Roles of immunoglobulin-like loops of junctional celladhesion molecule 4; involvement in the subcellular
localization and the cell adhesion
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of JAM4
Blackwell
Oxford,Genes
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1356-9597
2003
Blackwell
toUK
Cells
Publishing
Publishing
Ltd.
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Makiko Tajima1,2, Susumu Hirabayashi1, Ikuko Yao1, Madoka Shirasawa1, Junichi Osuga2,
Shun Ishibashi4, Toshiro Fujita2,3 and Yutaka Hata1,*
1

Department of Medical Biochemistry, Graduate School of Medicine,Tokyo Medical and Dental University, 1-5-45Yushima, Bunkyo-ku,Tokyo
113-8519, Japan;
2
Department of Metabolic Diseases and 3Department of Nephrology and Endocrinology, Faculty of Medicine, University of Tokyo, 7-3-1 Hongo,
Bunkyo-ku, Tokyo 113-8655, Japan;
4
Department of Internal Medicine, Jichi Medical School, 3311-1 Yakushiji, Minamikawachi-machi, Kawachi-gun,Tochigi 329-0498, Japan

Abstract
Background: Membrane-associated guanylate kinasewith inverted domain structure-1 (MAGI-1) is a
scaffolding protein at tight junctions (TJs). We haverecently identified junctional adhesion molecule 4(JAM4) as a MAGI-1-interacting protein. JAM4 belongs
to the immunoglobulin superfamily and mediates
Ca2+-independent adhesion. In this study, we examined the subcellular localization of JAM4 in various
tissues and the involvement of JAM4 in the localizationof MAGI-1. Moreover, we investigated into roles of
immunoglobulin-like loops (Ig-loops) of JAM4.
Results: JAM4 was localized at TJs but also on apicalmembranes of epithelial cells in jejunum, ileum, andrenal proximal tubules. In Madine Darby canine kidney

Introduction
Junctional cell adhesion molecule 1 (JAM1) belongs to
the immunoglobulin superfamily and consists of two
V-type immunoglobulin-like loops (Ig-loops), a single
transmembrane region, and a short cytoplasmic tail(Martin-Padura et al. 1998; Ozaki et al. 1999). It ispresent at TJs in epithelial and endothelial cells. JAM1
mediates cell adhesion via homophilic interactions andregulates paracellular permeability (Martin-Padura et al.
1998; Liu et al. 2000). It is involved in the process of
inflammation by mediating transmigration of monocytes
and neutrophils through endothelial cells (Martin-Padura
Communicated by: Tadashi Yamamoto*Correspondence: E-mail: yuhammch@med.tmd.ac.jp© Blackwell Publishing Limited(MDCK) cells, the localization of JAM4 at TJs
depended on the first Ig-loop and did not require
the MAGI-1-interacting region. JAM4 determined
the subcellular localization of MAGI-1 in MDCK
cells. In ileum, however, MAGI-1 was localized at
TJs where JAM4 was not detected. Both of Ig-loops
were necessary for homophilic interactions, but cis
interactions depended on the first Ig-loop.
Conclusion: JAM4 may be primarily targeted to apical membranes, and subsequently recruited to TJs
through the first Ig-loop–mediated molecular interaction. JAM4 determines the localization of MAGI1 in MDCK cells, but the in vivo localization of
MAGI-1 does not necessarily depend on JAM4.

et al. 1998; Del Maschio et al. 1999). It is also expressedin peripheral blood platelets and thought to be involvedin primary haemostasis (Naik et al. 1995; Sobocka et al.
2000; Naik et al. 2001). Subsequently, two homologs of
JAM1 were reported and named JAM2 (also called
VE-JAM and JAM-3) and JAM3 (also called JAM-2)(Cunningham et al. 2000; Palmeri et al. 2000; AurrandLions et al. 2001a,b; Arrate et al. 2001). Like JAM1,
JAM2 and JAM3 possess two Ig-loops, but the second
Ig-loop proximal to the membrane is the C2-type
instead of the V-type. JAM2 is preferentially expressed in
the intercellular boundaries of the endothelium, particularly in heart and placenta, and function as an adhesive
ligand for T, NK, and dendritic cells (Palmeri et al. 2000;
Cunningham et al. 2000; Liang et al. 2002). JAM3 shows
endothelial expression in many tissues, predominantly inGenes to Cells (2003) 8, 759–768

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placenta, brain and kidney (Arrate et al. 2001). JAM3 isalso found in T cells and is up-regulated by T-cell activation. JAM2 adheres to T cells through the heterotypicinteraction with JAM3. The engagement of JAM2 with
JAM3 facilitates the interaction between JAM2 and α4β1
integrin in T cells (Cunningham et al. 2002).Thus, JAM2
and JAM3 are considered to take parts in leucocytetransmigration.
Recently, we have identified a novel protein which
belongs to the immunoglobulin superfamily as a binding
partner of membrane-associated guanylate kinase withinverted domain structure-1 (MAGI-1) (also called brain
angiogenesis inhibitor 1-associated protein 1) (Dobrosotskaya
et al. 1997; Shiratsuchi et al. 1998; Hirabayashi et al.
2003). Because the protein shows 14.0%, 10.4% and13.5% amino acid homology to JAM1, JAM2 and JAM3,
respectively, we named it JAM4. MAGI-1 has six PDZ
domains, is localized at TJs in polarized epithelial cells
and may be involved in the assembly of TJ components. Consistently, JAM4 is localized at TJs in Madine
Darby canine kidney (MDCK) cells and mouse mammary gland epithelial cells. JAM4 contains two extracellular Ig-loops of the V-type and binds to the second andthe fifth PDZ domains of MAGI-1 by its C-terminal
PDZ-binding motif. JAM4 exhibits Ca2+-independent
cell adhesion activity and reduces the paracellularpermeability in the monolayer of CHO cells. MAGI-1
strengthens the cell adhesion activity of JAM4. We also
revealed that MAGI-1 is recruited to JAM4-based cell
contacts in mouse fibroblastic L cells. In this model system, other TJ proteins such as ZO-1 and occludin are
also accumulated to JAM4-based cell contacts. All thesefindings support the theory that the interaction between
JAM4 and MAGI-1 is important for the organization of
TJs.
For the previous study, we examined the subcellular
localization of JAM4 in tissues of several organs including kidney, small intestine, and mammary gland. As in
MDCK cells, JAM4 was detected at TJs in epithelial
cells of small intestine and mammary gland ducts. In
kidney, JAM4 was most concentrated in glomeruli
and its localization at slit diaphragm was confirmed
by immunoelectron microscopy. However, we also foundthe signal on apical membranes of epithelium of renal
proximal tubules. We here extended the previous study
to determine the localization of JAM4 in various tissues
and compare it with that of MAGI-1. We attempted toelucidate the TJ-targeting mechanism of JAM4 andits role in the recruitment of MAGI-1. We are alsointerested in the structure of JAM4.The crystal structure
determination of JAM1 revealed the structural basis
for homophilic interaction (Kostrewa et al. 2001; Prota
760 Genes to Cells (2003) 8, 759–768

et al. 2003). Inspired by these studies, we have examined
the role of each Ig-loop of JAM4 for the cell adhesion
activity.

Results
JAM4 is localized at TJs in various tissues

In the previous study, we reported that JAM4 is localized
at slit diaphragm in kidney glomeruli and confirmed
the localization using immunoelectron microscopy(Hirabayashi et al. 2003). We also found that JAM4 isco-localized with ZO-1, a tight junction marker protein, in small intestine and mammary gland epithelial
cells on the immunofluorescence level and concluded
that JAM4 is a tight junction protein. However, JAM4
immunoreactivity was also detected on apical membranes of renal proximal tubular epithelial cells. Thesefindings prompted us to perform a study to determine
the subcellular localization of JAM4 in various rat
tissues. First, we reexamined the localization of JAM4
in various portions of small intestines. JAM4 was colocalized with ZO-1 in duodenum, although somesignal was detected on apical membranes (Fig. 1Aa).
However, in jejunum and ileum, JAM4 was detected
mainly on apical membranes (Fig. 1Ab and c). The
absorption with the antigen abolished the apical signals,
indicating that these signals are specific (Fig. 1B). Immunogold labelling of ultrathin sections of rat duodenal
epithelium revealed that gold particles were accumulated
at TJs, while they were absent from AJs and desmosomes(Fig. 1C). JAM4 was localized at TJs in epithelial cellsof submandibular glands and eccrine sweat glands (Fig. 2,
A and B). In kidney, JAM4 was detected at TJs of collecting ducts (Fig. 3A). JAM4 immunoreactivity on apical membranes in renal proximal tubular epithelial cells
was also reproduced (Fig. 3B).
The first Ig-loop is required for the recruitment of
JAM4 to TJs

Based on the in vivo localization of JAM4 on apicalmembranes and at TJs, we raised a question how JAM4
was targeted to TJs.To address this question, we preparedstable transformants of MDCK cells expressing various
FLAG-tagged JAM4 proteins (Fig. 4). FLAG-JAM4-1
was concentrated at cell junctions, co-localized with
ZO-1, but also slightly detected on apical membranes(Fig. 5A). FLAG-JAM4-2 lacking the first Ig-loop
mostly appeared on apical membranes (Fig. 5B). We also
confirmed the apical localization of FLAG-JAM4-1 and-2 using the surface protein biotinylation (Fig. 5E).
© Blackwell Publishing Limited

Roles of Ig-loops of JAM4

Figure 1 JAM4 in small intestine. (A)Immunohistochemical analysis of JAM4
in small intestine. (a) JAM4 and ZO-1 induodenum. (b) JAM4 and ZO-1 injejunum. (c) JAM4 and ZO-1 in ileum. Bar,20 µm. (B) Immunofluorescence of ileum
after absorption with the immunogen.
Apical signals were abolished. Bar, 20 µm.
(C) Immunogold analysis of JAM4 in rat
duodenal epithelial cells. The ultrathin
section of the adult rat small intestine wasstained with rabbit polyclonal anti-JAM4
antibody, followed by immunoelectron
microscopy with the immunogold method.
Gold particles were detected at TJs (arrows)
and also decorated the apical side to TJs.
Bar, 100 nm.

Figure 2 JAM4 in salivary and sweat
glands. (A) JAM4 and ZO-1 in epithelial
cells of submandibular glands. The
demarcated area in (a) is shown at higher
magnification in (b). (a) Bar, 20 µm (b)Bar, 10 µm. (B) JAM4 and ZO-1 inepithelial cells of eccrine sweat glands. The
demarcated area in (a) is shown at higher
magnification in (b). (a) Bar, 20 µm (b) Bar,
5 µm.

FLAG-JAM4-1 and -2 were biotinylated, only when
the biotinylation reagent was added to the apical chambers. Like FLAG-JAM4-1, FLAG-JAM4-3 lacking the Cterminal PDZ-binding motif was mainly localized at TJs,
although some of FLAG-JAM4-3 was detected on apicalmembranes (Fig. 5C). We also noticed that the junctional staining of FLAG-JAM4-3 appeared coarse, compared to that of FLAG-JAM4-1. FLAG-JAM4-4 lacking
the second Ig-loop was localized at TJs, but was also distributed along lateral membranes outside TJs (Fig. 5D).
These data imply that the targeting of JAM4 to TJs primarily depends on the first Ig-loop, and that the PDZbinding motif, which is involved in the interaction with
MAG-1, is not required.
© Blackwell Publishing Limited

Over-expressed JAM4 is localized on apicalmembranes and interferes with the localization of
MAGI-1In the next sets of experiments, we examined the effectof JAM4 on the localization of MAGI-1. Because FLAGJAM4-1 was mostly co-localized with MAGI-1 in stabletransformants of MDCK cells, we made use of FLAGJAM4-2. First, we determined the localization of endogenous MAGI-1 in stable transformants of MDCK cells
expressing FLAG-JAM4-2 (Fig. 6A). As described forFig. 5B, FLAG-JAM4-2 was localized on apical membranes. MAGI-1 was still concentrated at TJs (arrowheads), but it was also detected on the apical membranes.
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Figure 3 JAM4 in kidney. (A) JAM4 incollecting ducts.The demarcated area in (a)is shown at higher magnification in (b). Bar,20 µm. (B) JAM4 in glomerulus and renal
proximal tubular epithelial cells. The
demarcated area in (a) is shown at higher
magnification in (b). Bar, 20 µm.

JAM4 recruits MAGI-1 to TJs in MDCK cells

Figure 4 Schematic model for various FLAG-tagged JAM4
constructs. The extracellular region of JAM4 contains twodomains with intramolecular disulphide bonds typical of
immunoglobulin-like loops (Ig-loops) of the V-type. The closed
rectangle at the C-terminus and the grey rectangle at the Nterminus of each construct indicate the PDZ-binding motif and
the FLAG-tag, respectively. Black dots are putative N-linked
glycosylation sites. The numbers show the first and the last aminoacid residue number of JAM4 for each construct.

Transient expression of FLAG-JAM4-2 showed a similar
but more remarkable effect on MAGI-1 (Fig. 6B). In
MDCK cells expressing FLAG-JAM4-2, MAGI-1 wasrecruited to apical membranes, while it remained at TJs
in parent MDCK cells (arrow). As described for Fig. 5C,
FLAG-JAM4-3 without the MAGI-1-binding region
mostly localized to TJs in stable transformant cells. However, when it was transiently over-expressed in MDCK
cells, the expression level was much higher and FLAGJAM4-3 was overflowed from TJs to be localized mainlyon the apical membranes (Fig. 6C). Even so, it did notaffect the localization of endogenous MAGI-1.
762 Genes to Cells (2003) 8, 759–768

To obtain another line of evidence that JAM4 determines
the subcellular localization of MAGI-1, we employed stabletransformants of MDCK cells expressing green fluorescent protein (GFP)-tagged MAGI-1 (MDCK-GFPMAGI-1). GFP-MAGI-1 showed diffuse distribution in
the cytoplasm and was slightly concentrated at cell contacts(Fig. 7A). Unlike endogenous MAGI-1, over-expressed
GFP-MAGI-1 was localized along lateral membranes.
When FLAG-JAM4-1 was transiently introduced to
MDCK-GFP-MAGI-1 cells, FLAG-JAM4-1 was colocalized with ZO-1 at TJs (Fig. 7B). Simultaneously,
the cytoplasmic signal of GFP-MAGI-1 disappeared.
Moreover, the signal at cell contacts was accumulated
exclusively to TJs (arrows). In contrast, FLAG-JAM4-3
lacking the MAGI-1-interacting domain did not affectthe localization of GFP-MAGI-1 (data not shown).Thesefindings overall suggest that JAM4 is a molecular determinant of the localization of MAGI-1 in MDCK cells.
MAGI-1 is localized at TJs in tissues where JAM4 isdetected on apical membranes

Subsequently, we examined how MAGI-1 was localized
in renal proximal tubules and ileum, where JAM4
showed apical localization. MAGI-1 was not detected inrenal proximal tubular epithelium (data not shown) thus
we could not evaluate how apical JAM4 affected the
localization of MAGI-1. In ileum, we detected MAGI1 on apical membranes (Fig. 7C, arrowheads). This isconsistent with the results described for MDCK cells.
However, MAGI-1 was also localized at TJs in ileum,
although JAM4 was not detected at TJs (arrows). It suggests that the localization of MAGI-1 is not determined
only by JAM4 in vivo.
© Blackwell Publishing Limited

Roles of Ig-loops of JAM4

Homophilic interactions of JAM4 depend on both of
the first and the second Ig-loops

As described above, the first Ig-loop is essential for JAM4
to be recruited to TJs.We next examined the function of
Ig-loops. We previously reported that JAM4 promotes
Ca2+-independent cell adhesion, and that this activity
does not require the PDZ-binding motif (Hirabayashi
et al. 2003).To evaluate how the Ig-loops are involved inthis activity, we prepared stable transformants of L cells
expressing FLAG-JAM4-1, -2, -3, -4 and -control (LFLAG-JAM4-1, L-FLAG-JAM4-2, L-FLAG-JAM4-3,
L-FLAG-JAM4-4 and L–FLAG-control cells). Aggregation assays demonstrated that L-FLAG-JAM4-2 and LFLAG-JAM4-4 cells formed aggregates in a timedependent manner, but these aggregates were consistedof fewer L cells and were smaller in size than those of
L-FLAG-JAM4-1 and L–FLAG-JAM4-3 cells (Fig. 8A
and B). Furthermore, L-FLAG-Control cells expressing
a JAM4 variant with no Ig-loop also showed someaggregation activity, suggesting that the aggregation of
L-FLAG-JAM4-2 and L-FLAG-JAM4-4 cells weremediated by the FLAG-tags rather than Ig-loops. All
FLAG-proteins were expressed at similar levels in cells
that we used for this experiment (Fig. 8C). Therefore,
we concluded that JAM4 requires both of Ig-loops forthe adhesion activity.
Cis interactions of JAM4 depend on the first Ig-loop

Figure 5 Subcellular distribution of various FLAG-JAM4
proteins in MDCK cells. Stable transformants of MDCK cells
expressing various FLAG-JAM4 proteins were immunostainedwith mouse anti-FLAG, rabbit anti-ERBIN, and rat anti-ZO-1
antibodies. (A) FLAG-JAM4-1 was mainly co-localized with ZO1, but some signal was also detected on apical membranes. (B)FLAG-JAM4-2 without the first Ig-loop was distributed on apicalmembranes. (C) FLAG-JAM4-3 without the C-terminal PDZbinding motif was co-localized with ZO-1, but some signal was
also detected on apical membranes. (D) FLAG-JAM4-4 without
the second Ig-loop was localized on basolateral membranes andpartially overlapped with both of ZO-1 and ERBIN. Bar, 10 µm.
(E) Surface protein biotinylation of FLAG-JAM4-1 and FLAGJAM4-2. MDCK cells expressing FLAG-JAM4-1 and FLAGJAM4-2 were cultured on Transwell plates and biotinylated fromthe upper (Apical) and lower (Basolateral) chambers. Biotinylated
proteins were precipitated with avidin-beads. Original cell lysates(ori) and precipitates (ppt) on avidin-beads were immunoblotted© Blackwell Publishing Limited

In the last sets of experiments, we investigated which Igloop is necessary for cis interactions of JAM4.We treated
L cells expressing various JAM4 mutants with a chemical
cross-linker and performed Western blotting to analyse
the dimer formation. FLAG-JAM4-1 and -3 formed
dimers when treated with a chemical cross-linker(Fig. 9). Consistent with this, the amount of FLAGJAM4-1 and -3 monomers decreased. FLAG-JAM4-2
lacking the first Ig-loop did not form dimers, although
the amount of FLAG-JAM4-2 seemed to decrease. We
consider that this decrease may be due to the nonspecificcross-linking of FLAG-JAM4-2 with other proteins. FLAGJAM4-4 lacking the second Ig-loop formed dimers.
with anti-E-cadherin or anti-FLAG antibody. (a) Immunoblot
with anti-E-cadherin antibody of the precipitates from MDCKFLAG-JAM4-1 cells. Although we do not show the data,
immunoblot of the precipitates from MDCK-FLAG-JAM4-2
cells gave a similar result. (b) Immunoblot with anti-FLAG
antibody of the precipitates from MDCK-FLAG-JAM4-1 cells.
(c) Immunoblot with anti-FLAG antibody of the precipitates from
MDCK-FLAG-JAM4-2 cells. Protein standard markers are shownon the left.

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Figure 6 Effect of JAM4 on the subcellular localization of
MAGI-1 in MDCK cells. (A) MAGI-1 in stable transformants of
MDCK cells expressing FLAG-JAM4-2. FLAG-JAM4-2 was
localized on apical membranes in MDCK cells. MAGI-1 wasconcentrated at TJs and was also distributed on apical membranes(arrow heads). Lines marked by open arrows indicate where the zsection was obtained. (B) MAGI-1 in MDCK cells in which
FLAG-JAM4-2 was transiently introduced. Endogenous MAGI1 moved to apical membranes and was co-localized with FLAGJAM4-2. In contrast, MAGI-1 remained at TJs in MDCK cells
that did not express FLAG-JAM4-2 (arrow). (C) MAGI-1 in
MDCK cells in which FLAG-JAM4-3 was transiently introduced.
FLAG-JAM4-3 lacking the PDZ-binding motif did not affect the
localization of endogenous MAGI-1. Bar, 10 µm.

Discussion
We previously described the localization of JAM4 atslit diaphragm in kidney and at TJs in small intestineand mammary gland ducts (Hirabayashi et al. 2003).
During the study, we detected JAM4 on apical membranes in renal proximal tubular epithelial cells.
Moreover, we found that JAM4 was localized on apicalmembranes in jejunum and ileum, while it was concentrated at TJs in duodenum, eccrine sweat glands,
764 Genes to Cells (2003) 8, 759–768

and submandibular glands. Thereby, we consider thatboth of the localizations at TJs and on apical membranes
are physiologically relevant. We tried to elucidate themechanism that determines the subcellular localizationof JAM4. Though JAM4 has not been detected in distal
tubules of rat kidneys in the immunohistochemical
study, we employed MDCK cells, a widely used epithelial cell line for analysis of TJ assembly and regulation.
The mutant of JAM4 lacking the first Ig-loop was not
recruited to TJs and was localized on apical membranes.
Furthermore, even the full-length of JAM4, when overexpressed, was observed not only at TJs but also on apicalmembranes. The immunoelectron microscopy of small
intestinal epithelium showed that gold particles weredetected on the apical side to TJs. The surface protein
biotinylation experiment also indicated that JAM4 was
localized on the apical side to TJs in MDCK cells. Nglycans are known as apical sorting signals in epithelial
cells and JAM4 is heavily modified with N-glycosylation(Scheiffele et al. 1995; Hirabayashi et al. 2003). Taken
together, we speculate that JAM4 would be first targeted
to apical membranes and then accumulated to TJs via thefirst Ig-loop–mediated molecular interaction. Because
the first Ig-loop is involved in cis interactions, therecruitment of single JAM4 molecule to TJs would
result in the further accumulation of JAM4. The
molecule that directly tethers JAM4 to TJs is currently
unknown. It should have the extracellular domain
and may be a JAM4-like cell adhesion molecule thatinteracts with JAM4 in cis- or trans- interactions. In cells
that lack the counterpart, JAM4 may remain on theapical membranes.
Another explanation is that JAM4 detected on apicalmembranes may be a splicing variant that lacks the signal
sequence essential for the TJ targeting. One form ofmurine vascular cell adhesion molecule-1 (VCAM-1)
called VCAMTM is expressed on basolateral membranes, whereas its splicing variant VCAMGPI is locatedon apical membranes (Pirozzi et al. 1994). Carcinoembryonic antigen-related cell adhesion molecule 1(CEACAM1) has two isoforms with different cytoplasmic domains and CEACAM1-L appears on both theapical and the lateral surfaces of epithelial cells, while
CEACAM1-S is exclusively recruited to apical membranes (Sundberg & Obrink 2002).There may be a splicing variant of JAM4 that shows the apical localization in
kidney and small intestine.
JAM4 recruits MAGI-1 to the cell contacts in L cells.
Likewise, JAM4 accumulates the over-expressed GFPMAGI-1 to TJs in MDCK cells. These data imply that
JAM4 is a molecular determinant for the TJ localizationof MAGI-1. However, in renal distal tubular epithelial© Blackwell Publishing Limited

Roles of Ig-loops of JAM4
Figure 7 Recruitment of GFP-MAGI-1 by
JAM4 to TJs. (A) GFP-MAGI-1 in MDCK
cells. Stable transformants of MDCK cells
expressing GFP-MAGI-1 (MDCK-GFPMAGI-1) was double-stained with anti-βcatenin and anti-ZO-1 antibodies. GFPMAGI-1 was localized along lateral membranes
and was also detected in the cytosol. (B) FLAGJAM4-1 was introduced into MDCK-GFPMAGI-1 and stained with anti-FLAG andanti-ZO-1 antibodies. In cells with FLAGJAM4, GFP-MAGI-1 was co-localized with
ZO-1 (arrows). In MDCK-GFP-MAGI-1
cells without FLAG-JAM4-1, GFP-MAGI1 was not concentrated at TJs (arrowheads).
(C) MAGI-1 in epithelial cells of ileum.
MAGI-1 was detected on apical membranes(arrow heads). However, some MAGI-1
was co-localized with ZO-1 (arrows). Bar,20 µm.

cells, jejunum, and ileum, MAGI-1 is present even at TJs
where JAM4 is absent.This fact rather contradicts to themodel. There may be some other protein besides JAM4
that recruits MAGI-1 to TJs.
Recently, the roles of each Ig-loop in immunoglobulinlike cell adhesion molecules have been characterized.
The first Ig-loop of JAM1 is responsible for cis interactions (Kostrewa et al. 2001). Concerning murine JAM1,
homo-cis-dimers on the surface of one cell engage
homo-cis-dimers on the adjacent cell and form thehomo-trans-dimers. For human JAM1, a different modelis proposed (Prota et al. 2003). The trans-dimerization ofhuman JAM1 may depend on the presence of additional
proteins, or a human JAM1 monomer may directlyinteract with a monomer on apposing cells and formhomo-trans-dimers. In nectin, which has three Ig-loops,
the first Ig-loop is involved in trans-dimerization, and thesecond one is necessary for cis-dimerization (Miyahara
et al. 2000; Momose et al. 2002). In case of JAM4, bothof two Ig-loops are indispensable for trans interactionsof JAM4. On the other hand, only the first Ig-loop isnecessary for cis interactions.
We conclude that JAM4 may be primarily targeted toapical membranes and subsequently recruited to TJs via
the interactions of the first Ig-loops. We also proved that
JAM4 determines the localization of MAGI-1 in
MDCK cells, but the in vivo localization of MAGI-1
does not always depend on JAM4. The counterpart of
JAM4 at TJs remains to be clarified. It is also an interesting question whether JAM4, like other JAM proteins,
takes part in the process of inflammation, possibly as amediator of leucocyte migration.
© Blackwell Publishing LimitedExperimental proceduresConstruction of expression vectors pLN vector was previously
described (Hirabayashi et al. 2003). pFLAG-CMV-1 was purchased from Eastman Kodak Company. The constructs of JAM4
used in this study are summarized in Fig. 4. They contain the following regions of JAM4; pFLAG and pLN FLAG JAM4, 21-370
of JAM4; pFLAG and pLN FLAG JAM4-2, 128-370 of JAM4;
pFLAG and pLN FLAG JAM4-3, 21-361 of JAM4; pFLAG andpLN FLAG JAM4-4, 21-127 and 223-370 of JAM4; and pFLAG
Control, 223-370 of JAM4.

Antibodies
Rabbit anti-ERBIN and anti-JAM4 antibodies were previously
described (Ohno et al. 2002; Hirabayashi et al. 2003). Rabbit antibody against the N-terminal region of synaptic scaffolding molecule was used to detect MAGI-1 in immunohistochemical andimmunocytochemical studies as in the previous study (Hirabayashi
et al. 2003). Mouse anti-ZO-1 antibody (Zymed), mouse anti-βcatenin antibody (BD Transduction), mouse and rabbit anti-FLAG
antibodies (Sigma-Aldrich Fine Chemicals), rat anti-ZO-1 antibody (Chemicon) and rhodamine-conjugated, FITC-conjugatedand Cy5-conjugated second antibodies for dual labelling (Chemicon) were purchased from commercial sources.

Cells
Phoenix ampho, Madine Darby canine kidney II (MDCK) andmouse fibroblastic L cells were cultured in DME medium supplemented with 10% FBS, 100 U/mL penicillin and 100 µg/mL
streptomycin under 5% CO2 at 37 °C. MDCK cells were transfected with Effectene Transfection Reagent (Qiagen). Retrovirus
system was used to generate stable transformants of MDCK and L
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Figure 9 Chemical cross-linking of various FLAG-JAM4
proteins. Stable transformants of L cells expressing various FLAGJAM4 proteins were treated with the mock or BS3 for crosslinking. The cell lysates were immunoblotted with anti-FLAG
antibody. Arrows and arrowheads indicate monomers and dimersof FLAG-JAM4 proteins, respectively. Protein standard markers
are shown on the left.

cells as described (Nishimura et al. 2000). Briefly, MDCK and L
cells were infected with the retrovirus and cultured in the selective
medium with 1 mg/mL G418 (Calbiochem). The resistant colonies were tested for the expression of each protein and cloned.

Immunocytochemistry and immunofluorescencemicroscopy
MDCK and L cells were fixed with 4% (w/v) formaldehyde inphosphate buffered saline (PBS) for 15 min, blocked with 50 mm
glycine in PBS for 30 min, and incubated with 0.2% (w/v) Triton
X-100 in PBS for 15 min and subsequently with 1% (w/v) bovine
serum albumin (BSA) in PBS for 30 min. These processes werecarried out at room temperature. The samples were incubatedwith various first antibodies and visualized with rhodamine-,
FITC-, or Cy5-conjugated second antibodies. The images wereobtained by a confocal microscopy (Zeiss LSM 510).

Immunohistochemistry

Figure 8 Cell adhesion activity of various FLAG-JAM4 proteins.
(A) Cell aggregation assays were performed using wild-type L cells
and stable transformants of L cells expressing various FLAG-JAM4
proteins. The extent of cell aggregation was represented by ratioof the total particle number at indicated time point (Nt) to theinitial particle number (N0). The Nt/N0 values are indicated by
the mean ± SE of three independent experiments.  wild-type of
L cells;  L-FLAG-JAM4-1;  L-FLAG-JAM4-2;  L-FLAG-766 Genes to Cells (2003) 8, 759–768

All procedures related to the care and treatment of animals werein accordance with institutional and National Institutes of Healthguidelines. 4- to 6-week-old Wistar rats were deeply anaesthetizedwith sodium pentobarbital (60 mg/kg i.p) and perfused with 4%(w/v) paraformaldehyde in 0.1 m phosphate buffer (PB) pH 7.4.
For small intestines, skins and kidneys, tissues were removed, postfixed in the same fixative, blocked with 50 mm glycine in 0.1 m
JAM4-3;  L-FLAG-JAM4-4; ∆ L-FLAG-Control. (B) Cellaggregates of wild-type of L cells (L-Wt) and various L-JAM4 cells(L-FLAG-JAM4-1, -JAM4-2, -JAM4-3, -JAM4-4 and -control)
at 30 min in cell aggregation assay. Bar, 200 µm. (C)Immunoblottings by the anti-FLAG antibody of various L-JAM4cell lysates. Protein standard markers are shown on the left.

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Roles of Ig-loops of JAM4
PB, immersed with 10% (w/v), 20% (w/v), and 30% (w/v)
sucrose in 0.1 m PB sequentially and frozen at −80 °C. 5 µm sections were prepared, blocked with PBS containing 5% goat serum
and 0.2% (w/v) Triton X-100 for 2 h and incubated with firstantibodies at 4 °C overnight. After washed with PBS four times,
the samples were incubated with second antibodies at room temperature for 2 h and bound antibodies were detected. Images wereobtained by confocal microscopy (Olympus FV300-BX or Zeiss
LSM 510). For submandibular glands, tissues were obtained fromanaesthetized rats without perfusion and immediately frozen on
powdered dry ice. 10 µm sections were then prepared directlyfrom frozen tissues and fixed with −20 °C methanol for 15 min.
Other procedures for the preparation of samples were the same asabove. Images were obtained by using Olympus IX71 CCD
microscope.

Immunoelectron microscopy
Small intestines from anaesthetized adult rats were fixed by perfusion of 0.5% (w/v) glutaraldehyde in PBS. Tissues were cut insmall pieces and fixed in the same fixative for 1 h on ice, washedwith PBS and dehydrated with a graded series of ethanol.Two mm
cubes of intestinal epithelium were embedded in LR white resin(London Resin Company Ltd, London), polymerized with LR
white accelerator and stored at −20 °C. Ultrathin sections were
mounted on 300-mesh nickel grids and treated with 3% H2O2 for
10 min. After blocking with 1% (w/v) BSA in PBS for 30 min,grids were incubated overnight at 4 °C with affinity-purified rabbit anti-JAM4 antibody, washed with PBS, and incubated with
10-nm gold-conjugated goat anti-rabbit IgG (Amersham Pharmacia Biotech) with the 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 Hitachi 7100 electron microscopeat × 30 000 or × 8000 magnification.

Chemical cross-linking
Various L-FLAG-JAM4 cells were treated with 0.2% (w/v) trypsin
and 1 mm EDTA at 37 °C for 5 min and dispersed by pipetting toobtain single-cell suspensions (1 × 106 cells/mL). Bis(sulfosuccinimidyl) suberate (BS3) (Pierce) stock solution (10 mm BS3 in PBS)
was freshly prepared. Appropriate amount of the BS3 stock solution was added to the cells to the final concentration of 1 mm andincubated at room temperature for 30 min. Reactions werequenched by the addition of 1 m Tris/HCl pH 7.4 to a final concentration of 20 mm. After 15 min, cells were homogenized in
40 µL of the buffer containing 20 mm HEPES/NaOH pH 7.4,
100 mm NaCl, and 1% (w/v) Triton X-100 and centrifuged at
100 000 g for 15 min at 4 °C. The supernatant was analysed in
SDS-PAGE and immunoblotted with anti-FLAG antibody.

Cell surface biotinylation
2 × 106 cells were cultured on 24-mm Transwell (Costar) with
uncoated polycarbonate filters at 37 °C in 5% CO2. After 5 days
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when cells were grown to confluency, the medium was removed.
Cells were rinsed with cold PBS-CM (PBS with 1 mm MgCl2 and
0.1 mm CaCl2) and biotinylated with 0.5 mg/mL sulfo-NHSbiotin (Pierce) in PBS-CM at 4 °C for 60 min.The reactions werequenched by the addition of 50 mm NH4Cl. Cells were harvested,homogenized in 400 µL of the buffer containing 20 mm Tris/HCl
pH 8.0, 100 mm NaCl, and 1% (w/v) NP-40 and centrifuged at
100 000 g for 15 min at 4 °C.The supernatant was incubated withavidin-agarose beads (Sigma-Aldrich Fine Chemicals), and theprecipitated proteins were immunoblotted with appropriate
antibodies.

Cell aggregation assay
Wild-type or various stable transformants of L cells were treatedwith 0.2% (w/v) trypsin and 1 mm EDTA at 37 °C for 5 min anddispersed by pipetting. Cells were resuspended in Hanks’ balanced
salt solution with either 1 mm CaCl2 or 5 mm EDTA at 1 × 106
cells/mL, placed in 12-well plates precoated with BSA and rotatedon a gyratory shaker at 37 °C for indicated periods of time.Aggregation was quenched by adding 2% (v/v) glutaraldehyde. The
extent of aggregation was described as the ratio of the total particle
number after incubation to the initial particle number before
incubation. Images were obtained with Olympus IX70 at ×10
magnification with ×1.5 zoom and printed on papers withdimensions of 29 × 21 cm. Any cells that were in contact withanother were regarded as one particle.

Acknowledgements
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, Scienceand Technology.
We thank Dr S.Tsukita (Kyoto University) for MDCK cells, Dr
Y. Nakamura (University of Tokyo) for cDNA of human MAGI1, Dr G. Noran (Stanford University) for phoenix ampho cells,
and Dr S. Ichinose (Tokyo Medical and Dental University) for valuable advice.We also thank Ms. C. Rokukawa and Ms. M. MiyaharaTenkatsu for skilful technical assistance.

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Received: 12 May 2003
Accepted: 7 July 2003

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