From the Department of Molecular Biology and
Biochemistry and § Department of Surgery and Clinical
Oncology, Osaka University Graduate School of
Medicine/Faculty of Medicine, Suita 565-0871, Japan and the
¶ KAN Research Institute Inc., 93 Chudoji-Awatamachi,
Shimogyo-ku, Kyoto 600-8815, Japan
Received for publication, December 27, 2002
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ABSTRACT |
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PAR-3 is a cell polarity protein that
localizes at tight junctions (TJs) by direct binding to an
immunoglobulin (Ig)-like cell-cell adhesion molecule JAM-1 in mammalian
epithelial cells. Another Ig-like cell-cell adhesion molecule nectin
plays a role in the localization of JAM-1 at TJs in epithelial cells.
Nectin furthermore plays a role in the organization of adherens
junctions (AJs) and TJs. Nectin comprises a family of four members,
nectin-1, -2, -3, and -4. Nectins are associated with the actin
cytoskeleton through afadin, of which the PDZ domain binds to nectins
through their C-terminal four amino acids. We show here that PAR-3
binds to nectin-1 and -3 in neuroepithelial cells of the embryonic
telencephalon, which are equipped with AJs, but not with typical TJs.
Nectin-1, -2, -3, and afadin, but not JAM-1, were concentrated at AJs
in neuroepithelial cells of the embryonic telencephalon at E13.5 and
PAR-3 co-localized with nectins. PAR-3 was co-immunoprecipitated with
nectin-1 and -3, but not with nectin-2 or JAM-1, from the mouse whole
brain at E13.5. Recombinant PAR-3 stoichiometrically bound to
recombinant nectin-1 and -3. The first one of the three PDZ domains of
PAR-3 bound to the C-terminal four amino acids of nectin-1 and -3. The
affinities of PAR-3 and afadin for nectin-1 and -3 were similar.
Cadherin-deficient L cells expressing nectin-1 and -3 formed nectin-1-
and -3-based cell-cell junctions, respectively, where PAR-3 as well as
afadin was recruited. These results indicate that nectin-1 and -3 are
involved in the localization of PAR-3 at AJs in the neuroepithelial
cells of the embryonic telencephalon.
Cell polarity is fundamental not only for cell functions but also
for development and tissue maintenance (1, 2). Studies in mammalian
epithelial cells have revealed that establishment of cell polarity
depends upon cell adhesion, intracellular signaling, cytoskeletal
organization, and protein sorting. Mechanisms of establishment of cell
polarity have been studied most extensively in Caenorhabditis
elegans and Drosophila (3). PAR proteins, PAR-1 to -6, were first identified in C. elegans as
indispensable proteins that are involved in establishment of the
anterior-posterior cell polarity of the one-cell embryo (4).
Thereafter, homologous proteins have been identified in
Drospophila and mammals (5). In mammals, PAR-3, a mammalian
homologue of the par-3 gene product, was first identified as
an atypical protein kinase C
(aPKC)1 PAR-3 localizes at tight junctions (TJs) and forms a ternary complex
with PAR-6 and aPKC In epithelial cells, TJs constitute a junctional complex with adherens
junctions (AJs) and desmosomes (9). These junctional structures are
typically aligned from the apical to the basal side, although
desmosomes are independently distributed in other areas. The formation
and maintenance of TJs are dependent on the cell-cell adhesion activity
of E-cadherin. E-Cadherin is a key Ca2+-dependent cell-cell adhesion molecule at
AJs (11, 12). E-Cadherin is associated with the actin cytoskeleton
through peripheral membrane proteins, including In addition to the localization of the PAR-3-aPKC-PAR-6 complex at TJs
in epithelial cells, this complex localizes at AJs in fibroblasts (6)
and in the neuroepithelial cells of the embryonic telencephalon during
E10.5 to E14.5 (20). Neuroepithelial cells at these stages contain only
AJs and gap junctions, but no typical TJs (21, 22), and the mechanism
of the localization of the PAR-3-aPKC-PAR-6 complex remains unknown in
this cell type. We show here that nectin-1 and -3, but not JAM-1, are
involved in the localization of PAR-3 at AJs in the neuroepithelial
cells of the embryonic telencephalon.
Antibodies--
Rat anti-nectin-1, -2, and -3 monoclonal
antibodies (mAbs) were prepared as described (18, 23, 24). Rabbit
anti-nectin-1 Construction and Purification--
pCAGIPuro-nectin-3 Cell Culture and Establishment of Transformants--
L cells
were kindly supplied by Dr. S. Tsukita (Kyoto University, Kyoto,
Japan). L cell lines were maintained in Dulbecco's modified Eagle's
medium supplemented with 10% fetal calf serum. Various L cell lines,
nectin-1 Immunoprecipitation--
The whole brain was dissected from
mouse embryo at E13.5, and the sample was incubated in 0.75 mM dithiobis (succinimidylpropionate) in PBS at 4 °C for
2 h. The cross-linking reaction was stopped by adding 1 M glycine at pH 7.5 in PBS. The sample was homogenized in a
homogenization buffer (20 mM Tris-HCl at pH 8.0, 150 mM NaCl, 10% glycerol, 5 mM CaCl2,
5 mM MgCl2, 10 µg/ml leupepitin, 1.5 µg/ml
aprotinin, and 10 µM
p-amidinophenylmethanesulfonyl fluoride). An equal volume of
the homogenization buffer containing 2% Nonidet P-40 was added to the
homogenate, and the sample was subjected to centrifugation at
20,000 × g for 20 min. The extract (2 mg of protein)
was incubated with protein G-Sepharose beads (Amersham Biosciences)
coated with the anti-PAR-3 pAb or control IgG at 4 °C for 16 h.
After the beads were extensively washed with the homogenization buffer
containing 1% Nonidet P-40, the bound proteins were eluted by boiling
the beads in the SDS sample buffer (60 mM Tris-HCl at pH
6.7, 3% SDS, 2% 2-mercaptoethanol, and 5% glycerol) for 5 min and
subjected to SDS-PAGE, followed by Western blotting.
Affinity Chromatography--
To determine the binding domains of
PAR-3 and nectin-1
To compare the affinities of PAR-3 and afadin for nectin-1 Other Procedures--
Immunofluorescence microscopy of frozen
sections of mouse embryonic neuroepithelial cells and cultured cells
was done as described (23, 29). Immunoelectron microscopy of mouse
embryonic neuroepithelial cells was done using the silver enhancement
technique as described (30). SDS-PAGE was done as described (31).
Protein concentrations were determined with bovine serum albumin as a
reference protein as described (32).
In neuroepithelial cells of the embryonic telencephalon at E13.5,
the immunofluorescence signals for nectin-1, -2, and -3 and afadin were
highly concentrated at the lumenal surface of neuroepithelial cells
(Fig. 1, A-C). All of these
signals apparently co-localized. The signal for PAR-3 colocalized with
the signal for nectin-3 (Fig. 1D). The signals for ZO-1 and
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-binding protein
(6).
in mammalian epithelial cells (5). These three
proteins directly interact with each other and play a critical role in
the apico-basal polarization of mammalian epithelial cells (5). This
PAR-3-aPKC-PAR-6 complex is an evolutionarily conserved cell
polarization machinery that works ubiquitously in a variety of
biological context from warm embryos to differentiated mammalian cells
(5). PAR-3 has recently been shown to be directly bound to junctional
adhesion molecule-1 (JAM-1), a Ca2+-independent
immunoglobulin (Ig)-like cell-cell adhesion molecule that localizes at
TJs in epithelial cells (7, 8). PAR-3 has three PDZ domains and one
aPKC-binding domain (6), and this binding to JAM-1 is mediated through
the first PDZ domain of PAR-3 and the C-terminal four aa (amino acids)
of JAM-1 (7). The PAR-3-aPKC-PAR-6 complex is tethered to TJs through
its binding to JAM-1. At TJs, claudin is a key cell-cell adhesion
molecule which forms TJ strands (9). Claudin is associated with the actin cytoskeleton through ZO-1. ZO-1 furthermore binds both to the
C-terminal four aa of JAM-1 through the third PDZ domain of ZO-1 (8)
and to the cytoplasmic tail of claudin through the first PDZ domain of
ZO-1 (10). Through these interactions, JAM-1 might be recruited and
tethered to TJ strands.
-,
-, and
-catenins,
-actinin, and vinculin (11, 12). Nectin and afadin
constitute another cell-cell adhesion unit at cell-cell AJs (13).
Nectin is a Ca2+-independent Ig-like cell-cell adhesion
molecule which comprises a family of four members, nectin-1, -2, -3, and -4. Most of the nectin family members have a C-terminal conserved
motif of four aa residues (E/A-X-Y-V), which interacts with the PDZ
domain of afadin. Afadin is an actin filament-binding protein with one
PDZ domain and four other domains and connects nectin to the actin cytoskeleton. The nectin-afadin unit has roles in the organization of
E-cadherin-based AJs and claudin-based TJs in epithelial cells (14-17), in the organization of AJs in fibroblasts (14, 15, 17), in
the formation of synapses in neurons (18), and in the formation of
Sertoli cell-spermatids junctions in the testis (19), in cooperation
with or independently of cadherin.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and -3
polyclonal antibodies (pAbs) were prepared
as described (23, 24). A rabbit anti-afadin pAb and a mouse anti-afadin mAb were prepared as described (25, 26). A rabbit anti-PAR-3 pAb was
kindly provided by Dr. S. Ohno (Yokahama City University, Yokohama,
Japan) (6). A rabbit anti-occludin pAb was purchased from
Zymed Laboratory Inc. A mouse anti-ZO-1 mAb was
purchased from Sanko-junyaku. A rabbit pAb was raised against the
cytoplasmic tail of human JAM-1. A rat anti-JAM-1 mAb (H202-106-7-4)
was kindly provided by Dr. M. Aurrand-Lions (27).
C,
containing aa 1-545 (deletion of the C-terminal four aa residues), was
constructed according to the standard molecular biology methods (28).
Prokaryote expression vectors were constructed in pGEX (Amersham
Biosciences) and pMAL-C2 (New England Biolabs Inc.). Various
constructs of mouse nectin-1
, -2
, and -3
and rat afadin
contained the following aa: glutathione S-transferase
(GST)-nectin-1
-CP, aa 431-515; GST-nectin-1
-CP
C, aa 431-511;
GST-nectin-2
-CP, aa 387-467; GST-nectin-3
-CP, aa 433-549;
GST-nectin-3
-CP
C, aa 433-545; and maltose-binding protein (MBP)-afadin-PDZ, aa 1007-1125 (PDZ domain) (23, 24). The cDNA of
rat PAR-3 was kindly provided by Dr. S. Ohno (Yokohama City University,
Yokohama, Japan). Various constructs of PAR-3 contained the following
aa: MBP-PAR-3-PDZ1-3, aa 257-717; MBP-PAR-3-PDZ1, aa 257-460;
MBP-PAR-3-PDZ2, aa 360-590; and MBP-PAR-3-PDZ3, aa 534-717. The GST
and MBP fusion proteins were expressed in RosettaBlue (Novagen) and
purified by the use of glutathione-Sepharose beads (Amersham
Biosciences) and amylose resin beads (New England Biolabs Inc.), respectively.
-L, -1
C-L, -2
-L, and -3
-L cells were prepared as
described (14, 23, 24). An L cell line stably expressing C-terminal
four-aa-deleted nectin-3
(nectin-3
C-L cells) was also
similarly prepared with pCAGIPuro-nectin-3
C.
, -2
, or -3
, various MBP-fusion fragments of
PAR-3 (200 pmol each) were immobilized on amylose resin beads.
GST-nectin-1
-CP, -1
-CP
C, -2
-CP, -3
-CP, or -3
-CP
C
(2 nmol each) was applied to the MBP-fusion protein-immobilized beads
equilibrated with PBS containing 0.1% Triton X-100. After the beads
were extensively washed with the same buffer, elution was performed
with the same buffer containing 10 mM maltose. The eluate
was then subjected to SDS-PAGE, followed by staining with Coomassie
Brilliant Blue.
or -3
,
GST-nectin-1
-CP or -3
-CP (200 pmol) was immobilized on
glutathione-Sepharose beads. Various amounts (200 pmol or 2 nmol) of
MBP-PAR-3-PDZ1-3 and MBP-afadin-PDZ were mixed and applied to the GST
fusion protein-immobilized beads equilibrated with PBS containing 0.1%
Triton X-100. After the beads were extensively washed with the same
buffer, elution was performed with the same buffer containing 20 mM glutathione. The eluate was then subjected to SDS-PAGE,
followed by staining with Coomassie Brilliant Blue.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-catenin colocalized with the signal for afadin as described (20)
(data not shown). However, the signal for occludin or JAM-1 was not
detected (Fig. 1, E and F).
View larger version (25K):
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Fig. 1.
Immunofluorescence microscopy of
neuroepithelial cells. The localization of nectins, afadin,
ZO-1, occludin, and JAM-1 at the neuroepithelial cells of the embryonic
telencephalon at E13.5 was analyzed by double-label immunofluorescence
microscopy. LV, lateral ventricular; VZ,
ventricular zone. Bars, 10 µm. The results shown are
representative of three independent experiments.
We then examined by the immunoprecipitation assay whether
PAR-3 binds to nectins in the neuroepithelial cells of the embryonic telencephalon at E13.5. When PAR-3 was immunoprecipitated by its Ab
from the embryonic mouse whole brain at E13.5, including telencephalon, nectin-1 and -3, but not nectin-2 or JAM-1, were co-immunoprecipitated with PAR-3 (Fig. 2A). These
results suggest that PAR-3 binds to nectin-1 and -3 in the
neuroepithelial cells of the embryonic telencephalon.
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It has been shown that AJs, but not typical TJs, are observed in
neuroepithelial cells (21, 22). Consistently, AJs, which are
undercoated with actin filament bundles, but not typical TJs, were
observed (Fig. 3). Immunogolds of
nectin-3, PAR-3, and afadin localized at AJs. Immunogolds of nectin-1
also localized at AJs (data not shown). These results, together with
the result obtained by the co-immunprecipitation experiment, suggest
that PAR-3 binds to nectin-1 and -3 and co-localize with them at AJs of
neuroepothelial cells of the embryonic telencephalon.
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We next examined whether PAR-3 directly binds to nectin-1 and -3, using
the recombinant PAR-3 fragment containing all three PDZ domains
(PAR-3-PDZ1-3) and the cytoplasmic tails of recombinant nectin-1, -2, and -3 (nectin-1-CP, -2-CP, and -3-CP, respectively). PAR-3-PDZ1-3
stoichiometrically bound to nectin-3-CP (Fig. 2B). PAR-3-PDZ1-3 also stoichiometrically bound to nectin-1-CP, but not to
nectin-2-CP (data not shown). These results indicate that the binding
of PAR-3 to nectin-1 and -3 is direct. We then examined the binding
sites of PAR-3 and nectin-1 and -3. We prepared pure samples of each
PDZ domain of recombinant PAR-3 and the C-terminal four aa-deleted
cytoplasmic tails of nectin-1 and -3 (nectin-1-CPC and -3-CP
C,
respectively). PAR-3-PDZ1-3 did not bind to nectin-3-CP
C under the
conditions where PAR-3-PDZ1-3 bound to nectin-3-CP (Fig. 2B). The first PDZ domain of PAR-3 bound to nectin-3-CP
(Fig. 2C). PAR-3-PDZ1-3 did not bind to nectin-1-CP
C
under the conditions where PAR-3-PDZ1-3 bound to nectin-1-CP, and the
first PDZ domain bound to nectin-1-CP (data not shown). These results
indicate that the first PDZ domain of PAR-3 directly binds to the
C-terminal four aa of nectin-1 and -3.
Thus, both afadin and PAR-3 bind to the C-terminal four aa of nectin-1 and -3. We therefore compared the affinities of afadin and PAR-3 for nectin-1 and -3. Various amounts of either afadin or PAR-3 alone or both were incubated with nectin-3-CP. When the same amounts of afadin and PAR-3 were mixed with nectin-3-CP, apparently the same amounts of afadin and PAR-3 bound to it, and 1 mol of each maximally bound to 2 mol of nectin-3-CP (Fig. 2D). The essentially same results were obtained for nectin-1 (data not shown). These results indicate that the affinities of PAR-3 and afadin for nectin-1 and -3 were apparently similar.
In the last set of experiments, we confirmed whether PAR-3 binds to
nectin-1 and -3 in an intact cell assay system, using cadherin-deficient L cells stably expressing full-length nectin-1 or -3 (nectin-1-L or -3-L cells, respectively) or the C-terminal four
aa-deleted nectin-1 or -3 (nectin-1C-L or -3
C-L cells, respectively). Nectin-3-L cells and nectin-3-L cells formed cell-cell junctions, and PAR-3 was concentrated there (Fig.
4). Afadin was also concentrated there
(data not shown). Nectin-3
C-L cells and nectin-3
C-L cells formed
cell-cell junctions, but PAR-3 was not concentrated there. Afadin was
not concentrated there, either (data not shown). Nectin-1-L cells and
nectin-1-L cells formed cell-cell junctions, and PAR-3 as well as
afadin was concentrated there (data not shown). Nectin-1
C-L cells
and nectin-1
C-L cells formed cell-cell junctions, but PAR-3 or
afadin was not concentrated there (data not shown). Nectin-3 forms not
only homo-trans-dimers with nectin-3 but also
hetero-trans-dimers with nectin-1 and -2 (13). PAR-3 as well
as afadin was concentrated at the junctions formed between nectin-3-L
cells and L cells stably expressing nectin-1 or -2 (data not shown).
These results are consistent with the above biochemical results and
indicate that PAR-3 binds to nectin-1 and -3 in intact cells.
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DISCUSSION |
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We have shown here that PAR-3 directly binds to nectin-1 and -3 and localizes with them at AJs of the neuroepithelial cells of the embryonic telencephalon. It has been shown that PAR-3 binds to JAM-1 and localizes with it at TJs in epithelial cells (7, 8). In the neuroepithelial cells of the embryonic telencephalon, TJs are not well developed, and we could not significantly observe the immunofluorescence signal for occludin or JAM-1 at the cell-cell junction area. Furthermore, JAM-1 was not co-immunoprecipitated with PAR-3 under the conditions where nectin-1 and -3 were co-immunoprecipitated with PAR-3 from the embryonic mouse whole brain at E13.5 including telencephalon. It has been shown that PAR-3 forms a ternary complex with aPKC and PAR-6 (5) and that this complex localizes at AJs in the neuroepithelial cells of the embryonic telencephalon (20). Taken together, it is likely that the PAR-3-aPKC-PAR-6 complex localizes at AJs in the neuroepithelial cells by directly binding of PAR-3 to nectin-1 and -3. The physiological function of this direct binding remains unknown, but it may recruit aPKC and PAR-6 and play a role in the formation and maintenance of polarity of the neuroepithelial cells.
We have shown here that all nectin-1, -2, and -3 localize at AJs of the neuroepithelial cells of the embryonic telencephalon, but that the first PDZ domain of PAR-3 directly binds to the C-terminal four aa of nectin-1 and -3, but not to that of nectin-2. All nectin-1, -2, and -3 have a C-terminal consensus motif with four aa for the binding to PDZ domains and bind afadin. The aa sequences of the motif of nectin-1 and -3 are the same, whereas that of nectin-2 is different from them (24). This difference may determine the specific binding of PAR-3 to nectin-1 and -3.
All nectins directly bind afadin, and this binding is mediated through the PDZ domain of afadin and the C-terminal four aa of nectins (13). This paper demonstrates for the first time that nectin-1 and -3 directly bind not only afadin but also PAR-3. We have shown here that the affinities of PAR-3 and afadin for nectin-1 and -3 are roughly similar and that 1 mol of each afadin and PAR-3 binds to 2 mol of nectin-1 and -3. All nectins first form cis-dimers, followed by the formation of trans-dimers, eventually inducing cell-cell adhesion (13). It remains unknown whether both afadin and PAR-3 bind to the same cis-dimer or either afadin or PAR-3 alone binds to the different cis-dimer.
Nectin-1 and -3 are expressed in many cell types in addition to the
neuroepithelial cells (13). They may be involved in the localization of
the PAR-3-aPKC-PAR-6 complex in cells where JAM-1 is not expressed. We
have previously shown that the nectin-afadin unit plays a role in the
localization of JAM-1 at TJs (16), where the PAR-3-aPKC-PAR-6 complex
is associated with JAM-1. Another possible function of nectin-1 and -3 is that PAR-3 first binds to nectin-1 or -3 and then transferred to
JAM-1 during the organization of the junctional complex in the
epithelial cells equipped with TJs. Further studies are necessary for
our understanding of the physiological role of the direct binding of
PAR-3 to nectin-1 and -3 in the neuroepithelial cells and other cell types.
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ACKNOWLEDGEMENTS |
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We thank Dr. S. Ohno (Yokohama City University, Yokohama, Japan) for providing us with the anti-PAR-3 pAb and the cDNA of rat PAR-3, Dr. M. Aurrand-Lions (Centre Medical Universitaire, Geneva) for providing us with the anti-JAM-1 mAb, and Dr. S. Tsukita (Kyoto University, Kyoto, Japan) for providing us with L cells.
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FOOTNOTES |
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* The work at Osaka University was supported by grants-in-aid for Scientific Research and for Cancer Research from the Ministry of Education, Culture, Sports, Science, and Technology, Japan (2001, 2002).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of Molecular
Biology and Biochemistry, Osaka University Graduate School of
Medicine/Faculty of Medicine, Suita 565-0871, Osaka, Japan. Tel.:
81-6-6879-3410; Fax: 81-6-6879-3419; E-mail:
ytakai@molbio.med.osaka-u.ac.jp.
Published, JBC Papers in Press, January 5, 2003, DOI 10.1074/jbc.C200707200
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ABBREVIATIONS |
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The abbreviations used are: aPKC, atypical protein kinase C; TJs, tight junctions; JAM-1, junctional adhesion molecule-1; Ig, immunoglobulin; aa, amino acid(s); AJs, adherens junctions; Ab, antibody; mAb, monoclonal antibody; pAb, polyclonal antibody; GST, glutathione S-transferase; MBP, maltose-binding protein; PBS, phosphate-buffered saline.
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