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The Journal of Neuroscience, February 1, 2002, 22(3):757-765
Interaction of Synaptic Scaffolding Molecule and  -Catenin
Wataru
Nishimura1, 2,
Ikuko
Yao1,
Junko
Iida1,
Noriaki
Tanaka2, and
Yutaka
Hata1
1 Department of Medical Biochemistry, Graduate School
of Medicine, Tokyo Medical and Dental University, Tokyo
113-8519, Japan, and 2 First Department of Surgery, Okayama
University Medical School, Okayama 700-8558, Japan
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ABSTRACT |
Synaptic scaffolding molecule (S-SCAM) is a synaptic
membrane-associated guanylate kinase with inverted domain organization (MAGI) that interacts with NMDA receptor subunits and neuroligin. In
epithelial cells, the non-neuronal isoform of S-SCAM (MAGI-1) is
localized at tight or adherens junctions. Recent studies have revealed
that the polarized targeting of MAGI-1 to the lateral membrane is
mediated by its C-terminal region and that MAGI-1 interacts with
 -catenin in epithelial cells. In this article, we report that S-SCAM
interacts with -catenin in neurons. -Catenin is
coimmunoprecipitated with S-SCAM from rat brain. Both S-SCAM and
-catenin are localized at synapses and are partially colocalized. The C-terminal region of S-SCAM binds to the C-terminal region of
-catenin. We have tested how the interaction between S-SCAM and
-catenin plays a role in the synaptic targeting of S-SCAM and
-catenin. S-SCAM is targeted to synapses via the C-terminal postsynaptic density-95/Dlg-A/ZO-1 (PDZ) domain. -Catenin is targeted to synapses with armadillo repeats. The overexpressed C-terminal region of -catenin blocks the synaptic targeting of S-SCAM. The overexpressed C-terminal region of S-SCAM is partially targeted to synapses and forms a small number of clusters. In the
presence of overexpressed -catenin, the C-terminal region of S-SCAM
forms more clusters at synapses. These data suggest that the synaptic
targeting of S-SCAM is mediated by the interaction with
-catenin.
Key words:
-catenin; synaptic scaffolding molecule; membrane-associated guanylate kinase with inverted domain organization; NMDA receptor; armadillo repeats; PDZ domain
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INTRODUCTION |
Synaptic scaffolding molecule
(S-SCAM) has been identified as a protein interacting with a synaptic
cytoskeleton adapter protein known as guanylate kinase (GK)-associated
protein (GKAP) (also called synapse-associated protein 90/postsynaptic
density-95-associated protein or discs-large tumor
suppressor-associated protein) (Kim et al., 1996 ; Satoh et al., 1997;
Takeuchi et al., 1997; Hirao et al., 1998). The same molecule is known
as an atrophin-1-interacting protein or an activin receptor-interacting
protein (Wood et al., 1998; Shoji et al., 2000). S-SCAM has six
postsynaptic density-95/Dlg-A/ZO-1 (PDZ) domains (PDZ0 to PDZ5), one
GK domain, and two WW domains. S-SCAM interacts with various
molecules using these domains. GKAP binds to the GK domain. A neuronal
cell adhesion molecule, neuroligin, and a GDP/GTP exchange factor for
rap1 bind to the second PDZ domain (PDZ1) (Hirao et al., 1998; Ohtsuka
et al., 1999). Protein tyrosine phosphatase and tensin homolog binds to
the third PDZ domain (PDZ2) (Wu et al., 2000). NMDA receptor subunits,
membrane-associated GK-interacting protein, neural plakophilin-related
armadillo-repeat protein/ -catenin, and activin receptor bind to the
last PDZ domain (PDZ5) (Hirao et al., 1998; Ide et al., 1999a; Yao et
al., 1999; Shoji et al., 2000). Atrophin-1 possibly interacts with the
WW domains (Wood et al., 1998). S-SCAM also forms homodimers via the
region containing PDZ4 and PDZ5 (Hirao et al., 2000). In epithelial cells, membrane-associated GK with inverted domain organization (MAGI)-1 (also called brain-specific angiogenesis inhibitor-associated protein-1), an isoform of S-SCAM, is localized at tight or adherens junctions (Dobrosotskaya et al., 1997; Shiratsuchi et al., 1998; Ide et
al., 1999b; Dobrosotskaya and James, 2000; Nishimura et al., 2000).
Recent studies have revealed that MAGI-1 interacts with -catenin,
megalin, and Mus musculus neuroepithelial cell transforming
gene 1 (Dobrosotskaya and James, 2000; Dobrosotskaya, 2001; Patrie et
al., 2001). The interaction with -catenin is involved in the
membrane association of MAGI-1 (Dobrosotskaya and James, 2000). These
findings suggest that -catenin determines the localization of MAGI-1
at epithelial junctions and subsequently recruits various molecules
interacting with MAGI-1 to cell-cell contacts. In neurons, -catenin
is enriched at synapses and interacts with N-cadherin and
other cadherin-like molecules (Uchida et al., 1996; Benson and Tanaka,
1998; Miskevich et al., 1998). Therefore, -catenin may be involved
in the synaptic targeting of S-SCAM. We actually obtained -catenin
from the yeast two-hybrid screening using S-SCAM as bait (Ide et al.,
1999a). Recently, KIAA0707, which is homologous to S-SCAM, has been
reported to bind to the glutathione S-transferase
(GST)--catenin affinity column (Kawajiri et al., 2000). Here, we
reveal that S-SCAM interacts with -catenin in neurons and that the
interaction with -catenin is involved in the synaptic targeting of
S-SCAM.
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MATERIALS AND METHODS |
Construction of expression vectors. Various
expression vectors were constructed by conventional molecular biology
techniques and the PCR method using pClneoMyc, pSind green
fluorescent protein (GFP), and pSindMyc. The PCR product using pLGFPC
(Clontech, Palo Alto, CA) as a template was ligated into
XbaI/SphI sites of pSindRep5 (Invitrogen, San
Diego, CA) to generate pSindGFP. A linker was ligated to
XbaI/SphI sites of pSindRep5 to generate
pSindMyc. pClneoMyc S-SCAM-1, -4, -8, and -10 and pGex4T-1 S-SCAM-10
have been described previously (Hirao et al., 1998, 2000). pSindGFP S-SCAM-1, -4, -8, and -10 contain the same amino acid residues as
pClneoMyc S-SCAM-1, -4, -8, and -10. pSindGFP S-SCAM-13 contains the
amino acid residues 772-1277. pSindGFP S-SCAM-19 contains the amino
acid residues 573-906 and 1134-1277. pSindMyc S-SCAM-1 contains the
same amino acid residues as pClneoMyc S-SCAM-1. The constructs below
contain the following amino acids of mouse -catenin: pClneoMyc and
pSindGFP -catenin-1, 1-781; pClneoMyc -catenin-3, 131-781;
pClneoMyc -catenin-4, 1-699; pClneoMyc -catenin-5, 1-555;
pClneoMyc -catenin-6, 555-781; pClneoMyc -catenin-7, 131-699;
pSindGFP -catenin-2, 131-781;pSindGFP -catenin-6, 131-696; pSindGFP -catenin-8, 696-781; pSindGFP -catenin-9, 437-781; pSindGFP -catenin-10, 1-367; and pSindGFP -catenin-11,
368-696. pSindGFP NMDA receptor subunit 2A (NR2A) contains amino acid
residues 839-1464 of rat NR2A.
Antibodies. The rabbit polyclonal anti-S-SCAM antibody
against the WW domain has been described previously (Hirao et al., 1998; Ide et al., 1999b). Sheep polyclonal anti-Myc antibody was raised
against the synthetic peptide
Glu-Gln-Lys-Ile-Ser-Glu-Glu-Asp-Leu-Asn-Ser-Ala-Val-Asp. Other
antibodies used in this study were as follows: mouse monoclonal anti-Myc-tag 9E10 (American Type Culture Collection, Manassas, VA);
mouse monoclonal anti-synaptophysin (Roche Molecular Biochemicals, Basel, Switzerland); mouse monoclonal anti--catenin (BD
Transduction Laboratories, San Diego, CA); and rhodamine-,
fluorescein isothiocyanate-, and Cy5-conjugated second antibodies for
dual labeling (Chemicon International Inc., Temecula, CA).
Immunoprecipitation from rat brain. S-SCAM was
immunoprecipitated from the Triton X-100 extract of rat crude
synaptosomes as described previously (Hirao et al., 2000). The
immunoprecipitates were immunoblotted with either the anti-S-SCAM or
the anti--catenin antibody.
Immunoprecipitation of various Myc-tagged constructs of S-SCAM
from COS cells. COS cells were cultured
in DMEM supplemented with 10% (w/v) FBS, 100 U/ml penicillin, and 100 µg/ml streptomycin at 37°C under 5% CO2 and were
transfected with DEAE dextran with various constructs of S-SCAM. COS
cells from one 10 cm plate were homogenized in 200 µl of 20 mM HEPES/NaOH, pH 8.0, containing 200 mM NaCl,
1% (w/v) NP-40, and 2 mM EGTA, and were centrifuged at
100,000 × g for 15 min at 4°C. The supernatant
was precleared with 7.5 µl of protein G Sepharose 4 fast-flow beads
and then incubated with 2.5 µl of the anti-Myc ascites fixed on 5 µl of protein G Sepharose 4 fast-flow beads. After the beads were
washed four times with 400 µl of 20 mM HEPES/NaOH, pH
8.0, containing 200 mM NaCl, 0.3% (w/v) NP-40, and 2 mM EGTA, the immunoprecipitates were immunoblotted with
either the anti-Myc or the anti--catenin antibody.
Pull-down assay. COS cells were transfected with various
constructs of -catenin, and the extracts were prepared as described above. For each extract, 160 µl was incubated with either 200 pmol of
GST-S-SCAM-10 containing PDZ4 and PDZ5 or 200 pmol of GST fixed on
14.5 µl of glutathione beads. After the beads were washed, the
proteins on the beads were immunoblotted with the anti-Myc antibody.
Hippocampal neuron culture and hippocampal slice
culture. Hippocampal neuron cultures were performed from
embryonic day 18 embryos as described previously (Takeuchi et al.,
1997; Goslin et al., 1998). Hippocampal slices were obtained from
postnatal day 6 (P6) or P8 rats and cultured on Millicell CM culture
plate inserts (Millipore, Bedford, MA) in Eagle's MEM containing 25% (v/v) HBSS, 6.5 gm/l glucose, 100 U/ml penicillin, 100 µg/ml
streptomycin, and 25% (v/v) horse serum at 32°C under 5%
CO2.
Sindbis virus production and infection. Baby hamster
kidney cells were cultured in MEM supplemented with 5% (v/v) FBS, 100 U/ml penicillin, and 100 µg/ml streptomycin. Capped in
vitro transcripts and helper RNA were synthesized from various
linearized pSindGFP constructs and DH(26S) template (Invitrogen) using
a RiboMAX large-scale RNA production system (Promega, Madison, WI) and
were transfected into baby hamster kidney cells by electroporation with
a GenePulser (Bio-Rad, Hercules, CA). Two days later, the medium was
collected and centrifuged at 400 × g for 5 min.
The supernatant was centrifuged at 113,000 × g for
90 min. The pellet was collected, resuspended in 200 µl of the
medium, and stocked at  80°C. Primary cultured hippocampal neurons
were infected with 3-5 µl of the virus stock per 500 µl of the
culture medium 10 d after plating. For the double infection, 3-5
µl of each virus stock was added to 500 µl of the culture medium at
the same time. Hippocampal slices were infected with 1 µl of the
virus stock per slice 7 d after plating.
Immunocytochemistry. Hippocampal neurons were fixed with
4% (w/v) paraformaldehyde for 15 min, blocked with 50 mM
glycine in PBS for 30 min, and permeabilized with 0.25% (w/v) Triton
X-100 in PBS for 5 min. After they had been blocked with PBS containing 10% (w/v) BSA, cells were incubated with the first antibody in PBS
containing 3% (w/v) BSA overnight, washed with PBS, and incubated with
the second antibody in PBS containing 3% (w/v) BSA for 1 hr. The
images were obtained with confocal microscopes (Olympus FV300-BX;
Olympus Optical, Tokyo, Japan) (Zeiss LSM510; Zeiss, Thornwood, NY).
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RESULTS |
Identification of -catenin as a S-SCAM-interacting molecule
To identify S-SCAM-interacting molecules, we performed a yeast
two-hybrid screening using S-SCAM as bait, as described previously (Ide
et al., 1999a). We obtained 34 positive independent clones from a rat
brain cDNA library. Among them, we found one clone encoding
-catenin. The interaction of -catenin with MAGI-1 or KIAA0705 in
epithelial cells has been reported previously (Dobrosotskaya et al.,
2000; Kawajiri et al., 2000). To confirm the interaction of S-SCAM with
-catenin in neurons, we immunoprecipitated S-SCAM from rat brains.
-Catenin was coimmunoprecipitated with S-SCAM (Fig.
1A). We subsequently
determined whether S-SCAM was colocalized with -catenin in neurons.
In the primary cultured hippocampal neurons, S-SCAM formed clusters on
dendrites apposed to those of synaptophysin, although some clusters of
synaptophysin were free of S-SCAM (Fig. 1B).
Similarly, S-SCAM was colocalized with -catenin, but some clusters
of -catenin were free of S-SCAM (Fig. 1C). We determined
how great a population of S-SCAM clusters was associated with
-catenin. We counted the clusters from three neurons. Of the S-SCAM
clusters, 56.7 ± 7.1% were colocalized with -catenin, whereas
of the -catenin clusters, 47.6 ± 9.8% were colocalized with
S-SCAM.
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Figure 1.
Interaction of S-SCAM and -catenin in neurons.
A, Coimmunoprecipitation of S-SCAM and -catenin.
S-SCAM was immunoprecipitated from rat brains, and the
immunoprecipitate was immunoblotted with either the anti-S-SCAM
antibody (lanes 1-3) or the anti--catenin antibody
(lanes 4-6). Lanes 1, 4, The
original rat brain extracts (Extract); lanes 2, 5, the immunoprecipitates with the preimmune serum
(Preimmune-IP); lanes 3, 6, the
immunoprecipitates with the anti-S-SCAM antibody
(Immune-IP). B, Laser confocal image of a
hippocampal neuron stained with the anti-S-SCAM and the
anti-synaptophysin antibodies. Insets, Areas demarcated
at higher magnification. S-SC, S-SCAM
(arrows); Syn, synaptophysin
(arrowheads); Merge, a superimposed image
of S-SCAM and synaptophysin. Scale bar, 10 µm. C,
Laser confocal image of a hippocampal neuron stained with the
anti-S-SCAM and the anti--catenin antibodies. Insets,
Areas demarcated at higher magnification. S-SC, S-SCAM
(arrows); -Cat, -catenin
(triangles); Merge, a superimposed image
of S-SCAM and -catenin. Scale bar, 10 µm.
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-Catenin-interacting region of S-SCAM and S-SCAM-interacting
region of -catenin
To determine the -catenin-interacting region of S-SCAM, we
prepared various Myc-tagged constructs of S-SCAM (Fig.
2A). We expressed these
proteins in COS cells, immunoprecipitated them with the anti-Myc
antibody, and examined whether the endogenous -catenin was
coimmunoprecipitated. -Catenin was detected in the
immunoprecipitates from COS cells expressing S-SCAM-1, -10, and -12 but
did not associate with S-SCAM-8 (Fig. 2B). To
determine the S-SCAM-interacting region of -catenin, we prepared
various Myc-tagged constructs of -catenin (Fig.
3A). We expressed these proteins in COS cells and examined which constructs interacted with
GST-fusion protein containing the C-terminal region of S-SCAM. -Catenin-1, -3, and -6 interacted with S-SCAM, whereas
-catenin-4, -5, and -7 did not (Fig. 3B). These data
suggest that the C-terminal region of S-SCAM interacts with the
C-terminal region of -catenin.
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Figure 2.
-Catenin-interacting domain of S-SCAM.
A, Schematic of various constructs of S-SCAM.
Gray boxes, PDZ domains; white box, the
GK domain; black boxes, WW domains. The numbers
of the first and the last amino acid residues of each construct
are shown. B, Interaction of the C-terminal region of
S-SCAM with -catenin. Various Myc-tagged S-SCAM proteins were
expressed in COS cells and were immunoprecipitated with the anti-Myc
antibody. The original extracts and the immunoprecipitates were
immunoblotted with either the anti-Myc antibody
(a) or the anti--catenin antibody
(b). Lanes 1, 2, Transfected with
mock; lanes 3, 4, pClneoMyc S-SCAM-1; lanes 5, 6, pClneoMyc S-SCAM-8; lanes 7, 8, pClneoMyc
S-SCAM-10; lanes 9, 10, pClneoMyc S-SCAM-12.
Lanes 1, 3, 5, 7, 9, The original extracts
(E); lanes 2, 4, 6, 8, 10, the
immunoprecipitates (P).
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Figure 3.
S-SCAM-interacting domain of -catenin.
A, Schematic of various Myc-tagged constructs of
-catenin. Boxes, Armadillo repeats. The numbers of
the first and the last amino acid residues of each construct are shown.
B, Interaction of the C-terminal region of -catenin
with the C-terminal region of S-SCAM. The extracts of COS cells
expressing various Myc-tagged -catenin proteins were incubated with
either the GST-S-SCAM-10 containing PDZ4 and PDZ5 or the GST fixed on
glutathione beads. The proteins on the beads were immunoblotted with
the anti-Myc antibody. Lanes 1-3, pClneoMyc
-catenin-1; lanes 4-6, pClneoMyc -catenin-3;
lanes 7-9, pClneoMyc -catenin-4; lanes
10-12, pClneoMyc -catenin-5; lanes 13-15,
pClneoMyc -catenin-6; lanes 16-18, pClneoMyc
-catenin-7. Lanes 1, 4, 7, 10, 13, 16, The original
extracts (E); lanes 2, 5, 8, 11, 14, 17, the precipitates with the GST-S-SCAM-10
(S); lanes 3, 6, 9, 12, 15, 18,
the precipitates with the GST (G).
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Localization of various GFP-S-SCAM constructs in rat
hippocampal slice and primary cultured hippocampal neurons
To determine the region involved in the synaptic targeting of
S-SCAM, we prepared GFP-tagged constructs containing various regions of
S-SCAM (Fig. 4A). We
expressed GFP-S-SCAM-1 in rat hippocampal slice neurons using Sindbis
virus. The protein began to appear at 9 hr after the infection and was
diffusely distributed in the soma and neurites (Fig.
4B, a). At 12 hr after the infection, the
clusters appeared on the dendrites, and the number of clusters increased (Fig. 4B, b and c).
We also tested other GFP constructs. GFP-S-SCAM-8 lacking PDZ5 was
diffusely distributed in the soma and neurites (Fig. 4C,
a). GFP-S-SCAM-10 (PDZ4 and PDZ5) was diffusely distributed
in most parts, although small clusters were observed in some parts
(Fig. 4C, b). GFP-S-SCAM-4 (PDZ2, PDZ3, PDZ4,
and PDZ5), -13 (PDZ3, PDZ4, and PDZ5), and -19 (PDZ2, PDZ3, and PDZ5) formed more clusters on dendrites (data not shown and Fig.
4C, c and d). Therefore, PDZ5 is
necessary for S-SCAM to form clusters on dendrites, although other
regions may also be involved in the formation of clusters. We
subsequently expressed GFP-S-SCAM-1, -10, -13, and -19 in rat primary
cultured hippocampal neurons and examined whether these proteins
were localized at synapses. The clusters of GFP-S-SCAM-1, -13, and
-19 were apposed to those of synaptophysin, suggesting synaptic
localization (Fig. 5A,C,D). GFP-S-SCAM-10 formed a few clusters in primary cultured hippocampal neurons, and these clusters were also apposed to those of synaptophysin (Fig. 5B).
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Figure 4.
Various GFP-S-SCAM proteins expressed in
hippocampal slice neurons. A, Schematic of various GFP
constructs of S-SCAM. Gray boxes, PDZ domains;
white box, GK domain; black boxes, WW
domains. B, Temporal profile of the expression of
GFP-S-SCAM-1 in hippocampal slice neurons. a, 9 hr
after the infection; b, 12 hr after the infection;
c, 24 hr after the infection. Insets,
Demarcated areas at higher magnification to show the clusters of
GFP-S-SCAM-1. Scale bar, 10 µm. C, Various
GFP-S-SCAM proteins in hippocampal slice neurons.
Insets, Demarcated areas at higher magnification.
a, GFP-S-SCAM-8; b, GFP-S-SCAM-10;
c, GFP-S-SCAM-13; d, GFP-S-SCAM-19.
Scale bar, 10 µm.
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Figure 5.
Various GFP-S-SCAM proteins expressed in primary
cultured hippocampal neurons. A, GFP-S-SCAM-1.
GFP-S-SCAM-1 formed clusters apposed to those of synaptophysin.
B, GFP-S-SCAM-10. C, GFP-S-SCAM-13.
D, GFP-S-SCAM-19. GFP-S-SCAM-10, -13, and -19 also
formed clusters. Insets, Demarcated areas at higher
magnification. S-SC, GFP-S-SCAM
(arrows); Syn, synaptophysin
(arrowheads); Merge, a superimposed image
of S-SCAM and synaptophysin. Scale bars, 10 µm.
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Localization of various GFP--catenin constructs in rat
hippocampal slice and primary cultured hippocampal neurons
To determine the region involved in the synaptic targeting of
-catenin, we prepared GFP-tagged constructs containing various regions of -catenin (Fig.
6A). We expressed
GFP--catenin-1 in rat hippocampal slice neurons using Sindbis
virus. The protein began to express at 6 hr after the infection (Fig.
6B). At that time, the protein was already clustered
on dendrites. We also expressed other GFP constructs.
GFP--catenin-2 lacking the N-terminal region was clustered on
dendrites (Fig. 6C, a). GFP--catenin-6 containing all armadillo repeats also formed clusters on dendrites (Fig. 6C, b). In contrast, GFP--catenin-8
containing the C-terminal region and GFP--catenin-9 containing the
last 4.5 armadillo repeats and the C-terminal region were diffusely
distributed (Fig. 6C, c and d).
GFP--catenin-10 containing the N-terminal region and the first five
armadillo repeats was localized in the nucleus (Fig. 6C,
e). GFP--catenin-11 containing the last six armadillo repeats was diffusely distributed (Fig. 6C,
f). We compared the localization of
GFP--catenin-1 and -6 with that of synaptophysin in rat primary
cultured hippocampal neurons. Both GFP--catenin-1 and
GFP--catenin-6 were colocalized with synaptophysin, suggesting that
the C-terminal region involved in the interaction with S-SCAM is not
necessary for the synaptic targeting of -catenin (Fig. 7A,B).
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Figure 6.
Various GFP--catenin proteins expressed in
hippocampal slice neurons. A, Schematic of various
GFP-tagged constructs of -catenin. Boxes, Armadillo
repeats. B, Temporal profile of the expression of
GFP--catenin-1 in hippocampal slice neurons.
a, 6 hr after the infection; b, 9 hr after the infection; c, 24 hr after the infection.
Inset, Demarcated area at higher magnification. Scale bar,
10 µm. C, Various GFP--catenin proteins in
hippocampal slice neurons. Insets, Demarcated areas at
higher magnification. a, GFP--catenin-2;
b, GFP--catenin-6; c,
GFP--catenin-8; d, GFP--catenin-9;
e, GFP--catenin-10; f,
GFP--catenin-11. Scale bar, 10 µm.
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Figure 7.
GFP--catenin proteins containing the armadillo
repeats in primary cultured hippocampal neurons. A,
GFP--catenin-1. B, GFP--catenin-6. Both
GFP--catenin-1 and GFP--catenin-6 formed clusters colocalized
with those of synaptophysin. Insets, Demarcated areas at
higher magnification. -Cat, GFP--catenin
(triangles); Syn, synaptophysin
(arrowheads); Merge, a superimposed image
of GFP--catenin and synaptophysin. Scale bars, 10 µm.
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The C-terminal region of -catenin blocks the synaptic targeting
of S-SCAM on dendrites, and the C-terminal region of S-SCAM was
recruited to synapses with -catenin
Next, we examined the effect of the overexpressed
S-SCAM-interacting region of -catenin on the localization of S-SCAM
and that of the overexpressed -catenin-interacting region of S-SCAM on the localization of -catenin. For this purpose, we prepared the
pSindMyc-S-SCAM-1 and -10. Myc-S-SCAM-1 containing full-length S-SCAM
was concentrated at synapses such as GFP-S-SCAM-1 (Fig. 8A). GFP--catenin-8
was diffusely distributed (Fig. 8B). When coexpressed
with GFP--catenin-8, Myc-S-SCAM-1 became diffusely distributed
(Fig. 8C). We also tested the effect of GFP--catenin-8 on the synaptic targeting of the endogenous S-SCAM. Forty-eight hours
after the infection, we immunostained neurons with the anti-S-SCAM and
the anti-synaptophysin antibodies and counted the clusters from three
neurons. In neurons expressing GFP--catenin-8, the number of the
clusters of S-SCAM decreased from 47.6 ± 5.5 to 30.3 ± 2.5 per 280 µm of the dendrite. Furthermore, 74.2 ± 5.1% of S-SCAM
clusters were colocalized with synaptophysin in neurons without
GFP--catenin-8, whereas only 32.3 ± 9.1% of the clusters were colocalized with synaptophysin in neurons expressing
GFP--catenin-8 (Fig. 8D). In contrast,
GFP--catenin-8 did not affect the colocalization of the clusters of
NR2A with synaptophysin (Fig. 8E) or the number of
the clusters of synaptophysin (65.0 ± 7.9 and 65.3 ± 8.1 per 280 µm of the dendrite in neurons with and without
GFP--catenin-8, respectively). Myc-S-SCAM-10 containing PDZ4 and
PDZ5 formed small clusters like GFP-S-SCAM-10 in some parts but did
not form clusters in most parts (Fig.
9A). When coexpressed with
GFP--catenin-1, Myc-S-SCAM-10 did not block the synaptic targeting
of GFP--catenin-1 (Fig. 9B, a). Conversely,
Myc-S-SCAM-10 formed more and larger clusters apposed to those of
synaptophysin (Fig. 9B, b). These data suggest
that -catenin is involved in the synaptic targeting of S-SCAM.
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Figure 8.
The C-terminal region of -catenin inhibits the
synaptic targeting of S-SCAM. A, Myc-S-SCAM-1 in a
primary cultured hippocampal neuron. Hippocampal neurons expressing
Myc-S-SCAM-1 were stained with the polyclonal sheep anti-Myc and the
anti-synaptophysin antibodies. Myc-S-SCAM-1 formed clusters apposed to
those of synaptophysin, such as GFP-S-SCAM-1. The large
image shows the merged image of Myc-S-SCAM-1 and synaptophysin.
Insets, Demarcated areas at higher magnification.
S-SC, Myc-S-SCAM-1 (arrows);
Syn, synaptophysin (arrowheads);
Merge, a superimposed image of Myc-S-SCAM-1 and
synaptophysin. Scale bar, 10 µm. B, GFP--catenin-8
in a primary cultured hippocampal neuron. GFP--catenin-8 was
diffusely distributed as in hippocampal slice neurons. The large
image shows the merged image of GFP--catenin-8 and
synaptophysin. Insets, Demarcated areas at higher
magnification. -Cat, GFP--catenin-8;
Syn, synaptophysin (arrowheads). Scale
bar, 10 µm. C, Myc-S-SCAM-1 coexpressed with
GFP--catenin-8 in a primary cultured hippocampal neuron.
Myc-S-SCAM-1 became diffusely distributed when coexpressed with
GFP--catenin-8. The large image shows the merged
image of Myc-S-SCAM-1 and GFP--catenin-8. Insets,
Demarcated areas at higher magnification. S-SC,
Myc-S-SCAM-1; -Cat, GFP--catenin-8;
Syn, synaptophysin (arrowheads). Scale
bar, 10 µm. D, The endogenous S-SCAM in neurons
expressing GFP--catenin-8. The clusters of the endogenous S-SCAM
were not apposed to those of synaptophysin in neurons expressing
GFP--catenin-8. S-SC, S-SCAM; Syn,
synaptophysin; -Cat, GFP--catenin-8;
Merge, a superimposed image of S-SCAM, synaptophysin,
and GFP--catenin-8. Scale bar, 1 µm. E, NMDA
receptors in neurons expressing GFP--catenin-8. The clusters of
NMDA receptor subunit 2A were apposed to those of synaptophysin even in
neurons expressing GFP--catenin-8. Syn,
Synaptophysin; -Cat, GFP--catenin-8;
Merge, a superimposed image of NMDA receptor subunit 2A,
synaptophysin, and GFP--catenin-8. Scale bar, 1 µm.
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[in a new window]
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Figure 9.
-Catenin facilitates the accumulation of the
C-terminal region of S-SCAM at synapses. A,
Myc-S-SCAM-10 containing PDZ4 and PDZ5 in a primary cultured
hippocampal neuron. Neurons expressing Myc-S-SCAM-10 were stained with
the polyclonal sheep anti-Myc and the anti-synaptophysin antibodies.
The distribution of Myc-S-SCAM-10 was similar to that of
GFP-S-SCAM-10. The number of clusters formed by Myc-S-SCAM-10 was
small. a, Myc-S-SCAM-10 (arrows);
b, synaptophysin (arrowheads);
c, a superimposed image of Myc-S-SCAM-10 and
synaptophysin. Scale bar, 10 µm. B, Myc-S-SCAM-10
coexpressed with GFP--catenin-1. Primary cultured hippocampal
neurons expressing Myc-S-SCAM-10 and GFP--catenin-1 were
immunostained with the sheep anti-Myc and the mouse anti-synaptophysin
antibodies. a, Merged image of GFP--catenin-1
(triangles) and synaptophysin
(arrowheads); b, merged image of
Myc-S-SCAM-10 (arrows) and synaptophysin
(arrowheads). Scale bar, 1 µm.
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The cytoplasmic region of NMDA receptor subunit 2A did not block
the synaptic targeting of S-SCAM
Because PDZ5 of S-SCAM binds various molecules besides
-catenin, other molecules may also be involved in the synaptic
targeting of S-SCAM. We tested whether the cytoplasmic region of NR2A
blocked the synaptic targeting of S-SCAM. The distribution of the
GFP-tagged cytoplasmic region of NR2A (GFP-NR2A) was similar to that
of GFP-S-SCAM-10 (Fig.
10A). It was
diffusely distributed in most parts, although some clusters were
observed. However, this protein did not block the synaptic targeting of
Myc-S-SCAM-1, unlike GFP--catenin-8 (Fig. 10B).
Thus, the cytoplasmic region of -catenin is distinct from that of
NR2A in the inhibition of the synaptic targeting of S-SCAM.
View larger version (25K):
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|
Figure 10.
The cytoplasmic region of NMDA receptor subunit
2A does not inhibit the synaptic targeting of S-SCAM. A,
GFP-tagged cytoplasmic region of NR2A (GFP-NR2A) in a primary cultured
hippocampal neuron. The distribution of GFP-NR2A was similar to that
of GFP-S-SCAM-10. The number of clusters formed by GFP-NR2A was
small. a, GFP-NR2A (asterisk);
b, synaptophysin (arrowheads);
c, a superimposed image of GFP-NR2A and synaptophysin.
Scale bar, 10 µm. B, Myc-S-SCAM-1 coexpressed with
GFP-NR2A. Primary cultured hippocampal neurons expressing
Myc-S-SCAM-1 and GFP-NP2A were immunostained with the sheep anti-Myc
and the mouse anti-synaptophysin antibodies. GFP-NR2A did not
inhibit the synaptic targeting of Myc-S-SCAM-1 and formed more
clusters when coexpressed with Myc-S-SCAM-1. a,
Myc-S-SCAM-1 (arrows); b, synaptophysin
(arrowheads); c, GFP-NR2A
(asterisks); d, a superimposed image of
Myc-S-SCAM-1, synaptophysin, and GFP-NR2A. Scale bar, 10 µm.
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DISCUSSION |
In epithelial cells, -catenin links E-cadherin to the
cytoskeleton and regulates gene transcription. -Catenin also binds lin-7 in Madin-Darby canine kidney cells, which fixes GABA transporter to the plasma membrane by blocking endocytosis (Perego et al., 1999,
2000). In neurons, -catenin is enriched at synapses and binds to
N-cadherin and other cadherin-like molecules (Uchida et al.,
1996; Benson and Tanaka, 1998; Miskevich et al., 1998). The role of
-catenin at synapses, however, is unclear. In this study, we reveal
that S-SCAM interacts with -catenin. -Catenin is
coimmunoprecipitated with S-SCAM from rat brain. -Catenin is
partially colocalized with S-SCAM in neurons. In vitro
binding assays indicate that the C-terminal region of S-SCAM binds to the C-terminal region of -catenin. We have tried to clarify the meaning of this interaction.
We have determined which region of S-SCAM is involved in its synaptic
targeting. The deletion construct of S-SCAM lacking the sixth PDZ
domain (PDZ5) is not localized at synapses, whereas the constructs
containing PDZ5 are localized at synapses. Because GFP-tagged PDZ5 did
not express, we could not conclude that PDZ5 is sufficient for the
synaptic targeting. Our findings indicate, however, that PDZ5 is
necessary for the synaptic targeting. This result is consistent with
the hypothesis that MAGI-1 is targeted to the lateral epithelial
membrane via the C-terminal region (Nishimura et al., 2000). Because
-catenin binds to PDZ5, we hypothesized that the interaction with
-catenin is involved in the synaptic targeting of S-SCAM. The
overexpressed C-terminal region of -catenin is diffusely distributed
in neurons and blocks the synaptic targeting of the coexpressed S-SCAM,
supporting this model. We immunostained the endogenous -catenin and
S-SCAM with the antibodies at various stages of synaptogenesis to
determine which molecule is targeted to synapses earlier, but we did
not reach any conclusions because of the different sensitivities of the
antibodies. However, GFP--catenin is concentrated at synapses
earlier than GFP-S-SCAM when expressed in neurons, suggesting that
-catenin is targeted to synapses before S-SCAM. The C-terminal
region of -catenin did not inhibit the synaptic localization of NR2A
or decrease or increase the number of the clusters of synaptophysin.
These findings do not support the possibility that the C-terminal
region of -catenin affects synaptogenesis. It should be noted that
we expressed GFP--catenin-8 in neurons 10 d after plating and
could maintain them for only 2 d. If GFP--catenin-8 is expressed in
neurons at an earlier stage and if neurons expressing
GFP--catenin-8 are cultured for a longer time, the effect on the
localization of other synaptic proteins may be detected.
The protein with PDZ4 and PDZ5 of S-SCAM (GFP-S-SCAM-10 or
Myc-S-SCAM-10) can accumulate at synapses but is resistant to being targeted to synapses, compared with other constructs such as
GFP-S-SCAM-1 (the full length), GFP-S-SCAM-13 (PDZ3, PDZ4, and PDZ5),
and GFP-S-SCAM-19 (PDZ2, PDZ3, and PDZ5). Even so, we consider that
the region covering PDZ4 and PDZ5 is sufficient for the synaptic
targeting, because Myc-S-SCAM-10 is clustered effectively at synapses
when coexpressed with -catenin. The reason why the synaptic
targeting of S-SCAM-10 depends on the overexpressed -catenin while
the targeting of other constructs, including GFP-S-SCAM-13 and -19, does not is currently unclear. PDZ3 may bind some molecule
interacting with -catenin, and the affinity of S-SCAM-10 for
-catenin may be lower than that of proteins with both PDZ3 and PDZ5.
PDZ5 of S-SCAM binds various molecules. We have raised the question of
whether -catenin is specific in the inhibition of the synaptic
targeting of S-SCAM. We have tested the C-terminal region of NR2A. Most of GFP-NR2A is diffusely distributed in neurons, but GFP-NR2A does
not affect the synaptic localization of S-SCAM. This result supports
the proposition that the C-terminal region of -catenin has a
specificity to block the synaptic localization of S-SCAM.
We have also determined that the region of the armadillo
repeats of -catenin is involved in the synaptic
targeting. Among the constructs that we have tested, only those with
all of the repeats are clustered at synapses. A recent study revealed
that the cytoplasmic domain of E-cadherin interacts with all 12 armadillo repeats of -catenin (Huber and Weis, 2001). Therefore,
-catenin is recruited to synapses by interaction with molecules such
as N-cadherin, which requires all of the armadillo repeats
to bind -catenin, and the interaction with S-SCAM does not play an
essential role in the synaptic localization of -catenin. Our data
establish a major role for -catenin in recruiting S-SCAM to
synapses. Although neuroligin and GKAP bind S-SCAM, the interactions
with these molecules are not necessary for the synaptic targeting of
S-SCAM.
| |
FOOTNOTES |
Received Aug. 6, 2001; revised Nov. 2, 2001; accepted Nov. 5, 2001.
This study was supported by grants-in-aid for Scientific Research and
by special coordination funds for Promoting Science and Technology from
the Ministry of Education, Culture, Sports, Science, and Technology; by
a grant from the Novartis Foundation (Japan) for the Promotion of
Science, 1999; and by a grant from the Yamanouchi Foundation for
Research on Metabolic Disorders, 2000. We thank Prof. Shigetada
Nakanishi (Kyoto University, Kyoto, Japan) for cDNA of rat NMDA
receptor subunit 2A and Prof. Masatoshi Takeichi (Kyoto University) for
cDNA of mouse -catenin. pClneoMyc constructs of S-SCAM and
-catenin were prepared by Y.H. in the Takai Biotimer Project,
Exploratory Research for Advanced Technology, headed by Prof. Yoshimi
Takai (Osaka University, Osaka, Japan).
Correspondence should be addressed to Dr. Yutaka Hata, Department
of Medical Biochemistry, Graduate School of Medicine, Tokyo Medical and
Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8519, Japan.
E-mail: yuhammch{at}med.tmd.ac.jp.
| |
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TOP
ABSTRACT
INTRODUCTION
