1 Institute of Cell Biology, ZMBE, University of Münster, Germany
2 Department of Pathology, Centre Medical Universitaire, CH-1211 Geneva,
Switzerland
3 Department of Molecular Biology, Yokohama City University School of Medicine,
Yokohama 236-004, Japan
4 Max-Planck-Institute of Vascular Biology, D-48149 Münster, Germany
* Author for correspondence (e-mail: ebnetk{at}uni-muenster.de)
Accepted 9 June 2003
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Summary |
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Key words: Cell polarity, Endothelium, JAMs, PAR-3, Tight junction, ZO-1
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Introduction |
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Three types of tight junction-associated integral membrane proteins have
been identified so far. These are occludin
(Furuse et al., 1993), the
claudins (Furuse et al.,
1998a
) and several immunoglobulin (Ig) superfamily members,
including junctional adhesion molecule (JAM-1)
(Martin-Padura et al., 1998
),
endothelial cell-selective adhesion molecule (ESAM)
(Nasdala et al., 2002
) and the
coxsackie- and adenovirus receptor (CAR)
(Cohen et al., 2001
). Among
these, occludin and claudins seem to form the molecular basis of the tight
junction strands, as antibodies against occludin exclusively label TJ strands
and the intensity of occludin staining correlates with the number of tight
junction strands (Saitou et al.,
1997
), and the expression of claudin-1 or claudin-2 in L cell
fibroblasts results in the formation of tight junction strands
(Furuse et al., 1998b
). This
is not the case when JAM-1 is expressed in L cells
(Itoh et al., 2001
),
suggesting a function for JAM-1 that differs from the functions of occludin
and claudins.
Recently, progress has been made in understanding the molecular mechanisms
underlying the formation of TJs. Accumulating evidence supports the idea that
a molecular complex consisting of the cell polarity proteins PAR-3 and PAR-6,
as well as atypical protein kinase C (aPKC), plays a central role in the
generation of TJs in vertebrate epithelial cells
(Ohno, 2001). These molecules
are localized at TJs of epithelial cells and form a ternary complex in which
PAR-3 and PAR-6 are linked through aPKC
(Joberty et al., 2000
;
Lin et al., 2000
;
Suzuki et al., 2001
). In
addition, the small GTPases Cdc42 and Rac1 can be part of the complex through
their association with PAR-6 (Joberty et
al., 2000
; Johansson et al.,
2000
; Lin et al.,
2000
; Qiu et al.,
2000
; Yamanaka et al.,
2001
). The requirement of this molecular complex for tight
junction formation is suggested by the observations that first, overexpression
of a PAR-6 mutant that lacks the aPKC binding domain leads to aberrant PAR-3
and aPKC (aPKC)
localization, as well as to mislocalization of TJ
proteins like occludin, claudin-1 and ZO-1
(Yamanaka et al., 2001
); and
second, overexpression of a dominant-negative mutant of aPKC (aPKCkn) induces
a mislocalization of PAR-3 and PAR-6 as well as occludin, claudin-1 and ZO-1.
More importantly, the overexpression of both mutants disrupts the function of
TJs as development of transepithelial electrical resistance (TER),
paracellular permeability and membrane polarity are severely affected
(Suzuki et al., 2001
;
Yamanaka et al., 2001
). An
intriguing finding in these studies, however, is that the effects of aPKCkn
overexpression are observed in cells that are in the process of developing TJs
but not in fully polarized cells, suggesting a central role for the
PAR-3/PAR-6/aPKC complex in the biogenesis, rather than maintenance, of TJs
(Suzuki et al., 2001
;
Yamanaka et al., 2001
).
One component of the PAR-3/PAR-6/aPKC complex, PAR-3, directly associates
with JAM-1 (Ebnet et al.,
2001; Itoh et al.,
2001
). During cell contact formation JAM-1 colocalizes with
E-cadherin and ZO-1 in primordial spot-like adherens junctions or puncta
(Ebnet et al., 2001
),
indicating that JAM-1 is among the first tight junction-associated proteins
appearing at cell-cell contacts during junction formation. PAR-3, as well as
aPKC, appear after spot-like adherens junctions have been formed
(Suzuki et al., 2002
). This
supports the idea that the PAR-3/PAR-6/aPKC complex is targeted to nascent
cell-cell contacts through the association of PAR-3 with JAM-1. Although
direct evidence is still missing, it seems conceivable that the concomitant
activation of Cdc42 in response to E-cadherin-mediated cell adhesion
(Kim et al., 2000
) results in
the activation of the complex-associated aPKC activity through the binding of
active Cdc42 to PAR-6 (Yamanaka et al.,
2001
). The downstream targets of aPKC activity are still unknown.
In this scenario, JAM-1 would play an important role in recruiting/localizing
a signalling complex to sites of cell-cell adhesion and thus in promoting the
formation of tight junctions from spot-like adherens junctions. Despite the
evolutionary conservation of the PAR-3/aPKC/PAR-6 complex from
Caenorhabditis elegans and Drosophila to vertebrates,
integral membrane proteins through which the complex is targeted to the
membranes in the former two species have not been identified.
JAM-1 belongs to a subfamily of the Ig superfamily, which is characterized
by the presence of two Ig-like domains, a membrane-distal V-type and a
membrane-proximal C2-type Ig-like domain, the CTX family
(Aurrand-Lions et al., 2001a;
Chretien et al., 1998
). The
closest relatives of JAM-1 are JAM-2 and JAM-3
(Arrate et al., 2001
;
Aurrand-Lions et al., 2000
;
Cunningham et al., 2000
;
Liang et al., 2002
;
Palmeri et al., 2000
) (see
footnote* for the nomenclature of
JAM-2 and JAM-3); all three JAMs share a canonical type II PDZ domain
targeting motif at their C-termini
(Songyang et al., 1997
). In
multicellular tissues, JAM-1 is widely expressed by endothelial and epithelial
cells (Liu et al., 2000
;
Martin-Padura et al., 1998
;
Ozaki et al., 1999
), whereas
JAM-2 and JAM-3 are largely confined to endothelial cells
(Aurrand-Lions et al., 2001b
;
Liang et al., 2002
;
Palmeri et al., 2000
), with
JAM-3 also being identified in a squamous cell carcinoma cell line of
epithelial origin (Aurrand-Lions et al.,
2001a
). The subcellular localization of JAM-2/-3 has not been
analysed yet at the ultrastructural level. Ectopic expression of JAM-2 in MDCK
(Madin-Darby Canine Kidney) epithelial cells results in colocalization of
JAM-2 with ZO-1, suggesting that JAM-2 is TJ-associated
(Aurrand-Lions et al., 2001b
).
The two other more distantly related members of the CTX family, which were
described to be localized at tight junctions, i.e. ESAM and CAR, are expressed
in endothelial cells or both endothelial and epithelial cells, respectively
(Carson et al., 1999
;
Cohen et al., 2001
;
Nasdala et al., 2002
). Despite
a similar overall organization, the latter two molecules differ from
JAM-1/-2/-3 in the size of the cytoplasmic domains and in their C-termini,
which end in canonical type I PDZ domain targeting motifs
(Bergelson et al., 1997
;
Hirata et al., 2001
),
suggesting differences in the nature of cytoplasmically associated
proteins.
To date, peripheral membrane components of tight junctions associating with JAM-2 and JAM-3 have not been identified. The structural similarities between JAM-1 and JAM-2/-3 prompted us to address whether JAM-2 and JAM-3 associate with the cell polarity protein PAR-3. We report that PAR-3 strongly associates with JAM-2 and JAM-3 but not with CAR or ESAM. In addition, we found that the tight junction protein ZO-1 associates with both JAM-2 and JAM-3. The localization of JAM-2 at cell-cell contacts is regulated by serine phosphorylation, and JAM-2 at cell contacts recruits both PAR-3 and ZO-1. Our findings support the idea of a general role for all three members of the JAM family in the regulation of tight junction formation and cell polarity.
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Materials and Methods |
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Rabbit polyclonal antibodies against PAR-3 (C2-3) and AF-6 were described
previously (Ebnet et al.,
2000; Izumi et al.,
1998
). The anti-JAM-2 monoclonal antibody (CRAM XIXH36, rat
IgG2a) was purified from serum-free Ultroser HY 0.75% medium
(Biosepra, France) by ammonium sulfate precipitation and protein G
immunoaffinity column. A polyclonal antibody against JAM-2 (ke738) was
generated by immunizing rabbits with a fusion protein consisting of the
extracellular domain of JAM-2 fused to the Fc-part of human IgG. The
antibodies were affinity-purified by adsorption on the same fusion protein
covalently coupled to cyanogen bromide-activated sepharose beads
(Amersham-Pharmacia Biotech, Freiburg, Germany), and antibodies directed
against the Fc-portion were depleted by adsorption on human IgG coupled to
cyanogen bromide-activated sepharose beads. The following commercially
available antibodies were used: rat mAb against ZO-1 (Chemicon, Hofheim,
Germany), rabbit pAb against ZO-1 (Zymed, Berlin, Germany), rat mAb against
PECAM-1 and mouse mAb against the heat-shock protein HSP-90 (BD Pharmingen,
Heidelberg, Germany); rabbit polyclonal antiserum against von Willebrand
factor (DAKO, Hamburg, Germany) and rat mAb MECA-79 against peripheral node
addressin (ATCC, Manassas, VA). Mouse anti-T7 tag mAb was purchased from
Calbiochem-Novabiochem (Schwalbach, Germany). Secondary antibodies were
purchased from Dianova (Hamburg, Germany).
Expression vectors
For the generation of GST fusion proteins pGEX expression vectors (Amersham
Pharmacia Biotech) were used. GST-JAM-1 expression vectors were described
elsewhere (Ebnet et al.,
2000). Expression vectors encoding GST-JAM-2 and GST-JAM2
5
were generated by cloning the cytoplasmic tail (aa 261-310) or a C-terminal
truncation mutant (aa 261-305) of JAM-2 in pGEX-5X-2 or pGEX-6P-2,
respectively. Expression vectors encoding GST-JAM-3 and GSTJAM-3
5 were
generated by cloning the cytoplasmic tail (aa 259-298) or a C-terminal
truncation mutant (aa 259-293) of JAM-3 in pGEX-5X-2 or pGEX-6P-2,
respectively. GST-ESAM was generated by cloning the cytoplasmic tail of ESAM
(aa 278-394) into pGEX-KG (Nasdala et al.,
2002
). GST-CAR was generated by cloning the cytoplasmic tail of
murine CAR (aa 259-345; GenBank accession number Y10320) into pGEX-4T-1. The
expression vector encoding murine JAM-2 has been previously described
(Aurrand-Lions et al., 2001a
;
Aurrand-Lions et al., 2001b
).
The point mutation S281A was generated by a PCR-based approach using
PfuTurbo® DNA polymerase (Stratagene, Netherlands). Expression vectors
encoding PAR-3 and truncation mutants of PAR-3 or ZO-1 were described
previously (Ebnet et al.,
2001
).
Generation of GST fusion proteins and in vitro binding assays
The purification of GST fusion proteins and in vitro GST-pulldown assays
were performed essentially as described previously
(Ebnet et al., 2000;
Ebnet et al., 2001
).
In vivo labelling, phosphoamino acid analysis and phosphotryptic
peptide mapping
CHO cells stably expressing JAM-2 wild-type or JAM-2 S281A were washed in
phosphate-free DMEM and subsequently metabolically labelled for 12 hours in
phosphate-free DMEM containing [32P]orthophosphate (0.5 mCi/ml).
Cells were lysed in lysis buffer (50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 0.5%
(v/v) Triron X-100, 12.5 mM NaF, 10 mM NaPPi, 10 mM
VO43-, 0.07 trypsin inhibitory units/ml aprotinin, 1 mM
PMSF (phenylmethyl sulphonyl fluoride), 1 mM dithiothreitol) and JAM-2 was
immunoprecipitated using affinity-purified polyclonal rabbit antibodies.
Phosphorylated proteins were resolved by SDS-PAGE. For phosphoamino acid
analysis proteins were transferred to PVDF membranes and visualized by
autoradiography. After excision of the bands corresponding to JAM-2, amino
acids were released by acid hydrolysis and separated by two-dimensional
electrophoresis on thin-layer cellulose plates using a Hunter HTLE 7000
apparatus. For phosphotryptic peptide mapping, the bands corresponding to
JAM-2 were eluted from the polyacrylamide gels, digested, separated and
visualized according to published protocols
(Boyle et al., 1991).
Transient transfection
For transient transfection, COS-7 cells were grown to a density of
approximately 80% confluency. Cells were incubated with a mixture of 2
µg/ml circular plasmid DNA and 12 µl/ml GeneJammer transfection reagent
(Stratagene Europe, Amsterdam, The Netherlands) for 3 hours. Cells were then
supplemented with complete medium and incubated under standard culture
conditions. Forty hours after transfection cells were harvested and lysates
were prepared as described (Ebnet et al.,
2001).
Immunohistochemistry and immunocytochemistry
For cryosections, organs and tissues from Balb/c mice were embedded in
Tissue Tek OCT compound (Miles, Elkhart, IN), snap frozen and stored at
-80°C. Sections of 7 µm were cut on a freezing microtome, mounted on
slides coated with poly-L-lysine (Menzel-Gläser, Nußloch, Germany)
and dried. For immunoperoxidase staining, the sections were fixed in acetone
for 10 minutes at 4°C; this was followed by a reduction of endogenous
peroxidase activity with 0.1% hydrogen peroxide, 20 mM sodium azide, for 30
minutes at room temperature. Nonspecific binding was blocked by incubation
with 2% bovine serum albumin in PBS for 30 minutes. Tissue sections were
incubated with the primary antibodies diluted in PBS/1% bovine serum albumin
for 1 hour, followed by incubation with affinity-purified
peroxidase-conjugated secondary antibodies. After visualization of the
reaction with 3-amino-9-ethylcarbazole the sections were counterstained with
Mayer's hematoxylin and mounted. All steps were performed in a humidified
chamber at room temperature. For control purposes sections were treated in the
same way but with the primary antibodies being omitted; these controls
consistently gave negative results.
For immunofluorescence analysis cells were grown on LabTec chamber slides
(Nalge-Nunc, Wiesbaden, Germany). Alternatively, cells were plated at low
density (1x103/cm2) on glass coverslips coated
with matrigel 1/20 (Becton-Dickinson) and grown for four days. This results in
islets of cells, which can be analysed individually for JAM-2 localization by
immunocytochemistry. Stainings were performed as previously described
(Ebnet et al., 2001).
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Results |
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|
PAR-3 associates with JAM-2 and JAM-3 through its first PDZ
domain
PAR-3 contains three PDZ domains, for which binding partners have been
described only for the first, i.e. JAM-1 and PAR-6
(Ebnet et al., 2001;
Lin et al., 2000
). Therefore,
it seemed possible that JAM-2 and JAM-3 associate with PAR-3 through PDZ
domains 2 and/or 3. To test this possibility, we generated individual PDZ
domains of PAR-3 by in vitro translation and incubated these with GST-JAM
fusion proteins immobilized on glutathione-Sepharose beads. As a positive
control we used a PAR-3 construct comprising all three PDZ domains. PDZ1
domain of PAR-3 strongly bound to both JAM-2 and JAM-3; PDZ2 domain did not
associate with either, whereas the PDZ3 domain weakly associated with both JAM
molecules (Fig. 2). In all
cases, the association was drastically reduced or abolished when the
C-terminal five amino acids of the JAM molecules were deleted. These findings
suggested that PAR-3 associates with JAM-2 and JAM-3 predominantly through
PDZ1 domain and weakly through PDZ3 domain.
|
When we used PAR-3 fragments containing all three PDZ domains with
individual PDZ domains inactivated by replacement with the inactive PDZ domain
present in the secreted form of interleukin 16 (IL-16)
(Ebnet et al., 2001;
Muhlhahn et al., 1998
), we
found that the inactivation of the PDZ1 domain completely abolished the
association between PAR-3 and JAM-2 or JAM-3, whereas the inactivation of the
PDZ2 domain had no effect on the binding, and inactivation of PDZ3 domain
reduced, but did not completely abolish the association (data not shown).
These findings complemented the observation with individual PDZ domains and
confirmed that PAR-3 associates in vitro with both JAM-2 and JAM-3
predominantly through PDZ1 domain and that PDZ3 domain might contribute to the
association.
PAR-3 can be affinity-isolated from COS-7 cell extracts
To analyse whether JAM-2 and JAM-3 associate with PAR-3 generated in vivo,
we transiently transfected COS-7 cells with PAR-3 expression vectors
containing either full-length PAR-3 or truncated PAR-3 constructs comprising
the C-terminal half of PAR-3, which includes PDZ3 domain and the aPKC-binding
domain (amino acids 583-1337) or a central part of PAR-3, including PDZ
domains 1-3 and the aPKC-binding domain (aa 258-936). The lysates of the
transfected cells were then incubated with immobilized GST-fusion proteins
containing the cytoplasmic domains of JAM-2 and JAM-3, and with immobilized
GST alone. Bound proteins were detected by western blot analysis using
antibodies against the T7-tag fused to the PAR-3 constructs. Under these
conditions full-length PAR-3, as well as the PAR-3 construct comprising all
three PDZ domains (aa 258-936), could be affinity-isolated from COS-7 cell
lysates, whereas the PAR-3 construct lacking PDZ domains 1 and 2 (aa 583-1337)
could not be affinity-isolated (Fig.
3). These findings indicate that PAR-3 constructs generated in
vivo associate with JAM-2 as well as with JAM-3 in vitro, and further support
the notion that this association is mediated predominantly through the PDZ1
domain of PAR-3.
|
PAR-3 associates exclusively with members of the JAM family among
tight junction-associated immunoglobulin-like transmembrane proteins
We have shown recently that among integral transmembrane proteins present
at tight junctions, which include JAMs, occludin and claudins
(Tsukita et al., 2001), PAR-3
associates exclusively with JAM-1 but not with occludin, claudin-1, claudin-4
or claudin-5 (Ebnet et al.,
2001
). Recently, two additional members of the immunoglobulin
superfamily, ESAM (Hirata et al.,
2001
) and CAR (Bergelson et
al., 1997
), were described to be localized at tight junctions of
endothelial cells and epithelial cells, respectively
(Cohen et al., 2001
;
Nasdala et al., 2002
). Both
molecules carry canonical PDZ domain targeting motifs at their C-termini,
which fit to the type I PDZ domain binding motif
(Songyang et al., 1997
). To
address the possibility that PAR-3 binds to ESAM or CAR we performed GST
binding experiments with GST-ESAM and GST-CAR fusion proteins and in vitro
translated, [35S]methionine-labelled PAR-3 constructs comprising
either the three PDZ domains of PAR-3 or full-length PAR-3. Both PAR-3
constructs associated exclusively with the three JAM molecules but not with
ESAM or CAR (Fig. 4). As
described recently (Ebnet et al.,
2001
), PAR-3 did not associate with claudin-1 or claudin-5. These
findings suggest a striking selectivity of PAR-3 for the JAM molecules among
all tight junction-associated integral membrane proteins.
|
JAM-2 and JAM-3 associate with ZO-1 in vitro
Besides PAR-3, JAM-1 associates with the tight junction-associated MAGUK
(membrane-associated guanylate kinase) protein ZO-1
(Bazzoni et al., 2000;
Ebnet et al., 2000
;
Itoh et al., 2001
). To
determine whether JAM-2 and JAM-3 also bind to ZO-1 we perfomed GST binding
assays with immobilized GST-JAM fusion proteins and in vitro-generated ZO-1
fragments that comprise the three PDZ domains of ZO-1. As shown in
Fig. 5A, both JAM-2 and JAM-3
bind to ZO-1. This association requires an intact C-terminal PDZ binding
motif, suggesting a PDZ domain-dependent association. To further show an
interaction between JAM-2 and JAM-3 with ZO-1, lysates derived from CMT
epithelial cells were incubated with immobilized GST-JAM fusion proteins, and
bound proteins were analysed by immunoblotting with antibodies directed
against ZO-1. Similarly to JAM-1, both JAM-2 and JAM-3 precipitated a protein
species of approximately 220 kDa that reacted with the ZO-1 mAb and that
comigrated with a protein detected in the lysate of CMT cells by the same
antibody (Fig. 5B). This
protein band probably represents the 220 kDa isoform of ZO-1. We also analysed
the interaction of ZO-1 with all integral membrane proteins of the
immunoglobulin superfamily described so far to be present in tight junctions
by GST binding assays. We found that both ESAM and CAR did not associate with
a ZO-1 fragment comprising the three PDZ domains of ZO-1
(Fig. 5C). However, a ZO-1
fragment comprising aa residues 6 to 1256 bound to immobilized GST-CAR but not
to immobilized GST-ESAM. These findings suggested that CAR might directly bind
to ZO-1 in a non PDZ domain-dependent manner. In summary, these experiments
indicated that ZO-1 binds to all three JAMs but, in contrast to PAR-3, ZO-1
associates with several other integral membrane proteins present at tight
junctions, including CAR, claudins and occludin
(Cohen et al., 2001
;
Furuse et al., 1994
;
Itoh et al., 1999
).
|
PAR-3 localizes at cell-cell contacts of endothelial cells
So far, our data suggest that JAM-2 and JAM-3 associate with both PAR-3 and
ZO-1 in a similar manner to JAM-1. A major difference between JAM-1 and JAM-2
or JAM-3 is in their expression patterns in multicellular tissues. JAM-2 and
JAM-3 are predominantly expressed in endothelial cells, whereas JAM-1 is
expressed by both endothelial cells and epithelial cells
(Aurrand-Lions et al., 2001b;
Liang et al., 2002
;
Martin-Padura et al., 1998
;
Palmeri et al., 2000
). To
determine whether PAR-3 is localized at cell-cell contacts of endothelial
cells we analysed human umbilical vein endothelial cells (HUVEC) by indirect
immunofluorescence with PAR-3 antibodies. As shown in
Fig. 6A, PAR-3 localizes at
cell-cell contacts of HUVEC in a similar way to AF-6 and ZO-1. Double
immunofluorescence labelling indicated that PAR-3 colocalizes with JAM-2 in
these cells when the stainings were performed within 48 hours after plating
(Fig. 6B). Interestingly, the
junctional staining for JAM-2 was lost over time, although the level of JAM-2
surface expression as analysed by flow cytometry was not changed (data not
shown). This suggests that JAM-2 might be involved in the early events of
interendothelial junction formation rather than in the stabilization of cell
contacts. Taken together, these findings show that PAR-3 localizes at
cell-cell contacts of endothelial cells and colocalizes with JAM-2 early
during cell contact formation.
|
PAR-3 is expressed by endothelial cells in various tissues
To analyse PAR-3 expression by endothelial cells in vivo, cryostat sections
of various mouse tissues were analysed by immunohistochemistry. Endothelial
cells were identified using endothelial cell-specific markers such as PECAM-1,
von Willebrand factor or the MECA-79 epitope, which is selectively expressed
in high endothelial venule (HEV) endothelial cells of peripheral and
mesenteric lymph nodes. PAR-3 immunoreactivity was identified in endothelial
cells lining capillaries in the tongue, the heart endocardium and the heart
arteries (Fig. 7). By contrast,
PAR-3 was absent in HEV endothelial cells. These data indicate that PAR-3 is
expressed by endothelial cells in various organs but is absent from HEV
endothelial cells.
|
PAR-3 and ZO-1 are recruited by JAM-2 to cell-cell contacts in CHO
cells
To determine whether JAM-2 influences the subcellular distribution of
PAR-3, we generated stable CHO cell lines expressing JAM-2. Surprisingly, only
few of these cells showed JAM-2 localization at cell contacts, despite high
levels of JAM-2 expression at the cell surface as analysed by flow cytometry
(Fig. 8A, left panel). On the
basis of this result and the observation of the regulated junctional
localization of JAM-2 in HUVECs, we reasoned that the clustering of JAM-2 at
cell-cell contacts may be affected by post-translational modifications such as
phosphorylation. Therefore, we generated various mutants of JAM-2 with
individual putative phosphorylation sites present in the cytoplasmic tail
mutated into alanine residues. These mutants were used to generate stable CHO
cell lines. One of them (aa residue 281 changed from serine to alanine, S281A
JAM-2) showed strong JAM-2 localization at cell contacts
(Fig. 8A, middle panel),
although the overall surface expression level was comparable to that of
wild-type JAM-2 as assessed by flow cytometric analysis (data not shown).
Mutation of the threonine residue at postion 296 had no effect on junctional
localization of JAM-2 (data not shown). Because the PDZ domain targeting motif
at the C-terminus of JAM-2 (aa 306-310) was unaffected by the S281A mutation,
we reasoned that endogenous PAR-3 and ZO-1 might be recruited to cell-cell
contact sites with intensive JAM-2 staining. As shown in
Fig. 8B, PAR-3 as well as ZO-1
colocalized with S281A JAM-2 at cell contact sites. HSP-90 was used as
negative control and did not colocalize with S281A JAM-2 at cell-cell
contacts. In cells transfected with wt JAM-2 the few cell contact sites
positive for JAM-2 (Fig. 8A,
left panel) were also positive for PAR-3 or ZO-1 (data not shown), indicating
that the S281A point mutation affects the subcellular localization at
cell-cell contacts of JAM-2 and does not influence the association between
JAM-2 and PAR-3 and ZO-1. These findings have two implications: first, JAM-2
localization at cell contacts seems to be a regulated process, possibly
through phosphorylation of the serine residue at position 281; second, JAM-2
actively recruits PAR-3 and ZO-1 to cell-cell contacts. The latter observation
also points to an association between JAM-2 and both PAR-3 and ZO-1 in living
cells.
|
JAM-2 is phosphorylated at the S281 residue in CHO cells
As outlined in the previous paragraph, the S281A mutation strongly
increased the localization of JAM-2 at cell-cell contacts, suggesting that the
junctional localization of JAM-2 is negatively regulated by phosphorylation of
the S281 residue. This was further supported by the observation that when we
mutated the S281 residue into aspartic acid, thus mimicking constitutive
phosphorylation of S281 (JAM-2 S281D), JAM-2-positive cell-cell contacts were
only sparsely observed and the frequency of junctional localization was
comparable to wild-type JAM-2 (Fig.
8A, right panel). To determine directly whether JAM-2 is
phosphorylated, we performed phosphoamino acid analyses of JAM-2
immunoprecipitated from stably transfected CHO cells. This revealed that both
JAM-2 wt and JAM-2 S281A were phosphorylated exclusively on serine residues
but not on threonine or tyrosine residues
(Fig. 9A). A phosphotryptic
peptide analysis revealed two phosphorylated peptides derived from JAM-2 wt
(Fig. 9B, right panel). One of
these two phosphopeptides was absent in tryptic digests derived from JAM-2
S281A (Fig. 9B, left panel).
These findings indicate that JAM-2 is phosphorylated on S281 in CHO cells and
make a strong case for a negative regulation of cell-cell contact localization
of JAM-2 by phosphorylation of the S281 residue.
|
![]() |
Discussion |
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![]() ![]() ![]() ![]() ![]() ![]() ![]() |
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In this study we report that PAR-3 associates with both JAM-2 and JAM-3.
The association between PAR-3 and JAM-2/JAM-3 is PDZ-domain-mediated and
involves predominantly the first PDZ domain of PAR-3. Thus, all three JAMs
behave very similarly regarding the domain through which they associate with
PAR-3. The physiological meaning of this similar behaviour is not clear, yet.
Because, in some cell types, two or all three JAMs are simultaneously
expressed [e.g. JAM-1 and JAM-3 are expressed by microvessels in the brain or
by KLN205 epithelial cells (Aurrand-Lions
et al., 2001a) and all three JAMs are expressed by glomerular
endothelial cells in the kidney
(Aurrand-Lions et al., 2001a
)],
the possibility that different tissues use different JAM molecules to regulate
TJ formation can be excluded. Rather, it seems possible that all JAMs present
in a given cell type are part of large molecular complexes involving the
association of PAR-3 with all JAMs present. A similar scenario has been
proposed for claudins. As in the case of JAMs, certain cell types express more
than one claudin (e.g. endothelial cells express claudin-1 and claudin-5)
(Liebner et al., 2000
), and
all claudins tested so far associate with ZO-1, ZO-2 and ZO-3 by a PDZ
domain-mediated interaction through the first PDZ domains of the respective ZO
proteins (Itoh et al., 1999
).
The same binding behaviour of all claudins towards ZO-1, ZO-2 and ZO-3 might
result in a strong attraction of these proteins to TJs and thus perhaps in the
formation of large protein clusters at the cytoplasmic plaque
(Itoh et al., 1999
).
PAR-3 associates exclusively with JAM-1/-2/-3
We have shown previously that PAR-3 does not bind to occludin or claudin-1,
-4 or -5 (Ebnet et al., 2001).
In this study we found that PAR-3 does not directly associate in vitro with
the two Ig-like proteins ESAM or CAR. Both proteins are present in TJs of
endothelial cells and/or epithelial cells. Their C-termini fit to the class I
PDZ domain consensus binding sequence
(Harris and Lim, 2001
;
Songyang et al., 1997
), and
therefore it is less likely that they associate with PAR-3 through a PDZ
domain-dependent interaction because all PAR-3 PDZ domains are predicted to
bind class II ligands (Izumi et al.,
1998
). However, we cannot exclude the possibility of an indirect
association in cells via other proteins. Thus, JAM-1/-2/-3 are the only
currently known integral membrane proteins at tight junctions to which PAR-3
binds directly. This makes them distinct from the other proteins and further
underlines their putative role in cell polarity formation.
ZO-1 associates with various integral membrane proteins in tight
junctions including JAM-2 and JAM-3
We also found that ZO-1 associates with JAM-2 and JAM-3. ZO-1 belongs to
the family of MAGUKs, which are associated with the plasma membrane
(Anderson, 1996). ZO-1
associates with claudins through PDZ domain 1
(Itoh et al., 1999
), with
JAM-1 through PDZ domain 3 (Ebnet et al.,
2000
; Itoh et al.,
2001
) and with occludin through the guanylate kinase (GK) domain.
The association of ZO-1 with all three families of integral membrane proteins
in TJs (i.e. occludin, claudins and JAM-1) is mediated through nonoverlapping
domains, which makes it conceivable that the association of ZO-1 with the
various integral membrane proteins serves to cluster these at TJs.
As in the case of JAM-1, the association with JAM-2 and JAM-3 is PDZ domain
mediated (Fig. 5A). We are
currently in the process of identifying the PDZ domain of ZO-1 involved in
binding to JAM-2 and JAM-3. We also found a weak association between ZO-1 and
CAR. As described by others, ZO-1 co-immunoprecipitates with CAR, and ZO-1 is
recruited to sites of homophilic CAR interaction in transfected CHO cells
(Cohen et al., 2001). Our data
support the view that ZO-1 and CAR can associate directly with each other.
This association, however, is not mediated through one of the three ZO-1 PDZ
domains because GST-CAR did not associate with the construct comprising the
ZO-1 PDZ domains (Fig. 5C).
This is in line with the prediction that all three ZO-1 PDZ domains do not
bind class I PDZ domains ligands (Harris
and Lim, 2001
; Willott et al.,
1993
). We did not observe an association between ESAM and
ZO-1/PDZ1-3 or ZO-1/6-1256 but we cannot rule out the possibility of a
PDZ-independent association between ESAM and ZO-1 through a region in the
C-terminal domain that is not present in the ZO-1/6-1256 construct.
PAR-3 is expressed by endothelial cells
Consistent with a predominant expression of JAM-2 and -3 in endothelial
cells, we found that PAR-3 is localized at intercellular junctions of cultured
HUVEC and is expressed by endothelial cells of certain tissues such as the
tongue and the heart. The strong signal of PAR-3 in vessels of the heart and
the endocardium correlates with JAM-2 and JAM-3 expression in the heart artery
and endocardium, as well as with JAM-2 expression in cultured endothelial
cells derived from the aorta (Arrate et
al., 2001; Palmeri et al.,
2000
; Phillips et al.,
2002
). In other tissues such as skin or the brain, the expression
of PAR-3 in vessels was less pronounced, which made it difficult to
distinguish between specific staining in vessels and unspecific background
staining (data not shown). By contrast, in endothelial cells lining the high
endothelial venules in secondary lymphoid organs, PAR-3 expression was
completely absent, although all three JAMs show expression in HEV endothelial
cells (Aurrand-Lions et al.,
2001a
; Aurrand-Lions et al.,
2001b
; Malergue et al.,
1998
; Palmeri et al.,
2000
). This indicates that JAM expression does not necessarily
correlate with PAR-3 expression in endothelial cells. The endothelium in HEVs
is characterized by a high rate of constitutive lymphocyte transmigration,
suggesting that the organization of TJs is less complex than in the
endothelium of other tissues. In fact, the complexity of interendothelial TJs
varies along the vascular tree and the lowest complexity is found in
postcapillary venules, the sites of leukocyte transmigration
(Bowman et al., 1992
;
Schneeberger, 1982
). So, it
seems possible that the absence of PAR-3 expression in endothelial cells
lining postcapillary venules such as the HEVs of secondary lymphoid organs
helps to prevent the formation of highly complex TJs, thus allowing a high
rate of paracellular transendothelial migration of lymphocytes. The expression
of the three JAMs in HEV endothelial cells, despite the absence of PAR-3
expression, is in line with several reports describing a role for JAMs in the
regulation of leukocyte-endothelial interactions by way of homophilic and/or
heterophilic JAM/JAM interactions (Arrate
et al., 2001
; Del Maschio et
al., 1999
; Johnson-Leger et
al., 2002
; Liang et al.,
2002
; Martin-Padura et al.,
1998
; Ostermann et al.,
2002
).
The junctional localization of JAM-2 is regulated by serine
phosphorylation
CHO cells stably expressing wt JAM-2 showed only sparse JAM-2 localization
at cell-cell contacts (Fig.
8A). By contrast, a point mutation that abolishes phosphorylation
of the S281 residue (S281A) dramatically increased JAM-2 localization at cell
contacts, suggesting that JAM-2 localization is negatively regulated by
phosphorylation. In addition to the S281 residue, we mutated the only
threonine residue present in the cytoplasmic tail of JAM-2 into alanine
(T296A), but this mutation had no effect on the junctional localization of
JAM-2 (data not shown). Consistent with these findings, we found
phosphorylation exclusively on serine residues
(Fig. 9A). Interestingly, in
addition to the peptide harbouring the S281 residue, we identified a second
phosphopeptide of JAM-2, suggesting that additional serine residues can be
phosphorylated. The identity, as well as the functional role, of this
additional serine residue has not yet been analysed.
The mechanism underlying the enhanced localization of JAM-2 S281A at cell contact sites is not clear. The possibility that phosphorylation of JAM-2 influences the association with PAR-3 and ZO-1 is rather unlikely. This is based on our observation that, despite the sparse localization of JAM-2 at cell-cell contacts in JAM-2 wt-transfected CHO cells (Fig. 8A), the few cell-cell contacts positive for JAM-2 were also positive for PAR-3 and ZO-1, indicating that JAM-2 wt is as effective as JAM-2 S281A in associating with PAR-3 and ZO-1. The possibility that increased JAM-2 localization at cell-cell contacts is the result of an increased protein stability can also be excluded. This is based on two observations: first, both JAM-2 wt and JAM-2 S281A CHO cells had similar levels of JAM-2 surface expression as analysed by FACS analysis (data not shown); second, when cells were surface-biotinylated for 1 hour ('pulse') and analysed for the amounts of surface-expressed JAM-2 by immunoprecipitation at various time periods up to 48 hours after replating ('chase'), we found no significant difference between JAM-2 wt- and JAM-2 S281A-transfected CHO cells (data not shown). Therefore, phosphorylation at S281 does not influence the stability of the protein at the surface, and it seems that the S281 phosphorylation specifically regulates the localization at sites of cell-cell contact in a negative manner.
A role for JAMs in cell polarity
One possible physiological relevance for the association between JAMs and
PAR-3 is to anchor the PAR-3/aPKC/PAR-6 complex at TJs. As the
PAR-3/aPKC/PAR-6 complex is localized at TJs of fully polarized epithelial
cells (Johansson et al., 2000;
Suzuki et al., 2001
), and as
no other membrane protein of TJs has been described yet for any of the three
components of the complex, it is conceivable that the association between
PAR-3 and JAM-1 serves to localize the whole complex to TJs. In addition to
this function, the association between JAMs and PAR-3 might have a role that
relates to TJ biogenesis. In the process of wounding-induced cell-cell contact
formation JAM-1 appears together with E-cadherin and ZO-1 very early in
primordial, spot-like adherens junctions
(Ebnet et al., 2001
).
Spot-like adherens junctions or `puncta' represent sites of initial cell-cell
contact mediated by E-cadherin homophilic interactions at tips of filopodia
(Adams et al., 1996
;
Yonemura et al., 1995
). At
this stage of cell contact formation, occludin or claudins are not present at
cell contacts (Suzuki et al.,
2002
). Also, both aPKC and PAR-3 are absent from cell junctions at
this stage (Suzuki et al.,
2002
). These observations open the possibility that early JAM-1
localization at spot-like structures is necessary to subsequently recruit the
PAR-3/aPKC/PAR-6 complex, all components of which have been implicated in TJ
formation (Nagai-Tamai et al.,
2002
; Suzuki et al.,
2001
; Suzuki et al.,
2002
; Yamanaka et al.,
2001
). Whether JAM-2 and JAM-3 are present at the tips of
filopodia or lamellipodia and colocalize with VE-cadherin and ZO-1 in
endothelial cells is currently being investigated in our lab. JAM-2 shows
predominant cell-cell contact localization in HUVEC when cells are
subconfluent and contact staining gradually decreases on contact maturation
(Aurrand-Lions et al., unpublished observations). In addition, as suggested by
our observations with the S281A JAM-2 mutant in CHO cells, the junctional
localization of JAM-2 seems to be negatively regulated through phosphorylation
of the S281 residue. One could envisage a scenario whereby nonphosphorylated
JAM-2 is localized at cell contacts early during cell contact formation where
it recruits PDZ domain-containing scaffolding proteins like PAR-3 and ZO-1,
which are necessary for further junctional maturation. The simultaneous
recruitment of serine kinases could lead to JAM-2 phosphorylation and its
subsequent delocalization from cell-cell contact sites. Once the scaffolding
complexes are recruited to cell-cell contacts they might be stabilized by
other proteins constitutively present at cell-cell contacts, e.g. JAM-1, and
JAM-2 would become dispensable. Taken together, these findings open the
possibility that a regulated targeting of JAM-2 to nascent cell-cell contact
sites might further promote TJ formation by recruiting the PAR-3/aPKC/PAR-6
complex to cell contacts.
In summary, our findings of a direct association between JAM-1/-2/-3 and the polarity proteins PAR-3 and ZO-1 make a strong case for JAMs as being involved in the formation and maintenance of TJ in epithelial and endothelial cells. Our findings further underline the functional dichotomy of JAM proteins as regulators of leukocyte recruitment as well as cell polarity formation.
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