MOLECULAR AND CELLULAR BIOLOGY, June 2003, p. 4267–4282
0270-7306/03/$08.00⫹0 DOI: 10.1128/MCB.23.12.4267–4282.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Vol. 23, No. 12

JAM4, a Junctional Cell Adhesion Molecule Interacting with a Tight
Junction Protein, MAGI-1
Susumu Hirabayashi, Makiko Tajima, Ikuko Yao, Wataru Nishimura,
Hiroki Mori, and Yutaka Hata*
Department of Medical Biochemistry, Graduate School of Medicine, Tokyo Medical and
Dental University, Bunkyo-ku, Tokyo 113-8519, Japan
Received 9 December 2002/Returned for modification 30 January 2003/Accepted 26 March 2003

MAGI-1 is a membrane-associated guanylate kinase protein at tight junctions in epithelial cells. It interacts
with various molecules and functions as a scaffold protein at cell junctions. We report here a novel MAGI-1binding protein that we named junctional adhesion molecule 4 (JAM4). JAM4 belongs to an immunoglobulin
protein family. JAM4 was colocalized with ZO-1 in kidney glomeruli and in intestinal epithelial cells. Biochemical in vitro studies revealed that JAM4 bound to MAGI-1 but not to ZO-1, whereas JAM1 did not bindto MAGI-1. JAM4 and MAGI-1 interacted with each other and formed clusters in COS-7 cells when coexpressed. JAM4 mediated calcium-independent homophilic adhesion and was accumulated at cell-cell contacts
when expressed in L cells. MAGI-1, ZO-1, and occludin were recruited to JAM4-based cell contacts. JAM4 also
reduced the permeability of CHO cell monolayers. MAGI-1 strengthened JAM4-mediated cell adhesion in L
cells and sealing effects in CHO cells. These findings suggest that JAM4 together with MAGI-1 provides anadhesion machinery at tight junctions, which may regulate the permeability of kidney glomerulus and small
intestinal epithelial cells.
The human MAGI-1 is called brain angiogenesis inhibitor
1-associated protein 1 (37). The synaptic scaffolding molecule(S-SCAM) is the neural isoform of MAGI-1 (also called
MAGI-2, atrophin-interacting protein, and activin receptorinteracting protein) and interacts with various molecules atsynapses (20, 38, 40). MAGI-1 is localized at TJs in epithelial
cells (21). Like S-SCAM, MAGI-1 binds various molecules,
including ␤-catenin, mNET1, RapGEP, synaptopodin, ␣-actinin-4, and megalin (9, 10, 30, 35, 36). ␤-Catenin is presumably
involved in the polarized targeting of MAGI-1 to lateral membranes of epithelial cells (10, 32). Because ␤-catenin is not
localized at TJs in polarized epithelial cells, the interaction
with ␤-catenin is not sufficient for the recruitment of MAGI-1
to TJs. mNET1 and RapGEP are regulators for small GTPbinding proteins, and MAGI-1 may accumulate these molecules to TJs. In kidneys, MAGI-1 is highly expressed in podocytes (36). Synaptopodin and ␣-actinin-4 are actin-binding
proteins and are expressed in podocytes (31, 36). MAGI-1 may
be linked to the actin cytoskeleton through these proteins in
podocytes. Among MAGI-1-interacting proteins, only megalin
is an integral membrane protein. Megalin is a transmembrane
endocytic receptor glycoprotein which belongs to the low-density lipoprotein receptor family (for reviews, see reference 6).
In kidneys, megalin and MAGI-1 are colocalized in podocytes,
where MAGI-1 may modulate the endocytic activity of megalin. However, megalin is localized on apical membranes inpolarized epithelial cells and is unlikely to interact with
MAGI-1 at TJs.
In this study, we tried to identify an integral membranecomponent of TJs that interacts with MAGI-1 and found anovel transmembrane protein. The protein is similar to JAM1
in molecular structure, and we call this molecule JAM4. JAM4
has cell adhesion activity. We have also discovered that the celladhesion activity of JAM4 is regulated by MAGI-1.

Tight junctions (TJs) play essential roles in the maintenanceof a physical barrier between external and internal environments in the body (for reviews, see references 2 and 39).
Recent studies have revealed the molecular architecture of
TJs. Like other cell junctions, TJs have integral membranecomponents and membrane-associated proteins. Occludin andclaudin are integral membrane proteins involved in the formation of TJ strands (14, 15, 16). In addition, junctional adhesion
molecule 1 (JAM1) is an integral membrane protein of TJs(29). JAM1 belongs to the immunoglobulin (Ig) superfamily
and does not directly constitute TJ strands. The interaction
between membrane-associated proteins and integral membrane proteins might be crucial for the organization of TJs.
The most representative membrane-associated proteins at TJs
are ZO-1 and its isoforms, ZO-2 and ZO-3 (1, 18, 24). ZO-1,-2, and -3 belong to membrane-associated guanylate kinases(MAGUKs) and bind to the C termini of claudins (5, 22; forreviews, see reference 13). ZO-1 also binds to JAM1 (11). Theinteractions are mediated by PDZ domains and PDZ-binding
motifs. Similarly, MUPP1, another TJ component that has 13
PDZ domains, binds to claudin-1 and JAM1 (17). Mammalian
homologue of PAR-3 is also concentrated at TJs, and its PDZ
domain binds to JAM1 (12, 23).
TJs have another member of the MAGUK family, MAGUKwith inverted domain structure (MAGI-1) (8). MAGI-1 wasoriginally identified as a protein interacting with Ki-ras in rats.
It has a unique molecular organization composed of one guanylate kinase domain, two WW domains, and six PDZ domains.

* Corresponding author. Mailing address: Department of Medical
Biochemistry, Graduate School of Medicine, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8519, Japan.
Phone: 81 3 5803 5164. Fax: 81 3 5803 0121. E-mail: yuhammch@med.tmd.ac.jp.
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VOL. 23, 2003

MAGI-1-BINDING JUNCTIONAL ADHESION MOLECULE
MATERIALS AND METHODS

Construction of expression vectors. Using 5⬘-ACGCGTTACAGAGATTTA
CCTGCC-3⬘ and 5⬘-GTCGACTACACTAAAGTCACATTTCT-3⬘ and 5⬘-AAG
CTTGTACAAGGCAAGGGTTCGG-3⬘ and 5⬘-GTCGACTACACCAGGAACGACGAGGT-3⬘ as primers, cDNAs of mouse JAM1 and mouse JAM4,
respectively, were obtained by PCR on mouse lung cDNA (BioChain, Inc.) andkidney cDNA (Clontech). pGex4T-1 and pFLAG-CMV-1 were purchased fromAmersham Pharmacia Biotech and Eastman Kodak Company, respectively. A
linker (5⬘-AGCTTCGACGCGTAGGATCCTCGCGAATGCATGTCGACGC
GGCCGC-3⬘ and 5⬘-GATCGCGGCCGCGTCGACATGCATTCGCGAGGATCCTACGCGTCGA-3⬘) was ligated into HindIII/BamHI sites of pLGFPN(Clontech) to generate pLGFPN-2, which was subsequently digested with NotI
and religated to generate pLN. pMXpuro Myc was generated by ligating a linker(5⬘-GATCCGCCCCACCATGGAGCAGAAACTTATCAGCGAGGAGGAC
CTGACGCGTGGATTCG-3⬘ and 5⬘-AATTCGAATCCACGCGTCAGGTCC
TCCTCGCTGATAAGTTTCTGCTCCATGGTGGGGC-3⬘) into BamHI/EcoRI
sites of pMXpuro. PCR was performed on pLGFPC, with 5⬘-GGATCCCTACCGGTCGCCACCATGGTG-3⬘ and 5⬘-GTCGACGGTACCACGCGTCGAGA
TCTGAGTCCGGCCGGACT-3⬘ as primers. The PCR product was digested
with BamHI/SalI and ligated into the BglII/SalI site of pShuttle cytomegalovirus(CMV) (a gift of Tong-Chuan He and Bert Vogelstein, Johns Hopkins Oncology
Center) to generate pShuttle CMV green fluorescent protein (GFP). The following constructs expressed the corresponding amino acids: pLN JAM4, aminoacids 1 to 370 of JAM4; pLN JAM4⌬C, 1 to 361 of JAM4; pFLAG JAM4, 21 to
370 of JAM4; pFLAG JAM4⌬C, 21 to 361 of JAM4; pGex4T-1 JAM4-C, 262 to
370 of JAM4; pFLAG JAM1, 25 to 300 of JAM1; pClneo Myc MAGI-1, pMXpuro Myc MAGI-1, and pShuttle CMV GFP MAGI-1, 1 to 1256 of human
MAGI-1; pBTM116 MAGI-1-8, 429 to 1256 of human MAGI-1; pGexKG SSCAM-1, 303 to 405 of rat S-SCAM; pGex4T-1 S-SCAM-7, 1 to 421 of rat
S-SCAM; pClneo Myc CASK, 1 to 909 of rat CASK; pClneo Myc ZO-1-1, 1 to920 of mouse ZO-1; and pClneo Myc Lin-7, 1 to 207 of rat Lin-7A. Using
5⬘-ACGCGTTAATTCACCATGTCTGCAC-3⬘ and 5⬘-CTCGAGAAGCTGGC
CGCGTACCC-3⬘ as primers, PCRs were performed on pFLAG JAM4, pFLAG
JAM4⌬C, and pFLAG JAM1. PCR products were digested with MluI/SalI andligated into MluI/SalI sites of pLN to generate pLN FLAG JAM4, pLN FLAG
JAM4⌬C, and pLN FLAG JAM1. MluI/SalI fragments from pLN FLAG JAM4
were ligated into the same sites of pClneo to generate pClneo FLAG JAM4 forin vitro transcription and translation. Throughout this study, we use the term
MAGI-1 to describe all constructs that are derived from human brain angiogenesis inhibitor protein 1-associated protein 1. The constructs of JAM4 used in thisstudy are summarized in Fig. 1E.
Antibodies. The rabbit anti-JAM4 antibody was raised against the product ofpGex4T-1 JAM4-C. The rabbit antibody against the product of pGexKG SSCAM-1 was used for the immunoprecipitation of MAGI-1. For immunohistochemistry, the affinity-purified rabbit antibody against the product of pGex SSCAM-7 was used to detect MAGI-1. The rabbit anti-ERBIN and the sheep
anti-Myc antibodies were described previously (33). The rat monoclonal antioccludin antibody was a gift from Shoichiro Tsukita (Kyoto University). Mouseanti-Myc-tag monoclonal antibody 9E10 was obtained from the American Type
Culture Collection. Mouse and rabbit anti-ZO-1 antibodies (Zymed), mouseanti-␤-catenin antibody (BD Transduction), mouse anti-GFP antibody (Santa
Cruz), mouse anti-FLAG antibody (Sigma-Aldrich Fine Chemicals), rat antiZO-1 antibody (Chemicon), and rhodamine-conjugated, fluorescein isothiocya-

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nate (FITC)-conjugated, and Cy5-conjugated second antibodies for dual labeling(Chemicon) were purchased from commercial sources.
Cells. Phoenix ampho, mouse mammary gland epithelial MTD-1A, MadinDarby canine kidney II (MDCK), mouse fibroblastic L, COS-7, and porcine
kidney LLC-PK1 cells were grown in cultures in Dulbecco modified Eagle medium supplemented with 10% fetal bovine serum, 100 U of penicillin/ml, and 100
␮g of streptomycin/ml under 5% CO2 at 37°C. CHO cells were grown in culturesin the medium containing 2 mM L-glutamine. COS-7 cells were transfected witha DEAE-dextran method. L cells were transfected with Effectene transfection
reagent (Qiagen). A retrovirus system was used to generate stable transformants
of MDCK, L, CHO, and LLC-PK1 cells. Briefly, Phoenix ampho cells weretransfected using a calcium phosphate coprecipitation method with pLN FLAG
JAM4, pLN FLAG JAM4⌬C, pLN JAM4, pLN JAM4⌬C, or pMXpuro Myc
MAGI-1. The medium was collected 48 h later as a virus stock. MDCK, L, CHO,
and LLC-PK1 cells were grown in cultures in the medium containing retrovirus
for 48 h and then in selective medium with 1 mg of G418 (Calbiochem)/ml or 2
␮g of puromycin (Invitrogen)/ml. To generate L-FLAG-JAM4/Myc-MAGI-1
and L-FLAG-JAM4⌬C/Myc–MAGI-1 cells, L-FLAG-JAM4 and L-FLAGJAM4⌬C cells were infected with retrovirus to express Myc–MAGI-1 and selected by G418 and puromycin. The resulting resistant colonies were tested forthe expression of each protein and cloned.
Collagen culture. L-FLAG-JAM4, L-FLAG-JAM4⌬C, L-FLAG-JAM4/Myc–
MAGI-1, and L-FLAG-JAM4⌬C/Myc–MAGI-1 cells were isolated with trypsin
and triturated into a single-cell suspension. Cells were diluted to 104 cells/ml ina type I collagen-neutral solution (Cellegen, Koken, Tokyo). The cultures wereincubated at 37°C to allow collagen to gel and then added with the medium. Cells
were grown in cultures and observed by confocal microscopy (Olympus EV
300-BX).
Yeast two-hybrid screening. Yeast two-hybrid screening was performed using
a human lung cDNA library (Clontech) and yeast strain L40. Histidine selection
plates contained 8 mM 3-amino-1,2,4-triazole, 180 mg of isopropyl-␤-D-1-thiogalactopyranoside/liter, and 360 mg of 5-bromo-4-chloro-3-indolyl-␤-D-galactopyranoside/liter. After 6 days of incubation, blue colonies were picked up for
further analysis.
Immunofluorescence microscopy. COS-7, L, and MDCK cells were fixed with
4% (wt/vol) formaldehyde in phosphate-buffered saline (PBS) at room temperature for 15 min and blocked with 50 mM glycine in PBS for 30 min. MTD-1A
cells were fixed with cold methanol for 15 min at ⫺20°C. The samples wereincubated with 0.2% (wt/vol) Triton X-100 in PBS for 15 min and subsequently
with 1% (wt/vol) bovine serum albumin (BSA) in PBS at room temperature for
30 min. The samples were incubated with various first antibodies and visualizedwith rhodamine-, FITC-, or Cy5-conjugated second antibodies. The images wereobtained by confocal microscopy (Zeiss LSM 510).
Immunohistochemistry. All procedures related to the care and treatment ofanimals were in accordance with institutional and National Institutes of Healthguidelines. Wistar rats (4 weeks old) were deeply anesthetized with pentobarbital
sodium (60 mg/kg of body weight, intraperitoneally administered) and perfusedwith 4% (wt/vol) paraformaldehyde in 0.1 M phosphate buffer (PB; pH 7.4).
Kidneys and small intestines were removed and postfixed in the same fixative,
immersed sequentially with 10% (wt/vol), 20% (wt/vol), and 30% (wt/vol) sucrose in 0.1 M PB (pH 7.4), and frozen at ⫺80°C. Sections (5 ␮m) were prepared,
washed in 0.1 M PB, blocked with 0.1 M PB containing 5% goat serum and 0.2%
(wt/vol) Triton X-100 for 2 h, and incubated with the first antibodies at 4°C

FIG. 1. Mouse JAM4 amino acid sequence. (A) Alignment of the amino acid sequences of pPrey 10427 and mouse BC004806. Amino acidresidues are shown by single-letter codes. pPrey 10427 has 76 amino acids. Among them, 61 amino acids are identical to the C-terminal region of
BC004806. (B) Structural model of JAM4 and mouse BC004806. The extracellular region of JAM4 contains two domains with intramoleculardisulfide bonds typical of Ig-like loops of the V type. BC004806 has only the first loop. JAM4 and BC004806 have the C-terminal PDZ-binding
motif. (C) Phylogenetic tree indicating the relationship between JAM4 and other Ig protein family members. Sequences were obtained from
GenBank. The alignment was analyzed with Phylotree (http://cbrg.inf.ethz.ch/Server/AllAll.html). Accession numbers are as follows: for Nectin-1␣,AAF76195; for Nectin-2, P32507; for Nectin-3␣, AAF63685; for Nectin-4, AAL79833; for coxsackievirus and adenovirus receptor (CAR),
AAH16457; for intestinal A33 antigen, AAH08528; for endothelial cell-selective adhesion molecule (ESAM), AAK51504; for Nephrin,
AAK38483; for receptor for advanced glycation end product (RAGE), AAB82007; for JAM1, AAC32982; for JAM2, NP076333; and for JAM3,
NP075766. (D) Amino acid sequence of mouse JAM4 and alignment with mouse JAM1, JAM2, and JAM3. The putative hydrophobic signal
peptide (indicated with double solid underlines) and transmembrane (dashed single underline) sequences of mouse JAM4 are marked. Cysteines
likely to form disulfide bonds are indicated by asterisks. Putative N-linked glycosylation sites are indicated by boldface dots. Numbers indicate thenumbers of amino acid residues of each protein. Amino acid residues conserved among more than three proteins are shown on a black background.
(E) Constructs of JAM4 used in this study. SP, signal peptide; FLAG, FLAG tag. Solid ovals correspond to the C-terminal 9 amino acids containing
the PDZ-binding motif.

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overnight. After the mixture was washed with 0.1 M PB four times, boundantibodies were detected with the second antibodies at room temperature for 3 h.
Images were obtained with a confocal microscope (Olympus FV300-BX or Zeiss
LSM 510).
Immunoelectron microscopy. Kidneys were isolated from anesthetized adultrats, which were fixed by perfusion of 0.5% (wt/vol) glutaraldehyde in PBS.
Tissues were cut in small pieces and fixed in the same fixative for 1 h on ice,
washed with PBS, and dehydrated with a graded series of ethanol. Cubes of renal
cortex (2 mm) were embedded in LR white resin (London Resin Company Ltd.,
London, United Kingdom) and polymerized with LR white accelerator andstored at ⫺20°C. Ultrathin sections were mounted on 300-mesh nickel grids andtreated with 3% H2O2 for 10 min. After blocking with 1% (wt/vol) BSA in PBS
for 30 min, grids were incubated overnight at 4°C with the affinity-purified rabbit
anti-JAM4 or anti-MAGI-1 antibody, washed with PBS, and incubated with
10-nm gold-conjugated goat anti-rabbit IgG (Amersham) at a dilution of 1:20 for
1 h at room temperature. Grids were washed with distilled water and stained withuranyl acetate for 20 min. After air drying at room temperature, the sections
were photographed on a Hitachi 7100 electron microscope at ⫻10,000 magnification.
Northern blotting. A multiple-tissue Northern blot of mouse tissues (Seegene)containing 20 ␮g of total RNA was hybridized with uniformly labeled probe
derived from the full coding sequence of JAM4.
Cell aggregation assay. Wild-type L cells or L-JAM4 cells were treated with
0.2% (wt/vol) trypsin and 1 mM EDTA at 37°C for 5 min and dispersed by
pipetting. Cells were resuspended in Hanks balanced salt solution with either 1
mM CaCl2 or 5 mM EDTA at 106 cells/ml, placed in 12-well plates precoatedwith BSA, and rotated on a gyratory shaker at 37°C for the indicated periods oftime. Aggregation was stopped by adding 2% (vol/vol) glutaraldehyde. The
extent of aggregation was defined as the ratio of the total particle number after
incubation to the initial particle number before incubation. We took the image
with an Olympus IX70 apparatus at ⫻10 magnification with a 1.5⫻ zoom andprinted it on paper 29 by 21 cm. We regarded any group of cells that contacted
each other as one particle.
Cell dissociation assay. Cells were plated at 106 cells/60-mm dish and grown in
cultures for 3 days to confluency, and the medium was removed. When indicated,
cells were treated with dimethyl sulfoxide (DMSO), 1 ␮g of nocodazole/ml, or 5
␮M latrunculin A in fresh medium for 60 min. After being washed with PBS, cells
were scraped off by rotating the plate one round under a 3-cm-wide silicone
blade cell scraper (Sumitomo Bakelite Co.). Cells were subsequently dissociatedin 500 ␮l of PBS by pipetting with disposable 1-ml plastic tips.
Subcellular fractionation. Various L cells were collected on one 10-cm-diameter plate and homogenized in 200 ␮l of 20 mM HEPES–NaOH (pH 7.4)
containing 100 mM NaCl and 1% (wt/vol) Triton X-100. The homogenates were
centrifuged at 100,000 ⫻ g for 15 min. The supernatant and pellet were designated as Triton X-100-soluble and -insoluble fractions, respectively. Comparable
amounts of the fractions were immunoblotted with appropriate antibodies.
Permeability assay. Cells were plated at 5 ⫻ 104 cells/well in Transwells(Costar Corp.; polycarbonate filter [0.4-␮m pore size, 6.5-mm diameter]) andgrown in cultures for 5 days to confluency. The culture medium was then changed
to serum-free medium, and 1 mg of FITC-dextran/ml with an average molecularmass of 40,000 Da (Sigma-Aldrich Fine Chemicals) was added to the upper
chamber. At 2 h later, 100-␮l aliquots were collected from the lower chamber
and assayed by fluorimetry (Luminescence Spectrometer LS50B; Perkin-Elmer)(excitation at 492 nm and emission at 520 nm). The value for wild-type CHO cells
was set at 100%.
Adenovirus production and infection. Adenovirus to express GFP-tagged
MAGI-1 was prepared using an AdEasy system (19). Briefly, pShuttle CMVGFP-MAGI-1 was transformed into BJ5183 derivatives containing AdEasy-1
plasmid. The recombinant adenoviral plasmid was transfected into 911 cells(Introgene, Leiden, The Netherlands) with Lipofectamine (Invitrogen). Viral
supernatants were obtained by freezing and thawing infected cells. The infectionof cells was repeated for further amplification. High-titer viral stock was prepared by CsCl gradient. CHO cells were infected to express either control GFPor GFP–MAGI-1. At 24 h later, CHO cells were harvested and plated on
Transwells for the permeability assay. Infected cells were grown in cultures onregular plates to examine the cell viability and harvested to confirm the expression of proteins.
Immunoprecipitation. Using the DEAE-dextran method, COS-7 cells weretransfected with pClneo Myc MAGI-1 and pFLAG JAM4, pFLAG JAM4⌬C, or
pFLAG JAM1. Cells from two 10-cm-diameter plates were homogenized in 400
␮l of lysis buffer A (20 mM HEPES–NaOH [pH 7.4], 100 mM NaCl, 1% [wt/vol]
Triton X-100) and centrifuged at 100,000 ⫻ g for 15 min at 4°C. The supernatant
was incubated with 1.0 ␮l of the anti-MAGI-1 serum or the preimmune serum

MOL. CELL. BIOL.
fixed on 7.5 ␮l of protein G-Sepharose 4 fast-flow beads. After the beads were
washed, the precipitates were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotted with either the antiMyc or the anti-FLAG antibody.
GST pulldown assay. COS-7 cells were transfected with the indicated constructs using the DEAE-dextran method. Cells from one 10-cm plate were
homogenized in 200 ␮l of the lysis buffer A and centrifuged at 100,000 ⫻ g for
15 min at 4°C. The supernatant (175 ␮l) was incubated with 250 pmol of various
glutathione S-transferase (GST) proteins fixed on 7.5 ␮l of glutathione-Sepharose 4B beads. After the beads were washed, the precipitates were analyzed by
SDS-PAGE and immunoblotted with the appropriate antibodies.
In vitro transcription and translation. Using 5 ␮g of pClneo FLAG-JAM4 as
a template in 50 ␮l of the reaction buffer according to the manufacturer’s
protocol, [35S]methionine-labeled FLAG-JAM4 was prepared with in vitro transcription and translation (TnT T7 quick-coupled transcription/translation system; Promega). The product was analyzed by SDS-PAGE, and the image wasobtained with an imaging analyzer (FLA-3000; Fujifilm).
Deglycosylation of JAM4. Stable transformants of L, MDCK, LLC-PK1, andCHO cells expressing FLAG-JAM4 and COS-7 cells transfected with pFLAG
JAM4 were harvested. Cell lysates were digested for 60 min at 37°C with Nglycosidase F (New England BioLabs) in the presence of 1% (wt/vol) NP-40
according to the manufacturer’s recommendations.
Cell surface biotinylation. COS-7 cells that were transfected with pFLAGJAM4 or pFLAG-JAM4⌬C, L-FLAG-JAM4, and L-FLAG-JAM4⌬C were biotinylated with 0.5 mg of sulfo-N-hydroxysuccinimide–biotin (Pierce)/ml in PBS
containing 1 mM MgCl2 and 0.1 mM CaCl2 at 4°C for 60 min. After the reaction
was stopped by adding 50 mM NH4Cl, membrane fractions were collected fromharvested cells and extracted in buffer containing 20 mM HEPES–NaOH (pH7.4) containing 100 mM NaCl and 1% Triton X-100. The extracts were incubatedwith avidin-agarose beads (Sigma-Aldrich Fine Chemicals), and the precipitatedproteins were immunoblotted with the appropriate antibody.
Nucleotide sequence accession number. The nucleotide sequence encoding
JAM4 has been deposited in GenBank (accession number AF537215).

RESULTS
Identification of JAM4. Using the PDZ domains of human
MAGI-1 as bait to identify a protein interacting with MAGI-1,
we performed yeast two-hybrid screening with a human lung
cDNA library. We screened 2 ⫻ 106 clones and obtained positive results for 65 clones. Sequence analysis of these clones
identified previously uncharacterized genes. Among them, we
focused on a particular clone, pPrey 10427. The encoded peptide sequence was composed of 76 amino acids and terminatedwith a PDZ-binding motif (Fig. 1A). A search of the GenBank
database revealed that the amino acid sequence had 80% homology to the C terminus sequence of the product of mouse
BC004806. Mouse BC004806 encodes a putative type I transmembrane glycoprotein with one V-type Ig loop (Fig. 1B).
Using sense and antisense primers that were designed based on
the sequence of BC004806, we subsequently performed PCR
on mouse lung cDNA. The PCR product encoded a protein
composed of 370 amino acids (Fig. 1D). The protein included
all amino acids of mouse BC004806 protein and one additional
V-type Ig-like loop (Fig. 1B). Because the gene product issimilar to JAM1, -2, and -3 among the members of the Ig
protein family, we named it JAM4 (Fig. 1C). It shows 14.0,
10.4, and 13.5% amino acid homology to mouse JAM1, mouse
JAM2, and mouse JAM3, respectively (Fig. 1D).
To examine which tissues express JAM4, we hybridized amultitissue mouse RNA blot with a JAM4 cDNA probe. A
single 1.6-kb transcript was present in liver, kidney, stomach,small intestine, and skeletal muscle cells (Fig. 2A). The weaksignal was also detected in lung cells. We raised a rabbit polyclonal antibody against the intracellular domain of JAM4. The
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FIG. 2. (A) Northern blot analysis of JAM4. A uniformly labeled probe corresponding to the full length of JAM4 was prepared. Blots with 20
␮g of total RNA from each mouse tissue were hybridized with the probe (3,600,000 cpm) and exposed for 8 days. The mobilities of molecular mass
standards (in kilobases) are indicated at the left. (B) Western blot with the anti-JAM4 antibody of rat kidney, intestine, spleen, and MTD-1A cells.
Homogenates (total protein, 20 ␮g) from rat kidney, small intestine, spleen, and MTD-1A cells were resolved by SDS-PAGE and immunoblottedwith the affinity-purified antibody against JAM4. Lane 1, spleen cells; lanes 2 and 4, kidney cells; lanes 3 and 5, small intestine cells; lanes 6 and
7, MTD-1A cells. For lanes 4, 5, and 7, the antibody was preincubated with 5 ␮M immunogen. The mobilities of molecular mass standards (inkilodaltons) are indicated at left. (C) FLAG-JAM4 in various cells. (a) Homogenates (total protein, 20 ␮g) from various stable transformants
expressing FLAG-JAM4 were resolved by SDS-PAGE and immunoblotted with the anti-FLAG antibody. (b) Homogenates of stable transformants
were treated with N-glycosidase F and charged onto SDS-PAGE gel with the in vitro transcription and translation product. The lane for the in vitrotranscription and translation product was separately analyzed with the image analyzer. The remaining lanes were immunoblotted with theanti-FLAG antibody. The mobilities of molecular mass standards (in kilodaltons) are indicated at the left.

and small intestine cells but not in spleen cells (Fig. 2B). A
weak signal of the same size was detected in MTD-1A cells too.
All signals disappeared after the antibody was adsorbed by the
immunogen (Fig. 2B, lanes 4, 5, and 7). The calculated molecular mass of JAM4 is 40 kDa, but FLAG-JAM4 was detected
as a protein of about 93 kDa in various cells (Fig. 2Ca). FLAG-

JAM4 was smaller in COS-7 cells. After the treatment with
N-glycosidase F to remove N-glycan chains, FLAG-JAM4
from all cells migrated at the same site as the in vitro transcription and translation product (Fig. 2Cb). Therefore, we
conclude that the 93-kDa protein recognized by the antibody is
JAM4 with N-glycosylation.

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FIG. 3. Immunohistological analysis of JAM4. (A) JAM4 in kidney cells. The sections of rat kidney were stained with the affinity-purified
antibody in the absence (a) or the presence (b) of 5 ␮M immunogen. Signals were detected in glomeruli (arrow) and on apical membranes ofproximal tubules (arrowheads). (a2) The demarcated area shown in panel a1 at higher magnification. Bars, 100 ␮m (a1 and b) and 20 ␮m (a2).
(B) Colocalization of JAM4 and MAGI-1 with ZO-1 in kidney glomeruli. Sections of rat kidney were double stained with various antibodies. (a)JAM4 and ZO-1; (b) MAGI-1 and anti-ZO-1. Bar, 20 ␮m. (C) Electron microscopy localization of JAM4 in podocytes. (a) JAM4; (b) MAGI-1.
Bar, 0.1 ␮m. (D) Colocalization of JAM4 with ZO-1 in small intestine epithelial cells. (a) JAM4 and ZO-1; (b) JAM4 and ␤-catenin. Bar, 50 ␮m.

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FIG. 4. Immunofluorescence analysis of JAM4 in MTD-1A and MDCK cells. (A) MTD-1A cells were fixed and immunostained with
anti-JAM4, anti-MAGI-1, anti-␤-catenin, and anti-ZO-1 antibodies. JAM4 and MAGI-1 were detected by anti-rabbit antibodies. ␤-Catenin and
ZO-1 were detected by anti-mouse monoclonal and anti-rat monoclonal antibodies, respectively. (a) JAM4, ␤-catenin, and ZO-1 in MTD-1A cells;(b) MAGI-1, ␤-catenin, and ZO-1 in MTD-1A cells. Bar, 20 ␮m. (B) MDCK-FLAG-JAM4 cells were fixed and immunostained with anti-FLAG,
anti-MAGI-1, anti-ERBIN, and anti-ZO-1 antibodies. MAGI-1 and ERBIN were detected by anti-rabbit antibodies. FLAG and ZO-1 weredetected by anti-mouse monoclonal and anti-rat monoclonal antibodies, respectively. (a) FLAG-JAM4, MAGI-1, and ZO-1 in MDCK cells; (b)FLAG-JAM4, ERBIN, and ZO-1 in MDCK cells. Bar, 20 ␮m.

JAM4 is localized at TJs in epithelial cells. We examinedthe distribution of JAM4 in rat kidney cells. At low magnification, the image showed the intense signal in glomeruli and at
apical membranes of proximal tubules (Fig. 3Aa). After incubation of the antibody with 5 ␮M antigen, the signals of glomeruli and apical membranes of proximal tubules disappeared,suggesting that both signals were specific (Fig. 3Ab). The double staining with the rabbit anti-JAM4 and mouse anti-ZO-1
antibodies indicated that JAM4 and ZO-1 were colocalized inglomeruli (Fig. 3Ba). MAGI-1 was also concentrated in glo-

meruli as described previously and colocalized with ZO-1 (Fig.
3Bb) (35). Immunogold labeling of ultrathin sections of glomeruli revealed that JAM4 and MAGI-1 were localized at slit
diaphragms of podocytes (Fig. 3C). An immunofluorescencestudy using small intestine cells showed that JAM4 was colocalized with ZO-1 in intestinal epithelial cells (Fig. 3Da). In
contrast, ␤-catenin, an adherens junction marker protein,showed a more diffuse distribution along the lateral membraneof cells (Fig. 3Db). The signal colocalized with ZO-1 was adsorbed by the excess amount of the immunogen (data not

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FIG. 5. Interaction of JAM4 with MAGI-1. (A) Coimmunoprecipitation of JAM4 and MAGI-1. Myc–MAGI-1 with either FLAG-JAM4 or
FLAG-JAM4⌬C was coexpressed in COS-7 cells. Anti-MAGI-1 antibody coprecipitated FLAG-JAM4 but not FLAG-JAM4⌬C with Myc–
MAGI-1. The mobilities of molecular mass standards (in kilodaltons) are indicated at the left. Input, cell lysates; PIS IP, immunoprecipitate withthe preimmune serum; MAGI IP, immunoprecipitate with the anti-MAGI-1 serum. (B) GST pulldown assay (using GST fusion proteins containing
various PDZ domains of MAGI-1) of FLAG-JAM4. Lysates of COS cells expressing FLAG-JAM4 were incubated with GST alone or GST–
MAGI-1–PDZ1, –PDZ2, –PDZ3, –PDZ4, or –PDZ5 immobilized on glutathione-Sepharose beads. The resulting complexes were analyzed byimmunoblotting with the anti-FLAG antibody. The mobilities of molecular mass standards (in kilodaltons) are indicated at the left. (C) Specificityof the interaction of MAGI-1 with JAM4. Myc–MAGI-1 was coexpressed with either FLAG-JAM4 or FLAG-JAM1 in COS-7 cells. Lysates wereincubated with either preimmune serum or anti-MAGI-1 serum immobilized on protein G-Sepharose beads. The resulting complexes wereanalyzed with either the anti-Myc or anti-FLAG antibody. MAGI-1 did not bind JAM1. The mobilities of molecular mass standards (in kilodaltons)
are indicated at the left. (D) Specificity of the interaction of JAM4 with MAGI-1. Lysates of COS-7 cells expressing Myc–MAGI-1, Myc–ZO-1-1,
Myc-CASK, or Myc–Lin-7 were incubated with GST alone or GST–JAM4-C immobilized on glutathione-Sepharose beads. The resultingVOL. 23, 2003

shown). Cytosolic staining was also detected but did not completely disappear even in the presence of the immunogen,suggesting that it was nonspecific (data not shown). JAM4 wasalso detected at TJs of luminal epithelial cells in ducts ofmammary glands (data not shown). The immunofluorescencestudy revealed that JAM4 and MAGI-1 were colocalized with
ZO-1, a TJ marker protein, in mouse mammary gland epithelial MTD-1A cells (Fig. 4A). We also confirmed directly thecolocalization of FLAG-JAM4 and MAGI-1 by immunostaining stable transformant MDCK cells expressing FLAG-JAM4(Fig. 4B). These data indicate that JAM4 is a TJ protein invarious epithelial cells and is colocalized with MAGI-1.
JAM4 binds to MAGI-1 in vitro. To confirm the interaction
between JAM4 and MAGI-1, we first attempted to coimmunoprecipitate JAM4 and MAGI-1 from rat kidney cells. We
excised kidneys and extracted glomeruli from minced renal
cortex by sieving techniques. MAGI-1 was highly concentrated
in the glomerular fraction (data not shown) as described in aprevious report (36). JAM4 was also slightly enriched in thesame fraction. Therefore, we made use of the glomerular fraction as starting material for the immunoprecipitation. MAGI-1
was, however, highly resistant to detergent extraction, and onlya small amount of MAGI-1 was extracted in the detergentsincluding Triton X-100 and deoxycholic acid (data not shown).
Even after treatment with 6 M urea, a significant amount of
MAGI-1 remained in the insoluble fraction. JAM4 was also
Triton X-100 insoluble in kidney, small intestine, and lungcells, although it was soluble in deoxycholic acid (data notshown). Therefore, we overexpressed Myc–MAGI-1 and
FLAG-JAM4 in COS-7 cells and immunoprecipitated Myc–
MAGI-1 with the anti-MAGI-1 antibody from the lysates. The
overexpressed Myc–MAGI-1 and FLAG-JAM4 were partially
recovered in Triton X-100-soluble fractions in COS-7 cells.
FLAG-JAM4 was coprecipitated with Myc–MAGI-1 (Fig. 5A).
In contrast, FLAG-JAM4⌬C lacking the PDZ-binding motif
was not coprecipitated, suggesting that JAM4 binds to
MAGI-1 by its C-terminal PDZ-binding motif.
To identify the JAM4-binding region of MAGI-1, we prepared GST fusion proteins of MAGI-1–PDZ1, –PDZ2,–PDZ3, –PDZ4, and –PDZ5 and performed pulldown assays.
FLAG-JAM4 was isolated with GST-PDZ1 and GST-PDZ4
but not with GST-PDZ5 (Fig. 5B). GST-PDZ2 and GSTPDZ3 trapped a trace amount of FLAG-JAM4. FLAGJAM4⌬C did not bind to any of GST proteins (data notshown). These findings suggest that JAM4 binds to PDZ1 and
PDZ4 of MAGI-1.
JAM4 does not bind to ZO-1 and JAM1 does not bind to
MAGI-1. JAM1 is known to bind to the PDZ domains ofseveral proteins, including ZO-1 and CASK, through its Cterminal PDZ-binding motif (5, 11, 28). We tested whether
MAGI-1 specifically bound to JAM4. We coexpressed FLAGtagged JAM1, as well as FLAG-JAM4, with Myc–MAGI-1 in

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COS-7 cells and immunoprecipitated Myc–MAGI-1 with theanti-MAGI-1 antibody. FLAG-JAM4 was coimmunoprecipitated with Myc–MAGI-1, while FLAG-JAM1 was not (Fig.
5C). Using GST pulldown assays, we also examined whether
the PDZ-binding motif of JAM4 bound to other PDZ proteins.
GST–JAM4-C containing the C-terminal PDZ-binding motif
did not bind Myc–ZO-1-1 under the conditions in which it
bound MAGI-1 (Fig. 5D). GST–JAM4-C did not bind MycCASK or Myc–Lin-7 either, suggesting that JAM4 specificallyinteracts with MAGI-1.
MAGI-1 induces clustering of JAM4. To obtain independent
evidence for the interaction between JAM4 and MAGI-1, an
immunofluorescence study was performed with COS-7 cells
expressing Myc–MAGI-1 and FLAG-JAM4. MAGI-1 was diffusely distributed in cells when expressed alone (Fig. 5Ea).
FLAG-JAM4 and FLAG-JAM4⌬C were also diffusely distributed (Fig. 5Eb and c). In cells expressing both Myc–MAGI-1
and FLAG-JAM4, Myc–MAGI-1 and FLAG-JAM4 formedclusters and were colocalized with each other (Fig. 5Ed). Myc–
MAGI-1 did not colocalize with FLAG-JAM4⌬C and did not
induce its clustering (Fig. 5Ee). After latrunculin A treatment
to disrupt the actin cytoskeleton, the clusters of FLAG-JAM4
and Myc–MAGI-1 were no longer detected (Fig. 5Ef). The cellsurface biotinylation revealed that FLAG-JAM4 and FLAGJAM4⌬C were expressed on cell surfaces (data not shown).
This suggests that the clustering of JAM4 takes place on thecell surface.
Transfected JAM4 promotes cell adhesion and recruits
MAGI-1 to intercellular contacts. In the next set of experiments, we evaluated the ability of JAM4 to mediate cell adhesion. We performed an aggregation assay with stable transformants of L cells expressing JAM4 (L-JAM4). JAM4 showed
cell aggregation activity in a time-dependent manner (Fig. 6A
and C). L-JAM4 cells formed aggregates in the presence of 5mM EGTA, suggesting that this activity did not require Ca2⫹.
We also prepared L-JAM4⌬C cells, which expressed JAM4
without the PDZ-binding motif. The cell surface biotinylation
revealed that JAM4 and JAM4⌬C were expressed on the cellsurface at similar levels (Fig. 6B). L-JAM4⌬C cells also formed
aggregates (Fig. 6A and C).
We prepared various single and double stable transformantsof L cells, including L-FLAG-JAM4, L-FLAG-JAM4⌬C, LMyc–MAGI-1, L-FLAG-JAM4/Myc–MAGI-1, and L-FLAGJAM4⌬C/Myc–MAGI-1, and grew them in cultures to generate cell contacts. FLAG-JAM4 and FLAG-JAM4⌬C wereaccumulated at cell contacts in L-FLAG-JAM4 and L-FLAGJAM4⌬C cells (Fig. 7Aa and b). Myc–MAGI-1 was not concentrated anywhere in cells (Fig. 7Ac). In L-FLAG-JAM4/
Myc–MAGI-1 cells, Myc–MAGI-1 was concentrated at cell
contacts and colocalized with FLAG-JAM4 (Fig. 7Ba). In
L-FLAG-JAM4⌬C/Myc–MAGI-1 cells, FLAG-JAM4⌬C did
not recruit Myc–MAGI-1 (Fig. 7Bb). We immunostained en-complexes were analyzed by immunoblotting with the anti-Myc antibody. JAM4 did not bind ZO-1, CASK, or Lin-7. The mobilities of molecularmass standards (in kilodaltons) are indicated at the left. (E) Interaction of JAM4 and MAGI-1 in COS-7 cells. Subcellular localization of
Myc–MAGI-1 (a), FLAG-JAM4 (b), and FLAG-JAM4⌬C (c) in COS-7 cells is shown. (d) Myc–MAGI-1 and FLAG-JAM4 were colocalized andformed clusters in COS-7 cells when coexpressed. (e) Myc–MAGI-1 and FLAG-JAM4⌬C did not interact with each other in COS-7 cells. (f) The
effect of latrunculin A on the clustering of FLAG-JAM4 and Myc–MAGI-1. COS-7 cells were transfected with pFLAG JAM4 and pClneo Myc
MAGI-1 and were treated (before fixation) with 5 ␮M latrunculin A for 5 min.

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FIG. 6. Cell aggregation activity of JAM4. (A) Cell aggregation
assays were performed using L cells or stable transformants of L cells
expressing JAM4 (L-JAM4) or JAM4⌬C (L-JAM4⌬C). The extent ofcell aggregation is represented by the ratio of the total particle number
at the indicated time point (Nt) to the initial particle number (N0). The
Nt/N0 values are representative of the means ⫾ standard errors of
three independent experiments. (B) Expression of JAM4 andJAM4⌬C. Cell surface proteins were biotinylated, precipitated by avidin-agarose beads, and then immunoblotted with the anti-JAM4 antibody. Lane 1, wild-type L cells; lane 2, L-JAM4 cells; lane 3,
L-JAM4⌬C cells. (C) Cell aggregation of L-JAM4 cells. Results forwild-type L cells (L-w.t.), L-JAM4 cells (L-JAM4), and L-JAM4⌬C
cells (L-JAM4⌬C) at 60 min in a cell aggregation assay are shown. Bar,200 ␮m.

dogenous ZO-1 in L-FLAG-JAM4, L-FLAG-JAM4/Myc–
MAGI-1, and L-FLAG-JAM4⌬C/Myc–MAGI-1 cells. ZO-1
was detected mainly in the nucleus in L-FLAG-JAM4 cells(Fig. 7Ca). ZO-1 was accumulated at JAM4-based intercellularcontacts in L-FLAG-JAM4/Myc–MAGI-1 cells but not in
L-FLAG-JAM4⌬C/Myc–MAGI-1 cells (Fig. 7Cb and c).
These findings imply that MAGI-1 recruits ZO-1 to JAM4based cell contacts. Moreover, another TJ protein, occludin,

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was also accumulated by Myc–MAGI-1 at JAM4-based cell
contacts in L-FLAG-JAM4/Myc–MAGI-1 cells (Fig. 7D).
MAGI-1 strengthens JAM4-mediated cell adhesion. We addressed the question of whether MAGI-1 regulated JAM4mediated cell adhesion. L-FLAG-JAM4 and L-FLAGJAM4/Myc–MAGI-1 cells showed different appearances in
the cultures. When plated at 105 cells/35-mm-diameter dish,both cell cultures were scattered on day 1 (Fig. 8Aa and c,left panels). On day 4, L-FLAG-JAM4 cells were still scattered while L-FLAG-JAM4/Myc–MAGI-1 cells formed aggregates (Fig. 8Aa and c, right panels). To avoid the bias
caused by the difference between clones, we transiently
transfected L-FLAG-JAM4 cells with pClneo Myc MAGI-1
and observed that cells expressing both FLAG-JAM4 and
Myc–MAGI-1 formed aggregates (data not shown).
L-FLAG-JAM4⌬C/Myc–MAGI-1 cells appeared to be similar to L-FLAG-JAM4⌬C cells even on day 4 (Fig. 8Ab andd). L-JAM4 cells at low density contacted each other by cell
protrusions, and FLAG-JAM4 was accumulated at cell contacts (Fig. 8Ba). In contrast, L-FLAG-JAM4/Myc–MAGI-1
cells formed wide contacts and FLAG-JAM4 was detected
as linear shapes (Fig. 8Bb). L-FLAG-JAM4/Myc–MAGI-1
cells appeared in round shapes. We also confirmed the difference between L-FLAG-JAM4 and L-FLAG-JAM4/Myc–
MAGI-1 in collagen cultures. L-FLAG-JAM4 cells dispersed in collagen, but L-FLAG-JAM4/Myc-MAGI-1 cells
did not (Fig. 8C). Based on these findings, we reasoned that
L-FLAG-JAM4/Myc–MAGI-1 cells displayed greater adhesive activity than L-FLAG-JAM4 cells. To compare thestrengths of cell adhesion, we performed a cell dissociation
assay. L-FLAG-JAM4 cells became a single cell suspension
after being pipetted five times (Fig. 8Da). L-FLAG-JAM4/
Myc–MAGI-1 cells remained as cell aggregates after being
pipetted five times (Fig. 8Db). We confirmed that both cells
expressed FLAG-JAM4 at similar levels (Fig. 8Dc).
MAGI-1 links JAM4 to Triton X-100-insoluble structures.
With regard to the mechanism underlying the reinforcement of
JAM4-mediated cell adhesion, we hypothesized that MAGI-1
might link JAM4-based cell contacts to the cytoskeleton. First,
we performed the subcellular fractionation of L-Myc–
MAGI-1, L-FLAG-JAM4, and L-FLAG-JAM4/Myc–MAGI-1
cells (Fig. 9A). Myc–MAGI-1 and FLAG-JAM4 were Triton
X-100 soluble when expressed alone. However, in L-FLAGJAM4/Myc–MAGI-1 cells, both Myc–MAGI-1 and FLAGJAM4 were recovered in the Triton X-100-soluble and -insoluble fractions. L-FLAG-JAM4/Myc–MAGI-1 cells treatedwith latrunculin A became easily dispersed in the cell dissoci-

FIG. 7. Recruitment of Myc–MAGI-1, ZO-1, and occludin to JAM4-based cell contacts. (A) Stable transformants of L cells expressing
FLAG-JAM4 (L-FLAG-JAM4) (a) and FLAG-JAM4⌬C (L-FLAG-JAM4⌬C) (b) were grown in cultures to generate cell contacts. FLAG-JAM4
and FLAG-JAM4⌬C were accumulated at cell contacts (arrows). (c) L-Myc–MAGI-1 cells. Myc–MAGI-1 was diffusely distributed. (B) Myc–
MAGI-1 in L-FLAG-JAM4/Myc–MAGI-1 (a) and L-FLAG-JAM4⌬C/Myc–MAGI-1 (b) cells. FLAG-JAM4 (arrows) and Myc–MAGI-1 (arrowheads) were accumulated at cell contacts in L-FLAG-JAM4/Myc–MAGI-1 cells. (C) (a) L-FLAG-JAM4 cells were double stained with anti-FLAG
and anti-ZO-1 antibodies. (b) L-FLAG-JAM4/Myc–MAGI-1 cells; (c) L-FLAG-JAM4⌬C/Myc–MAGI-1 cells. L-FLAG-JAM4/Myc–MAGI-1 cells
and L-FLAG-JAM4⌬C/Myc–MAGI-1 cells were triple stained with anti-FLAG, anti-Myc, and anti-ZO-1 antibodies. ZO-1 (arrowheads) was
recruited with Myc–MAGI-1 (arrows) to cell contacts in L-FLAG-JAM4/Myc–MAGI-1 cells. (D) (a) L-FLAG-JAM4 cells were double stainedwith anti-FLAG and anti-occludin antibodies. (b) L-FLAG-JAM4/Myc–MAGI-1 cells were triple stained with anti-FLAG, anti-Myc, and antioccludin antibodies. Occludin (arrowheads) was recruited with Myc–MAGI-1 (arrows) to cell contacts in L-FLAG-JAM4/Myc–MAGI-1 cells. Bar,
20 ␮m.

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FIG. 8. Effect of MAGI-1 on the cell adhesive activity of JAM4. (A) Phase-contrast images of L-FLAG-JAM4 (a), L-FLAG-JAM4⌬C (b),
L-FLAG-JAM4/Myc–MAGI-1 (c), and L-FLAG-JAM4⌬C/Myc–MAGI-1 (d) cells grown in cultures on plastic dishes. Bar, 50 ␮m. (B) Immunofluorescence of FLAG-JAM4 in L-FLAG-JAM4 (a) and L-FLAG-JAM4/Myc–MAGI-1 (b) cells. Bar, 10 ␮m. (C) Phase-contrast images of
L-FLAG-JAM4 (a) and L-FLAG-JAM4/Myc–MAGI-1 (b) cells in collagen cultures. Bar, 100 ␮m. (D) Dissociation assay of L-FLAG-JAM4 and
L-FLAG-JAM4/Myc–MAGI-1 cells. (a) L-FLAG-JAM4 cells after being pipetted five times; (b) L-FLAG-JAM4/Myc–MAGI-1 cells after being
pipetted five times. Bar, 200 ␮m. (c) Western blot with the anti-FLAG antibody of L-FLAG-JAM4 and L-FLAG-JAM4/Myc–MAGI-1 cells. Lane
1, L-FLAG-JAM4 cells; lane 2, L-FLAG-JAM4/Myc–MAGI-1 cells. The mobilities of molecular mass standards (in kilodaltons) are indicated at
the left.

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FIG. 9. Effect of disruption of the cytoskeleton on JAM4/MAGI-1-mediated cell adhesion. (A) Subcellular fractionation of L-Myc–MAGI-1,
L-FLAG-JAM4, and L-FLAG-JAM4/Myc–MAGI-1 cells. Comparable amounts of the fractions from various L cells were immunoblotted with theanti-Myc or the anti-FLAG antibody. Insol., insoluble. (a) L-Myc–MAGI-1 cells; (b) L-FLAG-JAM4 cells; (c) L-FLAG-JAM4/Myc–MAGI-1 cells.
(B) Dissociation assay of L-FLAG-JAM4/Myc–MAGI-1 cells after mock, nocodazole, and latrunculin A treatment. L-FLAG-JAM4/Myc–MAGI-1
cells were treated with DMSO (a), 1 ␮g of nocodazole/ml (b), or 5 ␮M latrunculin A (c) for 60 min before the dissociation assay. Upper photos,
cells after the treatment and before scraping; lower photos, cells after being pipetted five times. Bar, 200 ␮m.

ation assay (Fig. 9B). Nocodazole treatment did not show any
effect.
JAM4 reduced permeability of CHO cell monolayers and
MAGI-1 augmented the reduction. In the last set of experiments, we examined whether JAM4 controls the permeabilityof CHO cell monolayers as JAM1 does. The permeability of
FITC-dextran was reduced by 25% in stable transformant
CHO cells expressing FLAG-JAM4 (CHO-FLAG-JAM4 cells)(Fig. 10A). We expressed control GFP or GFP–MAGI-1 using
adenovirus in wild-type CHO and CHO-FLAG-JAM4 cells.
GFP–MAGI-1 further decreased the permeability in CHOFLAG-JAM4 cells but not in wild-type CHO cells (Fig. 10Ba).
We confirmed the expression of FLAG-JAM4, GFP, and
GFP–MAGI-1 in these cells (Fig. 10Bb).

DISCUSSION
For this study, we have reported a novel protein that interacts with MAGI-1. We have named this protein JAM4. Because we had only rabbit polyclonal antibodies against both
MAGI-1 and JAM4, we cannot directly show the colocalizationof MAGI-1 and JAM4. However, JAM4 is colocalized with
ZO-1 in kidney glomeruli and small intestinal epithelial cells,
whereas MAGI-1 is colocalized with ZO-1 (21). Moreover,
FLAG-JAM4 is colocalized with endogenous MAGI-1 in
MDCK cells. Although we could not perform a coimmunoprecipitation study using endogenous proteins, JAM4 is coprecipitated with MAGI-1 from COS-7 cells expressing JAM4 andMAGI-1. An immunofluorescence study showed that MAGI-1

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FIG. 10. Effect of JAM4 and MAGI-1 on the permeability of CHO cell monolayers. (A) Wild-type CHO and CHO-FLAG-JAM4 cells weregrown in cultures in Transwells. FITC-dextran was added to the upper chamber, and the permeability was measured. The permeability of wild-type
CHO cells was taken as 100%. Data are indicated as the means ⫾ standard errors of three independent experiments. (B) (a) Wild-type CHO andCHO-FLAG-JAM4 cells were infected by adenovirus to express either GFP or GFP–MAGI-1. At 2 h after the infection, these cells were platedon Transwells for the permeability assay. (b) Immunoblots (IB) of CHO and CHO-FLAG-JAM4 cells to confirm the protein expression. Left panel,
immunoblot with the anti-GFP antibody; right panel, immunoblot with the anti-FLAG antibody. Lanes 1, wild-type CHO cells with GFP; lanes
2, wild-type CHO cells with GFP–MAGI-1; lanes 3, CHO-FLAG-JAM4 cells with GFP; lanes 4, CHO-FLAG-JAM4 cells with GFP–MAGI-1. The
mobilities of molecular mass standards (in kilodaltons) are indicated at the left.

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and JAM4 cooperatively form clusters in COS-7 cells. These
data suggest that JAM4 interacts with MAGI-1 at TJs.
The amino acid sequence of JAM4 suggests that JAM4 has
two domains with intrachain disulfide bonds typical of Ig loopsof the V type. JAM4 has five potential sites of N-glycosylation.
JAM4 appears as a protein of about 93 kDa in various cells,
although the calculated size is 40 kDa. The result from theexperiment using N-glycosidase F indicates that JAM4 is indeed modified by N-glycosylation. Cell aggregation assays using L cells showed that JAM4 mediates calcium-independent
cell adhesion. L cells are suitable for this assay, because wildtype L cells do not express cadherins and do not form aggregates. JAM4 is similar to JAM1 in the arrangement of V-Vtype loops. JAM1 is concentrated at TJs of epithelial andendothelial cells (29, 34). JAM1 has a C-terminal PDZ-binding
motif and binds to PDZ domains of ZO-1, CASK, afadin/AF-6,
ASIP/PAR-3, and MUPP1 (11, 12, 17, 23, 28). Besides PDZ
proteins, cingulin also interacts with JAM1 directly or indirectly (5). All these interactions suggest that JAM1 plays central roles in the assembly of TJ components. By analogy, we
speculated that JAM4 is involved in the assembly of TJ components. First, we have shown that MAGI-1 is recruited to
JAM4-based cell contacts in L cells. Moreover, we have observed that ZO-1 and occludin are also accumulated to JAM4based cell contacts in a MAGI-1-dependent manner. Although
the molecular mechanism underlying the recruitment of ZO-1
and occludin remains to be clarified, the complex of JAM4 andMAGI-1 may be involved in the assembly of components of
TJs. It is necessary to examine how various mutants of JAM4
and MAGI-1 interfere with the formation of intact TJs. In
immunofluorescence assays, JAM4 is detected on apical membranes of proximal renal tubules. The molecular constituentsthat tether JAM4 to TJs in MTD-1A, MDCK, and intestinal
epithelial cells may be absent in proximal tubular epithelial
cells, with the result that JAM4 is not localized at TJs. It is also
important to determine the molecular mechanism that localizes JAM4 at TJs.
JAM4 is slightly enriched in the glomerular fraction, and it
appears to be colocalized with ZO-1 and MAGI-1 in kidney
glomeruli at the immunofluorescence level. Immunoelectron
microscopy revealed the localization of JAM4 in podocytes.
JAM1 is known to regulate TJ resealing in epithelial cells (27).
Nephrin, which is an Ig-like transmembrane protein localized
at the podocyte slit diaphragm, is essential for maintenance of
normal glomerular permeability and is involved in congenital
nephrotic syndrome (25). We showed that JAM4 reduced thepermeability of CHO cell monolayers. JAM4 may be relevantin the control of the physical barrier at epithelial cells andpodocytes.
Our data also indicate that MAGI-1 strengthens the celladhesion and sealing activities of JAM4. A simple explanationis that MAGI-1 may induce the clustering of JAM4 and concentrate JAM4 at cell-cell contacts. We also speculate thatcytoskeletons may contribute to the effect of MAGI-1. JAM4
becomes Triton X-100 insoluble when expressed with MAGI-1
in L cells. MAGI-1 also becomes Triton X-100 insoluble, depending on the presence or absence of JAM4. Treatments
disrupting the actin cytoskeleton abolish the clustering of
MAGI-1 and JAM4 in COS-7 cells and the effect of MAGI-1

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on the cell adhesion activity of JAM4 in L cells. The complexof JAM4 and MAGI-1 may form a scaffold which is linked tothe cytoskeleton. Consistent with this, endogenous MAGI-1
and JAM4 are Triton X-100 insoluble in kidney, small intestine, and lung cells. The attachment of the actin cytoskeleton
may be necessary for MAGI-1 and JAM4 to form tight adhesions.
Besides JAM1, two JAM-related proteins have been identified. JAM2 and JAM3 have one V-type and one C2-type Ig
loops (3, 4, 7, 26). JAM2 is expressed in endothelial and lymphatic cells and interacts with JAM3. JAM3 is expressed on T,
NK, and dendritic cells. The interaction between JAM2 andJAM3 may be important for the trafficking of these cells acrossthe endothelium (26). JAM1 is also expressed in endothelial
cells and is involved in regulating monocyte migration (29, 34).
We have not confirmed the localization of JAM4 in endothelial
cells. It remains to be determined whether JAM4 plays roles inendothelial cells as well as in epithelial cells.
ACKNOWLEDGMENTS
This study was supported by grants-in-aids for Scientific Researchand Special Coordination Funds for Promoting Science and Technology from the Ministry of Education, Culture, Sports, Science, andTechnology and a grant from Yamanouchi Foundation for Researchon Metabolic Disorders (2000).
We thank S. Tsukita (Kyoto University) for cDNA of mouse ZO-1,
rat anti-occludin antibody, and MDCKII cells, M. Takeichi for
MTD-1A cells, Y. Nakamura (University of Tokyo) for cDNA ofhuman MAGI-1, S. Uchida and S. Sasaki (Tokyo Medical and Dental
University) for LLC-PK1 cells, T. Kitamura (University of Tokyo) for
pMXpuro vector, G. Noran (Stanford University) for Phoenix ampho
cells, S. Hollenberg (Oregon Health Sciences University) for reagentsfor yeast two-hybrid screening, and T.-C. He and B. Vogelstein (Johns
Hopkins Oncology Center) for the pAdEasy system. We thank N.
Terada, S. Ichinose, and K. Terashima (Tokyo Medical and Dental
University) for valuable advice. We also thank C. Rokukawa and M.
Miyahara-Tenkatsu for skillful technical assistance.
REFERENCES
1. Anderson, J. M., B. R. Stevenson, L. A. Jesaitis, D. A. Goodenough, and
M. S. Mooseker. 1988. Characterization of ZO-1, a protein component of thetight junction from mouse liver and Madin-Darby canine kidney cells. J. Cell
Biol. 106:1141–1149.
2. Anderson, J. M., and C. M. van Itallie. 1999. Tight junctions: closing in on
the seal. Curr. Biol. 9:R922–R924.
3. Arrate, M. P., J. M. Rodriguez, T. M. Tran, T. A. Brock, and S. A. Cunningham. 2001. Cloning of human junctional adhesion molecule 3 (JAM3) and its
identification as the JAM2 counter-receptor. J. Biol. Chem. 276:45826–
45832.
4. Aurrand-Lions, M., L. Dunca, C. Ballestrem, and B. A. Imhof. 2001. JAM-2,
a novel immunoglobulin superfamily molecule, expressed by endothelial andlymphatic cells. J. Biol. Chem. 276:2733–2741.
5. Bazzoni, G., O. M. Martinez-Estrada, F. Orsenigo, M. Cordenonsi, S. Citi,
and E. Dejana. 2000. Interaction of junctional adhesion molecule with thetight junction components ZO-1, cingulin, and occludin. J. Biol. Chem.
275:20520–20526.
6. Christensen, E. I., and H. Birn. 2002. Megalin and cubilin: multifunctional
endocytic receptors. Nat. Rev. Mol. Cell Biol. 3:258–268.
7. Cunningham, S. A., M. P. Arrate, J. M. Rodriguez, R. J. Bjercke, P. Vanderslice, A. P. Morris, and T. A. Brock. 2000. A novel protein with homology to
the junctional adhesion molecule. J. Biol. Chem. 275:34750–34756.
8. Dobrosotskaya, I., R. K. Guy, and G. L. James. 1997. MAGI-1, a membraneassociated guanylate kinase with a unique arrangement of protein-protein
interaction domains. J. Biol. Chem. 272:31589–31597.
9. Dobrosotskaya, I. Y. 2001. Identification of mNET1 as a candidate ligand forthe first PDZ domain of MAGI-1. Biochem. Biophys. Res. Commun. 283:
969–975.
10. Dobrosotskaya, I. Y., and G. L. James. 2000. MAGI-1 interacts with betacatenin and is associated with cell-cell adhesion structures. Biochem. Biophys. Res. Commun. 270:903–909.
11. Ebnet, K., C. U. Schulz, M.-K. M. zu Bickwedde, G. G. Pendl, and D.

4282

HIRABAYASHI ET AL.

Vestweber. 2000. Junctional adhesion molecule interacts with the PDZ domain-containing proteins AF-6 and ZO-1. J. Biol. Chem. 275:27979–27988.
12. Ebnet, K., A. Suzuki, Y. Horikoshi, T. Hirose, M.-K. M. zu Brickwedde, S.
Ohno, and D. Vestweber. 2001. The cell polarity protein ASIP/PAR-3 directly associates with junctional adhesion molecule (JAM). EMBO J. 20:
3738–3748.
13. Fanning, A. S., and J. M. Anderson. 1999. Protein modules as organizers ofmembrane structure. Curr. Opin. Cell Biol. 11:432–439.
14. Furuse, M., T. Hirase, M. Itoh, A. Nagafuchi, S. Yonemura, S. Tsukita, and
S. Tsukita. 1993. Occludin: a novel integral membrane protein localizing attight junctions. J. Cell Biol. 123:1777–1788.
15. Furuse, M., K. Fujita, T. Hiiragi, K. Fujimoto, and S. Tsukita. 1998. Claudin-1 and -2: novel integral membrane proteins localizing at tight junctions
with no sequence similarity to occludin. J. Cell Biol. 141:1539–1550.
16. Furuse, M., H. Sasaki, K. Fujimoto, and S. Tsukita. 1998. A single geneproduct, claudin-1 or -2, reconstitutes tight junction strands and recruits
occludin in fibroblasts. J. Cell Biol. 143:391–401.
17. Hamazaki, Y., M. Itoh, H. Sasaki, M. Furuse, and S. Tsukita. 2002. MultiPDZ domain protein 1 (MUPP1) is concentrated at tight junctions throughits possible interaction with claudin-1 and junctional adhesion molecule.
J. Biol. Chem. 277:455–461.
18. Haskins, J., L. Gu, E. S. Wittchen, J. Hibbard, and B. R. Stevenson. 1998.
ZO-3, a novel member of the MAGUK protein family found at the tightjunction, interacts with ZO-1 and occludin. J. Cell Biol. 141:199–208.
19. He, T.-C., S. Zhou, L. T. Da Costa, J. Yu, K. W. Kinzler, and B. Vogelstein.
1998. A simplified system for generating recombinant adenoviruses. Proc.
Natl. Acad. Sci. USA 95:2509–2514.
20. Hirao, K., Y. Hata, N. Ide, M. Takeuchi, M. Irie, I. Yao, M. Deguchi, A.
Toyoda, T. C. Sudhof, and Y. Takai. 1998. A novel multiple PDZ domaincontaining molecule interacting with N-methyl-D-aspartate receptors andneuronal cell adhesion proteins. J. Biol. Chem. 273:21105–21110.
21. Ide, N., Y. Hata, H. Nishioka, K. Hirao, I. Yao, M. Deguchi, A. Mizoguchi,
H. Nishimori, T. Tokino, Y. Nakamura, and Y. Takai. 1999. Localization of
membrane-associated guanylate kinase (MAGI)-1/BAI-associated protein(BAP) 1 at tight junctions of epithelial cells. Oncogene 18:7810–7815.
22. Itoh, M., M. Furuse, K. Morita, K. Kubota, M. Saitou, and S. Tsukita. 1999.
Direct binding of three tight junction-associated MAGUKs, ZO-1, ZO-2,
and ZO-3, with the COOH termini of claudins. J. Cell Biol. 147:1351–1363.
23. Itoh, M., H. Sasaki, M. Furuse, H. Ozaki, T. Kita, and S. Tsukita. 2001.
Junctional adhesion molecule (JAM) binds to PAR-3: a possible mechanism
for the recruitment of PAR-3 to tight junctions. J. Cell Biol. 154:491–497.
24. Jesaitis, L. A., and D. A. Goodenough. 1994. Molecular characterization andtissue distribution of ZO-2, a tight junction protein homologous to ZO-1 andthe Drosophila discs-large tumor suppressor protein. J. Cell Biol. 124:949–
961.
25. Kestilä, M., U. Lenkkeri, M. Männikkö, J. Lamerdin, P. McCready, H.
Putaala, V. Ruotsalainen, T. Morita, M. Nissinen, R. Herva, C. E. Kashtan,
L. Peltonen, C. Holmberg, A. Olsen, and K. Tryggvason. 1998. Positionallycloned gene for a novel glomerular protein—nephrin—is mutated in congenital nephrotic syndrome. Mol. Cell 1:575–582.
26. Liang, T. W., H. H. Chiu, A. Gurney, A. Sidle, D. B. Tumas, P. Schow, J.
Foster, T. Klassen, K. Dennis, R. A. DeMarco, T. Pham, G. Frantz, and S.
Fong. 2002. Vascular endothelial-junctional adhesion molecule (VE-JAM)/
JAM2 interacts with T, NK, and dendritic cells through JAM3. J. Immunol.
168:1618–1626.

MOL. CELL. BIOL.
27. Liu, Y., A. Nustrat, F. J. Schnell, T. A. Reaves, S. Walsh, M. Pochet, and
C. A. Parkos. 2000. Human junction adhesion molecule regulates tight junction resealing in epithelia. J. Cell Sci. 113:2363–2374.
28. Marinez-Estrada, O. M., A. Villa, F. Breviario, F. Orsenigo, E. Dejana, and
G. Bazzoni. 2001. Association of junctional adhesion molecule with calcium/
calmodulin-dependent serine protein kinase (CASK/Lin-2) in human epithelial Caco-2 cells. J. Biol. Chem. 276:9291–9296.
29. Marin-Padura, I., S. Lostaligo, M. Schneemann, L. Williams, M. Romano,
P. Fruscella, C. Panzeri, A. Stoppacciaro, L. Ruco, A. Villa, D. Simmons, and
E. Dejana. 1998. Junctional adhesion molecule, a novel member of theimmunoglobulin superfamily that distributes at intercellular junctions andmodulates monocyte transmigration. J. Cell Biol. 142:117–127.
30. Mino, A., T. Ohtsuka, E. Inoue, and Y. Takai. 2000. Membrane-associatedguanylate kinase with inverted orientation (MAGI)-1/brain angiogenesis inhibitor 1-associated protein (BAP1) as a scaffolding molecule for Rap small
G protein GDP/GTP exchange protein at tight junctions. Genes Cells
5:1009–1016.
31. Mundel, P., H. W. Heid, T. M. Mundel, M. Kruger, J. Reiser, and W. Kriz.
1997. Synaptopodin: an actin-associated protein in telencephalic dendrites
and renal podocytes. J. Cell Biol. 139:193–204.
32. Nishimura, W., T. Iizuka, S. Hirabayashi, N. Tanaka, and Y. Hata. 2000.
Localization of BAI-associated protein1/membrane-associated guanylate kinase-1 at adherens junctions in normal rat kidney cells: polarized targeting
mediated by the carboxyl-terminal PDZ domains. J. Cell. Physiol. 185:358–
365.
33. Ohno, H., S. Hirabayashi, T. Iizuka, H. Ohnishi, T. Fujita, and Y. Hata.
2002. Localization of p0071-interacting proteins, plakophilin-related armadillo-repeat protein-interacting protein (PAPIN) and ERBIN, in epithelial
cells. Oncogene 21:7042–7049.
34. Ozaki, H., K. Ishii, H. Horiuchi, H. Arai, T. Kawamoto, K. Okawa, A.
Iwamatsu, and T. Kita. 1999. Cutting edge: combined treatment of TNF-␣
and IFN-␥ causes redistribution of junctional adhesion molecule in humanendothelial cells. J. Immunol. 163:553–557.
35. Patrie, K. M., A. J. Drescher, M. Goyal, R. C. Wiggins, and B. Margolis.
2001. The membrane-associated guanylate kinase protein MAGI-1 binds
megalin and is present in glomerular podocytes. J. Am. Soc. Nephrol. 12:
667–677.
36. Patrie, K. M., A. J. Drescher, A. Welihinda, P. Mundel, and B. Margolis.
2002. Interaction of two actin-binding proteins, synaptopodin and ␣-actinin-4, with the tight junction protein MAGI-1. J. Biol. Chem. 277:30183–
30190.
37. Shiratsuchi, T., M. Futamura, K. Oda, H. Nishimori, Y. Nakamura, and T.
Tokino. 1998. Cloning and characterization of BAI-associated protein 1: a
PDZ domain-containing protein that interacts with BAI1. Biochem. Biophys.
Res. Commun. 247:597–604.
38. Shoji, H., K. Tsuchida, H. Kishi, N. Yamakawa, T. Matsuzaki, Z. Liu, T.
Nakamura, and H. Sugino. 2000. Identification and characterization of a
PDZ protein that interacts with activin type II receptors. J. Biol. Chem.
275:5485–5492.
39. Tsukita, S., M. Furuse, and M. Itoh. 2001. Multifunctional strands in tightjunctions. Nat. Rev. Mol. Cell Biol. 2:285–293.
40. Wood, J. D., J. Yuan, R. L. Margolis, V. Colomer, K. Duan, J. Kushi, Z.
Kaminsky, J. J. Kleiderlein, A. H. Sharp, and C. A. Ross. 1998. Atrophin-1,
the DRPLA gene product, interacts with two families of WW domaincontaining proteins. Mol. Cell. Neurosci. 11:149–160.