Interaction of NE-dlg/SAP102, a Neuronal and Endocrine
Tissue-specific Membrane-associated Guanylate Kinase Protein, with
Calmodulin and PSD-95/SAP90
A POSSIBLE REGULATORY ROLE IN MOLECULAR CLUSTERING AT SYNAPTIC
SITES*
Norio
Masuko
§,
Keishi
Makino
,
Hiroaki
Kuwahara
,
Kohji
Fukunaga¶,
Tamotsu
Sudo
,
Norie
Araki
,
Hideyuki
Yamamoto¶,
Yuji
Yamada§,
Eishichi
Miyamoto¶, and
Hideyuki
Saya
From the Departments of
Tumor Genetics and Biology
and ¶ Pharmacology, Kumamoto University School of Medicine, 2-2-1, Honjo, Kumamoto 860-0811, Japan and the § Cancer Research
Laboratory, Hanno Research Center, Taiho Pharmaceutical Co., Ltd.,
1-27, Misugidai, Hanno, Saitama 357-8527, Japan
 |
ABSTRACT |
NE-dlg/SAP102, a neuronal and endocrine
tissue-specific membrane-associated guanylate kinase family protein, is
known to bind to C-terminal ends of
N-methyl-D-aspartate receptor 2B (NR2B) through
its PDZ (PSD-95/Dlg/ZO-1) domains. NE-dlg/SAP102 and NR2B colocalize at
synaptic sites in cultured rat hippocampal neurons, and their
expressions increase in parallel with the onset of synaptogenesis. We
have identified that NE-dlg/SAP102 interacts with calmodulin in a
Ca2+-dependent manner. The binding site for
calmodulin has been determined to lie at the putative basic
-helix
region located around the src homology 3 (SH3) domain of
NE-dlg/SAP102. Using a surface plasmon resonance measurement system, we
detected specific binding of recombinant NE-dlg/SAP102 to the
immobilized calmodulin with a Kd value of 44 nM. However, the binding of Ca2+/calmodulin to
NE-dlg/SAP102 did not modulate the interaction between PDZ domains of
NE-dlg/SAP102 and the C-terminal end of rat NR2B. We have also
identified that the region near the calmodulin binding site of
NE-dlg/SAP102 interacts with the GUK-like domain of PSD-95/SAP90 by
two-hybrid screening. Pull down assay revealed that NE-dlg/SAP102 can
interact with PSD-95/SAP90 in the presence of both Ca2+ and
calmodulin. These findings suggest that the Ca2+/calmodulin
modulates interaction of neuronal membrane-associated guanylate kinase
proteins and regulates clustering of neurotransmitter receptors at
central synapses.
 |
INTRODUCTION |
Over the past decades, many efforts were given to identify the
various proteins composing the postsynaptic sites in neurons. Postsynaptic densities
(PSDs),1 which are the
insoluble fractions of neurons in traditional biochemical detergents,
have been found to contain cytoskeletal proteins and their associated
proteins, such as actin (1), spectrin, tubulin, microtubule-associated
proteins (2), calmodulin (3), and calmodulin-dependent
protein kinase II (4). Isolated PSDs have also been shown to be
enriched in N-methyl-D-aspartate (NMDA) and
-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors. Recently, several proteins, including PSD-95/SAP90 (5), NE-dlg/SAP102 (6, 7), and chapsyn-110/PSD-93 (8), which belong to a novel protein
family called membrane-associated guanylate kinases (MAGUKs), have been
identified as components of PSD. These MAGUK proteins are homologous to
the Drosophila disc large (dlg) tumor suppressor protein (6,
7) in both sequence and structural organization and contain three
distinct domains: an N-terminal segment comprising one or three copies
of 80-90 amino acid motif called PDZ (PSD-95/Dlg/ZO-1) domain, a
src homology 3 (SH3) domain, and a region with high
similarity to guanylate kinases (9, 10). These domains have been shown
to be utilized as modules for interacting with various cellular proteins.
PSD-95/SAP90 has been reported to have an activity of clustering
shaker-type K+ channels (11) and NMDA receptor 2B (NR2B)
(12). The PDZ domains of PSD-95/SAP90 were found to interact with the C
terminus (Ser/Thr)-X-Val ((S/T)XV) motif of the
shaker-type K+ channel and NR2B, resulting in a clustering
of ion channels and neurotransmitter receptors on the plasma membrane.
Similarly, the PDZ domains of NE-dlg/SAP102 were shown to interact with
the C terminus (S/T)XV motif of NR2B (7) and tumor
suppressor protein APC (adenomatous polyposis coli) (6), suggesting
that NE-dlg/SAP102 also contributes to a clustering of the diverse
molecules in certain regions of the cells. In addition, the PDZ domain
of neuronal nitric oxide synthetase was reported to interact with the
PDZ domains of PSD-95/SAP90 and chapsyn-110/PSD-93 (13), suggesting that MAGUK proteins compose a heteromeric oligomer by means of their
PDZ domains.
Various proteins that are involved in intracellular signaling systems
have one or more SH3 domains; an SH3 domain is a protein-protein interaction module conferring a specific binding ability with a
proline-rich motif in a target protein (14-17). However, no specific binding protein interacting with the SH3 domain of the MAGUK family has
been identified yet. The GUK-like domains of PSD-95/SAP90, NE-dlg/SAP102, and chapsyn-110/PSD-93 have been recently reported to
interact with a novel family of proteins called SAP-associated proteins
(SAPAPs) (18-20). Although the function of SAPAPs has not been
clarified yet, this finding suggests that the GUK-like domains of MAGUK
family proteins also contribute to the protein-protein interaction.
We previously reported that NE-dlg/SAP102 is highly expressed in brain
and endocrine tissues, such as pancreas, thyroid, and trachea (6). This
tissue-specific expression suggests that NE-dlg/SAP102 plays specific
roles in these neuronal and endocrine tissues. In the neurons, we found
that NE-dlg/SAP102 is abundantly expressed in axons and dendrites of
matured neurons (6). Muller et al. (7) also reported that
the increase in NE-dlg/SAP102 mRNA levels parallels that of newly
formed synapses in the developing rat cerebral cortex. Therefore, it
has been speculated that NE-dlg/SAP102 contributes to the synapse
formation by clustering neurotransmitter receptors and other molecules
at synaptic sites. However, the molecular mechanisms responsible for
the regulation of clustering and maintenance of the receptors at
synaptic sites are largely unknown.
In the present study, we have first shown that NE-dlg/SAP102 protein
colocalizes closely with NR2B at synaptic sites in cultured hippocampal
neurons and increases in amount during postnatal development. To
elucidate the involvement of NE-dlg/SAP102 in synapse formation, we
searched for proteins that bind to NE-dlg/SAP102 by two-hybrid screening. We have found that calmodulin binds to the region containing the SH3 domain of NE-dlg in a calcium-dependent manner. A
calmodulin-binding region of NE-dlg/SAP102 is predicted to form a basic
amphiphilic
-helix, which was reported as a calmodulin-binding
structure. Moreover, we have identified interaction between
NE-dlg/SAP102 and PSD-95/SAP90 by two-hybrid screening, and this
interaction was observed in the presence of both calmodulin and
Ca2+. Our findings indicate that the association between
NE-dlg/SAP102 and PSD-95/SAP90 is modulated by a calcium signaling
pathway, and this mechanism may regulate the clustering of
neurotransmitter receptors, resulting in formation and/or structural
change in central synapses.
 |
EXPERIMENTAL PROCEDURES |
Neonatal Rat Hippocampal Cell Culture--
Neonatal rat
hippocampal cell cultures were prepared according to the method
described previously (21). For the Western blot analysis, cells were
harvested at the indicated days (Fig. 1A) in culture and
lysed in SDS sample buffer. The samples were resolved by 10-20%
SDS-polyacrylamide gel electrophoresis. After being blotted on a
nitrocellulose filter, the filter was blocked with phosphate-buffered
saline containing 10% skim milk for 1 h at room temperature and
then incubated with anti-NE-dlg polyclonal antibody (6) and anti-rat
NMDA receptor 2B monoclonal antibody (Transduction Laboratories,
Lexington, KY). The filter was then incubated with anti-rabbit and
anti-mouse IgG (Zymed Laboratories Inc., South San
Francisco, CA), and specific proteins were detected using
125I-labeled protein A (NEN Life Science Products).
For the immunofluorescence analysis, cells were fixed with methanol (10 min at
20 °C) followed by permeabilization with 0.05% Triton
X-100 in phosphate-buffered saline for 10 min. Fixed cells were stained
according to the previously described method by using anti-NE-dlg
polyclonal antibody and anti-NMDA receptor 2B monoclonal antibody as
the first antibody, and then fluorescein-conjugated goat anti-rabbit
IgG (Cappel, Aurora, OH) and rhodamine-conjugated goat anti-mouse IgG
(Cappel) as the second antibody. The stained cells were observed with
confocal laser microscopes (Fluoview, Olympus).
Yeast Two-hybrid Screening--
The bait plasmid pGAL4bd-NE-dlg
was constructed as described previously (6). The pGAL4bd-NE-dlg-SH3
plasmid was constructed as follows. A NE-dlg-SH3 fragment was amplified
by polymerase chain reaction using the following sets of
primers, 5'-TACAGTCCCATGGAATCGAAGATAC-3', 5'-GCCGGGAAGCTTGTGGCACACAGGATCC-3', digested with NcoI
and BamHI, and subcloned into pAS1-CYH2 vector (1). The bait
plasmid pGAL4bd-NE-dlg (6) or pGAL4bd-NE-dlg-SH3 was cotransfected into
yeast HF7c (22) with a prey plasmid containing a human adult brain
cDNA library fused to the GAL4 activation domain (GAL4ad) in the
pGAD 10 vector (CLONTECH, Palo Alto, CA) by
electroporation (23). The transformants were screened as described
previously (6).
Construction of GST Fusion Proteins--
The cDNA fragments
coding full-length-(I) and a region-(II) (as illustrated in Fig.
3A) of NE-dlg/SAP102 cDNA were amplified by polymerase
chain reaction using the following sets of primers (full-length-(I), 5'-CAGTGCCATGCACAAGCACCAGCACTGCTGTAA-3' and 5'-ATGGTGTCGACGGTACCTTCAGAGTTTTTCAGGGGATGGGAC-3'; region-(II), 5'-ATGGCGAATTCAAATATGAGGAAATCGTACTTGAG-3' and
5'-TTGTTCGGAGGGATCCAGACCCAG-3'), subcloned into a pCR2 TA cloning
vector (Invitrogen, San Diego, CA), digested by EcoRI and
subcloned into a pGEX-2TH bacterial expression vector (24). The
cDNA fragments coding region-(III) and region-(IV) were amplified
by PCR using the following sets of primers (region-(III),
5'-GCCAGGAATTCACCATTGTGGCCCAGTACAGA-3' and
5'-AGGCCAAGCTTAGTGAATTTCTTGCCGTGTCAC-3'; region-(IV),
5'-ATCGGTGGATCCCCCAGTAAGAAG-3' and 5'-CCGGGAAGCTTCTGTTAGACTCAATC-3'),
di- gested by EcoRI and HindIII for
region-(III), by BamHI and HindIII for
region-(IV), and subcloned into a pGEX-2TH vector. The cDNA
fragment of PSD-95/SAP90 was cut out from the two-hybrid clone
containing the C-terminal 284-amino acid region of PSD-95/SAP90
cDNA with EcoRI and subcloned into a pGEX-2TH vector.
The entire sequences of all inserted cDNA were confirmed to be
identical to the previously reported sequences by DNA sequencing
analysis. The expression and purification of the GST fusion proteins
were as described previously (24).
Solution Binding Assay--
The solution binding assay was
performed according to the method of Rexach and Blobel (25). Briefly,
0.5 µg of GST-fusion protein was mixed with 10 µl packed gel of
calmodulin-agarose (1.5 mg of calmodulin/ml of packed gel, Sigma) in
100 µl of binding buffer (10 mM Tris (pH 7.5), 150 mM NaCl, 0.1% Tween 20, 0.1% casamino acids, 10 mM DTT) containing the indicated amount of CaCl2 or EGTA at 4 °C for 1 h. Calmodulin-agarose
was sedimented by brief centrifugation at 4 °C, separated from the
supernatant, and washed three times with 100 µl of binding buffer
containing the same concentration of CaCl2 or EGTA as the
binding reaction. The fusion proteins retained in the supernatant and
bound to the calmodulin-agarose were resolved by SDS-polyacrylamide gel
electrophoresis analysis, transferred to a nitrocellulose membrane and
detected using anti-GST monoclonal antibody (MBL, Nagoya, Japan) for a primary antibody, anti-mouse horseradish peroxidase (Amersham Pharmacia
Biotech) for a second antibody and ECL (Amersham Pharmacia Biotech) for visualization.
Surface Plasmon Resonance Measurements--
Calmodulin was
purified from bovine brain as described previously (26). All of
reagents and sensor chip for BIAcore instrumentation were purchased
from BIAcore AB (Uppsala, Sweden). The specific interactions were
analyzed by Surface Plasmon Resonance responses in BIAcore instrument
(BIAcore AB). Four flow cells are placed in a surface of sensor chip
CM5. Calmodulin was immobilized to a flow cell of sensor chip by an
amine coupling procedure according to a guide method in the instruction
handbook. The amount of immobilized calmodulin was calculated from the
sensorgram of the immobilization. The immobilization procedure without
calmodulin was performed in another flow cell of sensor chip in order
to be made as blank control and measure the nonspecific responses of
analyte. GST-NE-dlg-(III) (analyte) was injected in both the control
cell and to the calmodulin-immobilized cell at the flow rate of 6 µl/min in the running buffer (10 mM CaCl2, 10 mM DTT, 10 mM Tris (pH 7.4), 0.15 M
NaCl, 3.4 mM EDTA, 0.05% surfactant P-20). The sensorgram
of the control cell was subtracted from that of the
calmodulin-immobilized cell. The dissociation constant was calculated
using BIAevaluation software, version 2.0 (Amersham Pharmacia Biotech)
according to a guide method in the instruction handbook. The apparent
binding rate was calculated by the linear fitting of the increase rate
of RU values using BIAevaluation software, version 2.0 (Amersham
Pharmacia Biotech). The indicated amount of calmodulin or soybean
trypsin inhibitor was coinjected with 0.25 µM of
GST-NE-dlg-(III) in the competition experiment. The biotinylated
peptides PEP7154 (biotin-NGHVYEKLSSIESDV-COOH) corresponding to the C
terminus of rat NMDA receptor 2B and PEP7153 (biotin-NGHVYEKLSSIESD-COOH) for a negative control were supplied by
Iwaki Glass Co. (Chiba, Japan). These peptides were immobilized on
sensor chip SA (Amersham Pharmacia Biotech). GST fusion protein and the
indicated amount of calmodulin were coinjected in both the
PEP7154-immobilized flow cell and the PEP7153-immobilized flow cell at
6 µl/min in the running buffer described above.
Pull Down Assay--
Hemagglutinin epitope-tagged NE-dlg-
GUK
expressing plasmid (pCGN-NE-dlg-
GUK) was constructed as follows. The
hemagglutinin epitope-tagged full-length NE-dlg expression plasmid
(pCGN-NE-dlg (1)) was digested with BamHI and self-ligated.
This procedure resulted in a removal of a C-terminal 164-amino acid
region, which corresponds to most of the GUK domain. Four µg of
pCGN-NE-dlg-
GUK plasmid was transfected into COS-7 cells grown in a
10-cm culture dish by using LipofectAMINETM (Life
Technologies, Inc.). Three days after the transfection, cells were
washed with phosphate-buffered saline and lysed with 500 µl of
extraction buffer (10 mM Tris-HCl at pH 7.5, 300 mM NaCl, 2% Triton X-100, 1 mM DTT, 1 mM 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride,
0.1 mM leupeptin, 1 µM pepstatin A, 54 µg/ml aprotinin) for 10 min on ice. After centrifugation at
12,000 × g for 20 min, the supernatant was diluted
with equal volume of dilution buffer (10 mM Tris-HCl at pH
7.5, 2% Triton X-100, 1 mM DTT, 1 mM
4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride, 0.1 mM leupeptin, 1 µM pepstatin A, 54 µg/ml
aprotinin). Fifty µl of GSH-agarose was incubated with 90 µg of GST
or GST-PSD-95 (C-terminal) for 3 h, washed with washing buffer (10 mM Tris-HCl at pH 7.5, 150 mM NaCl, 2% Triton
X-100, 1 mM DTT) three times, and divided into four tubes.
The cell lysate, supplemented with CaCl2 or EGTA at a
concentration of 1 mM and with or without calmodulin at a
concentration of 3 µM, was added to each tube, conjugated for 2 h at 4 °C, and washed three times with washing buffer
supplemented with 1 mM CaCl2 or EGTA. The
proteins bound to the GSH-agarose were analyzed by Western blotting by
using anti-hemagglutinin epitope antibody (12CA5) as described
previously (6).
 |
RESULTS |
Expression of NE-dlg/SAP102 in Cultured Neurons--
To
investigate the role of NE-dlg/SAP102 in neurons, we first analyzed the
expression of NE-dlg/SAP102 in the neonatal rat hippocampal cell
culture by Western blot analysis (Fig.
1). NE-dlg/SAP102 protein, as well as
NR2B, expressed at very low level until the 7th day in culture.
However, the expression levels of both NE-dlg/SAP102 and NR2B began to
elevate from the 10th day and reached a maximum at around 14 days of
culture. These elevation of NE-dlg/SAP102 and NR2B expression during
neuronal development appears to parallel the synapse formation in this
neonatal hippocampal cell culture system (27). Next, we examined the
subcellular localization of NE-dlg/SAP102 and NR2B in the rat
hippocampal cells (21 days in culture). The antibody against NR2B gave
punctate immunoreactivity along the dendrites (Fig.
2A), and the
anti-NE-dlg/SAP102 antibody also gave a similar punctate pattern along
the dendrites with diffuse staining in the cytoplasm of neurons (Fig.
2B). As shown in Fig. 2C, NE-dlg/SAP102
colocalized closely with NR2B in dendritic spines, at presumed synaptic
sites. These findings suggest that NE-dlg/SAP102 and NR2B accumulate
together at the synaptic sites during synapse formation in neuronal
cells.

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Fig. 1.
Expression of NE-dlg/SAP102 and NMDA receptor
2B in the cultured rat hippocampal cells. A, changes in
the protein expression level of NE-dlg/SAP102 and NMDA receptor 2B in
the neonatal rat hippocampal cell culture were analyzed by Western
blotting. The cells at indicated days in culture were harvested and
analyzed as described under "Experimental Procedures."
B, the intensity of the band indicating NMDA receptor 2B
( ) or NE-dlg ( ) detected from cells at 4 days in culture by the
Western blot analysis was taken as 100%. From this value, the relative
intensity of the band detected in cells at 1, 7, 10, 14, or 21 days in
culture was calculated as a percentage and plotted.
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Fig. 2.
Colocalization of NE-dlg/SAP102 and NMDA
receptor 2B in the cultured rat hippocampal cells. The subcellular
localization of NE-dlg/SAP102 and NMDA receptor 2B in the neonatal rat
hippocampal cell culture was analyzed by immunofluorescent staining.
The cells at 21 days in culture were fixed and stained using anti-NMDA
receptor 2B monoclonal antibody and rhodamine-conjugated goat
anti-mouse IgG (red) (A) or anti-NE-dlg
polyclonal antibody and fluorescein-conjugated goat anti-rabbit IgG
(green) (B). Composite images show colocalization
of signals (yellow) (C).
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Identification of Interaction between NE-dlg/SAP102 and
Calmodulin--
To obtain further insights into the action of
NE-dlg/SAP102 on the synapse formation, we have tried to find novel
NE-dlg/SAP102-interacting molecules by a yeast two-hybrid method using
a human brain cDNA library as the prey. We used a region of amino
acids 126-583, which contains the three PDZ domains and SH3 domain of
human NE-dlg/SAP102 as the bait. Previously, we identified interaction
between APC and NE-dlg by a two-hybrid screening using the same bait
and a human fetal brain cDNA library as the prey (6). In the
present study, we screened 105 clones and identified one
positive clone that included a part of calmodulin cDNA encoding the
region from the 14th to 142nd amino acid. This region contained all
four calcium binding segments of calmodulin and lacked only a part of
helices I and VII (28).
To identify the calmodulin binding site in NE-dlg/SAP102 protein, we
performed a solution binding assay using calmodulin-agarose and
NE-dlg/SAP102 deletion mutants fused to GST. GST-NE-dlg-(I), which
contains the full-length NE-dlg, and GST-NE-dlg-(III), which contains
the SH3 domain of NE-dlg, interacted with calmodulin in the presence of
Ca2+, whereas GST-NE-dlg-(II), which contained three PDZ
domains, and GST did not interact (Fig.
3, A and B). These
results indicate that a region responsible for calmodulin binding lies
at the SH3 domain and the intervening sequence around it in the
NE-dlg/SAP102 protein. The computational analysis using a protein
secondary structure prediction program (MacVector, Eastman Kodak) and
-helical modeling reveal that the region from the 560th to the 591st
amino acid is predicted to form an
-helix and that one side of this helix consists of only basic amino acids (Fig. 3C). This
characteristic structure is consistent with one of the known
calmodulin-binding structures (29, 30). Therefore, to assess whether
calmodulin binds to this small
-helix region of NE-dlg, we performed
a solution binding assay using calmodulin-agarose and a GST-fusion
protein containing 32 amino acids corresponding to this basic
-helix (GST-NE-dlg-(IV)). GST-NE-dlg-(IV) showed binding ability to
calmodulin-agarose regardless of the presence or absence of
Ca2+ at the NaCl concentration of 150 mM (Fig.
3D, a). However, because this region is highly basic, we
tried to use a more stringent washing condition for evaluating the
interaction. This small
-helical region bound calmodulin-agarose in
a Ca2+-dependent manner when the washing was
performed with 300 mM NaCl instead of the 150 mM NaCl used in the standard reaction (Fig. 3D,
b) This result suggests that the binding ability of the 32 amino
acids
-helical region of NE-dlg to calmodulin is also
Ca2+-dependent.

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Fig. 3.
Detection of interacting site of
NE-dlg/SAP102 with calmodulin by solution binding assay.
A, schematic presentation of association of calmodulin with
GST-NE-dlg/SAP102 deletion mutants. The structure of NE-dlg/SAP102 is
shown at the top. The PDZ domains, the SH3 domain, and the
GUK-like domains are indicated. Four boldface lines below
the model NE-dlg/SAP102 structure represent the portions of
NE-dlg/SAP102 retained in the deletion mutants. The numbers
under the blocks indicate the actual residues retained. The GST region
of the fusion protein is not shown. The column on the right
summarize binding activity to calmodulin as determined by the
experiments depicted in B and D. B,
solution binding assay of GST-NE-dlg deletion mutants with
calmodulin-agarose. Calmodulin-agarose was incubated with each of
GST-NE-dlg deletion mutant protein. Total bound fraction
(B), 25% of unbound fraction (U), and 25% of
input protein (IN) were analyzed by SDS-polyacrylamide gel
electrophoresis, followed by Western blotting with anti-GST monoclonal
antibody. Ca2+(+), 0.1 mM
CaCl2 was supplemented to the binding buffer;
Ca2+(-), 1 mM EGTA was supplemented to
the binding buffer. C, -helical modeling of the region
from the 564th to the 585th amino acids of NE-dlg/SAP102. The basic
residues clustering one side of the helical wheel are boxed.
D, solution binding assay of GST-NE-dlg-(IV) with
calmodulin-agarose. Calmodulin-agarose was incubated with
GST-NE-dlg-(IV), and the total bound fraction was washed with the
binding buffer containing 150 mM NaCl (a) and
300 mM NaCl (b). Total bound fraction
(B), 25% of unbound fraction (U), and 25% of
input protein (IN) were analyzed by SDS-polyacrylamide gel
electrophoresis, followed by Western blotting with anti-GST monoclonal
antibody. Ca2+(+), 0.1 mM
CaCl2 was supplemented to the binding buffer;
Ca2+(-), 1 mM EGTA was supplemented to
the binding buffer.
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We performed surface plasmon resonance measurements in order to
evaluate the specificity of the interaction between NE-dlg/SAP102 and
calmodulin. GST-NE-dlg-(III) produced a dose-dependent
increase of the apparent binding rate (dRU/s) to the
calmodulin-immobilized sensor chip (Fig.
4A). The apparent binding rate
was proportional to the concentration of fusion protein (Fig.
4B). Analysis of the association and dissociation phases of
these sensorgrams yielded an overall dissociation constant,
KD, of 44 nM (ka = 1.57 × 104 M
1
s
1 and kd = 6.94 × 10
4 s
1). Soluble calmodulin was coinjected
with GST-NE-dlg as the competitor against the immobilized calmodulin
(Fig. 5, A and C,
b). The relative binding rate was calculated by dividing the
binding rate in the presence of the competitor by that in the absence
of the competitor. Because the amount of immobilized calmodulin was
77.8 fmol and the volume of the flow cell was 60 nl, the average
concentration of immobilized calmodulin in the flow cell was 1.3 µM. It is noteworthy that the concentration at which the
competitor inhibits 50% of binding, IC50, of soluble
calmodulin (1.6 µM) is very close to the average
concentration of immobilized calmodulin. In contrast, the
IC50 of soybean trypsin inhibitor, which has a molecular
weight and acidity similar to those of calmodulin, was more than
10-fold higher than immobilized calmodulin (Fig. 5, B and
C, a). These results clearly indicate that NE-dlg/SAP102
specifically binds to calmodulin.

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Fig. 4.
Specific binding of calmodulin to
NE-dlg/SAP102. A, the binding of various concentrations
(a, 0.5 µM; b, 0.4 µM; c, 0.3 µM; d, 0.2 µM; e, 0.1 µM) of
GST-NE-dlg-(III) protein and 0.5 µM GST protein
(f) to calmodulin coupled to the sensor chip was monitored
with the use of BIAcore. B, the apparent binding rate
(dRU/s) of NE-dlg to calmodulin was calculated from the
boldface areas of each sensorgram shown in A and
plotted on the graph.
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Fig. 5.
Competitive inhibition of the interaction
between GST-NE-dlg and immobilized calmodulin by soluble
calmodulin. A, sensorgrams of GST-NE-dlg-(III) (0.25 µM) coinjected into the calmodulin-immobilized sensor
chip with soluble calmodulin at the following concentrations:
a, 0 µM; b, 0.25 µM;
c, 0.75 µM; d, 2.5 µM; e, 7.5 µM; f, 25 µM. B, sensorgrams of 0.25 µM
GST-NE-dlg-(III) coinjected to the calmodulin-immobilized sensor chip
with soybean trypsin inhibitor at the following concentrations:
a, 0 µM; b, 0.25 µM;
c, 2.5 µM; d, 25 µM.
C, relative binding rate of NE-dlg to immobilized calmodulin
was suppressed by addition of soluble calmodulin. a, soybean
trypsin inhibitor; b, soluble calmodulin. The apparent
binding rate (dRU/s) was calculated from the bold areas of each
sensorgram shown in A and B. The relative binding
rate was calculated as indicated under "Experimental
Procedures."
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Effect of Calmodulin Binding on Interaction between NE-dlg/SAP102
and (S/T)XV-COOH Motif--
We examined whether the interaction of
calmodulin with NE-dlg/SAP102 affects the binding between PDZ domains
of NE-dlg/SAP102 and the (S/T)XV-COOH sequence by using
surface plasmon resonance measurement. The biotinylated peptide PEP7154
(biotin-NGHVYEKLSSIESDV-COOH) corresponding to the C-terminal region of
rat NR2B was first immobilized on the sensor chip, and GST-NE-dlg-(I)
protein and calmodulin were coinjected into the flow cell. PEP7153
(biotin-NGHVYEKLSSIESD-COOH), which lacks the C terminus valine of
PEP7154, was used for the negative control experiment. GST-NE-dlg-(I)
bound to the immobilized NR2B peptide but not to the peptide lacking
the C-terminal Val residue (data not shown), and the coinjection with
calmodulin did not suppress but, rather, barely increased the binding
rate of GST-NE-dlg-(I) to the NR2B peptide (Fig.
6). This finding suggests that calmodulin
binding does not modulate interaction between NE-dlg and
(S/T)XV-COOH sequence.

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Fig. 6.
Calmodulin did not affect the interaction
between GST-NE-dlg and NR2B peptide. GST-NE-dlg-(I) (25 nM) was coinjected into the NR2B peptide-immobilized sensor
chip with calmodulin at the following concentrations: a, 0 µM; b, 1 µM; c, 3 µM; d, 10 µM. GST (500 nM) (e) was injected into the NR2B
peptide-immobilized sensor chip as a negative control.
|
|
Identification of Interaction between NE-dlg/SAP102 and
PSD-95/SAP90--
To elucidate the biological significance of
NE-dlg-calmodulin interaction in a neuronal cell, we attempted to
identify another cellular protein that binds to the region near the
calmodulin binding site of NE-dlg by two-hybrid screening using a human
brain cDNA library as the prey. Among 1.6 × 104
clones screened, a positive clone contained a 1.7-kilobase pair insert,
the sequence of which was identical to a part of human PSD-95/SAP90 cDNA. The insert encoded the C-terminal
284-amino acid fragment of the PSD-95/SAP90 protein. This region
contains a part of SH3 domain and an entire GUK-like domain. To test
whether calmodulin binding can modulate interaction between
NE-dlg/SAP102 and PSD-95/SAP90, we performed a pull down assay. Whole
cell lysate of COS-7 cells transfected with pCGN-NE-dlg-
GUK were
incubated with either GST or the GST-PSD-95 (C-terminal) bound
GSH-beads. In the presence of both Ca2+ and calmodulin,
NE-dlg-
GUK was strongly associated with PSD-95 (C-terminal) but not
with GST (Fig. 7, lanes 3 and
4). However, specific interaction between NE-dlg-
GUK and
GST-PSD-95 was not observed in the presence of calmodulin alone (Fig.
7, lanes 5 and 6) or Ca2+ alone (Fig.
7, lanes 7 and 8). These results indicate that
NE-dlg/SAP102 specifically interact with PSD-95/SAP90 in the presence
of Ca2+/calmodulin.

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Fig. 7.
Binding of NE-dlg to PSD-95. Lysates of
COS-7 cells transfected with pCGN-NE-dlg- GUK (lanes 3-8)
or parental nontransfected COS-7 cells (lanes 1 and
2) were incubated with either GST (lanes 1, 3, 5, and 7) or GST-PSD-95 (C term) (lanes 2, 4, 6, and 8) immobilized on GSH beads. Bound cellular
proteins were analyzed by Western blotting using anti-hemagglutinin
epitope monoclonal antibody. The binding assay was performed in the
presence of 1 mM CaCl2 (lanes 1-4,
7, and 8) or in the presence of 1 mM EGTA
(lanes 5 and 6) and in the presence (lanes
1-6) or absence (lanes 7 and 8) of
calmodulin.
|
|
 |
DISCUSSION |
In this study, we have shown that NE-dlg/SAP102 and NR2B
colocalize at putative synapses in cultured rat hippocampal neurons and
that their expression are coincident with synapse formation. Because
the expression of PSD-95/SAP90 protein is also known to parallel CNS
synaptogenesis (11), the clustering of neurotransmitter receptors by
multiple MAGUK proteins may play a role in the synaptic architecture
formation. To understand the mechanisms that underlie the clustering
and localization of membrane-associated proteins in neurons, we
performed two-hybrid screening using human brain cDNA library to
identify NE-dlg/SAP102-interacting proteins. Initially, we identified
the interaction between NE-dlg/SAP102 and calmodulin. The in
vitro solution binding assay using calmodulin-agarose showed that
a binding site for calmodulin lies at the basic amino acid-rich region
that flanks the SH3 motif of NE-dlg/SAP102. The surface plasmon
resonance measurement revealed that the interaction between calmodulin
and NE-dlg/SAP102 was specifically inhibited by the addition of soluble
calmodulin in the system.
Although the calmodulin-binding regions of the target proteins have not
shown a rigid consensus sequence, many of them are known to possess a
region that is characterized by a basic amphipathic
-helix
consisting of approximately 20 amino acids (29, 30). The
calmodulin-binding region of NE-dlg/SAP102 also contains basic amino
acids interspersed among hydrophobic residues and is predicted to form
a basic amphipathic
-helix based on the present computational analysis and
-helix modeling. Moreover, the positions of the basic
amino acids in this region are mostly conserved among hdlg-1/SAP97, chapsyn-110/PSD-93, PSD-95/SAP90, and Drosophila DLG-1,
suggesting that calmodulin may be able to interact with all of these
proteins (Fig. 8).

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Fig. 8.
Alignment of the putative calmodulin-binding
domains of MAGUK family proteins. The basic amino acid cluster
regions flanking the SH3 domains of NE-dlg/SAP102, hdlg-1/SAP97,
chapsyn-110/PSD-93, PSD-95/SAP90, and Drosophila DLG-1 were
aligned. The basic amino acids located on one side of the predicted helix are boxed.
|
|
The PDZ domains of NE-dlg/SAP102 are known to interact tightly with
(S/T)XV motifs located on the C terminus of NMDA receptor and APC tumor suppressor protein. The x-ray structure of the third PDZ
domain of hdlg-1/SAP97 alone (31) and PSD-95 binding to a 9-mer peptide
(32) revealed that the C-terminal hydrophobic side chain is buried
deeply in a hydrophobic pocket of the PDZ domain and that the
C-terminal carboxylate interacts with the GLGF motif, which is highly
conserved in most of the PDZ domains. Using a peptide library approach,
Songyang et al. (33) showed that the PDZ domain of dlg or
PSD-95 could selectively bind octapeptides having a consensus sequence
of (S/T)X(V/I) at the C terminus. In the present study, we
have tried to determine whether calmodulin can modulate the interaction
between the PDZ domains and (S/T)XV motif. When calmodulin
and GST-NE-dlg were applied simultaneously to the NR2B-peptide
immobilized sensor chip, the binding rate of GST-NE-dlg was not
affected by the addition of calmodulin. This finding suggests that
calmodulin does not negatively regulate the interaction between
NE-dlg/SAP102 and the (S/T)XV motif.
We have found that another MAGUK protein, PSD-95/SAP90, also
specifically interacts with the region flanking the SH3 motif of NE-dlg
protein in the presence of both Ca2+ and calmodulin. It is
noteworthy that the binding of NE-dlg/SAP102 and PSD-95/SAP90 is not
mediated by their PDZ domains. The two-hybrid screening demonstrated
that SH3 and intervening sequences of NE-dlg/SAP102 is associated with
a part of SH3 and GUK-like domain of PSD-95/SAP90. Because both
NE-dlg/SAP102 and PSD-95/SAP90 can bind to the C terminus of NR2B by
their PDZ domains, our finding suggests that heteromeric complex
formation of MAGUK proteins contributes clustering of NR2B at
postsynaptic membrane.
Ca2+/calmodulin has been reported to bind NMDA receptor
type 1 (NR1) and to disrupt the interaction between
-actinin and NR1 (34, 35). Wyszynski et al. (35) suggested that a
postsynaptic Ca2+ influx, perhaps through activated NMDA
receptors, may lead to a Ca2+-dependent
detachment of NMDA receptors from the actin cytoskeleton and their
redistribution during synaptic activity (35). This Ca2+/calmodulin-dependent detachment can
liberate the NMDA receptor from the anchor of actin filament and
facilitate the clustering of neighboring receptors through the binding
of PSD-95/SAP90 and NE-dlg/SAP102. Taken together, it can be speculated
that a Ca2+ entry from NMDA receptor may modulate
interaction among NE-dlg/SAP102, PSD-95, and NMDA receptors, and the
redistribution of those molecules may consequently play an important
role in the assembly of the synapses (Fig.
9).

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Fig. 9.
Schematic diagram illustrating proposed model
of calmodulin-dependent clustering of NMDA receptors by
heteromeric MAGUK protein complex. Activation of NMDA receptors by
glutamate leads to an entry of Ca2+ ions through the
channel. Binding of Ca2+ to calmodulin allows calmodulin to
interact with NR1, NE-dlg/SAP102, and PSD-95. The binding of
Ca2+/calmodulin to NR1 induces detachment of NMDA receptors
from the actin cytoskeleton and their redistribution (Ref. 35). The
binding of Ca2+/calmodulin to NE-dlg/SAP102 and PSD-95
results in heteromeric complex formation of these MAGUK proteins and
leads to clustering of the NMDA receptors during synaptic
activity.
|
|
There is considerable evidence supporting a functional involvement of
MAGUK proteins, NMDA receptor, and Ca2+ signaling in the
synaptogenesis (36). Mutation of the DLG-1 gene was shown to
disrupt the synaptic morphology of the Drosophila neuromuscular junction (37). Furthermore, the degree of axonal sprouting in cultured young Xenopus neurons is increased by
NMDA receptor blockade, and this effect can be mimicked by chelating Ca2+ (38). This finding suggests that NMDA receptor
activation may suppress inappropriate growth of axons so that they can
proceed with subsequent stages of appropriate synaptogenesis (39, 40). This evidence, together with our findings, indicates that
Ca2+/calmodulin-dependent regulation of NMDA
receptor clustering mediated by the neuronal MAGUK proteins may serve
as an initial step for synaptogenesis.
 |
ACKNOWLEDGEMENTS |
We thank Drs. Y. Sugimoto and K. Suzuki,
Cancer Research Laboratory, Hanno Research Center, Taiho Pharmaceutical
Co., Ltd., for technical advice regarding BIAcore system and T. Arino for secretarial assistance.
 |
FOOTNOTES |
*
This work was supported by a grant for Cancer Research from
the Ministry of Education, Science, Sports and Culture of Japan (to
H. S.).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. Tel.:
81-96-373-5116; Fax: 81-96-373-5120; E-mail:
hsaya{at}gpo.kumamoto-u.ac.jp.
 |
ABBREVIATIONS |
The abbreviations used are:
PSD, postsynaptic
density;
DTT, dithiothreitol;
NMDA, N-methyl-D-aspartate;
NR2B, NMDA receptor 2B;
dlg, Drosophila disc large;
GST, glutathione
S-transferase;
RU, resonance unit;
SH3, src
homology 3;
GUK, guanylate kinase;
MAGUK, membrane-associated GUK;
APC, adenomatous polyposis coli.
 |
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