From the Department of Pharmacology, University of Wisconsin,
Madison, Wisconsin 53706-1532 and the Neurobiology
Research Center, University of Alabama,
Birmingham, Alabama 35213-0021
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ABSTRACT |
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Rapid glutamatergic synaptic transmission is
mediated by ionotropic glutamate receptors and depends on their precise
localization at postsynaptic membranes opposing the presynaptic
neurotransmitter release sites. Postsynaptic localization of
N-methyl-D-aspartate-type glutamate receptors
may be mediated by the synapse-associated proteins (SAPs) SAP90,
SAP102, and chapsyn-110. SAPs contain three PDZ domains that can
interact with the C termini of proteins such as
N-methyl-D-aspartate receptor subunits that
carry a serine or threonine at the -2 position and a valine,
isoleucine, or leucine at the very C terminus (position 0). We now show
that SAP97, a SAP whose function at the synapse has been unclear, is
associated with -amino-3-hydroxy-5-methylisoxazole-4-propionic acid
(AMPA)-type glutamate receptors. AMPA receptors are probably tetramers
and are formed by two or more of the four AMPA receptor subunits
GluR1-4. GluR1 possesses a C-terminal consensus sequence for
interactions with PDZ domains of SAPs. SAP97 was present in AMPA
receptor complexes immunoprecipitated from detergent extracts of rat
brain. After treatment of rat brain membrane fractions with the
cross-linker dithiobis(succinimidylpropionate) and solubilization with
sodium dodecylsulfate, SAP97 was associated with GluR1 but not GluR2 or
GluR3. In vitro experiments with recombinant proteins
indicate that SAP97 specifically associates with the C terminus of
GluR1 but not other AMPA receptor subunits. Our findings suggest that SAP97 may be involved in localizing AMPA receptors at postsynaptic sites through its interaction with the GluR1 subunit.
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INTRODUCTION |
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The prevailing excitatory neurotransmitter in the mammalian brain
is glutamate (1, 2). Upon its release from presynaptic sites, this
neurotransmitter binds to ionotropic glutamate receptors that mediate
rapid excitatory synaptic transmission in the mammalian brain (1, 2).
Several immuno-electron microscopic studies have demonstrated that
ionotropic glutamate receptors are clustered at postsynaptic sites of
excitatory synapses (3-5). Two major glutamate receptor families
exist, namely N-methyl-D-aspartate (NMDA)1 receptors, which
mediate Ca2+ influx, and non-NMDA receptors, which are
usually not Ca2+-permeable (1, 2, 6). Non-NMDA receptors
are further divided into
-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors
and kainate receptors. At low frequency, synaptic transmission normally
depends nearly exclusively upon AMPA receptors. On the other hand,
kainate and NMDA receptors require higher frequencies for activation.
NMDA receptor-mediated Ca2+ influx is necessary for
different forms of synaptic plasticity, such as long term potentiation
(1, 7, 8). At different synapses in the hippocampus and in other brain
areas, a few bursts of high frequency electric stimulation that
activate NMDA receptors induce a long lasting increase in synaptic
transmission, the hallmark of long term potentiation.
Glutamate receptors are thought to be heterotetramers consisting of homologous subunits (39). AMPA receptors are formed by two or more of the four AMPA receptor subunits designated GluR1-4 (9). NMDA receptors are heterooligomers composed of one or two NR1 and two or three NR2 subunits. One NR1 subunit and four different NR2 subunits (NR2A-D) have been identified (6, 10, 11). Hydrophobicity plots indicated the presence of four hydrophobic regions termed M1-M4 in all glutamate receptor subunits. The N terminus is extracellular and is followed by the transmembrane region M1. There is now strong evidence that the hydrophobic region M2 of glutamate receptors loops only partially into the plasma membrane and then back into the cytosol. M3 and M4 are transmembrane regions. The C terminus is localized on the intracellular side of the plasma membrane (12, 13).
Synapse-associated proteins (SAPs) constitute a family of closely
related proteins that has been implicated in the process of clustering
NMDA receptors at postsynaptic sites (14-17). A prototypic SAP
consists of three PDZ domains in the N-terminal part followed by an SH3
domain and a guanylate kinase-like domain in the C-terminal region. The
guanylate kinase-like domain does not appear to be catalytically active
(18) but interacts with another family of structural proteins known as
guanylate kinase-associated proteins or SAP90/PSD-95-associated
proteins (19, 20). SAP90/PSD-95 (21, 22), chapsyn-110/PSD-93 (15, 23),
and SAP102 (24, 25) directly bind to the very C termini of NMDA
receptor subunits (14, 15, 24, 25). These interactions are mediated by
the first and second PDZ domain of these SAPs and require the presence of a valine, isoleucine, or leucine at the very C-terminal position (designated as the 0 position) of the NMDA receptor subunits (17, 26).
In addition, serine or threonine has to be present at the 2 position,
which is two amino acids upstream of the C-terminal 0 position (17,
26).
GluR1 possesses a threonine at the C-terminal 2 position and leucine
at the 0 position. The C terminus of GluR1, thereby, constitutes a
consensus site for SAP binding via interaction with PDZ domains (17,
26). SAP97, another member of the SAP family (27), does not
coimmunoprecipitate with solubilized NMDA receptors, in contrast to
SAP90/PSD-95, chapsyn-110/PSD-93, and SAP102 (28). In addition, the
synaptic function of SAP97 has been unclear. Here we show that SAP97 is
associated with AMPA receptors in vivo. It binds to the
GluR1 subunit and does not appear to directly interact with the GluR2
or GluR3 subunits. Our findings raise the possibility that SAP97 may be
important for AMPA receptor localization at synaptic junctions.
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EXPERIMENTAL PROCEDURES |
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Immunoprecipitation and Immunoblotting-- Crude membrane fractions were prepared from rat brains in the presence of protease inhibitors, washed by resuspension and re-centrifugation in buffer A (150 mM NaCl, 10 mM Tris-Cl, pH 7.4, 10 mM EDTA, 10 mM EGTA), solubilized in buffer A containing protease inhibitors and 1% of either deoxycholate or Triton X-100 or SDS, cleared by ultracentrifugation, and used for immunoprecipitation and subsequent immunoblotting as described (29). For immunoprecipitation after SDS solubilization, a 7-fold volume of a 1% Triton X-100 solution in buffer A was added. This procedure converts the pure SDS micelles into mixed micelles containing mainly Triton X-100 and a much smaller fraction of SDS. The result is a significant reduction in the potential of SDS to dissolve and denature proteins, allowing the addition of antibodies for immunoprecipitation (30, 31). For chemical cross-linking, membrane fractions were washed by resuspension in 10 mM HEPES-NaOH, pH 7.0, followed by centrifugation and then incubated for 10 min on ice with 200 µM of the cross-linking agent dithiobis(succinimidylpropionate) (DSP). The reaction was stopped by adding Tris-Cl, pH 7.4, to a final concentration of 100 mM before solubilization with SDS. Subsequent immunoprecipitation and immunoblotting were performed as described earlier (29).
NMDA receptors were precipitated using antibody 54.2 (Affinity Chromatography Using GST Fusion Proteins-- For in vitro interaction assays, SAPs and the C termini of GluR1, GluR2, and GluR4 were expressed as recombinant GST fusion proteins in E. coli. Expression constructs for the GST-SAP90, GST-SAP97, and GST-SAP102 fusion proteins are described in Muller et al. (24). SAP97 was also expressed in E. coli as recombinant protein without a tag fused to it using the pRK174 vector (27). To express the final 10 C-terminal amino acids of GluR1, GluR2, or GluR4 fused to the C terminus of GST, complimentary sense and antisense oligonucleotides encoding the respective sequences with engineered endonuclease restriction site overhangs for BamHI added on the 5' end and EcoRI on the 3' end were annealed and ligated into a BamHI- and EcoRI-digested expression vector pGEX-4T-1 (Amersham). The oligonucleotides used were: 5'-GA.TCC.TCA.GGG.ATG.CCC.TTG.GGA.GCC.ACA.GGA.TTG.TAG-3' (GluR1 sense); 5'-A.ATT.CTA.CAA.TCC.TGT.GGC.TCC.CAA.GGG.CAT.CCC.TGA.G-3' (GluR1 antisense); 5'-GA.TCC.AAC.GTA.TAT.GGC.ATC.GAG.AGT.GTT.AAA.ATT.TAG-3' (GluR2 sense); 5'-A.ATT.CTA.AAT.TTT.AAC.ACT.CTC.GAT.GCC.ATA.TAC.GTT.G-3' (GluR2 antisense); 5'- GA.TCC.GGA.TTG.GCT.GTC.ATT.GCA.TCG.GAC.CTA.CCA.TAG-3' (GluR4 sense); and 5'-A.ATT.CTA.TGG.TAG.GTC.CGA.TGC.AAT.GAC.AGC.CAA.TCC.G (GluR4 antisense). Please note that stop codons are underlined and that the ten codons immediately upstream of these stop codons encode the very C-terminal amino acids of each of the corresponding AMPA receptor subunits. All constructs were verified by DNA sequence analysis and transformed into the protease-deficient E. coli strain BL21 (Novagen) for protein production.
GST fusion proteins were expressed and purified according to the protocols suggested by the manufacturer (Amersham). Briefly, overnight cultures from single colonies were grown in 50 ml of LB medium containing 100 µg/ml ampicillin at 37 °C with aeration to saturation, diluted 1:10 with LB medium, and incubated under the same conditions for 2-4 h until the culture reached an A600 of about 1.0. For induction, isopropyl- ![]() |
RESULTS |
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Solubilization of SAPs and Glutamate Receptors--
NMDA receptor
complexes can be solubilized from synaptic membranes with ionic
detergents such as deoxycholate or SDS but not with the nonionic
detergent Triton X-100 (Fig. 1,
NR2A/B) (11). Similarly, SAP90/PSD-95, SAP102, and
chapsyn-110/PSD-93 can only be efficiently extracted from synaptic
membranes with SDS (25) or deoxycholate but not with Triton X-100 (Fig.
1 and data not shown). Therefore, we tested whether these SAPs are
associated with NMDA receptors after solubilization with deoxycholate.
Immunoprecipitation of NMDA receptors with the NR1 antibody resulted
in the coprecipitation of proteins immunoreactive with antibodies
against SAP102 (Fig. 2, lane
2) or chapsyn-110/PSD-93 (data not shown). Similarly, SAP90/PSD-95, SAP102, and chapsyn-110/PSD-93 immunoreactive proteins coimmunoprecipitate with NMDA receptors solubilized in SDS extracts (25, 28).2 These results
indicate that NMDA receptors can form complexes with SAP90/PSD-95,
SAP102, and chapsyn-110/PSD-93 that are resistant to dissociation by
SDS and deoxycholate.
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SAP97 Coimmunoprecipitates with the AMPA Receptor Complex Extracted
with Triton X-100--
The synaptic function of SAP97, the fourth SAP
family member, has not been elucidated. Like AMPA receptors, SAP97 is
readily extracted by Triton X-100 (Fig. 1). Furthermore, one of the
AMPA receptor subunits possesses an established C-terminal consensus sequence for PDZ domain binding. We, therefore, tested whether SAP97 is
associated with AMPA receptors. Triton X-100 solubilizes AMPA receptors
as a complex containing several different AMPA receptor subunits (9).
Immunoprecipitation of Triton X-100-solubilized AMPA receptor complexes
with either GluR1 or
GluR2/3 antibodies resulted in
coprecipitation of a protein immunoreactive to SAP97 antibodies (Fig.
2, lanes 4 and 5). Immunoprecipitation with
control antibodies (see "Experimental Procedures") did not yield
SAP97-immunoreactive bands (Fig. 2, lane 6), thus excluding
the possibility that SAP97 directly bound nonspecifically either to the
resin or to antibodies not directed against the AMPA receptor-SAP97
complex.
SAP97 Coprecipitates with GluR1 but Not GluR2 or GluR3 after
Cross-linking and Solubilization with SDS--
After solubilization of
brain extracts with SDS, GluR1 immunoprecipitated GluR1 (Fig.
3A, lane 1) but not
GluR2/3 (lane 4). Similarly, precipitation with
GluR2/3
yielded GluR2/3 but no GluR1 immunoreactivity (Fig. 3A,
lanes 5 and 2, respectively). Accordingly, SDS
dissociates the different AMPA receptor subunits from each other. To
investigate whether SAP97 may interact with GluR1, membrane fractions
were treated with DSP. DSP is a homobifunctional cross-linker
containing an internal disulfide bridge. The disulfide bridge allows
cleavage by reducing agents and subsequent detection of the DSP-linked
proteins at their normal apparent molecular masses observed without
cross-linking. After cross-linking with DSP, the membrane fractions
were extracted with SDS to dissociate GluR1 from GluR2 and GluR3. Under
these conditions, SAP97 immunoreactivity coprecipitated with GluR1 but
not with GluR2/3 nor with NMDA receptor immunocomplexes or with control
antibodies (Fig. 3B). These findings indicate that DSP
specifically linked SAP97 to the GluR1 subunit, suggesting that GluR1
directly interacts with SAP97 in vivo. In contrast, SAP102
immunoreactivity that coprecipitated with the NMDA receptor complex was
not associated with immunoprecipitated GluR1, GluR2, or GluR3 subunits
following the same protocol (Fig. 3C). Accordingly, DSP does
not cross-link AMPA receptor subunits to those SAPs that are known to
be associated with NMDA receptors rather than AMPA receptors. This
control corroborates the specificity of GluR1-SAP97 cross-linking by
DSP.
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GluR1 Directly Associates with SAP97--
To test whether GluR1
can directly bind to SAP97, AMPA receptor subunits were dissociated
with SDS, SDS was diluted with Triton X-100, and the extracts were
applied to affinity chromatography on glutathione-Sepharose loaded with
either GST-SAP90, GST-SAP97, or GST-SAP102. Similar amounts of these
three fusion proteins were present on the affinity resins as confirmed
by SDS-polyacrylamide gel electrophoresis and Coomassie staining (Fig.
4B). Only GluR1 but not GluR2
or GluR3 bound to GST-SAP97 (Fig. 4A, lanes 3 and 9, respectively). None of these AMPA receptor subunits
associated with GST-SAP90 or GST-SAP102, indicating that the
interaction between GluR1 and GST-SAP97 is specific with respect to the
AMPA receptor subunit as well as the SAP. Of note, when equal amounts of the same extract were probed by immunoblotting with GluR1 and
GluR2/3 antibodies, immunostaining of similar intensity was observed
on the same exposures (Fig. 4C). Thus, the detection sensitivity for GluR1 and for GluR2/3 was very similar, and the lack of
GluR2/3 immunoreactivity after affinity chromatography with the GST-SAP
fusion proteins was not due to a difference in sensitivity of the
respective antibodies.
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SAP97 Specifically Binds to the C-terminal End of GluR1--
GST
fusion proteins carrying the sequence of the 10 C-terminal amino acids
of GluR1, GluR2, or GluR4 at their C termini were produced and purified
(Fig. 5A). Note that GluR1
possesses a threonine in the 2 position and a leucine in the 0 position, a consensus sequence for interaction with SAPs. GluR2 carries
a serine in the
3 rather than the
2 position and in
vitro also binds to PDZ domains, namely to PDZ domain 4 and 5 of
glutamate receptor interacting protein (GRIP) (Ref. 34; see
"Discussion"). The C terminus of GluR2 was included in our studies
to test for the specificity of SAP97 association with C-terminal
sequences capable of binding to PDZ domains. The very C-terminal amino
acid of GluR4 is proline, and the C-terminal GluR4 sequence is not
expected to interact with PDZ domains. Therefore, the C terminus of
GluR4 was chosen as a likely nonspecific negative control.
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DISCUSSION |
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Evidence for the Specific Interaction between GluR1 and SAP97 in
Vivo--
Our studies show that AMPA receptor complexes solubilized
with Triton X-100 contain SAP97. After dissociation of AMPA receptor subunits with SDS, GluR1, but not GluR2 or GluR3, binds to recombinant SAP97 rather than to SAP90/PSD-95 or to SAP102, two SAPs that are known
to interact with NMDA receptor subunits. Similar to most other proteins
that have been shown to associate with PDZ domains, the interaction
between GluR1 and SAP97 is mediated by the C terminus of GluR1;
recombinant SAP97 specifically associated with a GST construct carrying
the C terminus of GluR1. The presence of SAP97 immunoreactivity in the
Triton X-100 solubilized AMPA receptor complex and the high degree of
specificity of the in vitro interactions between GluR1 and
SAP97 strongly argue that the interaction between GluR1 and SAP97
occurs in vivo. This interaction is corroborated by the
cross-linking experiments that demonstrate that SAP97 is closely
associated with GluR1 in the plasma membrane (Fig. 3; see below).
Furthermore, immunohistochemical characterization of SAP97 suggests
that it may be co-localized with AMPA receptors at the postsynaptic
site (27). Immuno-electron microscopy with SAP97 in combination with
a secondary antibody coupled to horseradish peroxidase showed that the
electron-dense reaction product of horseradish peroxidase was not only
associated with presynaptic nerve terminals but also to a significant
extent with postsynaptic sites. Accordingly, SAP97 may be present not
only at presynaptic but also at postsynaptic sites (27).
Physiological Relevance of GluR1-SAP97 Interaction--
Similar to
those SAPs that are associated with NMDA receptors, SAP97 may help to
localize GluR1-containing AMPA receptors at the synapse. GRIP has very
recently been identified as another structural protein that interacts
with AMPA receptors (34)2. GRIP contains seven PDZ domains
but is otherwise quite different from the SAPs described above. It
binds to the very C-terminal ends of GluR2 and GluR3 subunits, which
are nearly identical to each other (34). This binding is mediated by
the fourth and fifth PDZ domain of GRIP. The interaction between GRIP
and GluR2 or GluR3 requires that a serine or threonine is in the 3
position (Fig. 5A) (34). However, interactions between GRIP
and GluR2 or GluR3 have only been described in vitro or in
heterologous cell lines overexpressing these proteins (34). Therefore,
it is unclear whether GRIP is associated with AMPA receptors in
vivo.
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ACKNOWLEDGEMENTS |
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We thank Dr. R. Jahn, Max-Planck Institute for Biophysical Chemistry (Göttingen, Germany) for the antibody 54.2; Dr. R. J. Wenthold, National Institute of Deafness and Other Communication Disorders, National Institute of Health (Bethesda, MD) for the antibodies against GluR1, GluR2/3, GluR4, and NR2 subunits; Dr. J. W. Tracy, University of Wisconsin (Madison, WI) for the antibody against GST; and Dr. P. J. Bertics, University of Wisconsin (Madison, WI) for critically reading the manuscript.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Research Grant R01-NS35563 (to J. W. H.) and AG12978 (to C. C. G.), American Heart Association Research Grant 97-GS-74 (to J. W. H.), a Shaw Scientist Award (to J. W. H.), and a grant to the University of Wisconsin Medical School under the Howard Hughes Medical Institute Research Resources Program for Medical Schools.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: 3770 MSC, 1300 University Avenue, Dept. of Pharmacology, University of Wisconsin, Madison, WI 53706-1532. Tel.: 608-262-0027; Fax: 608-262-1257; E-mail: jwhell{at}facstaff.wisc.edu.
1
The abbreviations used are: NMDA,
N-methyl-D-aspartate; AMPA,
-amino-3-hydroxy-5-methylisoxazole-4-propionic acid; DSP,
dithiobis(succinimidylpropionate); GRIP, glutamate receptor-interacting
protein; GST, glutathione S-transferase; SAP, synapse-associated
protein.
2 A. S. Leonard and J. W. Hell, unpublished results.
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REFERENCES |
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