. Thus, the F-actin-binding protein, afadin, may serve as a scaffold molecule to organize various proteins including other actin-binding proteins, alpha -catenin, alpha -actinin, and profilin, at the nectin-based cell-cell adhesion sites (Fig. 9). The finding that the proper organization of AJs and TJs is impaired in epithelial cells of afadin (-/-)-mice and (-/-)-embryoid bodies (60), suggests that this afadin-based organization of the various proteins is important for the formation of AJs and TJs. It is of crucial importance to clarify the molecular mechanism of this organization in the formation of the junctional complex in epithelial cells.


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Fig. 9.   A schematic model for the interactions between the nectin-afadin and cadherin-catenin systems at AJs. Double arrows indicate direct interactions. F-actin and peripheral membrane proteins shown in the bottom cell are omitted in the top cell. See details under "Discussion."


    ACKNOWLEDGEMENTS

We thank Dr. T. Nagase for providing the cDNA of KIAA0923 and Dr. W. Birchmeier for providing the MDCK cells.

    FOOTNOTES

* The investigation at Osaka University Medical School was supported by grants-in-aid for Scientific Research and for Cancer Research from the Ministry of Education, Culture, Sports, Science, and Technology, Japan (2001 and 2002).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the DDBJ/GenBankTM/EBI Data Bank with accession number(s) AF532969 and AF532970.

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

Published, JBC Papers in Press, November 21, 2002, DOI 10.1074/jbc.M209832200

    ABBREVIATIONS

The abbreviations used are: TJ, tight junction; aa, amino acid(s); Ab, antibody; AJs, adherens junctions; DIL, dilute domain; F-actin, actin filament; GST, glutathione S-transferase; HA, hemagglutinin; mAb, monoclonal antibody; mADIP, mouse ADIP; MBP, maltose-binding protein; pAb, polyclonal antibody; rADIP, rat ADIP; PRR, poliovirus receptor-related protein; JAM, junctional adhesion molecule; MDCK, Madin-Darby canine kidney.

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