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The Journal of Neuroscience, October 1, 2001, 21(19):7506-7516
Synapse-Associated Protein 97 Selectively Associates with
a Subset of AMPA Receptors Early in their Biosynthetic Pathway
Nathalie
Sans,
Claudia
Racca,
Ronald S.
Petralia,
Ya-Xian
Wang,
Jennifer
McCallum, and
Robert J.
Wenthold
Laboratory of Neurochemistry, National Institute on Deafness and
other Communication Disorders, National Institutes of Health, Bethesda,
Maryland 20892-8027
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ABSTRACT |
The regulation of AMPA receptors at the postsynaptic membrane is a
fundamental component of synaptic plasticity. In the hippocampus, the
induction of long-term potentiation increases the delivery of
GluR1, a major AMPA receptor subunit in hippocampal pyramidal neurons,
to the synaptic plasma membrane through a mechanism that requires the
PDZ binding domain of GluR1. Synapse-associated protein 97 (SAP97), a member of the membrane-associated guanylate kinase family, is believed to associate with AMPA receptors (AMPARs) containing the GluR1 subunit, but the functional significance of these
interactions is unclear. We investigated the interaction of GluR1 with
SAP97, the only PDZ protein known to interact with GluR1. We find that
interactions involving SAP97 and GluR1 occur early in the secretory
pathway, while the receptors are in the endoplasmic reticulum or
cis-Golgi. In contrast, few synaptic receptors associate
with SAP97, suggesting that SAP97 dissociates from the receptor complex
at the plasma membrane. We also show that internalization of GluR1, as
triggered by NMDAR activation, does not require SAP97. These results
implicate GluR1-SAP97 interactions in mechanisms underlying AMPA
receptor targeting.
Key words:
SAP97; GluR1; trafficking; ER-cis-Golgi; postsynaptic density; hippocampus
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INTRODUCTION |
Ionotropic glutamate receptors
mediate most excitatory neurotransmission in the CNS. These receptors
are concentrated at postsynaptic densities (PSDs) of excitatory
synapses although a large population is also present in the cytoplasm
of neuron somata and dendrites (Petralia, 1997 ). AMPA-type ionotropic
glutamate receptors (AMPARs) include four subunits (GluR1-4) and form
functional heteromeric or homomeric complexes of four or five subunits
(Dingledine et al., 1999). GluR2 and GluR3 have a C-terminal
PDZ-binding site (-SVKI) through which they can associate with
PDZ domain-containing proteins such as glutamate
receptor-interacting protein (GRIP), AMPA-binding protein (ABP), and
PKC-interacting protein 1 (PICK1) (Dong et al., 1997; Srivastava
et al., 1998; Wyszynski et al., 1999; Xia et al., 1999). The last four
amino acids of the C terminus of GluR1 (-ATGL) interact with the PDZ
domains of SAP97 (Leonard et al., 1998). Also, GluR2 interacts with
N-ethylmaleimide-sensitive factor (Nishimune et al.,
1998; Osten et al., 1998; Song et al., 1998) and GluR1 with
cytoskeletal protein 4.1 homologs (Shen et al., 2000a). These
interacting proteins have been implicated in targeting and synaptic
clustering of AMPARs, although the mechanisms involved are poorly
understood. Native AMPARs, as heteromeric complexes, contain several
potential interacting domains exposed to the cytoplasm. This raises the
possibility of simultaneous, competing, or sequential interactions with
other proteins.
Current evidence indicates that AMPAR trafficking plays a prominent
role in regulating synaptic efficacy. The number and type of AMPARs
present at the PSD can be rapidly altered by synaptic activity through
endocytosis and exocytosis of receptors associated with the plasma
membrane or intracellular membranes. Understanding the coordination
between AMPAR trafficking events and the various known and unknown
interacting proteins is key to understanding the regulation of synaptic
function at excitatory synapses.
GluR1 is a major AMPAR subunit in hippocampal pyramidal neurons,
and by expressing tagged green fluorescent protein-GluR1 subunits in pyramidal cells of hippocampal slices, it was shown that
long-term potentiation (LTP) or increased activity of
Ca2+/calmodulin-dependent protein kinase II (CaMKII)
increased delivery of GluR1 to synapses (Shi et al., 1999; Hayashi et
al., 2000). This effect was diminished by mutating the PDZ interacting
domain of GluR1, although this same mutation had no effect on basal
synaptic transmission. Therefore, the interaction of GluR1 with a PDZ
domain-containing protein may be critical for activity-induced
increases in synaptic AMPARs but not required for constitutive addition
of receptors. SAP97 may play a role in regulating AMPAR addition
to hippocampal pyramidal cell synapses because it interacts with the
C-terminal PDZ-interacting domain of GluR1 (Leonard et al., 1998). The
report of this interaction initially appeared to have limited
functional significance because SAP97 was earlier reported to be
presynaptic (Müller et al., 1995), whereas GluR1 is predominantly
postsynaptic (Petralia, 1997). However, it was recently shown that
SAP97 is present at postsynaptic sites in cerebral cortex (Valtschanoff et al., 2000). In the present study we investigated the role of SAP97
in organizing and processing AMPARs in intact rat hippocampus. Although
both biochemical and immunocytochemical results show that SAP97 is
associated with the PSD, we find that most of the SAP97 is present in
the cytoplasm and that it preferentially co-immunoprecipitates with
immature AMPAR complexes. Our results support a model in which SAP97
associates with GluR1-GluR2 containing AMPARs while they are in
the endoplasmic reticulum-cis-Golgi (ER-CG), with SAP97
dissociating from the complex at the plasma membrane.
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MATERIALS AND METHODS |
Antibodies. The following primary antibodies were
used for immunoprecipitation (IP), immunoblotting, and
immunocytochemistry. Rabbit polyclonals to: SAP97 (JH62426, Sans et
al., 2000; JH62428, Valtschanoff et al., 2000); SAP97 (PA1-066,
PA1-741; Affinity BioReagents, Golden, CO); GluR1, GluR2/3, GluR3, and
GluR4 (Wenthold et al., 1992); GluR2 (Petralia et al., 1997); PSD-95
(T60, JH62092; Sans et al., 2000); and calnexin (Rubio and Wenthold,
1999a). Mouse monoclonals to: GluR1 and GluR2-3 (Ottiger et al.,
1995); NR1 (clone 54.1; PharMingen, San Diego, CA); NR2B, EEA1, and
Rab4 (Transduction Laboratories, Lexington, KY), PSD-95 (7E1-1B8;
Affinity BioReagents), synaptophysin and tubulin (Boehringer Mannheim, Indianapolis, IN); and BiP and Lamp 1 (StressGen, Victoria, British Columbia, Canada).
Preparation of membranes and subcellular fractionation.
Total hippocampal membranes were obtained from postnatal day 35 (P35) Sprague Dawley rats by homogenizing in buffer A [20
mM HEPES, pH 7.4, 150 mM
NaCl, containing a cocktail of protease inhibitors (2 mM EDTA, 1 mM
4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF), 50 µg/ml leupeptin, 10 µg/ml pepstatin, and 10 µg/ml aprotinin] and
centrifugation (14,000 × g; 30 min; 4°C).
Subcellular fractionation was performed following Gurd et al. (1974).
Synaptic membranes (SMs) were prepared according to the procedure of
Blackstone et al. (1992). Synaptic plasma membranes were recovered in
the layer between 1.0 and 1.2 M sucrose and
resuspended in PBS, pH 7.4, with protease inhibitors or in
buffer A as described above.
Immunoprecipitation. IP experiments were performed after
Triton X-100 solubilization as described previously (Blahos and
Wenthold, 1996).
Deglycosylation. Membrane homogenates were either treated
directly or were detergent solubilized and then incubated with SAP97 antiserum to immunoprecipitate SAP97 and associated AMPARs. Membranes or protein A-agarose pellets were resuspended in denaturing buffer (10 mM
NaH2PO4, pH 6, 0.5% SDS,
2% glycerol, and 1%  -mercaptoethanol), incubated for 3 min at
100°C, and diluted with 1% Nonidet P-40 in 10 mM
NaH2PO4, pH 6, containing
the above protease inhibitor mixture, and incubated with
endoglycosidase-H (Endo H) (15 mU) or N-glycosidase F
(PNGaseF) (3 U) for 4 hr at 37°C. The resulting material was diluted
with 5× sample buffer and subjected to SDS-PAGE on 8% acrylamide gels.
Magnetic bead fractionation. Magnetizable polystyrene beads
coated with recombinant Protein A (Dynabeads Protein A; Dynal, Lake
Success, NY) were used to isolate PSDs from the SM fraction. Beads were
coated with PSD-95 antibodies [a combination of polyclonal (JH62092)
and monoclonal (Affinity BioReagents) antibodies (12 µg of each
antibody/100 µl beads)] in 0.1 M Na-phosphate,
pH 8.1, according to the manufacturer's suggestions, then washed and
incubated with 250 µg of SM for 1 hr at 4°C. After incubation, the
beads were washed four times with 0.25 M sucrose
in PBS, pH 7.4, and either solubilized in protein sample buffer,
subjected to SDS-PAGE and analyzed by Western blotting, or fixed in 4%
paraformaldehyde (PFA) and 0.5% glutaraldehyde in 0.1 M phosphate buffer (PB) for 30 min, then 3× PBS,
1% OsO4 in PBS, 3× PBS, ethanol (10 min; 50, 75, 95, and 3× at 100%), 2× propylene oxide, propylene
oxide:Epon (1:1 and 1:2 for 30 min each), overnight in Epon in vacuum,
Epon in vacuum at 64°C for 24 hr, and stained with uranyl acetate and lead citrate.
SDS-PAGE and immunoblot analysis. Proteins were separated
with SDS-PAGE (8 or 4-20% gradient gels; Novex, San Diego, CA) and transferred to Immobilon-P membrane (Millipore, Bedford, MA) and treated as described in Sans et al. (2000). After chemiluminescence detection, films were scanned using a Molecular Dynamics (Sunnyvale, CA) densitometer.
Neuron cultures. Hippocampal or cortical primary neurons
were prepared as described (Standley et al., 2000) and were maintained in DMEM-F-12 and 10% FBS and N2 supplements or in NB-B27 (Life Technologies, Grand Island, NY). High-density cortical
neurons were cultured in 60 mm dishes coated with
poly-L-lysine.
Biotinylation of cell surface proteins. Cultured cortical
neurons were biotinylated with NHS-SS-biotin (1 mg/ml; Pierce,
Rockford, IL) as described (Mammen et al., 1997). Membranes were
resuspended in buffer A (600 µl/plate) and solubilized with either
2% Triton X-100 for 30 min at 37°C or 1% SDS, boiled 5 min and then
adjusted to 1% Triton X-100. Insoluble material was removed by
centrifugation, and the soluble fraction was immunoprecipitated using
antibodies to SAP97. Biotinylated proteins were isolated with
streptavidin agarose (Sigma, St. Louis, MO) (Huh and Wenthold, 1999).
For the double IP, biotinylated AMPARs were purified, after SAP97 IP, by elution of the affinity column with 50 mM
diethylamine, pH 11.5, containing 0.05% (w/v) sodium deoxycholate.
Four fractions of 0.5 ml were collected and immediately quenched to
neutral pH with 2 M glycine. The eluted
biotinylated AMPAR-containing fractions were pooled and applied to an
anti-streptavidin antibody affinity column for 2 hr at 4°C and
processed as described above.
Immunocytochemistry. To label surface AMPARs, neurons (2 weeks in culture) were incubated live for 1 hr with mouse GluR1-N antibody (1 µg/ml) at room temperature (RT) in 3% normal goat serum
(NGS)-PBS, then 2× PBS, 4% PFA-PBS (RT; 20 min), 10% NGS-PBS (1 hr), and Cy3 IgG (1:500; Jackson ImmunoResearch, West Grove, PA) in 3%
NGS-PBS (30 min). For double-labeling, neurons were then permeabilized
with 0.1% Triton X-100-PBS, incubated with 10% NGS-0.1% Triton
X-100-PBS (1 hr), primary antibody [rabbit SAP97 (JH62428; 1:100) in
3% NGS and 0.1% Triton X-100-PBS (1 hr)], and secondary antibody
(FITC; 1:500; Jackson ImmunoResearch) in 3% NGS-0.1% Triton
X-100-PBS (30 min). For localization of internalized AMPARs with
SAP97, neurons were incubated live 1 hr with GluR1-N antibody at RT in
growth medium before a 2 min application of control solution, 100 µM AMPA, and 50 µM APV
(to prevent NMDA receptor activation), or 20 µM
NMDA. Cells were then incubated for 10-15 min at 37°C in growth
medium to allow receptor endocytosis and recycling and fixed in 4% PFA
(RT; 20 min). Remaining surface GluR1-N antibody was blocked with
unconjugated rabbit anti-mouse IgG (Jackson ImmunoResearch), and cells
were permeabilized with 0.1% Triton X-100-PBS. Double-labeling was
performed as above. For colocalization of GluR1 or SAP97 with BiP
(1:200) and SAP97 with endosomal proteins [EEA1 (1:200), Rab4 (1:50),
and Lamp1 (1:50)], neurons were fixed, permeabilized, and
double-labeled as above. Coverslips were mounted with ProLong AntiFade
Kit (Molecular Probes, Eugene, OR). Fluorescent images of the neurons
were obtained using a confocal microscope (Zeiss) and processed with
Adobe Photoshop.
Preembedding light microscope methods for immunofluorescence and
immunoperoxidase labeling of rat brain have been described previously
(Petralia and Wenthold, 1992; Petralia et al., 1997). SAP97 serum was
used (1:200 for immunofluorescence; 1:100-1:200 for immunoperoxidase).
In control sections, PBS was substituted for primary antibody, and
labeling was absent. The postembedding method has been described
(Petralia et al., 1997, 1999a; Petralia and Wenthold, 1999; Sans et
al., 2000) and is modified from a previous study (Matsubara et al.,
1996). Briefly, rats were anesthetized and perfused with 4% PFA plus
0.5% glutaraldehyde in 0.12 M phosphate buffer. Sections
were cryoprotected in 30% glycerol and frozen in liquid propane in a
Leica electron microscopy (EM) CPC (Universal cryoworkstation) (Vienna, Austria), then processed in a Leica automatic
freeze-substitution system (AFS). An unfixed tissue method also
was used [Petralia et al., 1999b, 2001; based on a method of J. Moreira (Petralia and Wenthold, 1998)], to avoid potential artifacts
of tissue fixation. Rats were anesthetized, and the brain tissue was
removed and frozen in a Life Cell CF100 cryofixation unit (The
Woodlands, TX) within ~2 min, then placed in the AFS and processed as
above. Antibodies for single-labeling (10 nm gold) included SAP97 serum
(JH62426; 1:50) and affinity-purified SAP97 (JH62426; 6 µg/ml). 1%
NGS-TBST was substituted for primary antibodies in controls.
Double-labeling methods using antibodies made in the same species, and
criteria for synapse identification and gold counting were described
previously (Petralia et al., 1999a; Sans et al., 2000).
All experiments were performed in accordance with the National
Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals (NIH publication number 85-23) under protocol number
874-98.
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RESULTS |
SAP97 is associated with a subset of AMPARs in hippocampus
Co-IP of native proteins was used to establish that SAP97
interacts with AMPARs in the hippocampus. With 1% Triton X-100, 82 ± 5% of SAP97 was solubilized (data not shown), which is
similar to the degree of AMPAR subunit solubilization in hippocampus
using this detergent (Wenthold et al., 1996). With antibodies to SAP97, IP of SAP97 was ~100% (Fig.
1A). SAP97 antibodies
immunoprecipitated a portion of GluR1 and GluR2 but almost no GluR3
(Fig. 1B) and only a small amount of
GluR4. By quantifying subunits remaining in the unbound
fraction, we find that the following amounts of subunits
co-immunoprecipitate with SAP97: GluR1, 36%; GluR2, 44%; GluR3,
5%; and GluR4, 17% (Fig. 1C). Because SAP97 interacts
directly with GluR1 only (Leonard et al., 1998), co-IP of other
subunits, particularly GluR2, presumably indicates that they are
present as complexes with GluR1. These results are consistent with
previous findings showing that AMPARs in hippocampus are largely of two types, GluR1-GluR2 and GluR2-GluR3 (Wenthold et al., 1996). Controls showed no co-IP of AMPAR subunits with PSD-95 or SAP102 (data not
shown), and NR1 did not co-immunoprecipitate with SAP97 in 1% Triton
X-100-soluble extract (Fig. 1B) as shown previously with deoxycholatesoluble extracts (Sans et al., 2000).
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Figure 1.
Co-immunoprecipitation of AMPAR subunits
with SAP97 in rat hippocampus. Proteins were solubilized in 1% Triton
X-100, and SAP97 was immunoprecipitated using anti-SAP97
antibodies. A, AMPAR subunits remaining (unbound
fraction) after IP of SAP97 from hippocampus homogenate with SAP97
antibodies. The two left lanes of each panel show the
non-immunoprecipitated Triton-solubilized fraction (Tx
sol). The 100% lane represents 10 µl
of sample applied, whereas the 5% lane represents 0.5 µl applied (after 1:10 dilution with sample buffer). These lanes
represent the range of labeling for quantification of immunoreactivity
in the depleted fractions. For each gel, standards of 75, 50, 25, and
10% of the solubilized fraction were also analyzed (data not shown).
To determine the amount of immunoprecipitated proteins, 10 µl of the
depleted fraction was analyzed, equivalent to an equal volume of the
solubilized fraction (100%). B, IP of AMPAR subunits
(bound fraction) using anti-SAP97 antibodies. Ten microliters of bound
immunoprecipitate fractions were separated by SDS-PAGE, immunoblotted,
and incubated with antibodies against SAP97, GluR1, GluR2, GluR3, and
NR1. C, Percentage of immunostaining remaining in the
depleted fractions is shown on the graph (mean ± SEM of three
separate experiments).
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SAP97 is associated with immature GluR1 and GluR2
Our IP results indicated that SAP97 is associated with only a
subset of GluR1 and GluR2, but the results do not differentiate between
a temporal or spatial subset. Because SAP97 may serve as an anchor to
the PSD, the most likely explanation is that only synaptic AMPARs are
associated with SAP97. The percentage of GluR1 and GluR2 associated
with SAP97 is approximately similar to the percentage of AMPARs on the
cell surface of cultured neurons (Hall and Soderling, 1997). However,
PDZ proteins have been shown to interact with ion channel subunits
earlier in their biosynthesis. A PDZ protein interacts with the NR1
subunit while it is in the ER-CG (Standley et al., 2000), and SAP97
interacts with newly synthesized Kv1 channels in transfected COS
cells (Tiffany et al., 2000), raising the possibility of a role for
SAP97 in AMPAR processing before insertion of receptors into the plasma
membrane. In the biosynthesis of many glycoproteins, there is an
intermediate stage in which the protein contains N-linked high-mannose
carbohydrates while trafficking through the ER-CG. Glycoproteins
associated with ER-CG can be identified by their sensitivity to
Endo-H, which specifically cleaves high mannose carbohydrates. Membrane
homogenates of hippocampus were analyzed by Western blotting after
denaturation and treatment with Endo-H or PNGaseF, which enzymatically
removes all N-linked carbohydrates. Treatment with Endo-H showed that all four subunits are to some extent Endo-H-sensitive. This was particularly apparent for GluR2, which contained a small
Endo-H-sensitive population that comigrated with completely
deglycosylated protein obtained by treating with PNGaseF (Fig.
2A). For the other
subunits, the same treatment resulted in the appearance of lower
molecular weight bands that migrate at intermediate positions as well
as a small proportion that migrated at the level of the completely deglycosylated subunit (data not shown). The partial Endo-H resistance is indicative of differential processing of multiple glycosylation sites on the molecule (Standley et al., 1998).
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Figure 2.
SAP97 is associated with immature GluR1 and GluR2.
A, Glycosylation state of SAP97-associated AMPARs.
Left, Membrane homogenates
(H) from hippocampus were solubilized with
0.5% SDS in the presence of 1% -mercaptoethanol. After dilution
with 1% NP-40, soluble extracts were incubated in the absence of
enzyme (control samples) or in the presence of Endo-H or PNGaseF. GluR2
has a small, but distinct, population that is Endo-H-sensitive.
Right, Triton-solubilized AMPARs were first
immunoprecipitated with SAP97 and then deglycosylated with Endo-H or
PNGaseF. Equal amounts of sample were treated and subjected to
SDS-PAGE. Although SAP97 immunoprecipitated both Endo-H-sensitive and
Endo-H-insensitive GluR2, there is a substantial relative increase in
the Endo-H-sensitive component showing that SAP97 preferentially
associates with immature AMPARs. B, Glycosylation state
of the crude synaptic membrane (P2) and microsomal
(P3) fraction. The P3 fraction is enriched in
Endo-H-sensitive GluR1 and GluR2. P2 and P3 fractions of hippocampus
were treated with Endo-H or PNGaseF and analyzed by SDS-PAGE and
Western blotting. C, Glycosylation state of
SAP97-associated AMPARs. Almost all SAP97-associated GluR1 and GluR2 in
the P3 fraction is Endo-H-sensitive.
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The GluR1 and GluR2 associated with SAP97 contained both
Endo-H-sensitive and -insensitive forms of the receptor, but the Endo-H-sensitive form was substantially enriched compared with that
found in total homogenate (Fig. 2A). These results
suggest that SAP97 is preferentially associated with AMPARs that are
incompletely glycosylated and localized to ER-CG. Because
Endo-H-sensitive GluR2 is co-immunoprecipitated with SAP97, subunit
assembly has at least partially occurred when SAP97 is linked to GluR1.
Deglycosylation was completed while the receptor was linked to beads
used to immunoprecipitate SAP97. To verify that these conditions alone
were not responsible for the different Endo-H effects, we did a
corresponding control in which AMPARs were linked to beads using
antibodies to C termini. Treatment with Endo-H under these conditions
gave the same results as treating an entire homogenate (data not shown).
A preparation enriched in ER and other intracellular
membranes can be obtained by subfractionation. Subfractionating the
hippocampus into P3 (microsomes) and P2 (crude synaptic membranes)
shows that a much greater proportion of both GluR1 and GluR2 is
Endo-H-sensitive in P3 compared with P2 (Fig. 2B). IP
of SAP97 from the P3 fraction shows that most of the
co-immunoprecipitating GluR1 and GluR2 is Endo-H-sensitive (Fig.
2C).
AMPARs associated with SAP97 are concentrated in
intracellular membranes
Our results on Endo-H sensitivity suggested that a large amount of
SAP97-associated AMPARs resides in ER-CG. To further investigate the
subcellular distribution of this population, adult rat hippocampus was
subfractionated into P3, P2, and SMs. Western blots (Fig. 3A) showed that SAP97 and
AMPARs are present in all three fractions. However, solubilization
followed by IP with SAP97 showed that SAP97-associated GluR1 and GluR2
is enriched in the microsome fraction. We found that 54% of GluR1 and
61% of GluR2 were co-immunoprecipitated by SAP97 in the P3 fraction,
26% of GluR1 and 35% of GluR2 in the P2 fraction, and only 9% of
GluR1 and 11% of GluR2 in the SM fraction (Fig. 3B). This
represents a significant decrease in the percentage of AMPARs
associated with SAP97 in the synaptic compartment versus the
intracellular one.
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Figure 3.
AMPARs associated with SAP97 are concentrated in
intracellular membranes. The hippocampus was subfractionated into crude
synaptic membrane (P2) and microsomal
(P3) fraction and synaptic membrane
(SM). Triton-solubilized subfractions were
immunoprecipitated with anti-SAP97 antibodies. A, GluR1
and GluR2 remaining (unbound fraction) after IP. B,
Quantitation of immunostaining remaining in the depleted fractions
(mean ± SEM of three separate experiments) shows little
SAP97-associated GluR1 and GluR2 in the SM fraction.
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Interestingly, the SM fraction contained a relatively high amount of
SAP97 in addition to AMPARs, yet the amount of SAP97 linked to AMPARs
was low, indicating that the SAP97 present in the SM fraction was not
associated with AMPARs. Subfractionation based on gradient and
differential centrifugation is not quantitative, and substantial
contamination by other membrane fractions can occur. Furthermore, the
SM fraction contains presynaptic as well as postsynaptic membranes, and
SAP97 has been reported to be presynaptic (Müller et al., 1995).
To eliminate the possibility that the SAP97 present in the SM fraction
is presynaptic, it would be useful to isolate PSDs. The PSD fraction
can be obtained in a relatively pure form, based on its molecular
composition and ultrastructural characteristics, but traditional PSD
preparations require detergent extraction. We find that the bulk of
SAP97, as well as AMPARs, is detergent-soluble, making this approach
unusable for assessing SAP97 in the PSD fraction. To circumvent this
limitation and to determine whether SAP97 is present in PSDs and
associated with AMPARs, we used an affinity isolation method using
magnetic beads coated with PSD-95 antibodies. PSD-95 is highly enriched
in PSD with little associated with intracellular and presynaptic
membranes (Cho et al., 1992). The PSD isolated with anti-PSD-95 beads
were analyzed by SDS-PAGE and Western blotting or analyzed directly by
EM. Ultrastructural analysis of the fraction verifies that it is
enriched in PSDs with accompanying plasma membrane (Fig. 4A). Biochemical
analysis shows that the PSD-95 isolated fraction is enriched in AMPARs
and NMDARs but contains substantially less SAP97 than found in the SM
(Fig. 4B). This fraction contains relatively little
ER-CG and presynaptic vesicles based on the absence of markers for
these organelles.
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Figure 4.
Isolation of PSD from the SM fraction using
magnetic beads coated with antibodies to PSD-95. PSD were
immunoisolated in the absence of detergent as described.
A, Electron micrograph showing PSD
(arrowheads) binding to magnetic beads
(mb). Scale bar, 0.2 µm. B,
Distribution of SAP97 and proteins associated with the PSD (PSD-95,
SAP102, GluR1, GluR2-3, NR2B), synaptic vesicles (synaptophysin), and
ER (calnexin). IgG refers to a control in which magnetic beads were
coated with rabbit IgG. For P3, P2, and SM, 10 µg of protein was
applied to each lane. Protein was not quantified in the immunoisolated
fraction, but the same volume of sample was applied for each
immunoblot.
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Light and electron microscopy immunocytochemistry support both
synaptic and intracellular localizations of SAP97
Our biochemical results suggested that SAP97 is present both at
synapses and in cytoplasm, but unlike PSD-95, which is concentrated in
PSDs, SAP97 is predominantly cytoplasmic. To verify this and to
determine whether SAP97 is uniformly distributed at all postsynaptic sites or concentrated at a subpopulation of synapses, the distribution of SAP97 was determined on sections of P35 rat hippocampus with immunofluorescence, immunoperoxidase, and immunogold techniques. Labeling was found throughout the cytoplasm in cell bodies and dendrites in the CA1 region, as seen with immunofluorescence and immunoperoxidase (Fig. 5A).
With postembedding immunogold, SAP97 was found associated with PSDs as
well as in cytoplasm and decorated cisternae of the ER (Fig.
5Bh), as shown by Aoki et al. (2001) in the rat visual
cortex. In double-labeling experiments using two sizes of immunogold
particles, SAP97 could be found colocalized with both GluR1 and
GluR2-3 at synapses (Fig. 5Ba-d,j,k) and in clusters in
cytoplasm (Fig. 5Be-g,i,l,m). These clusters of gold particles were typically associated with presumptive vesicular or
tubulovesicular structures and ER cisternae (Rubio and Wenthold, 1999b;
Aoki et al., 2001). In general, synaptic labeling was low, and most
synapses appeared unlabeled. The apparent absence of labeling at
synapses may indicate either a true absence or small amount of SAP97 or
a statistical phenomenon. To address this, we optimized immunolabeling
by using 5 nm gold secondary antibody and analyzed the labeling pattern
at 204 synapses. Our results show that most synapses are unlabeled
(28% labeled; 0.6 gold per synapse and 2.1 gold per labeled synapse).
Of the labeled synapses, most have a single gold particle, but a subset
of synapses are heavily labeled with three or more gold particles (Fig.
5C), suggesting that a small population of synapses contains
a high level of SAP97.
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Figure 5.
GluRs from hippocampus and distribution
of SAP97 in the CA1 region using light (A) and
electron (B, C) microscopy. A, With light
microscopy (immunofluorescence-FITC and immunoperoxidase-DAB), labeling
is found throughout the cell body, apical dendrites, and neuropil in
strata oriens (so), pyramidale, and radiatum
(sr). B, EM (a-m),
colocalization of SAP97 (10 nm gold) with GluR1 (5 nm gold;
a-g, i) and GluR2-3 (5 nm gold:
j-m) and immunolabeling for SAP97 (10 nm gold;
h) in fixed (h) and unfixed
(a-g, i-m) sections of the stratum pyramidale and
radiatum of the CA1 region of the hippocampus, respectively.
h, Arrowheads indicate gold particles
decorating ER cisternae in the soma of a pyramidal neuron. a-g,
i-m, Arrows indicate colocalization of SAP97
and AMPARs at synapses. Arrowheads indicate presumptive
cytoplasmic vesicular or tubulovesicular structures that are double- or
single-labeled. These labeled structures are common in dendrites
(e-g, i, l, m) but also are seen in postsynaptic spines
(j, k). Three labeled vesicle-like structures are
seen in i, one double-labeled and one each labeled with
either 5 or 10 nm gold. p, Presynaptic terminal. Scale
bar: a-g, i-m, 0.2 µm; h, 0.4 µm.
C, Histogram showing immunogold labeling for SAP97 (5 nm
gold; stratum radiatum; fixed section), representing 28% of the total
synapses. Although zero and one counts are dominant, there seems to be
a great deal of unobserved heterogeneity. This leads to
overdispersion (highly significant; p < 0.0001); that is, the actual variance exceeds the nominal Poisson
variance. These results are consistent with there being a higher
density of SAP97 in some synapses.
|
|
SAP97 rarely colocalizes with surface expressed GluR1 and does not
localize to AMPAR degradation and recycling pathways
To investigate the relationship of SAP97 with AMPARs expressed at
the surface of neurons, we examined the distribution of SAP97 and
surface-expressed GluR1 in cultured hippocampal neurons (Fig.
6). Immunofluorescence techniques were
used to surface label live neurons for GluR1 (red), followed
by fixation and permeabilization to detect the intracellular SAP97
(green). In general, we observed that the SAP97
labeling is diffuse in the somatic cytoplasm of cultured neurons (data
not shown) and becomes more punctate within dendrites. Proximal
dendrites showed both diffuse and punctate labeling (Fig. 6C,
NMDA central panel, D), whereas distal dendrites or
dendrites of smaller caliber showed a punctate staining (Fig. 6A). Although SAP97 is present within the dendritic
cytoplasm as well as somata of cultured neurons (data not shown), it
appears to colocalize with GluR1 expressed at the surface of neurons
very rarely (Fig. 6A). Furthermore, SAP97 and GluR1
partially colocalize with ER compartments within the somata (data not
shown) as well as within dendrites, as shown by the colocalization with
BiP, a chaperone protein residing in the ER (Fig.
6B). To further determine whether SAP97 is involved
in degradation and recycling pathways of surface-expressed AMPARs, we
performed GluR1 internalization experiments (Beattie et al., 2000;
Ehlers, 2000; Lin et al., 2000). Internalization of GluR1 was
stimulated with application of control solution, AMPA and APV, or NMDA
as in Ehlers (2000). Regardless of the agonist used, internalized GluR1
(red) accumulated in puncta along dendrites and within
somata of neurons. SAP97 (green) also showed a
punctate distribution within the same cellular compartments. Nonetheless, GluR1 puncta corresponding to endocytosed AMPARs seldom
labeled for SAP97 (Fig. 6C). Recently it has been shown that
internalized GluR1 localizes to early and late endosomes (Ehlers, 2000;
Lin et al., 2000). To determine whether SAP97 is also present in these
compartments, internalization was induced, and neurons were
double-labeled for specific markers for early/recycling endosomes (EEA1
and Rab4) or late endosomes (Lamp1) and SAP97. Figure
6D shows that, irrespective of agonist, SAP97
(green) does not colocalize with any of these markers
(red). Thus, SAP97 rarely colocalizes with GluR1 expressed
at the plasma membrane or after internalization and does not label
membranous elements of degradation and recycling pathways used by
GluR1.
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Figure 6.
SAP97 is colocalized with a subpopulation of
surface GluR1 in cultured hippocampal neurons. A, Live
cultured hippocampal neurons were surface-labeled with antibodies to
the GluR1-N and subsequently double-labeled for SAP97. Surface GluR1
forms clusters distributed throughout the dendritic tree
(red). SAP97 immunofluorescence
(green) was observed along dendrites and in the
soma and showed a more diffuse pattern of distribution than GluR1 along
dendrites. SAP97 labeling rarely overlapped with AMPAR labeling
(arrows). B, Dendritic distribution of
GluR1 (green) and BiP (red)
(right panel) and SAP97
(green) and BiP (left
panel). Both GluR1 and SAP97 showed partial
colocalization (yellow) with BiP, an ER marker,
within dendrites. C, Live neurons were prelabeled with
antibodies to GluR1-N, stimulated with 100 µM AMPA and 50 µM APV, or 20 µM NMDA, returned to growth
medium for 10-15 min at 37°C, and internalized GluR1
(red) was detected together with SAP97
(green). Internalized GluR1 accumulated in puncta
along dendrites. SAP97 labeling is also punctate, but SAP97 and
internalized GluR1 very rarely colocalize. D, Stimulated
neurons were double-immunolabeled for SAP97
(green) and early/recycling endosomes (EEA1 and
Rab4) and late endosomes (Lamp1) (red). Under all
stimulation conditions (control shown), labeling for endosomes was
punctiform and observed within dendritic and somatic cytoplasm. The
endosomal compartments showed no SAP97 labeling. Scale bar, 2 µm.
|
|
Some surface AMPARs are associated with SAP97 in
cortical cultures
Although SAP97 is much reduced in affinity-purified SM-PSDs, some
AMPARs were co-immunoprecipitated with SAP97 in the SM fraction. Furthermore, EM studies on cerebral cortex (Valtschanoff et al., 2000)
and hippocampus (this study) show that SAP97 is found in the PSD. To
investigate further the relationship between AMPARs on the surface of
neurons and SAP97, we biotinylated surface AMPARs in cortical cultures.
To preserve or disrupt protein-protein interactions, membranes were
solubilized in Triton X-100 or SDS, respectively. Biotinylated
solubilized proteins were bound to streptavidin agarose beads or were
immunoprecipitated with SAP97 antibodies and evaluated by Western blot
analysis. Co-IP analysis of biotinylated receptors indicated that there
is an association between SAP97 and biotinylated proteins (Fig.
7A). SAP97 was detected in the
Triton X-100-solubilized preparation but not in the SDS preparation in
which protein interactions are disrupted, indicating that SAP97 itself
is not biotinylated but is included through an interaction with another
protein. Moreover, we immunoprecipitated SAP97 complexes and probed for
biotinylated proteins. SAP97 immunoprecipitated unbiotinylated AMPAR
subunits along with biotinylated ones (Fig. 7B). A minor
band stained with streptavidin-HRP co-migrated with GluR1 and GluR2
(Fig. 7B, arrow). SAP97 does not associate with NMDARs, as
shown by the lack of biotinylated NR1 co-immunoprecipitating with
SAP97. However, neither of these two approaches proves that SAP97 is
associated with surface AMPARs. In Figure 7A, SAP97 could be
associated with an unknown biotinylated surface protein. In Figure
7B, the streptavidin-HRP-labeled band that co-migrates with
GluR1 and GluR2 could be an unrelated biotinylated protein that
migrates at the same position. To show that surface AMPARs are
associated with SAP97, we did sequential purification first with SAP97
antibodies followed by streptavidin binding of the bound fraction.
Staining of the double bound fraction with antibodies to GluR1 and
GluR2 showed the presence of the AMPARs (Fig. 7C). We also
analyzed the surface-biotinylated AMPAR subunits isolated with
streptavidin. Treatment of the surface-biotinylated protein, isolated
with streptavidin, with Endo-H had no effect on GluR2 subunits, showing
that surface GluR2 subunits are Endo-H-resistant (Fig.
7D).
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Figure 7.
SAP97 is associated with some surface
receptors in cultured cortical neurons. To determine if surface AMPARs
are associated with SAP97, cortical cultures were biotinylated with
NHS-SS-biotin, and presence of biotinylated AMPARs associated with
SAP97 was assayed. Three experimental approaches were used to assess
the relationship between SAP97 and biotinylated receptors.
A, Two-week-old cortical cultures were biotinylated, and
membranes were solubilized with 1% SDS (with boiling) or with 1%
Triton X-100. The detergent-soluble fraction was added to
streptavidin-conjugated beads and incubated at 4°C. Solubilized
membranes (S), IgG-precipitated (control), and
streptavidin-precipitated proteins, were loaded so that each lane
represents 1% of the material from the plate. The blots were probed
with antibodies to GluR1, GluR2, SAP97, and PSD-95. BiP and tubulin
antibodies were used as controls. The presence of SAP97 in the
streptavidin fraction shows that some SAP97 is associated with
biotinylated surface receptors. Note that this is seen only with Triton
solubilization but not SDS solubilization, which disrupts
protein-protein interactions. The absence of SAP97 in the SDS
solubilized material is a control for biotinylation of intracellular
proteins. Tubulin or BiP, an ER protein, are not biotinylated.
B, Cultures of cortical neurons were biotinylated and
membranes were solubilized with 1% SDS or with 1% Triton X-100. The
detergent-soluble fraction was added to SAP97-conjugated protein A and
incubated at 4°C. Solubilized membranes (S),
IgG-precipitated (control), and SAP97-precipitated proteins were loaded
so that each lane represents 1% of the material from the plate. The
blots were probed with GluR1, GluR2, SAP97, and NR1 antibodies, and
with streptavidin-HRP. SAP97 immunoprecipitated unbiotinylated subunits
along with biotinylated ones. In the streptavidin-HRP-stained
panel, a band that co-migrates with GluR1 and GluR2 is seen
(arrow). SAP97 did not associate with NMDARs.
C, Co-migration of streptavidin-HRP with GluR1 and
GluR2, as shown in B, does not prove that the
biotinylated band is indeed GluR1-GluR2. To establish this, double
affinity purification of biotinylated AMPARs by sequential anti-SAP97
and streptavidin IP was done. Cortical neurons were exposed to 1 mg/ml
NHS-SS-biotin or to
PBS-Ca2+-Mg2+ (control) and
processed as described. Lanes 1 and 2,
Anti-SAP97 immunoprecipitated GluRs; lanes 3 and
4, anti-SAP97 and streptavidin-precipitated GluRs.
D, To show that Endo-H-sensitive receptors are not
present on the cell surface, surface receptors immunoprecipitated with
streptavidin-conjugated beads (IP), were treated with
Endo-H or PNGaseF and analyzed by SDS-PAGE and Western blotting.
Treatment of surface-biotinylated protein with Endo-H has no effect on
GluR1 or GluR2, showing that surface GluR2 are Endo-H-resistant. Total
homogenates (H) contain an
Endo-H-sensitive component.
|
|
| |
DISCUSSION |
Regulation of number and type of AMPARs present at the
postsynaptic membrane has emerged as one of the key issues in
understanding the molecular mechanisms underlying the expression of LTP
and long-term depression (LTD). In the hippocampus, induction of LTP increases delivery of GluR1, a major AMPAR subunit in hippocampal pyramidal neurons, to the synaptic plasma membrane through a mechanism that requires the PDZ binding domain of GluR1 (Shi et al., 1999; Hayashi et al., 2000). In the present study, we investigated the role
of SAP97, the only protein known to interact with the GluR1 PDZ binding
domain, in the biosynthesis and processing of AMPARs in the
hippocampus. Our results show that SAP97 interacts with GluR1 early in
the biosynthetic pathway of GluR1-containing receptors where it may
play a role in maturation of receptor complexes in the ER-CG and
delivery of receptors to synapses. However, at the SM, SAP97 is largely
dissociated from AMPARs, suggesting that it does not play a major role
in anchoring AMPARs at synapses.
SAP97 is associated with intracellular AMPARs in
the hippocampus
SAP97 is a member of the membrane-associated guanylate kinase
family that also includes PSD-95, PSD-93, and SAP102. Our biochemical and immunocytochemical analyses of the individual family members show
two general patterns of distribution: PSD-95 and PSD-93 are highly
concentrated at PSD, whereas SAP97 and SAP102, although found at
synaptic densities, are abundant in the cytoplasm and associated with
intracellular membranes (Sans et al., 2000; Standley et al., 2000).
This distribution may be determined by palmitoylation sites, present at
N termini of PSD-95 and PSD-93 but absent in SAP97 and SAP102. These
sites may anchor receptors to the plasma membrane (El-Husseini et al.,
2000). The intracellular localization of SAP102 and SAP97 is consistent
with a role in intracellular organization of membrane proteins. We find
that a subpopulation of AMPARs in the hippocampus co-immunoprecipitates
with SAP97. These receptors are composed primarily of GluR1 and GluR2
and essentially no GluR3. This finding is consistent with previous results revealing no apparent co-IP of GluR3 with GluR1 in the hippocampus and supports the conclusion that AMPARs in the hippocampus are primarily composed of GluR1-GluR2 and GluR2-GluR3 (Wenthold et
al., 1996). Because SAP97 has been shown to interact with GluR1 only
(Leonard et al., 1998), the co-immunoprecipitating GluR2 would be in a
heteromeric complex with GluR1. Less than 50% of the GluR1 and GluR2
is co-immunoprecipitated with SAP97, and this population is enriched in
immature receptor complexes, which are incompletely glycosylated.
The increasing number of examples of PDZ proteins interacting with ion
channels at the level of the ER-CG suggests that this interaction may
be an important part of a mechanism for regulating the distribution and
surface expression of ion channels in neurons. Kv1 channels expressed
in COS cells are present on the cell surface; however, coexpression
with SAP97 resulted in ER retention of Kv1 and a block of surface
expression (Tiffany et al., 2000). This effect is dependent on the PDZ
interaction because the ER distribution of Kv1 is abolished after
mutation of its C terminus. A PDZ interaction with NR1 splice variants
has an opposite effect and abolishes ER retention and results in
surface expression of the unassembled NR1 subunit (Standley et al.,
2000). The effect of SAP97 on GluR1 distribution has been investigated
in COS cells and expression of SAP97 inhibited surface expression of
GluR1 (Shen et al., 2000b). ER retention is often associated with
quality control and is used to ensure proper folding, subunit
interaction, and stoichiometry. Because of the limitations in obtaining
pure subcellular fractions, we cannot determine whether all GluR1
associates with SAP97 when it resides in the ER. However, the
significant enrichment of SAP97-associated GluR1 in P3, a fraction that
contains ER but also a range of other intracellular membranes,
indicates at least that most of the GluR1 interacts with SAP97. The
fact that a significant part of the SAP97-associated AMPARs are
Endo-H-insensitive, showing that they have cleared the ER-CG,
indicates that SAP97 has a role beyond the ER-CG. SAP97 is unlikely to
play a dominant role in the dendritic targeting of GluR1. SAP97 is
reported to be present both in axons and dendrites, so either SAP97
alone is not specifically targeted to one domain or its distribution is
determined by the protein with which it is associated, in this case
GluR1. The latter interpretation is consistent with results showing
that the axonal localization of PSD-95 requires an interaction of Kv1.4
(Arnold and Clapham, 1999). This interpretation is also supported by
data showing that the dendritic sorting of GluR1 is dependent on the
proximal region of the C terminus, and this does not involve PDZ
interactions (Ruberti and Dotti, 2000).
The presence of ER in dendrites raises the possibility that ER
processing of membrane proteins destined for the postsynaptic membrane
occurs in the dendrite. This would allow synaptic surface expression to
be directly regulated by exit from the dendritic ER and, thus,
associated proteins, such as SAP97, could directly regulate exit from
the ER. However, we find a relatively small amount of Endo-H-sensitive
GluR1 and GluR2 in the hippocampus. Therefore, if any AMPAR is
associated with the dendritic ER-CG, it would account for a very small
proportion of the pool of intracellular AMPARs found in dendrites.
Regardless of where AMPARs are ER associated in neurons, the regulation
of ER exit could be a critical step in receptor regulation by
controlling the amount of receptor present in a pool available for
surface expression. Because a significant fraction of SAP97-associated
receptor is Endo-H-insensitive and has passed through the ER-CG, our
results suggest that SAP97-associated AMPARs are released from the
ER-CG into an intracellular pool and that SAP97 remains associated
with GluR1 in this pool. The presence of such a pool is supported by
our EM immunogold results that show AMPARs in close proximity to SAP97
in dendrites.
The role of SAP97 in the synaptic delivery of AMPARs
Our data extend current models of AMPAR delivery to the SM (Fig.
8). Malinow et al. (2000) propose that
AMPARs are trafficked in two different ways based on the composition of
the receptor complex. Thus, receptors containing GluR1 are added only
through a regulated pathway, whereas those without GluR1 are added
through a constitutive pathway. In the hippocampus, GluR1-containing
receptors are mostly heteromeric complexes of GluR1 and GluR2, whereas
receptors without GluR1 are mostly heteromers of GluR2 and GluR3. We
find that SAP97 associates with GluR1 while it is in the ER-CG and remains with the GluR1 during delivery to the dendrite. Our results suggest that with synaptic stimulation, the SAP97-AMPAR complex is
delivered to the plasma membrane, whereupon the SAP97 soon dissociates
from the receptor. Our observation that a small population of synapses
has high levels of SAP97 would correspond to those synapses to which
GluR1 has been delivered recently. The absence of SAP97 from most PSDs
suggests that it is not primarily involved in anchoring the receptor
complex at the postsynaptic membrane. This may be achieved by one of
the other known interactors of AMPAR C termini, a yet unknown protein,
or the AMPARs may not be firmly anchored, which would be consistent
with the solubility of AMPARs in weak detergent, such as Triton X-100
(Wenthold et al., 1996).
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Figure 8.
Model for the addition of AMPARs to the synaptic
membrane. AMPARs with and without GluR1 are added to the SM following
different pathways. The predominant AMPAR subunits in the adult
hippocampus are GluR1 (shaded ovals), GluR2 (open
ovals), and GluR3 (striped ovals). SAP97 is
associated with GluR1 and plays a role only in initial delivery of
receptors. After internalization, GluR1-containing complexes enter the
constitutive receptor pool.
|
|
A number of studies have now established that synaptic AMPARs are
readily internalized following a variety of stimuli. This mechanism may
partly control the number of receptors at the SM and may also regulate
the composition of these receptors by selective internalization and
re-insertion (Liu and Cull-Candy, 2000). Malinow et al. (2000) propose
that receptors containing GluR1 are added only through a regulated
pathway, whereas those without GluR1 are added through a constitutive
pathway. Although it remains to be determined exactly when the
regulated and constitutive pathways would be operative, limiting GluR1
addition to the regulated pathway, as the Malinow's model proposes,
raises a number of questions. In the adult hippocampus, most synapses
contain GluR1; if GluR1 is added only through the regulated pathway, it
would indicate that most synapses have been recently activated or that
GluR1 is a stable component of the synapse after it is added. Studies on the cerebellum and spinal cord cultures indicate that total and
surface AMPARs have half-lives in the order of 1 d (Mammen et al.,
1997; Huh and Wenthold, 1999). Studies on hippocampal cultures show
that GluR1 is rapidly internalized and re-expressed on the surface
(Beattie et al., 2000; Ehlers, 2000; Lin et al., 2000). Our data
suggest that internalized GluR1 does not re-associate with SAP97.
Because most GluR1 in the hippocampus is in a complex with GluR2, the
internalized GluR1-GluR2 complex could now be transferred to the
constitutive pathway and be controlled by proteins that interact with
the GluR2 C terminus such as NSF and GRIP-ABP. A recent study has
shown that GRIP may play a role in retaining internalized AMPARs in an
intracellular store after induction of LTD (Daw et al., 2000). Such a
mechanism would parallel a role for SAP97 in maintaining an
intracellular store of newly synthesized GluR1. Our model would also
provide a separate mechanism for trafficking homomeric GluR1 receptors.
GluR1 homomeric complexes are relatively minor in the hippocampus
overall (~8% of the total AMPARs in the CA1-CA2) (Wenthold et al.,
1996), but are much more abundant in some specific cell types. Although
such complexes may be treated like heteromeric complexes during initial
delivery to the synapse, after internalization, they would not enter
the constitutive pathway because they lack the GluR2 C terminus. This
would provide a mechanism to transiently supply calcium-permeable
receptors to newly potentiated synapses that might increase
Ca2+ entry during LTP.
| |
FOOTNOTES |
Received May 9, 2001; revised June 20, 2001; accepted June 26, 2001.
This work was supported by the National Institute on Deafness and Other
Communication Disorders intramural program. We thank Drs. J. Hell
for antibodies to SAP97 and PSD-95 and P. Streit for antibodies to
GluR1 and GluR2/3. We also thank Dr. A. Dosemeci for sharing the method
for affinity purification of PSDs, G. P. Riordan and Dr. B. Kachar
for assistance in using the Life Cell slam-freezing device for freezing
unfixed tissue, Drs. C.-W. Ko and H. Hoffman for the statistical
analysis of the EM results, and members of the laboratory of R. J. Wenthold for assistance in preparing and critical reading of this manuscript.
Correspondence should be addressed to Dr. Nathalie Sans, National
Institute on Deafness and Other Communication Disorders, National
Institutes of Health, Building 50, Room 4146, Bethesda, MD, 20892-8027. E-mail: sansn{at}nidcd.nih.gov.
| |
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ABSTRACT
INTRODUCTION
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