Identification of an Intramolecular Interaction between the SH3
and Guanylate Kinase Domains of PSD-95*
Aaron W.
McGee
and
David S.
Bredt§
From the Department of Physiology and Program in Neuroscience,
University of California at San Francisco,
San Francisco, California 94143-0444
 |
ABSTRACT |
Postsynaptic density-95 (PSD-95/SAP-90) is a
member of the membrane-associated guanylate kinase (MAGUK) family of
proteins that assemble protein complexes at synapses and other cell
junctions. MAGUKs comprise multiple protein-protein interaction motifs
including PDZ, SH3 and guanylate kinase (GK) domains, and these binding sites mediate the scaffolding function of MAGUK proteins. Synaptic binding partners for the PDZ and GK domains of PSD-95 have been identified, but the role of the SH3 domain remains elusive. We now
report that the SH3 domain of PSD-95 mediates a specific interaction with the GK domain. The GK domain lacks a poly-proline motif that typically binds to SH3 domains; instead, SH3/GK binding is a bi-domain interaction that requires both intact motifs. Although isolated SH3 and
GK domains can bind in trans, experiments with intact PSD-95 molecules indicate that intramolecular SH3/GK binding dominates and prevents intermolecular associations. SH3/GK binding is conserved in the related Drosophila MAGUK protein DLG but is not
detectable for Caenorhabditis elegans LIN-2. Many
previously identified genetic mutations of MAGUKs in invertebrates
occur in the SH3 or GK domains, and all of these mutations disrupt
intramolecular SH3/GK binding.
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INTRODUCTION |
Postsynaptic density-95
(PSD-95/SAP-90)1 is a major
protein constituent of the postsynaptic density (PSD) at excitatory
synapses and participates in assembly of the PSD (1-3). PSD-95 is a
member of a large family of membrane-associated guanylate kinase
(MAGUK) proteins, which play critical roles in regulating both
structure and signal transduction at sites of cell-cell contact (4-7). These functional roles for MAGUKs have been established by genetic studies of invertebrates. Mutations of discs large
(dlg), which encodes a MAGUK in Drosophila,
disrupt epithelial cell septate junctions and epithelial cell polarity
and cause overgrowth of the imaginal discs (6). A related MAGUK, LIN-2
in Caenorhabditis elegans, is also essential in regulating
cell junctions and differentiation (7). lin-2 is required
for polarized sorting of a receptor tyrosine kinase in C. elegans vulval epithelial cell precursor cells (8). In
lin-2 mutant worms, vulval precursor cells do not respond to
an inductive signal appropriately, preventing vulval formation (7).
MAGUK proteins regulate cell junctions and cell differentiation by
functioning as molecular scaffolds that organize intracellular signaling pathways (9). This scaffolding function is explained by the
structure of MAGUK proteins, which comprise multiple protein-protein interaction domains. MAGUKs contain one or three PDZ domains, an SH3
domain, and a region homologous to yeast guanylate kinase (GK) (1, 2,
6, 7). PDZ domains are modular protein-protein interaction motifs (3,
5, 9, 10). Concatenated PDZ domains from PSD-95 can help specify and
accelerate signal transduction at the synapse by interacting with
multiple components of a signaling pathway (9). Although functions for
the PDZ domains of MAGUKs are established, physiological roles for the
GK and SH3 domains are less clear (11, 12). The GK domains of MAGUKs
are highly homologous to the enzyme guanylate kinase, but catalytic
activity has not been reported. Instead, the GK domains of certain
MAGUKs have been characterized as protein-protein interaction
interfaces (13-15). The GK domain of PSD-95 binds with high affinity
to both microtubule-associated protein 1A (MAP1A) (15) and to a novel family of guanylate kinase associated proteins (GKAPs or
synapse-associated protein associated proteins (SAPAPs)) (13, 14).
Although the functional implications of MAP1A and GKAP interaction with
the GK domain are uncertain, genetic screens have identified several mutations in the GK domain of MAGUKs in invertebrates (7, 16), indicating that this region is critical for gene function.
Genetic studies have determined that the SH3 domains of MAGUKs also are
essential (12, 16); however, the molecular functions for the SH3
domains remain elusive. SH3 domains classically mediate protein-protein
interactions by binding to proline-rich sequences (17-19). Although
this function would appear to fit well with the scaffolding role for
MAGUKs, binding partners for their SH3 domains are unknown. Recent
studies have shown that SH3 domains in certain tyrosine kinases not
only mediate protein-protein interactions, but can also regulate
protein function through intramolecular interactions (20, 21).
Accordingly, we now identify a specific interaction between the SH3 and
GK domains within PSD-95. Binding studies with protein fragments of
PSD-95 demonstrate that the SH3 and GK domains can bind in
trans. However, experiments with intact PSD-95 molecules
indicate that intramolecular SH3/GK binding dominates, preventing
intermolecular associations. SH3/GK interactions are conserved and
occur in both PSD-95 and DLG. All previously identified genetic
mutations of the SH3 or GK domains in dlg disrupt the
intramolecular binding, correlating the critical functional role
for these domains with the SH3/GK interaction.
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EXPERIMENTAL PROCEDURES |
Antibodies--
Monoclonal antibodies to PSD-95 (clone 7E3-18;
Affinity Bioreagents) and green fluorescent protein (GFP)
(CLONTECH) and polyclonal antibodies to GKAP (14),
actinin-associated LIM protein (ALP) (22), and the GAL4 DNA binding
domain (Santa Cruz Biotechnology) have been previously characterized. A
polyclonal antiserum to the PDZ domains of PSD-95 was raised by
immunizing a sheep with a GST fusion protein of amino acids 1-386 of
rat PSD-95. Polyclonal antisera to the GK domain of PSD-95 were raised
by injecting rabbits with a 14-mer peptide (amino acids 698-711 of
PSD-95) coupled to keyhole limpet hemocyanin (KLH). Polyclonal antisera
to GFP were raised by injecting guinea pigs with a GST fusion of GFP. All antisera were affinity purified on Affi-Gel-10 columns charged with
the immunizing antigen.
Construction of cDNA Plasmids--
For yeast two-hybrid
experiments, domains of PSD-95 were amplified by PCR with primers
containing endonuclease restriction sites, and PCR products were
restricted with the appropriate enzymes and ligated into pGBT9 or
pGAD424 (CLONTECH). All constructs were sequenced
to confirm that inserted nucleotide sequences were correct. The primers
for PSD-95 sequences are as follows: PSD-95 SH3 sense, 5'-CGG-GAA-CAG-CTC-ATG-AAT-3'; PSD-95 SH3 antisense,
5'-GGA-GCC-CCA-GTC-CTT-GGC-3'; PSD-95 GK sense,
5'-AAG-GCC-AAG-GAC-TGG-GGC-TCC-3'; PSD-95 GK antisense,
5'-CTA-GAG-TCT-CTC-TCG-GGC-TGG-3'; and PSD-95 GK611sense, 5'-ACC-AGC-GTC-CAG-TCT-GTG-3'. The constructs containing the W470F mutation were assembled by sequential PCR with a codon changed in both
the sense and antisense primer from W = TGG to F = TTT: sense, 5'-GAA-GAG-TTT-TGG-CAA-GCA-CGG-CGG-GTG-CAC-TCC-3'; and antisense, 5'-GGA-GTG-CAC-CCG-CCG-TGC-TTG-CCA-AAA-CTC-TTC-3'. The SH3
(408-560) constructs (with or without point mutations) were subcloned
by excising the SH3 domain at the EcoRI site in the
polylinker and the BamHI site in the GK domain from the
SH3GK construct and ligating into pGBT9 at
EcoRI/BamHI. The constructs containing the SH3
L460P mutation were made by inverse PCR using SAP90:pGEX4T-1 as a
template with the following primers: sense, 5'-CAT-GTC-ATC-GAT-GCT-GGT-GAC-GAA-GAG-TGG-TGG-CAA-GCA-3'; and antisense, 5'-ACC-AGC-ATC-GAT-GAC-ATG-AGG-CAC-ATC-CCC-GAA-GCG-GAA-3'. Yeast two-hybrid constructs were then made by using this plasmid as a
template for the 95SH3 sense and antisense primers described above.
GK
N109 was constructed by subcloning the DNA fragment between the
BamHI site in the PSD-95 GK domain and the BamHI
site in the polylinker into the BamHI site in pGBT9.
Sequences from DLG, LIN-2, GKAP, and nNOS were also amplified by PCR
with primers containing endonuclease restriction sites, restricted with
the appropriate enzymes and ligated into pGBT9 or pGAD424. The primers
for DLG sequences are as follows: DLG GK sense,
5'-GCA-GCT-AAT-AAT-AAT-CGT-GAT-AAG-3'; DLG GK antisense, 5'-TCA-TAG-AGA-TTC-CTT-GGA-AGG-3'; DLG SH3 sense,
5'-AAA-CAA-CAG-GCT-GCC-CTC-3'; and DLG SH3 antisense,
5'-CTA-CTT-ATC-CAG-ATT-ATT-ATT-AGC-TGC-3'. The primers for LIN-2
sequences are as follows: LIN-2 SH3 sense, 5'-GAT-GCT-CGA-GGT-CAA-GTC-3'; LIN-2 SH3 antisense,
5'-CTA-GAA-CCA-CAT-GCA-GTG-AGT-3'; LIN-2 GK sense,
5'-ACT-CAC-TGC-ATG-TGG-TTC-3'; and LIN-2 GK antisense, 5'-TCA-GTA-GAC-CCA-AGT-GAC-TGG-3'. The primers for GKAP sequences are
as follows: GKAP sense, 5'-ATG-ATC-GAC-CTT-TTT-AAG-GCT-3'; and GKAP
antisense, 5'-CTG-TAT-CCC-AAT-AGA-TAG-GCA-3'. The nNOS and PSD-95
PDZ1-3 constructs were previously described (4). The C-terminal GFP
fusion constructs were subcloned by ligating a PCR-amplified sequence
of PSD-95 SH3 or GK (with the primers described above) into EGFP-C2
(CLONTECH).
Yeast Two-hybrid Analysis--
Yeast co-transformation, colony
lift
-galactosidase filter assays, and ONPG liquid culture assays
were done as described in the Matchmaker Library protocol
(CLONTECH) with the yeast strain HF7c or SFY526.
Growth on His(
) was tested by streaking several individual colonies
to
LWH plates. Positives were scored by the presence of individual
colonies after 3 days at 30 °C.
Cell Transfection--
HEK 293 cells were transiently
transfected with EGFP-C2 constructs using LipofectAMINE Plus (Life
Technologies). Transfections were done in six-well culture plates with
cells at 50-80% confluence. Two µg of plasmid DNA were used for
each transfection. Six h after transformation, the media was changed to
Dulbecco's modified Eagle's medium-21 plus 10% heat-inactivated
fetal bovine serum, and the cells were harvested 48 h later.
In Vitro Binding Assays with GST Fusion Proteins--
To test
for interactions between SH3 and GK domains in vitro, GST
fusion proteins were incubated with HEK cell extracts expressing GFP
fusion proteins. GST fusion proteins were expressed and purified as
described previously (23). Extracts from transfected HEK 293 cells were
prepared from a six-well plate. Cells were washed with 1 ml of
phosphate-buffered saline and then scraped from the substrate into
resuspension buffer: 25 mM Tris-HCl (pH 7.5), 150 mM NaCl, 5 mM EDTA, 5 mM EGTA.
Cells were sheared with 10 passes through a 25-gauge needle.
Phenylmethylsulfonyl fluoride was added to 1 mM, and Triton
X-100 was added to 1%. The solution was gently agitated at 4 °C for
30 min and then centrifuged for 20 min at 14,000 rpm in a
microcentrifuge. The soluble fraction of the extracts was removed to
new tubes. Three µg of GST protein coupled to Sepharose beads was
added, and the samples were incubated at 4 °C for 60 min. The
protein-coupled GST beads were pelleted by centrifugation and then
washed 5 times with 1 ml of resuspension buffer. The beads were
resuspended in 5× protein loading buffer, and samples were separated
by SDS-polyacrylamide gel electrophoresis with 12% acrylamide gels,
transferred to polyvinylidene difluoride membranes (Millipore) and
analyzed by Western blotting as described previously (4).
Immunoprecipitations--
Adult rat brain was homogenized in 20 volumes of resuspension buffer. After centrifugation at 15,000 × g to remove the soluble fraction, membranes were solubilized
for 30 min in resuspension buffer + 0.5% Triton (Triton Extracts) or + 0.2% SDS (SDS extracts). Triton X-100 was then added to 1% to the SDS
extracts to sequester the ionic detergent. Solubilized proteins were
recovered by centrifuging the extracts at 100,000 × g
for 30 min. Immunoprecipitations were performed by first adding the
appropriate antibody, and then, after a 60-min incubation at 4 °C,
adding 20 µl of protein G-Sepharose to precipitate the antibodies.
The protein G beads were then washed five times with resuspension
buffer and immunoprecipitated proteins were recovered in 5× protein
loading buffer. Immunoprecipitates were resolved by 10%
SDS-polyacrylamide gel electrophoresis and analyzed by immunoblotting.
 |
RESULTS |
Because SH3 domains can regulate protein function through
intramolecular interactions (20, 21), we determined whether the SH3
domain of PSD-95 might associate with other domains from the protein.
We first evaluated this possibility using the yeast two-hybrid system.
Yeast strains HF7c and SFY526 were transformed with appropriate
GAL4-fusion expression vectors encoding the SH3 domain of PSD-95
together with either the PDZ, SH3, or GK domains. Using both an assay
for
-galactosidase activity and an assay for growth on His(
)
plates, we found that the SH3 domain specifically interacts with the GK
domain and that the SH3 domain does not interact with the PDZ or SH3
domains (Fig. 1A). A
quantitative liquid culture assay indicated that this association is
robust and appears similar in magnitude to the PSD-95 PDZ domain
interaction with the PDZ domain of neuronal nitric-oxide synthase (4)
and to the PSD-95 GK domain interaction with GKAP (13, 14). To verify
that the SH3/GK interaction is authentic, we also evaluated binding of
protein fragments in vitro. We found that the SH3 domain of
PSD-95 fused to GFP, expressed in HEK 293 cells, binds specifically to a bacterial GST fusion protein of the GK domain. Similarly, the GK
domain of PSD-95, expressed in HEK cells, interacts with a GST-SH3
fusion protein (Fig. 1B).

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Fig. 1.
Interaction of the SH3 and GK domains of
PSD-95. A, yeast strains SFY526 were transformed with
expression vectors encoding various GAL4 DNA binding domain and GAL4
activation domain fusion proteins. The amino acid residues from PSD-95
encoded in each construct are shown in parentheses. Each
transformation was plated on synthetic dextrose plates lacking
tryptophan and leucine. Interactions were quantified using an ONPG
liquid culture assay. Values are representative of an experiment that
was repeated twice with similar results. B,
glutathione-Sepharose beads (25 µl) were charged with 3 µg of GST,
GST-SH3, or GST-GK protein and were incubated with extracts from HEK
cells expressing either GFP alone, GFP fused to the GK domain of PSD-95
(GFP-GK), or GFP fused to the SH3 domain of PSD-95
(GFP-SH3). After washing the beads, bound proteins were
eluted and analyzed by immunoblotting. Note that GFP-GK binds
specifically to GST-SH3 and that GFP-SH3 binds to GST-GK. The input
lanes represent 5% of the cell extract used for the binding assays.
Positions of molecular mass marker proteins (in kDa) are on the
left.
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The SH3/GK interaction identified here is atypical because the GK
domain lacks a P-X-X-P motif that typically binds
to SH3 domains (17-19). We therefore characterized the structural
requirements within the GK domain that are essential for interaction.
Using the yeast two-hybrid system, we found that an intact GK domain is
required to bind the SH3 domain. The GK domain in PSD-95 comprises amino acids 534-712 (1). Yeast transformed with constructs encoding GK
domains that were missing 36 amino acids from the N terminus or 13 amino acids from the C terminus do not turn blue in
-galactosidase
assays nor do they form colonies on synthetic media plates lacking
histidine (Table I).
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Table I
Structural requirements for the PSD-95 SH3/GK domain interaction
Yeast SFY526 and HF7c cells were co-transformed with expression vectors
encoding various GAL4 binding domain and GAL4 activation domain fusion
proteins. Each transformation mixture was plated on two synthetic
dextrose plates, one lacking tryptophan and leucine and the other
lacking tryptophan, leucine, and histidine. -Galactosidase activity
was scored as the time required for colonies of co-transformed yeast to
turn blue at 30 °C: ++, <60 min; +, 60-240 min; , no significant
activity. Growth on His( ) plates was positive if yeast streaked to
LWH plates produced isolated colonies within 3 days.
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We also characterized features of the SH3 domain that mediate GK domain
binding. Three-dimensional structures of SH3 domains are well
characterized and are conserved (18). Trp-470 of PSD-95 corresponds to
an amino acid in other SH3 domains that mediates interaction with
proline-containing peptides (23). Mutation of this tryptophan to
phenylalanine typically disrupts SH3/proline-containing peptide
interactions without compromising the SH3 domain structure (23), but we
found it does not abolish SH3/GK binding (Table I). One allele of
dlg, a Drosophila MAGUK, contains a point
mutation that changes a conserved leucine in the SH3 domain to proline (16). We introduced the corresponding mutation, L460P, into the PSD-95
SH3 domain constructs and found it disrupts the SH3/GK domain
interaction (Table I).
We next asked whether the SH3/GK domain interaction found in PSD-95 is
unique or is conserved in other MAGUK proteins. We tested for SH3/GK
binding in both Drosophila DLG (6), which is highly
homologous to PSD-95, and in C. elegans LIN-2 (7), a MAGUK
that is more distantly related. We found that the SH3 domain from DLG
interacts with its GK domain but that the SH3 and GK domains from LIN-2
do not (Fig. 2). Furthermore, SH3/GK domain binding was detected in SH3 and GK combinations between PSD-95
and DLG (Fig. 2). Our inability to detect SH3/GK interactions with the
protein domains from LIN-2 either reflects the lower affinity for this
interaction within LIN-2 or suggests that this interaction is not a
general feature of more distantly related MAGUK proteins.

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Fig. 2.
The SH3/GK domain interaction is
conserved between PSD-95 and DLG, but is not detectable with
LIN-2. Yeast strains SFY526 were transformed with plasmids
encoding combinations of SH3 and GK domains from PSD-95, DLG, and
LIN-2. Constructs encoding SH3 domains were fusions with the GAL4
activation domain, and those encoding GK domains were fusions with the
GAL4 DNA binding domain. Interactions were assayed with the
-galactosidase colony filter-lift assay. -Galactosidase activity
was scored as the time required for colonies of transformed yeast to
turn blue at 30 °C: ++, <60 min; +, 60-240 min; , no significant
activity.
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Intermolecular SH3/GK domain binding could mediate multimerization of
MAGUKs, whereas intramolecular SH3/GK binding could mediate a
regulatory interaction. To determine which mode of binding predominates, we evaluated binding of isolated SH3 or GK domains to
intact PSD-95 in vitro. We found that GST fusions of either the SH3 or GK domain alone did not bind to a full-length PSD-95-GFP fusion expressed in HEK 293 cells (Fig.
3B). Further, in yeast two-hybrid experiments, no binding was detected between isolated SH3 or
GK domains and SH3GK (Fig. 3A). This absence of binding may
indicate that intramolecular SH3/GK binding in intact PSD-95 molecules
is highly favored and prevents intermolecular associations. This
interpretation predicts that introducing mutations that disrupt the
intramolecular binding would facilitate intermolecular binding of
isolated SH3 or GK domains to intact PSD-95 or SH3GK. Indeed, we found
that an isolated GK domain interacts only with an SH3GK construct that
contains a C-terminal GK domain truncation (Fig. 3A). We
also determined, by yeast two-hybrid analysis and with GST-fusion
protein binding experiments, that an isolated SH3 domain can bind
full-length PSD-95 or SH3GK constructs that contain a disruptive L460P
point mutation in the SH3 domain (Fig. 3B). This latter
interaction is specific because an SH3GK construct with both the L460P
SH3 mutation and a truncation of the GK domain does not bind to SH3
(Fig. 3B)

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Fig. 3.
An intramolecular SH3/GK domain interaction
within PSD-95 prevents similar intermolecular interactions.
A, yeast strains HF7c were transformed with two-hybrid
constructs encoding various combinations of the SH3, GK, and SH3GK
domains of PSD-95. The amino acid residues from PSD-95 encoded in each
construct are shown in parentheses. Interactions between hybrid
proteins were assayed with the -gal colony filter lift assay and
growth on His( ) plates. -Galactosidase activity was scored as the
time required for colonies of transformed yeast to turn blue at
30 °C: ++, <60 min; +, 60-240 min; , no significant activity.
Growth on His( ) plates was scored as positive if yeast streaked to
LWH plates produced isolated colonies within 3 days. Note that the
SH3GK constructs do not bind SH3 or GK domains unless the corresponding
domain is mutated in SH3GK. B, glutathione-Sepharose beads
(25 µl) were charged with 3 µg of GST, GST-SH3, or GST-GK protein
and were incubated with extracts from HEK cells expressing 1)
GFP-PSD-95, 2) GFP-PSD95(L460P) containing the SH3 domain mutation, 3)
GFP-SH3GK, 4) GFP-SH3(L460P)GK containing the SH3 domain mutation, or
5) GFP-SH3(L460P)GK C25 containing both the SH3 domain mutation and
the GK C-terminal truncation. After washing the beads, bound proteins
were detected by Western blotting. The input lanes represent 5% of the
cell extract used for the binding assays. Positions of molecular mass
marker proteins (in kDa) are on the left.
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As an additional tool to evaluate the SH3/GK domain interaction in
PSD-95 protein extracted from brain, we generated an antibody to the
last 14 amino acids of the GK domain (amino acids 698-711). We find
that this antibody immunoprecipitates PSD-95 from whole brain
homogenates solubilized with 0.2% SDS (SDS extracts) but does not
immunoprecipitate PSD-95 from brain homogenates solubilized with 1%
Triton X-100 alone (Fig. 4A).
In contrast, another antibody directed to the PDZ domains
immunoprecipitates PSD-95 equally from both the SDS and Triton extracts
(Fig. 4A).

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Fig. 4.
Immunoprecipitations from whole brain
homogenate and transfected HEK 293 cells. PSD-95 was detected with
a monoclonal PSD-95 antibody (Affinity Bioreagents, Inc.). GFP-fusion
constructs were detected with a monoclonal antibody
(CLONTECH). Positions of marker proteins are given
on the left (in kDa). A, rat brain extracts were
solubilized with 1% Triton (Triton extracts) or with 0.2% SDS (SDS
extracts) and were immunoprecipitated with antibodies against either
ALP (negative control), the GK domain of PSD-95, or the PDZ domains of
PSD-95. Note that the PDZ antibody immunoprecipitates PSD-95 from both
Triton and SDS extracts whereas the GK antibody only immunoprecipitates
from SDS extracts. Extract lanes represent 10 and 5% of the
volume of Triton and SDS extracts used for immunoprecipitations,
respectively. B, Triton and SDS extracts were prepared from
HEK 293 cells expressing the GK domain of PSD-95 fused to GFP.
Immunoprecipitations and analyses were done as in panel A.
The extract lanes contain 5% of the extract used for each condition.
C, immunoprecipitations were performed with Triton and SDS
extracts prepared from transfected HEK 293 cells expressing PSD-95,
PSD-95 containing the SH3 mutation L460P (PSD-95(*)),
GFP-SH3GK, or GFP-SH3(*)GK with the L460P point mutation. The extract
lanes contain 5% of the extract used for each condition.
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The selective immunoprecipitation of PSD-95 by the GK domain
antibody in SDS extracts may indicate that the antibody only interacts
with a denatured epitope. Alternatively, the antigenic epitope of the
GK domain may not be accessible to the antibody under native conditions
because of intermolecular protein interactions, such as with the GKAPs
or MAP1A, or because of intramolecular interactions, such as with the
SH3 domain. To help distinguish between these possibilities, we
evaluated immunoprecipitation of PSD-95, SH3GK-GFP, or GK-GFP expressed
in HEK 293 cells, which lack GKAPs and MAP1A. The GK antibody avidly
immunoprecipitates GK-GFP in both SDS and Triton extracts (Fig.
4B), indicating that the GK antibody indeed can react with
the GK domain in Triton extracts. However, we find that the transfected
PSD-95 proteins containing both the SH3 and GK domains
immunoprecipitate with the GK antibody only from SDS extracts and not
from extracts solubilized with Triton X-100 alone (Fig. 4C).
These results would seem to indicate that, under native conditions, the
SH3 domain from PSD-95 masks the GK domain epitope. We next repeated
these transfection/immunoprecipitation experiments with constructs that
contained the SH3 L460P point mutation, which disrupts SH3/GK binding.
Remarkably, introduction of this SH3 domain mutation into constructs
containing the SH3 and GK domains restores antigenic accessibility of
the GK domain in Triton extracts (Fig. 4C). Taken together
with other data in this study, the simplest model consistent with these
results is that intramolecular binding between the SH3 and GK domains
of PSD-95 is sensitive to SDS denaturation and that the SH3/GK
interaction blocks antigenic access of the GK domain antibody. This
parsimonious interpretation would indicate that the intramolecular
SH3/GK domain interaction occurs to a significant extent within PSD-95
from both brain and heterologous cells.
To determine whether the SH3 domain interaction might regulate GK
binding to GKAP or MAP1A, we evaluated the association of wild-type and
mutant PSD-95 with GKAP. We co-transfected HEK cells with expression
constructs encoding GKAP (13, 14) together with either full-length
PSD-95, GFP-SH3GK, or with PSD-95 or GFP-SH3GK constructs containing
the SH3 L460P mutation, which disrupts SH3/GK binding. In extracts from
these cells, we immunoprecipitated PSD-95 and evaluated association of
GKAP (Fig. 5). We found that GKAP co-immunoprecipitates equally with all these PSD-95 constructs, indicating that SH3/GK binding does not influence GK binding
activity.

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Fig. 5.
Co-immunoprecipitation of GKAP is
unaffected by the SH3 mutation L460P. HEK 293 were
co-transfected with expression constructs for GKAP and either PSD-95,
PSD-95 containing the SH3 mutation L460P (PSD-95(*)),
GFP-SH3GK, or GFP-SH3(*)GK with the L460P point mutation. Cell extracts
were solubilized with 1.0% Triton. Immunoprecipitations of these
extracts were analyzed by immunoblotting for GKAP. The extract
lanes represent 5% of the cell extract used for
immunoprecipitations. Positions of molecular mass marker proteins (in
kDa) are on the left.
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DISCUSSION |
This work identifies a specific intramolecular association between
the SH3 and GK domains within PSD-95 and shows that this interaction is
conserved in other MAGUK proteins. Although isolated SH3 and GK domains
of PSD-95 can bind one another in trans, this intermolecular
interaction is not detected between intact PSD-95 molecules or fusion
proteins that contain both the SH3 and GK domain. Instead, an
intramolecular SH3/GK association appears to predominate within
full-length PSD-95 molecules, suggesting a regulatory rather than
scaffolding role for the interaction.
Several lines of evidence suggest that the intramolecular interaction
between the SH3 and GK domains identified here is likely to play a
physiological role in MAGUK function. First, SH3/GK binding is
conserved even between the distantly related MAGUK proteins PSD-95 and
DLG. Second, genetically identified mutations of the SH3 and GK domains
in dlg disrupt the intramolecular SH3/GK association. A
point mutation in the SH3 domain that disrupts SH3/GK binding was
identified by its strong phenotype (16). Conversely, C-terminal GK
domain truncations that resemble genetic mutations of dlg
disrupt the SH3 domain interaction. These truncations of DLG in
Drosophila are lethal in the absence of maternal
contribution (16). It is also intriguing that certain genetic mutants
of the SH3 and GK domains of dlg complement one another in
mixed heterozygous flies, negating the lethality of the recessive
mutations (16). Our data suggest that a MAGUK protein with a mutant SH3 domain will bind in trans to a MAGUK with a GK domain
mutation. If such a mixed bimolecular complex restores dlg
function, this could explain the genetic complementation that has been observed.
The SH3 domain interactions described here are atypical. SH3 domains
have classically been shown to interact with proline-containing motifs
that contain the consensus sequence P-X-X-P (17).
This binding model cannot explain the SH3/GK interactions, as GK
domains lack a P-X-X-P motif. Moreover, mutation
of the SH3 domain of PSD-95 at the conserved Trp-470 that normally
mediates interaction with proline-containing peptides (23) does not
disrupt SH3/GK binding. Rather than the SH3 domain recognizing a short
peptide sequence within the GK domain, our data suggest that SH3/GK
binding represents a bi-domain interaction and requires proper folding of both intact motifs.
The intramolecular interactions described here are reminiscent of
recent studies showing that intramolecular SH3 domain associations mediate autoinhibition of Src and Tec family tyrosine kinases (20, 21).
In the Src family kinase, Hck, the SH3 motif binds to and blocks the
catalytic activity of the adjacent tyrosine kinase domain. This
intramolecular SH3 domain interaction within Hck is displaced, and
tyrosine kinase activity is restored when the Hck SH3 domain binds to
an appropriate protein ligand in trans. By analogy, the
intramolecular SH3 domain interaction within PSD-95 may regulate the GK
domain. While we find that this interaction does not alter GK domain
binding to either GKAP (Fig. 5) or MAP1A (data not shown), the SH3
domain may regulate an as yet unidentified catalytic activity of the GK
domain. It is also possible that the intramolecular SH3/GK interaction
mediates functional interactions between the SH3 domain and other
unknown proteins, as studies with DLG in Drosophila have
suggested that the GK domain may act as a negative regulator of DLG in
controlling cell proliferation (12). Recent studies indicate that PDZ
domains within PSD-95 can negatively regulate GK binding activity (15),
though the PDZ domains themselves do not bind to GK (15). As the SH3
domain is interposed between the PDZ and GK domains, the SH3/GK
interaction described here could play a role in autoinhibition of GK
binding by PDZ domains. Alternatively, while our data best support a
model in which intramolecular associations between the SH3 and GK
domains predominate, it does not eliminate the possibility that other factors present in vivo may facilitate intermolecular
interactions that could contribute to the scaffolding functions of
PSD-95.
 |
ACKNOWLEDGEMENTS |
We thank Dr. Morgan Sheng for the GKAP
antibody and expression constructs and Dr. Wendell Lim for designing
the SH3 domain mutants.
 |
FOOTNOTES |
*
This work was supported by grants (to D. S. B.) from the
National Association for Research on Schizophrenia and Depression, the
Culpeper and Beckman Foundations, and the National Institutes of Health
(R01, GM36017).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.
A Howard Hughes Predoctoral Fellow.
§
To whom all correspondence should be addressed: University of
California at San Francisco School of Medicine, 513 Parnassus Ave.,
San Francisco, CA 94143-0444. Tel.: 415-476-6310; Fax: 415-476-4929; E-mail: bredt{at}itsa.ucsf.edu.
 |
ABBREVIATIONS |
The abbreviations used are:
PSD-95, postsynaptic
density-95;
ALP, actinin-associated LIM protein;
MAGUK, membrane-associated guanylate kinase;
GK, guanylate kinase;
DLG, discs
large;
PSD, postsynaptic density;
GKAP, guanylate kinase associated
protein;
MAP1A, microtubule-associated protein 1A;
GFP, green
fluorescent protein;
HEK, human embryonic kidney;
GST, glutathione
S-transferase;
PCR, polymerase chain reaction;
ONPG, O-nitrophenyl
-D-galactopyranoside.
 |
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