Synaptic Targeting of PSD-Zip45 (Homer 1c) and Its
Involvement in the Synaptic Accumulation of F-actin*
Shinichi
Usui
§,
Daijiro
Konno
,
Kei
Hori
,
Hisato
Maruoka
,
Shigeo
Okabe¶,
Takashi
Fujikado§,
Yasuo
Tano§, and
Kenji
Sobue
From the Department of
Neuroscience (D13) and
§ Ophthalmology (E7), Osaka University Graduate School of
Medicine, 2-2 Yamadaoka, Suita, Osaka 565-0871 and the
¶ Department of Anatomy and Cell Biology, School of Medicine,
Tokyo Medical and Dental University, Bunkyo-ku,
Tokyo 113-8519, Japan
Received for publication, October 22, 2002, and in revised form, December 30, 2002
 |
ABSTRACT |
PSD-Zip45/Homer1c, which contains an enabled/VASP
homology 1 (EVH1) domain and leucine zipper motifs, is a postsynaptic
density (PSD) scaffold protein that interacts with metabotropic
glutamate receptors and the shank family. We studied the molecular
mechanism underlying the synaptic targeting of PSD-Zip45 in cultured
hippocampal neurons. The EVH1 domain and the extreme C-terminal leucine
zipper motif were molecular determinants for its synaptic targeting. The overexpression of the mutant of the EVH1 domain or deletion of the
extreme C-terminal leucine zipper motif markedly suppressed the
synaptic localization of endogenous shank but not PSD-95 or GKAP. In
contrast, an overexpressed GKAP mutant lacking shank binding activity
had no effect on the synaptic localization of shank. Actin
depolymerization by latrunculin A reduced the synaptic localization of
PSD-Zip45, shank, and F-actin but not of PSD-95 or GKAP. Overexpression
of PSD-Zip45 enhanced the accumulation of synaptic F-actin.
Additionally, overexpression of PSD-Zip45 and an isoform of shank
induced synaptic enlargement in association with the further
accumulation of synaptic F-actin. The EVH1 domain and extreme
C-terminal leucine zipper motif of PSD-Zip45 were also critical for
these events. Thus, these data suggest that the PSD-Zip45-shank and
PSD-95-GKAP complexes form different synaptic compartments, and
PSD-Zip45 alone or PSD-Zip45-shank is involved in the synaptic
accumulation of F-actin.
 |
INTRODUCTION |
As revealed by electron microscopy, the postsynaptic density
(PSD)1 is a dense structure
of the excitatory synapses in the central nervous system (1, 2).
Recently, numerous studies (3-5) have reported on the identification
of PSD scaffold proteins and their involvement in synaptic function. We
isolated PSD-Zip45, a novel member of the Homer family, using a
PSD-specific monoclonal antibody. This protein contains an enabled/VASP
homology 1 (EVH1) domain at its N- and C-terminal leucine zipper motifs
(6, 7). The same protein was independently reported as Homer 1c (8) and
vesl-1L (9). The EVH1 domain interacts with the PPXXF motif found in group 1 metabotropic glutamate receptors (mGluRs) (7, 8, 10),
shank family members (11, 12), inositol triphosphate receptors, and
ryanodine receptors (13). The extreme C-terminal leucine zipper motif
is involved in self-multimerization (7). We recently analyzed the
dynamic behavior of PSD-Zip45 within dendritic spines using time-lapse
confocal microscopy, and we demonstrated its high steady-state turnover
rate and assembly/disassembly dynamics, which depend on
Ca2+ signals from different sources (14).
The first-discovered PSD scaffold protein, PSD-95 (SAP90), interacts
with the NR2 subunits of the NMDA receptor (15-17) and other PSD
scaffold proteins such as GKAP (18, 19), signaling molecules, and cell
adhesion molecules (4, 20, 21). The shank family of proteins, which are
composed of multiple protein-protein interaction domains, were isolated
as GKAP-binding proteins (11) and have also been called ProSAP (22) and
synamon (23). One of the shank family proteins was originally reported
as cortactin-binding protein 1, CortBP1 (24). The PDZ and the SH3
domain of shank family members interact with the extreme C terminus of
GKAP (11) and the
-amino-3-hydroxyl-5-methyl-4-isoxazolepropionate (AMPA) receptor-interacting protein GRIP (25), respectively. The proline-rich region of shank includes binding sites for the Homer proteins (26) and
the actin-binding protein cortactin (11). Tu et al. (26)
demonstrated the macromolecular coupling of the mGluR-Homer and NMDA
receptor-PSD-95-GKAP complexes by shank family members in
vitro. Taking these observations together, Xiao et al.
(27) proposed that shank-dependent protein-protein
interactions in the postsynaptic protein lattice link together
different classes of glutamate receptors and further couple them to the
actin cytoskeleton and the Ca2+ sequestering machinery.
However, these protein-protein interactions were identified in a
piecemeal fashion in vitro. The molecular organization and
dynamic interactions of a postsynaptic protein lattice in
vivo therefore remain largely unknown. Here we focused on the
molecular mechanism underlying the synaptic targeting of PSD-Zip45 and
its effect on other PSD scaffold proteins. We demonstrated that both
the EVH1 domain and C-terminal leucine zipper motif of PSD-Zip45 are
critical for its synaptic targeting and that PSD-Zip45/shank and
PSD-95/GKAP form different compartments in the synapse. In addition,
the synaptic localization of shank is PSD-Zip45-dependent,
and their interaction is involved in the enhanced accumulation of
synaptic F-actin, and overexpressing these molecules induces synaptic
enlargement in association with the further accumulation of synaptic
F-actin.
 |
EXPERIMENTAL PROCEDURES |
Antibodies--
Polyclonal antibodies against GKAP and shank
were produced as follows: the cDNAs encoding amino acid residues
367-694 of GKAP clone2-2A (19) and 826-1259 of CortBP1 (22), an
isoform of the shank family proteins, were amplified by PCR and
subcloned into the GST fusion vector pGEX6P1 (Amersham Biosciences).
GST-fused GKAP and CortBP1 fragments expressed in Escherichia
coli BL21 were isolated using glutathione-conjugated Sepharose 4B
(Amersham Biosciences) and used to immunize New Zealand White rabbits.
The antisera against the GST-fused GKAP fragment were exhaustively preabsorbed with GST protein, followed by purification using a GST-GKAP
fragment-coupled Sepharose 4B gel matrix. The antibody against
GST-fused CortBP1 fragment was purified by the same procedure. The
antibody against CortBP1 fragment (Fig. 2B) thus obtained specifically cross-reacted with all the isoforms of the shank family
and was used as an anti-shank antibody. Rabbit polyclonal and mouse
monoclonal antibodies against GFP (Molecular Probes, Eugene, OR) and
other antibodies (anti-PSD-Zip45, anti-PSD-95, anti-synaptophysin,
anti-FLAG, and anti-GST antibodies) are described elsewhere (7). These
antibodies, a rabbit polyclonal antibody against inositol triphosphate
receptors (Oncogene Research Products, Boston, MA) and mouse monoclonal
antibodies against synaptophysin (Sigma) and GABAA receptor
(Upstate Biotechnology, Inc., Lake Placid, NY), were used for
immunocytochemistry and/or immunoblotting.
Plasmid Construction--
All constructs were amplified by PCR
and subcloned into the GST fusion vector pGEX-6P1 (Amersham
Biosciences) or the mammalian expression vector pEGFP
(Clontech, Palo Alto, CA), pcDNA3.1(+)-FLAG (modified from Invitrogen), or pCAGGS-GFP modified from pCAGGS (28).
Each construct was tagged with GFP or FLAG at the N terminus, as
indicated. A series of PSD-Zip45 constructs (PSD-Zip45WT,
PSD-Zip45
CC
ZipA, and PSD-Zip45
ZipB) was described previously
(7), and other PSD-Zip45 variants (PSD-Zip45G89A, PSD-Zip45Nterminus,
PSD-Zip45Cterminus, and PSD-Zip45NterminusG89A) were newly designed.
PSD-Zip45-Nterminus (residues 1-175) and PSD-Zip45Cterminus (residues
176-366) were amplified by PCR. A glycine-to-alanine point mutation
was introduced into PSD-Zip45WT and PSD-Zip45Nterminus at residue 89, yielding the mutants PSD-Zip45G89A and PSD-Zip45NterminusG89A. Each
member in a series of PSD-Zip45 variants was subcloned into the pEGFP vector to yield the following constructs: GFP-Zip45WT, GFP-Zip45G89A, GFP-Zip45Nterminus, GFP-Zip45Cterminus, GFP-Zip45
CC
ZipA, and GFP-Zip45
ZipB. PSD-Zip45WT, PSD-Zip45G89A, and PSD-Zip45
ZipB were
also subcloned into the pcDNA3.1(+)-FLAG vector to yield FLAG-Zip45WT,
FLAG-ZipG89A, and FLAG-Zip45
ZipB. PSD-Zip45Nterminus and
PSD-Zip45NterminusG89A were subcloned into the pGEX-6P1 vector to yield
GST-Zip45Nterminus and GST-Zip45NterminusG89A and were used for ligand
overlay assays (Fig. 2B). The following GKAP and CortBP1
constructs were also designed for this study. Full-length GKAP
clone2-2A was constructed and named GKAP-WT, and a leucine-to-alanine point mutant at residue 694, GKAP-L694A, was constructed by
site-directed mutagenesis. Each GKAP variant was subcloned into the
pEGFP vector to yield GFP-GKAP-WT and GFP-GKAP-L694A. Full-length
CortBP1 was constructed and subcloned into the pCAGGS-GFP vector and
named GFP-CortBP1.
Cell Cultures--
Hippocampal neurons were prepared from
embryonic rat brains at 18 days of gestation (E18). The hippocampi were
dispersed with 0.25% trypsin in Hanks' balanced salt solution, and
the cell suspension was plated at a density of 10,000-15,000
cells/cm2 onto cover glasses (Matsunami, Osaka, Japan)
coated with 1 mg/ml poly-L-lysine. Neurons were cultured in
neurobasal medium (Invitrogen) containing 2% B27 supplement
(Invitrogen) and 0.5 mM L-glutamine. One-half
of the medium was changed once a week. Cultured neurons were used for
microinjection or immunocytochemistry 16-21 days after plating.
Microinjection of Plasmid DNAs--
By using a micromanipulator
(Narishige, Tokyo, Japan), plasmid DNAs (low expression, 10 ng/µl;
overexpression, 100 ng/µl) were microinjected through glass
capillaries into the nuclei of rat hippocampal neurons. After 8-24 h,
the neurons were fixed for immunocytochemistry as described below.
Actin Depolymerization--
Latrunculin A (Molecular Probes) was
added directly to the culture medium from a concentrated
Me2SO stock solution to a final concentration of 5 µM and incubated with cultured hippocampal neurons for
24 h.
Immunocytochemistry--
Cultured hippocampal neurons were
simultaneously fixed and permeabilized in methanol for 15 min at
20 °C and then treated with blocking solution (10% normal goat
serum, 0.2% BSA, and 0.1% Triton X-100 in PBS) for 1 h at
37 °C. In the following experiments, all procedures were carried out
at 37 °C. The cultured neurons were then incubated with primary
antibodies in the same blocking solution for 2 or 3 h. After
incubation, the epitopes in the neurons were incubated with Alexa
FluorTM 488- and/or 546-conjugated secondary antibodies (2 µg/ml, Molecular Probes) in the blocking buffer for 40 min. For
triple labeling, the neurons were fixed with 4% paraformaldehyde and
4% sucrose in PBS for 20 min, followed by permeabilization with 0.25%
Triton X-100 in PBS for 5 min, treatment with the blocking solution
(10% BSA) for 30 min, and incubation with primary antibodies in 3% BSA for 3 h. After labeling with the primary antibodies, the
neurons were further incubated with Alexa FluorTM 350- and
488-conjugated secondary antibodies (2 µg/ml, Molecular Probes) and
Alexa FluorTM 568 phalloidin (2 µg/ml, Molecular Probes)
in 3% BSA for 40 min. Primary antibodies used for immunocytochemical
studies were as follows: anti-PSD-Zip45 (1:150), -PSD-95 (1:400), -GKAP
(1:400), -shank (1:2,000), -synaptophysin (1:400), -GABAAR
(1:200), -GFP (1:1,000), and -FLAG (1:10,000) antibodies. After being
washed with PBS, the cover glasses were mounted for observation.
Fluorescence images were acquired using a cooled CCD camera (Roper
Scientific, Tucson, AZ) mounted on an Olympus IX-70 microscope.
Quantification--
Quantitative measurements were performed
blind. Morphometric studies were performed using Metamorph imaging
software. The synapse/dendrite fluorescence intensities (Fig.
2D and Fig. 4B, a) were analyzed as
the ratios of the average fluorescence intensities of the indicated
areas in GFP-labeled synapses to those in the adjacent dendrites. For
analyses of the normalized fluorescence intensities (Fig.
3B, Fig. 4B, b, and Fig.
6B), the fluorescence intensities of labeled synaptic areas
in transfected neurons were randomly assessed and compared with those
in untransfected neurons. This was measured as the mean fluorescence
intensity normalized to the fluorescence intensity in untransfected
cells. In the actin depolymerization assays (Fig. 5B), the
number of clusters that was positive for the indicated protein
(PSD-Zip45, shank or GKAP) as well as for PSD-95, with or without
latrunculin A treatment, was counted, and the ratios of the number of
clusters positive for each protein to the number of PSD-95-positive
clusters were calculated. To determine the cluster sizes of PSD-95 and
GKAP with or without latrunculin A treatment, the areas of labeled clusters were measured. The data were exported to Microsoft Excel software. Statistical significance was determined using Student's t test (Fig. 5B) or one-way analysis of variance
with Bonferroni correction for multiple comparison (Fig.
6B).
Ligand Overlay Assays--
For the ligand overlay assay of
PSD-Zip45, the PSD fraction was prepared from the forebrain of
7-week-old Sprague-Dawley rats by the method of Wu et al.
(29). Twenty micrograms of each protein sample per lane were loaded
onto SDS-polyacrylamide gels and transferred to a nitrocellulose
membrane. GST, GST-Zip45Nterminus, or GST-Zip45NterminusG89A was used
as the ligand, and ligands bound to the membrane were detected by an
anti-GST antibody. For Western blot analysis, an anti-shank antibody
was used as the primary antibody and visualized using
peroxidase-conjugated secondary antibodies, followed by ECL (Amersham Biosciences).
 |
RESULTS |
Molecular Determinants of PSD-Zip45 for Synaptic
Targeting--
Previous immunocytochemical and immunoelectron
microscopic studies demonstrated the postsynaptic localization of NMDA
receptors and PSD scaffold proteins such as PSD-Zip45, shank, PSD-95,
and GKAP in excitatory synapses (7, 8, 11, 26, 30). To reveal the
molecular mechanism underlying the synaptic targeting of PSD-Zip45, a
small amount (10 ng/µl) of expression plasmids carrying GFP- or
FLAG-tagged wild-type PSD-Zip45 (GFP-Zip45WT or FLAG-Zip45WT) was
microinjected into the nuclei of cultured hippocampal neurons, and
their expression was localized by GFP or FLAG staining (Fig.
1, b and c).
GFP-labeled clusters along the dendrites were detectable within 8 h after microinjection. Double labeling for GFP-Zip45WT and endogenous
PSD scaffold proteins showed that the GFP immunoclusters along the
dendrites overlapped greatly with the PSD-95, GKAP, and shank clusters
(Fig. 1, d3-f3) but not with clusters of an inhibitory
postsynaptic molecule, GABAA receptor (Fig. 1,
h3). The GFP immunoclusters partially overlapped with
clusters of synaptophysin, a presynaptic molecular marker (Fig. 1,
g3). The same results were obtained using FLAG-Zip45WT (Fig.
1c and data not shown). Thus, these results indicate that exogenous PSD-Zip45WT proteins are precisely targeted to postsynaptic sites of excitatory synapses in a manner that is indistinguishable from
the endogenous protein.

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Fig. 1.
Immunocytochemical localization of
PSD-Zip45 and synaptic proteins in cultured hippocampal neurons.
Endogenous PSD-Zip45, GFP-Zip45WT, or FLAG-Zip45WT were localized using
anti-PSD-Zip45 (a), -GFP (b), or -FLAG
(c) antibodies, respectively. GFP-Zip45WT and synaptic
proteins were double-labeled for GFP (d1-h1) and PSD-95
(d2), GKAP (e2), shank (f2),
synaptophysin (g2), or GABAA receptor
(h2), respectively. The right panels
(d3-h3) show a merged image of the left and
middle panels. Scale bars, 10 µm.
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To analyze the molecular determinants of PSD-Zip45 that are required
for synaptic targeting, we generated a series of expression plasmids
bearing GFP-tagged PSD-Zip45 variants (Fig.
2A) and microinjected small
amounts (10 ng/µl) of them into hippocampal neurons. After they were
fixed, the neurons were double-labeled with anti-GFP and synaptophysin
antibodies (Fig. 2C, a1-a3). In the following experiments, synaptophysin was used to determine the orientation of the
synapse. PSD-Zip45 is composed of an N-terminal EVH1 domain and a
C-terminal domain containing a coiled-coil structure and two leucine
zipper motifs (ZipA and ZipB in Fig. 2A and in Refs. 6 and
7). We prepared four deletion constructs, lacking the EVH1 domain, the
coiled-coil structure, ZipA and/or ZipB, and a mutant of the EVH1
domain (Fig. 2A). An overlay binding assay showed that a
short form of PSD-Zip45WT, GST-Zip45Nterminus, bound to all the
isoforms of the shank family in the PSD fraction, whereas a point
mutation of the GST-Zip45Nterminus, GST-Zip45NterminusG89A, lost
the shank binding activity, indicating that the EVH1 domain of
PSD-Zip45 is the binding site for the shank family members (Fig.
2B).

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Fig. 2.
Molecular determinants of PSD-Zip45 for
synaptic targeting. A, schematic diagram of the
PSD-Zip45 variants used in this study. Each member of a series of
PSD-Zip45 variants was tagged with GFP at the N terminus. GFP-Zip45G89A
carried a point mutation in the EVH1 domain, which lacked the shank
binding activity. B, ligand overlay assays using PSD-Zip45
variants. The PSD fraction prepared from the rat forebrain was loaded
on a gel. GST alone, the GST-fused N terminus of PSD-Zip45 containing
the EVH1 domain (GST-Zip45Nterminus), and its mutant
(GST-Zip45NterminusG89A) were used as ligands. After ligand binding,
the ligands bound to the targets were detected with an anti-GST
antibody ( -GST). These results were compared with Western
blotting data using the anti-shank antibody ( -shank).
IB, immunoblotting. C, localization of exogenous
GFP-tagged PSD-Zip45 variants in hippocampal neurons. Expression
plasmids (10 ng/µl) carrying a series of GFP-tagged PSD-Zip45
variants as shown in A and GFP alone were microinjected into
cultured hippocampal neurons. The expressed proteins were
double-labeled with anti-GFP and synaptophysin antibodies. GFP-labeled
images are shown as follows: GFP-Zip45WT (a), GFP alone
(b), GFP-Zip45G89A (c), GFP-Zip45Nterminus
(d), GFP-Zip45Cterminus (e),
GFP-Zip45 CC ZipA (f), and GFP-Zip45 ZipB
(g). Synaptophysin was used to determine the orientation of
synapses. a1-a3 show a representative example.
Immunoclusters for GFP-Zip45WT (a1) and synaptophysin
(a2) were partially overlapped in the merged image
(a3), as shown enlarged in the boxed region
(a). Scale bars, 10 µm. D, summary
of the synaptic targeting intensities of the PSD-Zip45 variants. The
synaptic targeting intensities of the PSD-Zip45 variants as shown in
C are expressed as synapse/dendrite fluorescence
intensities. Four to six independent experiments were performed for
each construct, and 600 synapses from 10 to 20 neurons were randomly
selected.
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GFP alone was distributed diffusely through the dendrites
to their spines (Fig. 2C, b). Among the six
constructs examined, GFP-Zip45WT and GFP-Zip45
CC
ZipA formed very
bright clusters along the dendrites (Fig. 2C, a
and f), and almost all of these clusters partially
overlapped with the synaptophysin-positive clusters (Fig.
2C, a1-a3), suggesting a postsynaptic
localization of GFP-Zip45WT and GFP-Zip45
CC
ZipA. In contrast, the
GFP-Zip45Nterminus, -Cterminus, and
ZipB derivatives were
distributed diffusely (Fig. 2C, c-e and
g). To quantify the extent of synaptic targeting (synaptic targeting intensity), we measured the immunofluorescence intensities of
the indicated areas that were positive for the GFP-labeled PSD-Zip45
variants in the synapses and of the dendrites immediately adjacent to
the synapses (31) as summarized in Fig. 2D. These results
indicate that the EVH1 domain and the extreme C-terminal leucine zipper
motif (ZipB) of PSD-Zip45 are critical determinants for the synaptic targeting.
Effect of Overexpressed PSD-Zip45 Variants on the Synaptic
Localization of Other PSD Scaffold Proteins--
To test whether
PSD-Zip45 may play a role in the synaptic localization of other PSD
scaffold proteins, we microinjected excess amounts (100 ng/µl) of
expression plasmids carrying GFP-Zip45WT, GFP-Zip45G89A, and
GFP-Zip45
ZipB into hippocampal neurons. After they were fixed, the
neurons were double-labeled with anti-GFP and anti-PSD-95, GKAP, or
shank antibodies (Fig. 3A). The synaptic localization of
exogenously expressed proteins was determined by the orientation of the
labeled presynaptic molecule, synaptophysin, in advance (data not
shown). When GFP-Zip45WT was overexpressed, endogenous shank, PSD-95,
and GKAP clusters in the synapses were exactly co-localized with
expressed GFP-Zip45WT clusters (Fig. 3A, a3,
d3, and g3). Notably, when GFP-Zip45G89A or
GFP-Zip45
ZipB was overexpressed, the immunofluorescence intensity,
but not the number or size, of shank clusters markedly decreased (Fig.
3A, b2 and c2). In turn, overexpressed
GFP-Zip45WT, GFP-Zip45G89A, and GFP-Zip45
ZipB had no discernible
effects on the intensity, number, and size of endogenous PSD-95 and
GKAP clusters (Fig. 3A, d2-i2). The effects of
overexpressed PSD-Zip45 variants on the synaptic localization of
endogenous PSD scaffold proteins were also quantified by the ratios of
the immunofluorescence intensities of labeled synaptic areas in the
transfected neurons to those in untransfected neurons (Fig.
3B). Thus, overexpressed GFP-Zip45G89A and GFP-Zip45
ZipB
markedly and specifically suppressed the synaptic localization of shank
in a dominant-negative fashion.

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Fig. 3.
Overexpressed GFP-Zip45G89A and
GFP-Zip45 ZipB suppressed the synaptic
localization of shank, but not of PSD-95 or GKAP, in cultured
hippocampal neurons. Effects of overexpressed PSD-Zip45 variants
on the synaptic localization of shank, PSD-95, and GKAP (A).
Excess amounts of plasmids (100 ng/µl) carrying GFP-Zip45WT,
GFP-Zip45G89A, or GFP-Zip45 ZipB were microinjected into hippocampal
neurons. Each neuron was double-labeled for GFP (a1-i1) and
shank (a2-c2), PSD-95 (d2-f2), or GKAP
(g2-i2). GFP-labeled images are as follows: GFP-Zip45WT
(a1, d1, and g1), GFP-Zip45G89A
(b1, e1, and h1), and GFP-Zip45 ZipB
(c1, f1, and i1). The right
panels show merged images from the left and
middle panels (a3-i3). Enlargements of the
boxed regions are shown in the respective lower panels
(a1'-i1', a2'-i2', and a3'-i3').
Arrows indicate the synapses in transfected neurons, and
arrowheads show the synapses in untransfected neurons.
Scale bars, 10 µm. Changes in the synaptic localization of
the PSD scaffold proteins induced by the overexpression of PSD-Zip45
variants (B). Six to ten independent experiments were
performed for each construct, and 800 synapses from 10 to 20 neurons
were randomly selected. The histogram shows the mean
fluorescence intensity normalized to the fluorescence intensities of
clusters in untransfected neurons stained with the antibodies against
the indicated proteins.
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GKAP and the Shank Family Are Independently Localized to Synaptic
Sites--
The extreme C-terminal sequence (-QTAL) of GKAP has been
reported to interact with the PDZ domain of shank in vitro,
and their interaction is believed to be involved in the synaptic
localization of shank (11). However, the overexpression analysis shown
in Fig. 3 suggests that the synaptic localization of PSD-Zip45 and shank may not be directly linked to that of GKAP and PSD-95. To test
whether GKAP affects the synaptic localization of shank, we analyzed
the synaptic targeting of GFP-tagged wild-type GKAP (GFP-GKAP-WT) and
its C-terminal mutant (GFP-GKAP-L694A) that lacked the shank binding
activity (11). A small amount (10 ng/µl) of expression plasmids
carrying GFP-GKAP-WT or GFP-GKAP-L694A was microinjected into cultured
hippocampal neurons. The orientation of the expressed proteins was also
determined by the presynaptic localization of synaptophysin, as
demonstrated in Fig. 2. Like endogenous GKAP, GFP-GKAP-WT and
GFP-GKAP-L694A were specifically targeted to the synapses (Fig.
4A, a-c). The
synaptic targeting intensities of the GFP-GKAP variants were quantified
by the same method as described in Fig. 2D (Fig.
4B, a). The synaptic localization of the
endogenous shank was unaffected by overexpressed GFP-GKAP-WT or
GFP-GKAP-L694A (Fig. 4A, d2 and e2,
and B, b). These results suggest that GKAP and
shank are independently localized to the synaptic sites.

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Fig. 4.
Synaptic targeting of GKAP is independent of
shank. A, immunolocalization of GKAP variants and
shank. Endogenous GKAP was immunolabeled with an anti-GKAP antibody
(a). A small amount (10 ng/µl) of expression plasmids
carrying GFP-tagged wild-type GKAP (GFP-GKAP-WT) or its mutant
(GFP-GKAP-L694A) was microinjected into cultured hippocampal neurons.
The expressed GFP-GKAPWT (b) and GFP-GKAP-L694A
(c) were immunolabeled with an anti-GFP antibody. Neurons
overexpressing GFP-GKAP-WT (d1) or GFP-GKAP-L694A
(e1) were double-labeled with anti-GFP and shank antibodies
(d2 and e2). The right panels
(d3 and e3) show merged images from the
left (d1 and e1) and middle
(d2 and e2) panels. Enlargements of
the boxed regions are shown in the respective lower
panels (a'-e'). Arrows indicate the
synapses in transfected neurons, and arrowheads show the
synapses in untransfected neurons. Scale bars, 10 µm.
B, summary of the synaptic targeting of GKAP variants and
changes in the synaptic localization of shank induced by the
overexpression of GKAP variants. The synaptic targeting intensities of
the GKAP variants are expressed as the synapse/dendrite fluorescence
intensities as described in Fig. 2D. Four independent
experiments were performed for each construct, and 400 synapses from 10 neurons were randomly selected (B, a). Changes
in the synaptic localization of shank induced by the overexpression
of GFP-GKAP-WT or GFP-GKAP-L694A are expressed by the ratios of the
fluorescence intensities of shank clusters in transfected neurons to
the fluorescence intensities in untransfected neurons. The
histogram shows the mean fluorescence intensity normalized
to the intensities of the shank clusters in untransfected neurons. Four
independent experiments were performed for each construct, and 400 synapses from 10 neurons were randomly selected (B,
b).
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Involvement of the Actin Cytoskeleton in the Synaptic Localization
of PSD-Zip45 and Shank--
As described in the Introduction, some PSD
scaffold proteins and F-actin co-localize within the dendritic spines
(32). In addition, the proline-rich region of CortBP1 and other shank
family members directly interacts with an actin-binding protein,
cortactin (11, 24). The actin cytoskeleton is also involved in the
dynamic properties of dendritic spines (33, 34) and the functional regulation of glutamate receptors in postsynaptic sites (35). To test
the linkage between the actin cytoskeleton and four PSD scaffold
proteins in postsynaptic sites, we compared their synaptic localization
in neurons with or without the actin-depolymerizing reagent latrunculin
A. This drug stoichiometrically binds to the actin monomer, resulting
in the sequestration of G-actin by the depolymerization of actin
filaments. Triple staining of F-actin, PSD-Zip45, and shank in
untreated neurons demonstrated the co-localization of these proteins
within the synapse (data not shown). Allison et al. (32)
reported that treatment with 5 µM latrunculin A for
24 h depolymerizes most of the F-actin in hippocampal neurons. Consistent with our previous report (14), the same treatment markedly
reduced, but did not completely eliminate, the number of F-actin
clusters along the dendrites. This may be because of the limitation of
this treatment or different latrunculin A resistance of actin filaments
with or without actin-binding proteins. In association with this
reduction, the numbers of PSD-Zip45- and shank-positive clusters
decreased to ~40% (Fig.
5A). In both cases, the
latrunculin A-resistant F-actin clusters were co-localized with the
PSD-Zip45- and shank-positive clusters (Fig. 5A,
b1-b3). On the other hand, the synaptic localization of the
PSD-95- and GKAP-positive clusters was not affected by the latrunculin
A treatment, except for a slight reduction in cluster size (in
untreated neurons, PSD-95 clusters (0.93 ± 0.37 µm2) and GKAP clusters (0.84 ± 0.39 µm2); in latrunculin A-treated neurons, PSD-95 clusters
(0.59 ± 0.11 µm2) and GKAP clusters (0.57 ± 0.09 µm2), respectively) (Fig. 5A,
h1 and h2). These results were obtained by
double-labeling with anti-PSD-95 and anti-PSD-Zip45, shank, or GKAP
antibodies (Fig. 5A) and then quantifying the labeled clusters based on the number of PSD-95 clusters (Fig. 5B).
Thus, our data suggest that the organization of the actin cytoskeleton is closely linked to the synaptic localization of PSD-Zip45 and/or the
shank family but not of PSD-95 or GKAP.

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Fig. 5.
Changes in the synaptic localization of PSD
scaffold proteins induced by depolymerization of F-actin in cultured
hippocampal neurons. A, immunolocalization of four PSD
scaffold proteins with or without latrunculin A treatment. Neurons
treated with 5 µM latrunculin A for 24 h
(b1-b3) or untreated (a1-a3) were
triple-labeled for F-actin (a1 and b1), PSD-Zip45
(a2 and b2), and shank (a3 and
b3). After treatment with latrunculin A
(f1, f2, g1,
g2, h1, and h2) or without treatment
(c1, c2, d1, d2,
e1, and e2), the neurons were double-labeled for
PSD-95 (c1-h1) and PSD-Zip45 (c2 and
f2), shank (d2 and g2), or GKAP
(e2 and h2). Arrows indicate
PSD-95-positive and PSD-Zip45- or shank-positive clusters, and
arrowheads show the PSD-95-positive and PSD-Zip45- or
shank-negative clusters, respectively. Each channel is shown in gray
scale. Scale bars, 5 µm. The ratios of the numbers of
PSD-Zip45-, shank-, or GKAP-positive clusters to the numbers of
PSD-95-positive clusters with or without latrunculin A treatment are
summarized (B). Data are derived from the analysis of 600 PSD-95-positive clusters from 20 neurons (*, p > 0.05;
**, p < 0.001).
|
|
To analyze further the relationship between PSD-Zip45 and/or shank and
the actin cytoskeleton, we compared the effects of PSD-Zip45 variants
with or without overexpressed shank on the synaptic F-actin (Fig.
6A). In our preliminary
experiments, we also confirmed that almost all of actin clusters along
the dendrites were localized in the synapses as defined by the
synaptophysin labelings (data not shown). Overexpressed GFP-Zip45
variants, endogenous shank, and F-actin were triple-labeled with
anti-GFP and shank antibodies and phalloidin, respectively.
Overexpressed GFP-Zip45WT enhanced the synaptic localization of
F-actin, whereas overexpressed GFP-Zip45G89A and GFP-Zip45
ZipB did
not affect the synaptic F-actin (Fig. 6A,
a3-c3). Consistent with the results of Fig. 3, GFP-Zip45G89A and
GFP-Zip45
ZipB, but not GFP-Zip45WT, markedly suppressed the synaptic
localization of shank (Fig. 6A, a2-c2). Taken
together, these results indicate that an increased PSD-Zip45 level, in
the presence of shank, enhances the accumulation of synaptic F-actin,
an activity in which the EVH1 domain and the extreme C-terminal leucine
zipper motif of PSD-Zip45 are critically important.

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Fig. 6.
Enhancement of the synaptic accumulation of
F-actin by overexpression of PSD-Zip45 variants with or without the
co-expression of shank (GFP-CortBP1) in cultured hippocampal
neurons. A, the synaptic localization of shank and
F-actin induced by the overexpression of PSD-Zip45 variants with or
without shank. Neurons overexpressing GFP-Zip45WT (a1),
GFP-Zip45G89A (b1), or GFP-Zip45 ZipB (c1)
alone were triple-labeled for GFP (a1-c1), shank
(a2-c2), and F-actin (a3-c3) in cultured
hippocampal neurons. Neurons overexpressing FLAG-Zip45WT,
FLAG-Zip45G89A, or FLAG-Zip45 ZipB and GFP-CortBP1 were
triple-labeled for FLAG (d1-f1), GFP
(d2-f2), and F-actin (d3-f3).
Enlargements of the boxed regions are shown in the respective
lower panels (a1'-f1'). Each channel is shown in
gray scale. Scale bars, 10 µm. B, summary of
changes in the ratio of the fluorescence intensities of synaptic
F-actin in transfected neurons to the fluorescence intensities in
untransfected neurons as shown in A. Four to six independent
experiments were performed for each construct, and 400 synapses from 8 to 10 neurons were randomly selected. The histogram shows
the mean fluorescence intensities normalized to the fluorescence
intensities of F-actin-positive clusters in untransfected neurons (*,
p < 0.001).
|
|
Sala et al. (36) recently observed that overexpression of
both shank 1B and Homer 1b induces synaptic enlargement in association with an increase in the synaptic F-actin. To determine the domain of
PSD-Zip45 that was critical for the synaptic enlargement and the
increased localization of synaptic F-actin, we co-transfected PSD-Zip45
variants (FLAG-Zip45WT, FLAG-Zip45G89A, or FLAG-Zip45
ZipB) and
wild-type CortBP1 (GFP-CortBP1; shank family) in hippocampal neurons.
FLAG-Zip45WT and GFP-CortBP1, but not FLAG-Zip45G89A or
FLAG-Zip45
ZipB, were precisely targeted to the synapses (Fig. 1c and data not shown). Both overexpressed FLAG-Zip45 and
GFP-CortBP1 were highly concentrated in the enlarged synapses and
co-localized with F-actin (Fig. 6A, d1-d3). On
the other hand, in neurons that were co-transfected with FLAG-Zip45G89A
or FLAG-Zip45
ZipB and GFP-CortBP1, there were less significant
changes in the synaptic size and fluorescence intensity of F-actin
(Fig. 6A, e3 and f3). The F-actin
labeling in the synapse was quantified as demonstrated in Fig.
4C. Thus, these results indicate that both the EVH1 domain and the extreme C-terminal leucine zipper motif of PSD-Zip45 are critical for the PSD-Zip45/shank-dependent synaptic
enlargement in association with the enhanced accumulation of synaptic
F-actin, and that the synaptic targeting of PSD-Zip45 is prerequisite
for these events.
 |
DISCUSSION |
Recent studies (31, 37) have demonstrated the synaptic targeting
of some PSD scaffold proteins. The first example was the synaptic
targeting of PSD-95, in which the N-terminal palmitoylation, the first
two PDZ domains, and/or a C-terminal targeting motif are critical.
Naisbitt et al. (11) reported that the overexpression of
GKAP variant, which lacks the PDZ-binding sequence, caused a marked
reduction in the levels of the endogenous shank family members in the
synapse. Sala et al. (36) also identified the molecular
determinants of the shank family required for their synaptic targeting
as their PDZ domain and the flanking sequences and concluded that an
interaction between the PDZ domain of shank 1B and the C terminus of
GKAP was important for the synaptic localization of shank 1B. Ango
et al. (38) analyzed the distribution of exogenous type 5 mGluR (mGluR5) and Homer proteins using cerebellar granule cells in
which mGluR5 and Homer 1 proteins were absent, and their findings
suggested that the synaptic targeting of mGluR5 is Homer protein-dependent. However, they did not address the
molecular determinants of the Homer proteins. We recently reported (14) that PSD-Zip45 that is exogenously expressed in hippocampal neurons is
targeted to the synapses and that its targeting is dynamically regulated by Ca2+ influxes from different sources. Here, we
studied the molecular determinants required for the synaptic targeting
of PSD-Zip45 and the effects of PSD-Zip45 variants on the synaptic
localization of other PSD scaffold proteins and F-actin.
Synaptic Targeting of PSD-Zip45 and Compartmentalization of
Major PSD Scaffold Proteins--
Exogenous PSD-Zip45 showed a synaptic
localization with other major PSD scaffold proteins, including PSD-95,
GKAP, and shank (Fig. 1). We identified both the EVH1 domain and the
extreme C-terminal leucine zipper motif of PSD-Zip45 as the molecular
determinants for its synaptic targeting (Fig. 2). Furthermore,
overexpression of PSD-Zip45G89A or PSD-Zip45
ZipB markedly suppressed
the synaptic localization of endogenous shank, but not of PSD-95 or
GKAP, in a dominant-negative fashion (Fig. 3). These results suggest
that the synaptic localization of PSD-Zip45 is a prerequisite for that of shank, and both of PSD-Zip45G89A and PSD-Zip45
ZipB cannot recruit
shank to the synapses because neither of them is targeted to the
synapses. It has been reported that GKAP interacts with the shank
family via its PDZ domain and that overexpressed GKAP-L694A, which does
not bind shank, suppresses the synaptic localization of shank (11). In
contrast, we demonstrated here that whereas GKAP-WT and GKAP-L694A were
targeted precisely to the synapses, overexpressed GKAP-WT and
GKAP-L694A did not affect the synaptic localization of endogenous shank
(Fig. 4). Thus, there is a discrepant result regarding the interaction
between GKAP and shank within the synapses. The molecular length of
GKAP, clone2-2A, used by us is longer than that by Naisbitt et
al. (11). We also constructed GKAP-L694A, a mutant of clone2-2A,
that lacked the shank binding activity. Naisbitt et al. (11)
used a C-terminal deleted variant of GKAP lacking the shank binding
activity. Furthermore, we introduced the plasmids into cultured neurons
by the microinjection method, whereas Naisbitt et al. (11)
used the transfection method with calcium phosphate. However, it
remains unknown whether these different constructs and expression
methods might produce the above discrepant result. Our present results
indicate that the synaptic localization of shank depends on that of
PSD-Zip45 but not of GKAP. It might be inferred from these data that
PSD-Zip45-shank and PSD-95-GKAP interactions form different
compartments in the synapses. The currently accepted model for a
postsynaptic protein lattice is that the shank family members provide
molecular bridges between NMDA receptor-PSD-95-GKAP, mGluR-PSD-Zip45,
and
-amino-3-hydroxyl-5-methyl-4-isoxazolepropionate (AMPA)
receptor-GRIP complexes, the actin cytoskeleton, and the Ca2+-sequestering machinery (25, 27, 39). Almost all of
these interactions, however, have been demonstrated by in
vitro assays. We reported previously (14) that although PSD-95
clusters in the synapses of living hippocampal neurons are less
dynamic, PSD-Zip45 shows a highly dynamic and rapid redistribution that
is regulated by Ca2+ influxes from different
Ca2+ sources. Combining our results, we suggest that
PSD-95-GKAP and PSD-Zip45-shank complexes form different compartments
in the synapse with respect to their interactions and dynamics.
Linkages between PSD-Zip45, Shank, and the Actin Cytoskeleton in
the Synapse--
Because the shank family interacts in
vitro with cortactin through a cortactin-binding motif in the
proline-rich region of the shank proteins (11, 24), it has
been postulated that the actin cytoskeleton is involved in the synaptic
localization of PSD-Zip45 and shank. In support of this idea, our
recent studies (14) suggest that the synaptic localization of PSD-Zip45
is linked to the polymerization/depolymerization dynamics of the actin
cytoskeleton. In this paper, we further compared the linkages between
four PSD scaffold proteins and the actin cytoskeleton. Treating
hippocampal neurons with the actin-depolymerizing reagent latrunculin A
caused a decrease in the synaptic localization of F-actin, PSD-Zip45,
and shank but not of PSD-95 or GKAP (Fig. 5). These results suggest
that the synaptic organization of the actin cytoskeleton is closely
associated with PSD-Zip45 and shank.
The PSD-Zip45-shank complex may link to the actin cytoskeleton through
the cortactin-shank interaction or another actin-binding protein that
interacts with the PSD-Zip45-shank complex. The former possibility,
however, seems to be unlikely because the synaptic localization of
shank was quite different from that of cortactin (data not shown).
Alternatively, there are several pieces of evidence that support the
latter possibility. The N terminus of Cupidin/Homer2a has been reported
to interact with F-actin in vitro (40). We did not, however,
detect a direct interaction between PSD-Zip45 and F-actin (data not
shown). In contrast, we demonstrated that overexpression of PSD-Zip45WT
enhances the synaptic accumulation of F-actin without enhancing that of
shank (Fig. 6A, a1-a3). Sala et al.
(36) reported that overexpression of both shank 1B and Homer 1b induces
synaptic enlargement in association with an increase in synaptic
F-actin. Consistent with this, we found that both of overexpressed
PSD-Zip45 and CortBP1 were highly concentrated in the enlarged synapses
with F-actin (Fig. 6A, d1-d3). This enhanced accumulation of synaptic F-actin was also observed in the normal-sized synapses in neurons overexpressing PSD-Zip45WT alone (Fig.
6A, a1 and a3). In contrast,
overexpressed PSD-Zip45G89A and PSD-Zip45
ZipB did not affect the
synaptic localization of F-actin whether or not CortBP1 was also
overexpressed (Fig. 6). These results indicate that the synaptic
localization of PSD-Zip45 is a prerequisite for that of shank, and the
complex formation between PSD-Zip45 and shank is critically linked to
the enhanced accumulation of synaptic F-actin. Why did overexpressed
PSD-Zip45 alone enhance synaptic accumulation of F-actin without the
accumulation of shank? This may be due to the difference of endogenous
shank and actin pools, in which free shank is more limited than
F-actin. Actually, both of the overexpressed PSD-Zip45 and CortBP1 were
highly concentrated in the synapses with the enhanced accumulation of
F-actin. Our present results further indicate that increasing the
levels of synaptically targeted PSD-Zip45 and shank through
overexpression induces synaptic enlargement in association with the
enhanced accumulation of synaptic F-actin. Future study will be
required to elucidate the involvement of the PSD-Zip45-shank and actin interaction in the postsynaptic structure and function.
 |
FOOTNOTES |
*
This work was supported by grants-in-aid for Research on
Brain Science from the Ministry of Health and Welfare of Japan (to K. S.) and in part by grants-in-aid for Scientific Research from the
Ministry of Education, Science, Sports and Culture of Japan (to
K. 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: Dept. of
Neuroscience (D13), Osaka University Graduate School of Medicine, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan. Tel.: 81-6-6879-3680; Fax:
81-6-6879-3689; E-mail: sobue@nbiochem.med.osaka-u.ac.jp.
Published, JBC Papers in Press, January 10, 2003, DOI 10.1074/jbc.M210802200
 |
ABBREVIATIONS |
The abbreviations used are:
PSD, postsynaptic
density;
EVH1, enabled/VASP homology 1;
mGluRs, metabotropic glutamate
receptors;
GFP, green fluorescent protein;
WT, wild type;
GST, glutathione S-transferase;
BSA, bovine serum albumin;
PBS, phosphate-buffered saline;
GABAA,
-aminobutyric acid,
type A;
NMDA, N-methyl-D-aspartic acid.
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