From the Departments of Neurobiology,
§ Pathology, and ¶ Physical Medicine and
Rehabitilation, University of Alabama at Birmingham, School of
Medicine, Birmingham, Alabama 35294
Received for publication, January 18, 2001, and in revised form, March 9, 2001
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Neuregulin is a factor essential
for synapse-specific transcription of acetylcholine receptor genes at
the neuromuscular junction. Its receptors, ErbB receptor tyrosine
kinases, are localized at the postjunctional membrane presumably to
ensure localized signaling. However, the molecular mechanisms
underlying synaptic localization of ErbBs are unknown. Our recent
studies indicate that ErbB4 interacts with postsynaptic density
(PSD)-95 (SAP90), a PDZ domain-containing protein that does not
interact with ErbB2 or ErbB3. Using as bait the ErbB2 C terminus, we
identified Erbin, another PDZ domain-containing protein that interacts
specifically with ErbB2. Erbin is concentrated in postsynaptic
membranes at the neuromuscular junction and in the central nervous
system, where ErbB2 is concentrated. Expression of Erbin increases
the amount of ErbB2 labeled by biotin in transfected cells, suggesting
that Erbin is able to increase ErbB2 surface expression. Furthermore,
we provide evidence that Erbin interacts with PSD-95 in both
transfected cells and synaptosomes. Thus ErbB proteins can interact
with a network of PDZ domain-containing proteins. This interaction may
play an important role in regulation of neuregulin signaling and/or
subcellular localization of ErbB proteins.
The neuromuscular junction is a cholinergic synapse that conveys
signals rapidly from motoneurons to muscle cells. The fast and accurate
neurotransmission at this synapse is guaranteed by the high
concentration of acetylcholine receptors in the postsynaptic membrane,
which accounts for only 0.1% of total muscle surface (1, 2). Muscle
fibers are multinucleated cells. Remarkably, it is only the synaptic
nuclei that actively transcribe genes encoding acetylcholine receptor
subunits. Such synapse-specific transcription is mediated by
neuregulin, a factor used by motoneurons to stimulate acetylcholine
receptor synthesis at the neuromuscular junction. Neuregulin receptors
are transmembrane tyrosine kinases of the ErbB family: ErbB2, ErbB3,
and ErbB4. Stimulation by neuregulin of ErbB proteins leads to their
tyrosine phosphorylation (3-6) and subsequent activation of multiple
intracellular signaling cascades (6-9), essential for compartmental
synthesis of acetylcholine receptors. In the central nervous system,
neuregulin regulates expression of neuronal nicotinic acetylcholine
receptor (10), N-methyl-D-aspartate
receptor (11), and ErbB proteins are not expressed evenly on the surface of cells. On the
contrary, they are localized in subcellular compartments. In the
nervous system, ErbB proteins are concentrated in postsynaptic membranes both at the neuromuscular junction (3, 14-16) and in the
central nervous system (13, 17). In epithelial cells, ErbB2 appears to
be enriched in basolateral membranes (18). The mechanism by which ErbB
proteins are localized in the subcellular compartments remains largely
unknown. The intracellular portions of ErbB receptor tyrosine kinases
contain large C termini in addition to kinase domains. Thus, it is
conceivable that ErbBs may interact with proteins that regulate their
localization, surface expression, or kinase activity. Indeed, recent
studies demonstrated that ErbB4, via its C terminus, interacts with
postsynaptic density
(PSD)1-95 (or SAP90), a
PDZ domain-containing protein (13, 17). PDZ domains are motifs
of 80-90 amino acids which often bind to specific sequences at the
extreme C termini of target proteins (19-22). They were originally
identified in PSD-95, the Drosophila septate junction
protein discs large, and the epithelial tight-junction protein zona
occludens 1 (23-26). PDZ domain-containing proteins appear to
coordinate the assembly of functional subcellular domains. PSD-95 uses
multiple PDZ domains to cluster ion channels, receptors, and cytosolic
signaling proteins in subcellular domains including synapses and
cellular junctions (27). The interaction of PSD-95 with ErbB4
potentially may allow for a localized signaling complex at synapses
while minimizing unwanted cross-talk. Moreover, PSD-95 could enhance
neuregulin signaling probably by promoting dimerization of ErbB4
receptor tyrosine kinases (13).
However, PSD-95 interacts with ErbB2 poorly and does not interact with
ErbB3 (13, 17), which raises the possibility that other PDZ
domain-containing protein may exist. Using a yeast two-hybrid strategy,
we identified a novel PDZ domain-containing protein that interacts
specifically with ErbB2, but not ErbB3 or ErbB4. This protein was named
B2BP for ErbB2-binding protein. B2BP was a polypeptide of 180 kDa. It
had 16 leucine-rich repeats (LRRs) in the N terminus and a PDZ domain
in the C terminus. While the study was in progress, Borg et
al. reported the cloning of Erbin as an ErbB2-interacting protein
(18, 28). Sequence analysis suggests that B2BP is the mouse homolog of
Erbin. We will refer B2BP as Erbin in the manuscript. We demonstrate
that Erbin is concentrated at the neuromuscular junction and a
component of the PSD in the central nervous system. Erbin interacts
with ErbB2 in synaptosomes. Moreover, Erbin increases surface
expression of ErbB2 in transfected cells. Furthermore, Erbin interacts
with PSD-95 in synaptosomes and in mammalian cells. These results
suggest that ErbB receptor tyrosine kinases interact with a network of PDZ domain-containing proteins that may regulate neuregulin signaling and localization.
Two-hybrid Studies in Yeast--
To identify ErbB2-interacting
proteins, the ErbB2 carboxyl terminus (amino acids 1251-1260) was
generated by two complimentary oligonucleotides and subcloned into the
pGBT9 yeast vector containing the Gal4 DNA binding domain
(CLONTECH). The bait plasmid was then transformed
into the yeast strain Y190 and used to screen mouse cDNA libraries
in Antibody Production and Purification--
The GST fusion protein
containing the PDZ of Erbin (amino acid residues 1241-1371) was
produced, affinity purified, and concentrated as described previously
(29). Antiserum against the GST-Erbin/PDZ fusion protein was raised in
a New Zealand White rabbit by standard procedures (30). To purify the
antibodies, two affinity columns were prepared by coupling GST protein
and GST- Erbin/PDZ fusion protein to Affi-Gel 15 (Bio-Rad),
respectively, according to the manufacturer's instruction. The
columns, 2 ml each, were washed sequentially with 10 ml of 100 mM glycine, pH 2.5, 10 ml of 10 mM Tris/HCl, pH
8.8, 10 ml of 100 mM triethylamine, pH 11.5, and equilibrated with 10 ml of 10 mM Tris/HCl, pH 7.5. 2 ml of
antiserum was diluted in 18 ml of 10 mM Tris/HCl, pH 7.5, and passed through the GST-coupled Affi-Gel 15 column three times. The
flow-through was then loaded on the GST-Erbin/PDZ-coupled Affi-Gel 10 column three times. After washing the column with 20 ml of 10 mM Tris/HCl, pH 7.5, and 20 ml of 500 mM NaCl
in 10 mM Tris/HCl, pH 7.5, antibodies were eluted with
aliquots of 100 mM glycine, pH 2.5, and collected in vials
containing 500 µl of 1 M Tris/HCl, pH 8.0. The antibodies were dialyzed overnight against 10 mM Tris/HCl, pH 7.5, 0.9% saline and stored in 0.02% sodium azide. Affinity-purified
antibodies were used in all experiments unless otherwise specified.
Commercially available antibodies used were from Upstate Technology
(PSD-95), Santa Cruz (ErbB2, ErbB3, ErbB4, and Myc), and Sigma
(anti-FLAG antibodies).
Cell Culture and Transfection--
HEK 293T cells (13), C2C12
muscle cells (6, 9, 31), and hippocampal neurons (13) were cultured as
described previously.
Primary rat muscle cell cultures were prepared as described previously
(32) with minor modifications. Muscles were isolated from hind legs of
day 19 rat embryos. Muscles were tweezed apart in PBS and incubated in
PBS containing 0.25% trypsin at 37 °C for 50 min with frequent
trituration. Dissociated cells were filtered through a 20-mesh screen
and pelleted twice. They were resuspended in the growth medium
(Dulbecco's modified Eagle's medium containing 10% fetal bovine
serum, 10% horse serum, 2% chick embryo extract, and 25 µg/ml
gentamicin). The cells were plated on regular culture dishes for 30 min
to get rid of fibroblasts prior to being plated on collagen-coated
culture dishes at a density of 7.5 × 106 cells/10-cm
dish. By the 4th day in vitro, myotubes were formed, and the
cultures were treated with 3 µg/ml cytosine arabinoside for 24 h
to inhibit fibroblast proliferation.
Cells were transfected using the standard calcium phosphate
technique. 2 days after transfection, cells were washed with
PBS and lysed in the modified RIPA buffer (1 ml/100-mm plate),
containing 20 mM sodium phosphate, pH 7.4, 50 mM sodium fluoride, 40 mM sodium pyrophosphate,
1% Triton X-100, 2 mM sodium vanadate, 50 µM
phenylarsine, 10 mM p-nitrophenyl
phosphate, including protease inhibitors (6). Lysed cells were
incubated on ice for 30 min and centrifuged at 13,000 × g for 10 min at 4 °C. The supernatant was designated as
cell lysate.
Northern Blotting--
Northern blot was done using a membrane
containing mRNAs from multiple tissues
(CLONTECH). An Erbin cDNA fragment (nucleotides 4044-4651) was labeled with [ Subcellular Fractionation--
Adult brains were homogenized in
a homogenizing buffer containing 0.32 M sucrose, 4 mM HEPES/NaOH, pH 7.4, 5 mM EDTA, 5 mM EGTA, 20 units/ml Trasylol, and 0.1 mM
phenylmethylsulfonyl fluoride with a glass-Teflon homogenizer as
described previously (33). Briefly, the ground tissue was centrifuged
at 800 × g for 10 min, and the supernatant was
designated the homogenate (S1). The homogenate was centrifuged at
9,000 × g for 15 min, yielding P2 (the crude synaptosomal fraction) and S2. The P2 fraction was resuspended in the
homogenizing buffer and used for coimmunoprecipitation studies. To
purify PSDs, the resuspended P2 pellet was subjected to another
centrifugation at 10,000 × g for 15 min, and the
pellet was lysed by hypoosmotic shock in water, rapidly adjusted to 1 mM HEPES/NaOH, pH 7.4, and stirred on ice for 30 min. The
lysate was then centrifuged at 25,000 × g for 20 min,
yielding P3 and S3. The P3 pellet was resuspended in 0.25 M
buffered sucrose, layered onto a discontinuous sucrose gradient
containing 0.8 M/1.0 M/1.2 M
sucrose, and centrifuged for 2 h at 65,000 × g in
a Beckman SW-28 rotor. The gradient yielded a synaptosomal plasma
membrane (SPM) fraction at the 1.0 M/1.2 M
sucrose interface. The SPM fraction was solubilized with 0.4% Triton
X-100 in 0.5 mM HEPES/NaOH, pH 7.4, yielding an insoluble
PSD fraction and a soluble SPM extract after a centrifugation at
65,000 × g for 20 min. Synaptophysin, a synaptic
vesicle protein, was fractionated with the SPM fraction but was not
present in the PSD fraction (see Fig. 8).
Immunoprecipitation, Pull-down Assays, and
Immunoblotting--
Cell lysates (~400 µg of protein) were
incubated directly without or with indicated antibodies for 1 h at
4 °C. They were then incubated with protein A-agarose beads
overnight at 4 °C on a rotating platform. After centrifugation,
beads were washed four or five times with the modified RIPA buffer.
Bound proteins were eluted with SDS sample buffer and subjected to
SDS-PAGE. Immunoprecipitation of Erbin from rat brain P2 fraction was
done as described previously (13).
The GST fusion protein containing the PDZ of Erbin (amino acids
1241-1371) was induced in BL21 cells with 1 mM isopropyl
Proteins resolved on SDS-PAGE were transferred to nitrocellulose
membranes (Schleicher & Schuell). Nitrocellulose blots were incubated
at room temperature for 1 h in Tris-buffered saline with 0.1%
Tween (TBS-T) containing 5% milk followed by an incubation with 1%
milk with the indicated antibodies except the anti-phosphotyrosine antibody, which required 3% bovine serum albumin in the blocking buffer and 1% bovine serum albumin in the blotting buffer. After washing three times for 15 min each with TBS-T, the blots were incubated with horseradish peroxidase-conjugated donkey anti-mouse or
anti-rabbit IgG (Amersham Pharmacia Biotech) followed by washing. Immunoreactive bands were visualized with enhanced chemiluminescence substrate (Pierce). In some experiments, after visualizing an immunoactive protein, the nitrocellulose filter was incubated in a
buffer containing 625 mM Tris/HCl, pH 6.7, 100 mM Immunohistochemistry--
Normal or denervated (5 days
postdenervation) muscles were rapidly dissected, stretched on a board,
and frozen in isopentane cooled with dry ice. 10-µm sections were
prepared using a cryostat, thaw mounted on gelatin-coated slides, and
stored at Labeling of Surface Proteins--
To label surface proteins,
cells were washed with cold PBS containing 1 mM
MgCl2 and 0.1 mM CaCl2 and
incubated with 0.5 mg/ml sulfo-NHS-LC-biotin in the same buffer at room
temperature for 30 min. The labeling reaction was quenched by
incubation with 100 mM glycine for 10 min at room
temperature. Cells were then lysated in the modified RIPA buffer.
Lysates were incubated with streptavidin-agarose beads (Molecular
Probes) overnight at 4 °C. Bound proteins were subjected to
SDS-PAGE.
Protein Assay--
The protein was assayed according to the
method of Bradford (34) using bovine serum albumin as a standard.
Cloning of Erbin--
The C terminus (-DVPV*) of ErbB2 fits the
consensus site for PDZ binding. However, whereas ErbB4 binds strongly
to PSD-95, ErbB2 has little or no affinity for PSD-95 (13). To identify proteins that bind to ErbB2, we generated several bait constructs composed of the ErbB2 C terminus in various lengths. Most of the baits
showed autonomous transactivation activity in various yeast strains
except the one with the last 10 amino acid residues (amino acids
1251-1260). Screens using this bait of mouse muscle, mouse brain, and
human heart cDNA libraries led to isolation of cDNAs all of
which encoded partial sequences of an apparently same protein with a
PDZ domain in the C terminus. This protein was initially named as B2BP
for ErbB2-binding protein because it only interacted with ErbB2 and not
ErbB3 or ErbB4 (see below). While this study was in progress, Borg
et al. reported Erbin (18). Sequence analysis indicated that
B2BP was the mouse homolog of Erbin. Thus, this protein will be
referred as Erbin in the rest of this manuscript. Erbin showed high
homology to Densin-180, a protein identified previously as a
postsynaptic component (35, 36). Like Densin-180, Erbin had 16 LRR
domains in the N terminus. In the C terminus, there is a PDZ domain of
group I which is characterized by a conserved histidine residue (37).
The homology between Erbin and Densin-180 was 73% in LRR domains, 71%
in the PDZ domain, and 39% in the middle region.
Characterization of Erbin Binding to ErbB2 in Yeast--
Nice
clones were isolated from the libraries that encoded two fragments of
Erbin: Erbin965 and Erbin1254 (Fig.
1A). The binding of Erbin to
ErbB2 was dependent on the PDZ domain of Erbin because deletion of the
PDZ domain prevented the interaction. Furthermore, the PDZ alone was
sufficient to bind to ErbB2. Although Erbin showed high homology with
Densin-180, ErbB2 did not interact with the PDZ domain of Densin-180
(Fig. 1A), nor did it interact with the PDZ domains of
PSD-95 (Fig. 1A) and of Scribble, nNOS, or Interaction of Erbin with ErbB2, Not ErbB3 or ErbB4--
To
characterize further the interaction between Erbin and ErbB proteins,
we examined the ability of Erbin's PDZ domain to bind to ErbBs in
in vitro pull-down assays. Lysates from HEK 293T cells
transfected with ErbB2, ErbB3, or ErbB4 were incubated with GST-Erbin
fusion protein immobilized on agarose beads. Bound proteins were
resolved on SDS-PAGE and immunoblotted with individual anti-ErbB antibodies. Consistent with the results from yeast two-hybrid assays,
GST-Erbin was only able to pull down ErbB2 (Fig.
2A). In contrast, ErbB3 or
ErbB4 was undetectable in the Erbin complex. To determine whether ErbB2
interacts with Erbin in mammalian cells, we expressed ErbB proteins
with or without Myc-tagged Erbin in HEK 293T cells. Lysates of
transfected cells were incubated with individual anti-ErbB antibodies,
and the resulting immunocomplex was blotted with anti-Myc antibodies.
Erbin was detected in the immunoprecipitates from cells that had been
cotransfected with ErbB2 and Erbin (Fig. 2B), suggesting
that ErbB2 associates with Erbin in vivo. In contrast, Erbin
was not detected in the ErbB3 or ErbB4 immunoprecipitates (Fig.
2C).
Expression of Erbin mRNA--
Northern blot analysis
was used to study mRNA expression of Erbin. The membrane
loaded with mRNAs from multiple tissues was probed with a
32P-labeled Erbin DNA fragment (encoding amino acids
1241-1371 plus the 3'-noncoding region). A major transcript at 7.5 kilobases was detected in various tissues (Fig.
3, top panel). The expression was high in the lung, heart, and kidney, moderate in the brain, skeletal muscle, and testis, and little, if any, in the spleen and
liver. In contrast, expression of the Densin-180 mRNA was brain-specific as reported previously (35). The 7.4-kilobase transcript
of Densin-180 was detected only in the brain, but not in any of tested
periphery tissues (Fig. 3, middle panel). Note the exposure
time of blots for Densin-180 (10 days) and Erbin (1 day), whereas both
used a similar amount of probes (5 ng/ml, 5 ml) with same specific
activity (4 × 108 cpm/µg of DNA). These results
suggest that the expression level of Erbin may be at least five times
higher than that of Densin-180 in the brain.
Erbin Is a Protein Tightly Associated with Membrane--
To study
Erbin expression at the protein level, antibodies against Erbin were
generated using as antigen the Erbin PDZ domain (amino acids
1241-1371). When affinity purified, the antibody detected a 180-kDa
protein on Western blots of HEK 293T cell lysates (Fig.
4A, large arrow).
The interaction of the 180-kDa protein and the serum was specific
because it could be blocked by preincubation of serum with the antigen
GST-Erbin/PDZ fusion protein (Fig. 4A, lane 3).
The antibodies also recognized the transfected Erbin965 recombinant
protein in HEK 293T cells (Fig. 4A, small arrow), whose expression was evident by blotting with an anti-Myc antibody (Fig. 4A, lane 5). Although there is a high
homology between the PDZ domains of Erbin and Densin-180, the
anti-Erbin antibody did not cross-react with Densin-180 (Fig.
4B, lane 7). Taken together, these results
indicate that Erbin is not a human or mouse ortholog of Densin-180.
As observed with ErbB2, Erbin was present only in membrane, but not
soluble, fractions (Fig. 5A).
To determine how tightly Erbin associates with the membrane fraction,
we treated brain membrane (P2) fractions with high concentrations of
salt, high pH, or various detergents to solubilize Erbin. Solubilized
or insolubilized fractions were immunoblotted with anti-Erbin
antibodies. As shown in Fig. 5A, Erbin was resistant to wash
with 1 M NaCl or high pH buffer (0.1 M
NaCO3), which disrupted protein interactions and extracted
mainly peripheral membrane proteins, respectively. Except for partial
solublization by 3% Nonidet P-40, Erbin was virtually insoluble in
2.5% CHAPS or 1% Triton, conditions under which many membrane
proteins were solubilized. However, Erbin could be solubilized by 1%
deoxycholate as has been described for various PSD proteins (38, 39).
The solubility pattern of Erbin was similar to that of ErbB2 (Fig.
5A). These results suggest that Erbin may be associated with
the cytoskeletal structure, PSD. In a previous study, we have
demonstrated that ErbB2 is localized at the PSD of the brain (13). It
is interesting to note that Erbin seems to be a doublet in the brain in
a previous study (18) but a singlet using our antibody (Fig. 5). The
cause of this apparent difference in chromatographic behavior remains
unclear. It may reflect the difference of the antibodies used in the
two studies.
The finding that Erbin was present only in membrane fractions and
insoluble in various detergents was very interesting because the
hydrophobicity profile of the Erbin amino acid sequence did not predict
the presence of a transmembrane domain (data not shown). We speculated
that Erbin was associated with membranes by interacting with integral
proteins such as ErbB2. To test this hypothesis, we biotinylated
surface proteins in HEK 293T cells, which were then isolated with
streptavidin-agarose beads. As shown in Fig. 5B, only 8.7%
of total Erbin was present in the complex pulled down with the beads.
In contrast, biotin labeled 35% of total ErbB2, a protein known to
have a transmembrane domain. As a control, the amount of ERK1, a
cytoplasmic kinase, in the pull-down complex was barely detectable.
These results suggest that Erbin may not be as accessible to surface
biotinylation in intact cells as the transmembrane ErbB2. Only a
minimal amount of Erbin was present in the streptavidin-pull-down
complex. To determine whether the presence of Erbin in the
streptavidin-pull-down complex was caused by interaction with other
proteins, we expressed Erbin and a mutant with deletion of the PDZ
domain. As shown in Fig. 5C, the presence of transfected
Erbin in the streptavidin-pull-down complex was dependent on the PDZ
domain. Deletion of the PDZ domain in Erbin, which blocked binding to
ErbB2 (Fig. 1), abolished its presence in the complex. These results
suggested to us that Erbin may be a protein in the cytoplasm. It may be
tightly associated membranes in a manner dependent on the PDZ domain,
probably via interaction with ErbB2. These results, however, were
unable to exclude the possibility that Erbin is a transmembrane protein
with the extracellular domain somehow inaccessible to biotin labeling.
Localization of Erbin at the Neuromuscular Junction--
As with
in the brain, Erbin was expressed as a 180-kDa protein in the skeletal
muscle, C2C12 mouse muscle cells, and muscle cells in primary culture
(Fig. 6A). Expression of Erbin
was at similar level in myoblasts and in myotubes, suggesting that
differentiation of muscle cells had little effect on its expression.
ErbB proteins including ErbB2 are concentrated in the postjunctional
membrane at the neuromuscular junction (3, 14-16, 40) and are
required for neuromuscular junction formation (43). We proposed that Erbin could be enriched at the neuromuscular junction by interacting with ErbB2. To determine subcellular localization of Erbin in skeletal
muscle, we stained mouse diaphragm sections with affinity-purified anti-Erbin antibody by immunofluorescence techniques.
Rhodamine-conjugated
To determine that the Erbin staining signal at the neuromuscular
junction was from the postsynaptic instead of presynaptic components,
we studied the effect of denervation on Erbin expression. Denervation
of the skeletal muscle causes rapid degeneration of presynaptic nerves.
Until the development of pathological conditions (such as atrophy and
inflammation), expression of postsynaptic proteins is normal or
elevated to compensate the loss of presynaptic input (44). As shown in
Fig. 7A, the level of Erbin
protein was increased in denervated muscles, and so was that of ErbB2 as observed previously (5). These results suggest that Erbin expression
may be regulated by electric activity. Remarkably, Erbin was detectable
at the neuromuscular junction in denervated muscles (Fig.
7B), supporting the notion that Erbin was present in the
postsynaptic membrane because presynaptic components degenerate in
denervated muscles.
Erbin Is a Component of PSD in the Central Nervous
System--
Expression of Erbin was at similar levels in the cerebral
cortex, hippocampus, cerebellum, and brain stem (Fig.
8A). To determine whether
Erbin was present in the PSD fraction, we performed subcellular fractionation studies. As shown in Fig. 8B, ErbB2 and Erbin
were present in synaptosomes and copurified into PSD fractions. The degree of Erbin enrichment in the PSD fraction correlated strongly with
that seen with ErbB2. These biochemical studies demonstrate that Erbin
is present in the PSD fraction and suggest that it is appropriately
localized to form a protein complex in vivo with ErbB2. Next
we determined whether Erbin interacts with ErbB2 in the central nervous
system. The interaction between ErbB2 and Erbin was examined in rat
brain synaptosomes. Synaptosomes were solubilized with 1% deoxycholate
and incubated with antibodies against Erbin. As shown in Fig.
8C, immunoprecipitation of Erbin resulted in
coimmunoprecipitation of ErbB2. In the lane where antibodies were
missed, ErbB2 was absent in the precipitates (Fig. 8C).
Furthermore, preabsorption of Erbin antibodies with the antigen blocked
the coimmunoprecipitation of ErbB2 (data not shown). These results
suggest that Erbin is associated with ErbB2 in vivo.
Erbin Interacted with PSD-95 in Synaptosomes and in Transfected
Cells--
Both ErbB2 and ErbB4 are proteins in the central nervous
system synapses (13, 17) and at the neuromuscular junction (40). Considering that PSD-95 binds to ErbB4 (13, 17), we determined whether
Erbin and PSD-95 were in the same complex in the central nervous
system. Deoxylate-solubilized synaptosomes were incubated with
anti-Erbin antibodies to isolate Erbin immunocomplexes. As shown in
Fig. 9A, PSD-95 was detected
in Erbin immunoprecipitates, suggesting an interaction of Erbin with
PSD-95 in vivo. To identify the domains in PSD-95 and Erbin
which are required for the interaction, HEK 293T cells were
cotransfected with various constructs (Fig. 9B). The
interaction of PSD-95 with Erbin was dependent on the PDZ domains of
PSD-95 (Fig. 9D); in fact, the first and second PDZ domains
were sufficient for interaction with Erbin (Fig. 9C). On the
other hand, the Erbin interaction with PSD-95 did not appear to require
Erbin's PDZ domain (Fig. 9E). Further analysis suggested that Erbin may interact with PSD-95 via the region between amino acids
965 and 1241 because an Erbin mutant with a deletion of the N-terminal
1-964 amino acid residues was able to interact with PSD-95 (Fig.
9F).
Erbin Increased Surface Expression of ErbB2--
As an initial
step to identify the function of Erbin, we investigated the effect of
Erbin on ErbB2 surface expression. HEK 293T cells were transfected with
Myc-tagged Erbin or a PDZ domain deletion mutant. As shown in Fig.
10A, expression of Erbin had no effect on the total amount of ErbB2 in transfected cells. In contrast, Erbin increased the amount of biotin-labeled ErbB2. Such an
increase was dependent on the intact C terminus of Erbin. In cells
transfected with the Erbin mutant with deletion in the PDZ domain, the
amount of biotin-labeled ErbB2 was similar to that in mock transfected
cells (Fig. 10, A and B). These results suggest a
possible role of Erbin in the regulation of ErbB2 surface expression.
The major findings of this study are the following. First, the
novel PDZ domain-containing protein Erbin interacts specifically with
ErbB2, not ErbB3 or ErbB4. Second, Erbin may not be an integral protein
but is tightly associated with membranes. Third, this protein, like
ErbB2 receptor tyrosine kinase, is enriched both in the postjunctional
membrane at the neuromuscular junction and in the PSD of the brain.
Fourth, in addition to ErbB2, Erbin also interacts with PSD-95, another
PDZ domain-containing protein that interacts with ErbB4. Last,
expression of Erbin increases the amount of biotin-labeled ErbB2 in
mammalian cells, suggesting that Erbin was able to increase ErbB2
surface expression. Together with a recent study that suggests a role
of Erbin in basolateral localization of ErbB2 in epithelial cells (18),
our results suggest that ErbB receptor tyrosine kinases interact with a
network of PDZ domain-containing proteins. The interaction between
ErbBs and the intracellular PDZ domain-containing proteins may be
essential for localized neuregulin signaling in a subcellular
compartment including the neuromuscular junction and central synapses.
Moreover, the PDZ domain-containing proteins, via interacting with
ErbBs, may regulate neuregulin signaling.
Erbin belongs to a unique family of PDZ domain-containing proteins.
These proteins include Densin-180; LET-413, an Erbin ortholog in
Caenorhabditis elegans (45); and Scribble, a
Drosophila protein essential for epithelial integrity (46).
In addition to the PDZ domain in the C termini, the members of this
family contain 16 LRRs in the N termini and have thus been named as LAP
(for LRR and PDZ) proteins (18, 45). Among the members of the LAP family, Densin-180 shares a high homology with Erbin in amino acid
sequence and overall primary structure (18). Densin-180 is also a
postsynaptic component and enriched in the PSD of the brain (35).
However, the PDZ domain of Densin-180 does not interact with ErbB2,
indicating substrate specificity of PDZ domains of Densin-180 and Erbin
and suggesting a different role of these proteins in the brain. The
C. elegans ortholog of Erbin, LET-413, is critical for
normal assembly of adherens junctions. In LET-413 mutants, adherens
junctions are abnormal, cell polarity is affected, and actin
cytoskeleton is disorganized (45). Our results suggest that Erbin may
play an important role in regulation of neuregulin signaling.
Expression of Erbin increased biotin-labeled ErbB2 in transfected
cells, indicating that Erbin can promote ErbB2 surface expression. This
effect was specific in that it relied on the presence of the PDZ
domain, through which Erbin interacted with ErbB2. The Erbin mutant
without the PDZ domain did not increase ErbB2 surface expression.
At the neuromuscular junction, proteins essential for neurotransmission
are densely packed at the postsynaptic membrane (1, 2, 47). This is
caused and maintained at least in part by active transcription in
synaptic nuclei. Neuregulin is a molecule from motoneurons which
stimulates acetylcholine receptor synthesis (48). In fact, one
initially identified isoform of neuregulin is ARIA (for
acetylcholine receptor inducing
activity) (4). Neuregulin is synthesized in motoneurons (4)
released from motoneurons and deposited in the synaptic cleft (49, 50), activates ErbB receptors (3, 6, 51) and subsequent multiple signaling
pathways (6, 9, 52, 53) in the skeletal muscle to increase
acetylcholine receptor expression. Results from studies of neuregulin-1
and ErbB2 mutant mice indicate that neuregulin is essential for the
formation and maintenance of the neuromuscular junction (43, 54). In
support of this hypothesis are findings that ErbB protein tyrosine
kinases and downstream signaling molecules are concentrated at the
neuromuscular junction (3, 15, 16, 40, 55, 56). However, the mechanisms
underlying clustering of ErbB proteins in muscle cells remain unclear.
We believe that ErbBs are clustered at the neuromuscular junction by
interaction via the C termini with a network of anchoring proteins. Of
the family of membrane-associated guanyl kinase-like proteins (MAGUK), PSD-95, SAP97, and SAP102 interacted with ErbB4
(13).2 They are expressed in
skeletal muscle cells.2 Moreover, earlier studies
suggested that SAP97 (57) and PSD-95 (58) may be localized at the
neuromuscular junction. These proteins may play a role in clustering
ErbB4 at the neuromuscular junction. Erbin may be a protein that
anchors ErbB2 at the neuromuscular junction. The immunoreactivity of
Erbin was enriched at the neuromuscular junction. Furthermore,
denervation that destroyed presynaptic components had no apparent
effect on Erbin staining in the skeletal muscle, suggesting that Erbin
is present in the postsynaptic membrane of the neuromuscular junction
where ErbB2 is localized. The hypothesis was supported further by
results from the recent study of ErbB2 expression in polarized
epithelial cells. ErbB2 is localized at the basolateral side of
epithelial cells (18). This localization is dependent on the intact C
terminus, the site of interaction with Erbin.
Another interesting finding of this paper is that PSD-95 interacts with
Erbin. PSD-95 is a well characterized protein with three PDZ domains in
the N terminus, an inactive guanylate kinase domain in the C terminus,
and a SH3 domain in between (19-22). PSD-95, via distinct PDZ domains,
interacts with various proteins. In addition, it can form head-to-head
multimers via disulfide linkage of its N terminus (41). The interacting
proteins of PSD-95 include N-methyl-D-aspartate
receptors, potassium channels, neuroligin, SynGap, and CRIP (42). Thus
it is believed that PSD-95 is important for the assembly and
maintenance of anatomic and/or functional synaptic complex. In this
study, PSD-95 interacted with Erbin not only in the heterologous
expression system but also in synaptosomes. The interaction of Erbin
with PSD-95 did not depend on Erbin's PDZ domain, but a region between
amino acids 965 and 1241, suggesting that Erbin may interact
simultaneously with ErbB2 and PSD-95. Together with previous
observations that PSD-95 interacts with ErbB4 (13, 17), these results
suggest that ErbB proteins may interact with a network of PDZ
domain-containing proteins. It will be interesting to determine whether
ErbB clustering requires both Erbin and PSD-95 or a PSD-95-like protein
in central nervous system synapses and at the neuromuscular junction.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-aminobutyric acid receptor (12). Recent
studies suggest that in addition to an essential role during
development, neuregulin appears to regulate synaptic plasticity in the
adult brain (13).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
ACT2 or in pACT2 that contained the Gal4 transcription activation
domain. Positive clones were selected on plates lacking leucine,
tryptophan, and histidine and were confirmed further by a filter assay
for
-galactosidase activity as described previously (13). Constructs
containing PDZ domains of PSD-95, nNOS,
1-syntrophin,
1-syntrophin, or
2-syntrophin, or C termini of ErbB3 or ErbB4 have been described previously (13). C termini of ErbB2 mutants and
NR2A (amino acids 1416-1464) were generated by polymerase chain
reaction and subcloned in pGBT9. Sequences of all constructs were
confirmed by DNA sequencing. The yeast vectors are transformed into
HF7c and Y190. Interactions were characterized by growth without
leucine, tryptophan, and histidine and by a filter assay for
-galactosidase activity.
-32P]dCTP by a random
prime method. The membrane was hybridized in a buffer (total 5 ml)
containing 32P-labeled probe (~4 × 108
cpm/µg of cDNA), 5 × SSC, 5 × Denhardt's solution,
0.5% SDS, 50% formamide, and 100 µg/ml salmon sperm DNA at 42 °C
overnight. It was then washed with 0.5 × SSC, 0.5% SDS at
55 °C three times each for 30 min and exposed to Kodak X-Omat AR
film at
70 °C with an intensifying screen.
-D-thiogalactopyranoside and purified using
glutathione-agarose beads (Roche Molecular Biochemicals, Indianapolis).
Equal amounts of GST fusion protein beads (~50 µg of protein) were
incubated with cell lysates overnight at 4 °C on a rotating
platform. After centrifugation, beads were washed four or five times
with wash buffer (150 mM sodium chloride, 10 mM
sodium phosphate, 1% Triton X-100, pH 7.4). Bound proteins were eluted
with SDS sample buffer and subjected to SDS-PAGE and immunoblotting.
-mercaptoenthanol, and 2% SDS at 50 °C for 30 min, washed with 0.1% Tween 20 in 50 mM TBS at room
temperature for 1 h, and reblotted with different antibodies.
80 °C. Sections of adult rat muscles were incubated with
2% normal goat serum (Vector Laboratories, Burlingame, CA) in PBS for
1 h at room temperature to reduce background staining and then
incubated with the affinity-purified antibodies against Erbin or
preimmune serum in 2% normal goat serum in PBS overnight at 4 °C.
In some experiments, affinity-purified antibodies were preincubated
with 10 nM GST-Erbin/PDZ overnight at 4 °C prior
to immunohistochemical studies. After washing the sections five times
with PBS, each for 30 min, the sections were incubated with a
fluorescein isothiocyanate-conjugated anti-rabbit antibody
(Zymed Laboratories Inc., San Francisco) and
rhodamine-conjugated
-bungarotoxin (Molecular Probes, Eugene,
OR). Fluorescent images of cells were captured on a Sony CCD
camera mounted on a Nikon E600 microscope using Photoshop imaging software.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1-,
1-, and
2-syntrophin (data not shown). The binding of Erbin to ErbB2 was
dependent on the ErbB2 C terminus. Mutation of the valine residues at
the
1 and
3 positions to alanine prevented ErbB2 from interacting
with Erbin (Fig. 1B). On the other hand, Erbin interacted
specifically with the C terminus of ErbB2 and did not interact with C
termini of the ErbB3, ErbB4, or NR2A subunit of the
N-methyl-D-aspartate receptor (Fig.
1C).
View larger version (19K):
[in a new window]
Fig. 1.
Binding of ErbB2 with Erbin in yeast.
A, interaction of Erbin with ErbB2 depended on the PDZ
domain in the C terminus. The domain structure of Erbin is shown in the
schematic diagram. Erbin965 and Erbin1254 are original clones isolated
from a yeast two-hybrid screen encoding the C termini starting from the
indicated amino acid residue. Erbin195 PDZ encoded Erbin amino acid
residues 195-1279 without the PDZ domain. Densin-180/PDZ encoded amino
acid residues 1161-1495 containing the PDZ domain. PSD-95/PDZ contains
amino acid residues 65-393 with all three PDZ domains. These
constructs were fused with the Gal4AD and cotransformed with
Gal4DB/ErbB2-DVPV* in yeast. Asterisks indicate amino acid
residues prior to the stop codon. B, dependence of the
interaction between ErbB2 and Erbin on the ErbB2 C terminus. Yeast
cells were cotransformed with a vector encoding the Gal4DB fused to
different ErbB2 C-terminal constructs and Gal4AD/Erbin. C,
interaction between Erbin with C termini of ErbBs or NR2A. Yeast cells
were cotransformed with Erbin and ErbB2 C-terminal constructs.
Transformed yeast cells were seeded in His
plates and
scored for growth and for
-galactosidase (
-Gal)
activity.
View larger version (23K):
[in a new window]
Fig. 2.
Specific interaction of Erbin with ErbB2 but
not ErbB3 or ErbB4. A, analysis of Erbin binding to
ErbB2 by in vitro pull-down assays. HEK 293T cells were
transfected with ErbB2, ErbB3, or ErbB4. Cell lysates were incubated
with GST or the GST-Erbin fusion protein (containing amino acid
residues 965-1371) immobilized on agarose beads. Bound proteins were
resolved on SDS-PAGE and subjected to immunoblotting with the indicated
specific anti-ErbB2, ErbB3, or ErbB4 antibodies. B, analysis
of Erbin binding to ErbB2 by immunoprecipitation. ErbB2 was transfected
without or with Myc-Erbin in HEK 293T cells. Immunoprecipitates
(IP) with anti-ErbB2 antibodies (left panels) or
preimmune serum (right panels) were resolved on SDS-PAGE and
immunoblotted (IB) with an anti-Myc antibody. One-tenth of
the input was used in the pull-down and coimmunoprecipitation
experiments. C, analysis of Erbin binding to ErbB3 and ErbB4
by immunoprecipitation. ErbB3 or ErbB4 was transfected with or without
Myc-Erbin in HEK 293T cells. Immunoprecipitates with the indicated
specific anti-ErbB3 or ErbB4 antibodies were resolved on SDS-PAGE and
immunoblotted with an anti-Myc antibody. One-tenth of the input was
used in coimmunoprecipitation experiments.
View larger version (79K):
[in a new window]
Fig. 3.
Expression of Erbin mRNA in various
tissues. A rat multitissue mRNA blot from
CLONTECH was hybridized with
32P-labeled cDNA probes for Erbin (top
panel). After appropriate exposure was obtained, the blot was
reprobed sequentially for Densin-180 (middle panel) or actin
(bottom panel). Autoradiogram exposure times were 1 day
(top and bottom panels) or 10 days (middle
panel) at 70 °C. The amounts of mRNAs of different
tissues were similar as indicated by the autoradiogram obtained with
the
-actin probe. Molecular weight markers are indicated at the
right of panels in kilobases.
View larger version (28K):
[in a new window]
Fig. 4.
Characterization of the anti-Erbin
antibody. HEK 293T cells were mock transfected or transfected with
Myc-Erbin (containing amino acid residues 965-1371) or FLAG-Densin-180
(containing amino acid residues 1161-1495). Cell lysates (40 µg of
protein) were resolved on SDS-PAGE and subjected to immunoblotting with
the affinity-purified antibody against Erbin or the antibody that was
preabsorbed with the antigen (Abs) (A, left
panels). Expression of recombinant Myc-Erbin was indicated by
immunoblotting with an anti-Myc antibody (A, right
panels). In B, transfected FLAG-Densin-180 was probed
with anti-FLAG or anti-Erbin antibodies. The positions of
electrophoretic mobility standards are indicated in kDa. The anti-Erbin
antibody recognized specifically the transfected recombinant protein
and 180-kDa endogenous protein of Erbin but not Densin-180.
View larger version (27K):
[in a new window]
Fig. 5.
Erbin was a membrane-associated but not
integral protein. A, Erbin was tightly associated with
membrane fractions in the brain. Rat brain synaptic plasma membranes
(P2, 100 µg of protein) were resuspended in PBS containing the
indicated buffers with high concentration of salt, high pH, or various
detergents for 30 min on ice and spun at 10,000 × g
for 15 min. The resulting pellets (P) and supernatant
(S) were resolved on SDS-PAGE and subjected to
immunoblotting with the respective antibodies. B, Erbin was
not an integral protein. HEK 293T cells were labeled with
sulfo-NHS-LC-biotin and lysed as described under "Experimental
Procedures." Lysates (20 times of input) were incubated with
streptavidin-agarose beads to pull-down biotinolated proteins or those
associated with biotinolated proteins. Isolated proteins were subjected
to Western blot for the indicated proteins. The left panel
shows blots from a representative experiment; the right
panel shows the results of densitometric analysis (mean ± S.D. of three different samples) of autoradiograms, which were scanned
and analyzed with NIH Imaging. C, dependence of Erbin
presence in pull-down complexes on the PDZ domain. Cells were
transfected with green fluorescent protein (GFP)-Erbin or
GFP-Erbin PDZ and labeled with sulfo-NHS-LC-biotin and lysed as
described in B. Pull-down complexes were probed for
transfected proteins with the indicated antibodies.
-bungarotoxin was used as a marker of the
neuromuscular junction (31). The immunoreactivity of Erbin was
visualized with a fluorescein isothiocyanate-conjugated secondary
antibody. As shown in Fig. 6B, Erbin showed a pattern of
labeling strikingly similar to that of the
-bungarotoxin staining.
Merging the two images indicated that Erbin is localized in precise
register with the acetylcholine receptor at the neuromuscular junction.
In addition, the Erbin imunoreactivity was also present in the
sarcolemma of the skeletal muscle (Fig. 6B, large
arrows). Specificity of the staining of Erbin at the neuromuscular
junction and sarcolemma was demonstrated by the fact that the preimmune
serum produced no staining above the background (Fig. 6C).
Furthermore, the Erbin staining was diminished by preabsorbing the
antibody with the immunogen (Fig. 6D). These results
indicated that Erbin is enriched at the neuromuscular junction and
expressed at a low level in sarcolemma.
View larger version (45K):
[in a new window]
Fig. 6.
Expression of Erbin in muscle cells and at
the neuromuscular junction. A, Erbin was expressed in
C2C12 and primary myoblasts and myotubes. Homogenates (50 µg of
protein) of rat brain, skeletal muscles and lysates of C2C12, and rat
primary muscle cells were resolved on SDS-PAGE and subjected to
immunoblotting with antibodies against Erbin or ErbB2. MB,
myoblasts; MT, myotubes; Primary, primary muscle
cells. B, C, and D, colocalization of
Erbin with -bungarotoxin (
BTX) in skeletal
muscles. Mouse diaphragm sections were incubated with affinity-purified
anti-Erbin antibody (B), preimmune serum, or the anti-Erbin
antibody preabsorbed with the immunogen. Rhodamine-conjugated
-bungarotoxin was added to label the acetylcholine receptor. The
Erbin immunoactivity was visualized by fluorescein
isothiocyanate-conjugated secondary antibody. Small arrows
indicate acetylcholine receptor clusters; large arrows
indicate sarcolemma.
View larger version (13K):
[in a new window]
Fig. 7.
Effects of denervation on Erbin
expression at the neuromuscular junction. A, expression
of Erbin and ErbB2 was increased in denervated muscles. Denervated
(Den, 5 days postsurgery) or control (Inn,
sham-operated) muscles were homogenized. 50 µg of protein was probed
for Erbin, ErbB2, or ERK. B, colocalization of Erbin with
-bungarotoxin (
BTX) in denervated muscles.
Leg muscle sections were incubated with the anti-Erbin antibody that
was visualized by fluorescein isothiocyanate-conjugated secondary
antibody. On the immediate right is an image of
-bungarotoxin staining and on the far right, an image of
overlays. Small arrows indicate acetylcholine receptor
clusters; large arrows indicate sarcolemma.
View larger version (22K):
[in a new window]
Fig. 8.
Erbin expression in the PSD and
interaction with ErbB2 in the central nervous system.
A, expression of Erbin in various brain regions. Homogenates
(100 µg of protein) were resolved on SDS-PAGE and subjected to
Western blot using the anti-Erbin antibody. B, expression of
Erbin in PSD. Rat brain homogenates (H) were subjected to
sequential centrifugations to yield cytosol (S2) and
synaptosomes (P2). Washed synaptosomes (P3)
were fractionated further by discontinuous sucrose gradient
centrifugation to generate synaptosomal plasma membrane
(SPM) which was treated with 0.4% Triton X-100. The
insoluble SPM was designated as PSD. Samples were separated by SDS-PAGE
and subjected to immunoblotting with the respective antibodies.
C, interaction between Erbin and ErbB2 in the central
nervous system. Rat brain synaptosomes were solubilized with 1%
deoxycholate. The resulting detergent extract (Input) was
incubated with preimmune serum or antibodies against Erbin.
Immunoprecipitates (IP) were resolved on SDS-PAGE and
subjected to immunoblotting (IB) for ErbB2. 10 times of
input were used for immunoprecipitations.
View larger version (28K):
[in a new window]
Fig. 9.
Interaction between Erbin and PSD-95.
A, interaction between Erbin and PSD-95 in synaptosomes. Rat
brain synaptosomes were solubilized with 1% deoxycholate. The
resulting detergent extract (Input) was incubated with
preimmune serum, antibodies against Erbin, or anti-Erbin antibodies
that were preincubated with excess antigen. Immunoprecipitates
(IP) were resolved on SDS-PAGE and subjected to
immunoblotting (IB) for PSD-95 and Erbin. 10 times of input
were used for immunoprecipitations. B, schematic diagrams of
used expression constructs. All constructs were tagged with Myc epitope
except NPDZ123 of PSD-95, which was tagged with a FLAG epitope.
C and D, dependence of Erbin interaction with
PSD-95 on the PDZ domains of PSD-95. FLAG-tagged
NPDZ123 or
Myc-tagged PDZ123, NPDZ123, or PSD-95 was transfected into HEK 293T
cells. Erbin was immunoprecipitated and probed with anti-FLAG (in
C) or anti-PSD-95 antibodies, which recognize all three
PSD-95 recombinant proteins (in D). E and
F, independence of Erbin interaction with PSD-95 on the PDZ
domain of Erbin. PSD-95, Erbin, Erbin
PDZ, or Erbin965 (all
Myc-tagged) was transfected or cotransfected into HEK 293T cells. Cell
lysates were incubated with anti-PSD-95 antibodies, and the resulting
immunocomplexes were subjected to blotting for the indicated
proteins.
View larger version (21K):
[in a new window]
Fig. 10.
Increase in surface expression of ErbB2 in
cells expressing Erbin. A, increased surface expression
of ErbB2 in Erbin-transfected cells. Cells were transfected with
Myc-Erbin, Myc-Erbin PDZ, or an empty vector. Protein complexes were
pulled down with streptavidin-agarose beads and probed for ErbB2 and
transfected proteins. Shown were blots from a representative
experiment. B, densitometric analysis of data in
A. The ratios of intensity of the signals (intensity of
ErbB2 signals in pull-down complexes/intensity of signals in lysates)
were calculated and normalized to the mock-transfected cells. Data were
three experiments (in mean ± S.D.). *, p < 0.05.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
![]() |
ACKNOWLEDGEMENTS |
---|
We are grateful to Drs. M. Sliwkowski and Jean-Paul Borg for valuable reagents.
![]() |
FOOTNOTES |
---|
* This work was supported in part by a faculty development award from Howard Hughes Medical Institute at the University of Alabama at Birmingham and Grants NS34062 and NS40480 from the NINDS, National Institutes of Health.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
Neurobiology, University of Alabama at Birmingham, School of Medicine, 516 Civitan International Research Center, 1719 6th Ave. South, Birmingham, AL 35294-0021. Tel.: 205-975-5196; Fax:
205-975-9927; E-mail: lmei@nrc.uab.edu.
Published, JBC Papers in Press, March 12, 2001, DOI 10.1074/jbc.M100494200
2 Y. Z. Huang, Q. Wang, W. C. Xiong, and L. Mei, unpublished observation.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: PSD, postsynaptic density; B2BP, ErbB2-binding protein (Erbin); LRR, leucine-rich repeat; GST, glutathione S-transferase; HEK, human embryonic kidney; PBS, phosphate-buffered saline; SPM, synaptosomal plasma membrane; PAGE, polyacrylamide gel electrophoresis; CHAPS, 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonic acid.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Hall, Z. W., and Sanes, J. R. (1993) Cell 72, 99-121[Medline] [Order article via Infotrieve] |
2. | Sanes, J. R., and Lichtman, J. W. (1999) Annu. Rev. Neurosci. 22, 389-442[CrossRef][Medline] [Order article via Infotrieve] |
3. | Altiok, N., Bessereau, J.-L., and Changeux, J.-P. (1995) EMBO J. 14, 4258-4266[Abstract] |
4. | Falls, D. L., Rosen, K. M., Corfas, G., Lane, W. S., and Fischbach, G. D. (1993) Cell 72, 801-815[Medline] [Order article via Infotrieve] |
5. |
Jo, S. A.,
and Burden, S. J.
(1992)
Development
115,
673-680 |
6. |
Si, J.,
Luo, Z.,
and Mei, L.
(1996)
J. Biol. Chem.
271,
19752-19759 |
7. | Gassmann, M., and Lemke, G. (1997) Curr. Opin. Neurobiol. 7, 87-92[CrossRef][Medline] [Order article via Infotrieve] |
8. | Burden, S., and Yarden, Y. (1997) Neuron 18, 847-855[Medline] [Order article via Infotrieve] |
9. | Si, J., Wang, Q., and Mei, L. (1999) J. Neurosci. 19, 8489-8508 |
10. | Yang, X., Kuo, Y., Devay, P., Yu, C., and Role, L. (1998) Neuron 20, 255-270[Medline] [Order article via Infotrieve] |
11. | Ozaki, M., Sasner, M., Yano, R., Lu, H. S., and Buonanno, A. (1997) Nature 390, 691-694[CrossRef][Medline] [Order article via Infotrieve] |
12. |
Rieff, H. I.,
Raetzman, L. T.,
Sapp, D. W.,
Yeh, H. H.,
Siegel, R. E.,
and Corfas, G.
(1999)
J. Neurosci.
19,
10757-10766 |
13. | Huang, Y. Z., Won, S., Ali, D. W., Wang, Q., Tanowitz, M., Du, Q. S., Pelkey, K. A., Yang, D. J., Xiong, W. C., Salter, M. W., and Mei, L. (2000) Neuron 26, 443-455[Medline] [Order article via Infotrieve] |
14. | Zhu, X., Lai, C., Thomas, S., and Burden, S. J. (1995) EMBO J. 23, 5842-5848 |
15. | Moscoso, L. M., Chu, G. C., Gautam, M., Noakes, P. G., Merlie, J. P., and Sanes, J. R. (1995) Dev. Biol. 172, 158-169[CrossRef][Medline] [Order article via Infotrieve] |
16. |
Trinidad, J. C.,
Fischbach, G. D.,
and Cohen, J. B.
(2000)
J. Neurosci.
20,
8762-8770 |
17. |
Garcia, R. A. G.,
Vasudevan, K.,
and Buonanno, A.
(2000)
Proc. Natl. Acad. Sci. U. S. A.
97,
3596-3601 |
18. | Borg, J.-P., Marchetto, S., Le Bivic, A., Ollendorff, V., Jaulin-Bastard, F., Saito, H., Fournier, E., Adelaide, J., Margolis, B., and Birnbaum, D. (2000) Nature Cell Biol. 2, 407-413[CrossRef][Medline] [Order article via Infotrieve] |
19. | Bredt, D. S. (1998) Cell 94, 691-694[Medline] [Order article via Infotrieve] |
20. | Kennedy, M. B. (1997) Trends Neurosci. 20, 264-268[CrossRef][Medline] [Order article via Infotrieve] |
21. | Sheng, M., and Lee, S. H. (2000) Nat. Neurosci. 3, 633-635[CrossRef][Medline] [Order article via Infotrieve] |
22. | Garner, C. C., Nash, J., and Huganir, R. L. (2000) Trends Cell Biol. 10, 274-280[CrossRef][Medline] [Order article via Infotrieve] |
23. | Cho, K. O., Hunt, C. A., and Kennedy, M. B. (1992) Neuron 9, 929-942[Medline] [Order article via Infotrieve] |
24. | Woods, D. F., and Bryant, P. J. (1991) Cell 66, 451-464[Medline] [Order article via Infotrieve] |
25. |
Kistner, U.,
Wenzel, B. M.,
Veh, R. W.,
Cases-Langhoff, C.,
Garner, A. M.,
Appeltauer, U.,
Voss, B.,
Gundelfinger, E. D.,
and Garner, C. C.
(1993)
J. Biol. Chem.
268,
4580-4583 |
26. |
Willott, E.,
Balda, M. S.,
Fanning, A. S.,
Jameson, B.,
Itallie, C. V.,
and Anderson, J. M.
(1993)
Proc. Natl. Acad. Sci. U. S. A.
90,
7834-7838 |
27. |
Fanning, A. S.,
and Anderson, J. M.
(1999)
J. Clin. Invest.
103,
767-772 |
28. |
Jaulin-Bastard, F.,
Saito, H.,
Le Bivic, A.,
Ollendorff, V.,
Marchetto, S.,
Birnbaum, D.,
and Borg, J.-P.
(2001)
J. Biol. Chem.
276,
15256-15263 |
29. |
Mei, L.,
Doherty, C. A.,
and Huganir, R. L.
(1994)
J. Biol. Chem.
269,
12254-12262 |
30. | Harlow, E., and Lane, D. (1993) Antibodies: A Laboratory Manual , Cold Spring Harbor Laboratory, Cold Spring Harbor, NY |
31. | Tanowitz, M., Si, J., Yu, D.-H., Feng, G.-S., and Mei, L. (1999) J. Neuorsci. 19, 9426-9435 |
32. | Gilmour, B. P., Goldman, D., Chahine, K. G., and Gardner, P. D. (1995) Dev. Biol. 168, 416-428[CrossRef][Medline] [Order article via Infotrieve] |
33. | Blackstone, C. D., Moss, S. J., Martin, L. J., Levey, A. I., Price, D. L., and Huganir, R. L. (1992) J. Neurochem. 58, 1118-1126[Medline] [Order article via Infotrieve] |
34. | Bradford, M. M. (1976) Anal. Biochem. 72, 248-254[CrossRef][Medline] [Order article via Infotrieve] |
35. |
Apperson, M. L.,
Moon, I. S.,
and Kennedy, M. B.
(1996)
J. Neurosci.
16,
6839-6852 |
36. |
Walikonis, R. S.,
Oguni, A.,
Khorosheva, E. M.,
Jeng, C.-J.,
Asuncion, F. J.,
and Kennedy, M. B.
(2001)
J. Neurosci.
21,
423-433 |
37. |
Songyang, Z.,
Fanning, A. S.,
Fu, C.,
Xu, J.,
Marfatia, S. M.,
Chishti, A. H.,
Crompton, A.,
Chan, A. C.,
Anderson, J. M.,
and Cantley, L. C.
(1997)
Science
275,
73-77 |
38. |
Kim, E.,
Naisbitt, S.,
Hsueh, Y. P.,
Rao, A.,
Rothschild, A.,
Craig, A. M.,
and Sheng, M.
(1997)
J. Cell Biol.
136,
669-678 |
39. | Muller, B. M., Kistner, U., Kindler, S., Chung, W. J., Kuhlendahl, S., Fenster, S. D., Lau, L. F., Veh, R. W., Huganir, R. L., Gundelfinger, E. D., and Garner, C. C. (1996) Neuron 17, 255-265[Medline] [Order article via Infotrieve] |
40. | Zhu, X., Lai, C., Thomas, S., and Burden, S. J. (1995) EMBO J. 14, 5842-5848[Abstract] |
41. | Hsueh, Y. P., Kim, E., and Sheng, M. (1997) Neuron 18, 803-814[Medline] [Order article via Infotrieve] |
42. | Sheng, M., and Pak, D. T. (2000) Annu. Rev. Physiol. 62, 755-778[CrossRef][Medline] [Order article via Infotrieve] |
43. | Morris, J. K., Lin, W., Hauser, C., Marchuk, Y., Getman, D., and Lee, K. F. (1999) Neuron 23, 273-283[Medline] [Order article via Infotrieve] |
44. | Tanowitz, M. B., and Mei, L. (1996) Brain Res. 712, 299-306[CrossRef][Medline] [Order article via Infotrieve] |
45. | Legouis, R., Gansmuller, A., Sookhareea, S., Bosher, J. M., Baillie, D. L., and Labouesse, M. (2000) Nat. Cell Biol. 2, 415-422[CrossRef][Medline] [Order article via Infotrieve] |
46. | Bilder, D., and Perrimon, N. (2000) Nature 403, 676-680[CrossRef][Medline] [Order article via Infotrieve] |
47. | Froehner, S. C. (1993) Annu. Rev. Neurosci. 16, 347-368[CrossRef][Medline] [Order article via Infotrieve] |
48. | Fischbach, G. D., and Rosen, K. M. (1997) Annu. Rev. Neurosci. 20, 429-458[CrossRef][Medline] [Order article via Infotrieve] |
49. | Goodearl, A. D. J., Yee, A. G., Sandrock, A. W., Jr., Corfas, G., and Fischbach, G. D. (1995) J. Cell Biol. 130, 1423-1434[Abstract] |
50. | Jo, S. A., Zhu, X., Marchionni, M. A., and Burden, S. J. (1995) Nature 373, 158-161[CrossRef][Medline] [Order article via Infotrieve] |
51. | Corfas, G., Falls, D. L., and Fischbach, G. D. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 1624-1628[Abstract] |
52. | Tansey, M. G., Chu, G. C., and Merlie, J. P. (1996) J. Cell Biol. 134, 465-476[Abstract] |
53. |
Altiok, N.,
Altiok, S.,
and Changeux, J. P.
(1997)
EMBO J.
16,
717-725 |
54. |
Sandrock, A. W.,
Dryer, S. E.,
Rosen, K. M.,
Gozani, S. N.,
Kramer, R.,
Theill, L. E.,
and Fischbach, G. D.
(1997)
Science
276,
599-603 |
55. | Won, S., Si, J., Colledge, M., Ravichandran, K. S., Froehner, S. C., and Mei, L. (1999) J. Neurochem. 73, 2358-2368[CrossRef][Medline] [Order article via Infotrieve] |
56. |
Colledge, M.,
and Froehner, S. C.
(1997)
J. Neurosci.
17,
5038-5045 |
57. | Rafael, J. A., Hutchinson, T. L., Lumeng, C. N., Marfatia, S. M., Chishti, A. H., and Chamberlain, J. S. (1998) Neuroreport 9, 2121-2125[Medline] [Order article via Infotrieve] |
58. | Luck, G., Hoch, W., Hopf, C., and Blottner, D. (2000) Mol. Cell. Neurosci. 16, 269-281[CrossRef][Medline] [Order article via Infotrieve] |