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The Journal of Neuroscience, August 1, 1999, 19(15):6528-6537
Association of AMPA Receptors with a Subset of Glutamate
Receptor-Interacting Protein In Vivo
Michael
Wyszynski1,
Juli G.
Valtschanoff3,
Scott
Naisbitt1,
Anthone W.
Dunah2,
Eunjoon
Kim1, 4,
David G.
Standaert2,
Richard
Weinberg3, and
Morgan
Sheng1
1 Department of Neurobiology and Howard Hughes Medical
Institute and 2 Department of Neurology, Massachusetts
General Hospital and Harvard Medical School, Boston, Massachusetts
02114, 3 Department of Cell Biology and Anatomy, University
of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, and 4 Dept of Pharmacology, Pusan National University,
Pusan 609-735, South Korea
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ABSTRACT |
The NMDA and AMPA classes of ionotropic glutamate receptors are
concentrated at postsynaptic sites in excitatory synapses. NMDA
receptors interact via their NR2 subunits with PSD-95/SAP90 family
proteins, whereas AMPA receptors bind via their GluR2/3 subunits to
glutamate receptor-interacting protein (GRIP), AMPA receptor-binding
protein (ABP), and protein interacting with C kinase 1 (PICK1). We
report here a novel cDNA (termed ABP-L/GRIP2) that is virtually
identical to ABP except for additional GRIP-like sequences at the
N-terminal and C-terminal ends. Like GRIP (which we now term GRIP1),
ABP-L/GRIP2 contains a seventh PDZ domain at its C terminus. Using
antibodies that recognize both these proteins, we examined the
subcellular localization of GRIP1 and ABP-L/GRIP2 (collectively termed
GRIP) and their biochemical association with AMPA receptors. Immunogold
electron microscopy revealed the presence of GRIP at excitatory
synapses and also at nonsynaptic membranes and within intracellular
compartments. The association of native GRIP and AMPA receptors was
confirmed biochemically by coimmunoprecipitation from rat brain
extracts. A majority of detergent-extractable GluR2/3 was complexed
with GRIP in the brain. However, only approximately half of GRIP was
associated with AMPA receptors. Unexpectedly, immunocytochemistry of
cultured hippocampal neurons and rat brain at the light microscopic
level showed enrichment of GRIP in GABAergic neurons and in GABAergic
nerve terminals. Thus GRIP is associated with inhibitory as well as
excitatory synapses. Collectively, these findings support a role for
GRIP in the synaptic anchoring of AMPA receptors but also suggest that GRIP has additional functions unrelated to the binding of AMPA receptors.
Key words:
AMPA receptor; GRIP; excitatory synapse; PDZ domain; immunoprecipitation; postsynaptic density
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INTRODUCTION |
At excitatory synapses, ionotropic
glutamate receptors of the NMDA class are highly concentrated in the
postsynaptic density (PSD). The NR2 subunits of the NMDA receptors bind
through their C-terminal cytoplasmic tails to the PDZ domains of
PSD-95/SAP90 and related proteins, which are major components of the
PSD (Kornau et al., 1995 ; Müller et al., 1996; Niethammer et al.,
1996). The NR1 subunit of the NMDA receptor interacts with a distinct set of intracellular proteins, including  -actinin (Wyszynski et al.,
1997), neurofilament-L (Ehlers et al., 1998), and yotiao (Lin et al.,
1998). The interaction between NMDA receptors and PSD-95 family
proteins may be important for the localization of NMDA receptors at
postsynaptic sites. In Drosophila, the PSD-95 homolog
Discs-large is essential for the synaptic clustering of its
PDZ binding partners Shaker and Fasciclin II (Tejedor et al., 1997;
Thomas et al., 1997; Zito et al., 1997). In addition, because of its
multidomain structure, PSD-95 can also bind to several signaling and
cytoskeletal proteins, including neuronal nitric oxide synthase
(Brenman et al., 1996), synGAP (Chen et al., 1998; Kim et al., 1998),
and CRIPT (Niethammer et al., 1998), thereby potentially
bringing these proteins together in a complex (for review, see Craven
and Bredt, 1998). By acting as scaffold proteins, PSD-95 and related
molecules can organize a specific cytoskeletal-signaling complex that
is physically linked to the NMDA receptor.
Ionotropic glutamate receptors of the AMPA class (composed of GluR1-4
subunits) are also concentrated at postsynaptic sites. Although they
largely colocalize with NMDA receptors in excitatory synapses, AMPA
receptors do not interact with PSD-95 family proteins. Instead, the
AMPA receptor subunits GluR2/3 bind specifically to other PDZ proteins,
termed glutamate receptor-interacting protein (GRIP) (Dong et al.,
1997), AMPA receptor-binding protein (ABP) (Srivastava et al., 1998),
and protein interacting with C kinase 1 (PICK1) (Xia et al., 1999).
GRIP contains seven PDZ domains and no other recognizable motif, in
contrast to PSD-95, which has three PDZ domains plus an Src homology
3 domain and a guanylate kinase-like domain. ABP resembles GRIP
in primary sequence; it differs from GRIP most notably in lacking the
C-terminal seventh PDZ domain. The C-terminal sequence (-ESVKI) shared
by AMPA receptor GluR2/3 subunits is reported to bind selectively to
the forth and fifth PDZ domains (PDZ4/5) of GRIP (Dong et al., 1997)
and the third, fifth, and sixth PDZ domains of ABP (Srivastava et al.,
1998). However, neither Dong et al. (1997) nor Srivastava et al. (1998)
confirmed a biochemical association of GRIP and AMPA receptors in
brain. We have recently found that GRIP is only modestly enriched in
the PSD fraction when compared with PSD-95 (Wyszynski et al., 1998).
These observations beg the question of how substantially AMPA receptors
interact with GRIP and ABP in vivo.
We report here the cloning of a novel GRIP-like protein (termed
ABP-L/GRIP2) that is virtually identical to ABP but that contains additional N-terminal and C-terminal sequences, including a seventh PDZ
domain. ABP and ABP-L/GRIP2 appear to be splice variants of the same
gene containing six or seven PDZ domains, respectively; thus the domain
organizations, as well as the primary sequence, of the ABP and GRIP
genes are highly conserved. We will henceforth use the term GRIP1 for
the original GRIP polypeptide described by Dong et al. (1997). Using
antibodies that recognize both GRIP1 and ABP-L/GRIP2 (these related
proteins collectively termed GRIP), we show a wider subcellular
distribution of these proteins in neurons than previously appreciated,
including enrichment in GABAergic nerve terminals. Using a
semiquantitative coimmunoprecipitation assay, we show that a majority
of the detergent-extractable GluR2/3 is complexed with GRIP in adult
rat brain tissue; however, a considerable fraction of GRIP is not
associated with GluR2/3. Taken together, these results show for the
first time the biochemical association of AMPA receptors and GRIP
in vivo but suggest additional functions for GRIP other than
that of anchoring AMPA receptors at synapses.
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MATERIALS AND METHODS |
Cloning of GRIP1 and ABP-L/GRIP2. Yeast two-hybrid
screening was performed as described previously using the L40 yeast
strain harboring HIS3 and  -galactosidase as reporter genes
(Niethammer et al., 1996). Approximately 2 × 106 clones were screened of a rat brain cDNA library
constructed in the GAL4 activation domain vector pGAD10 (Clontech, Palo
Alto, CA). The two-hybrid bait consisted of a C-terminal peptide,
-GRISYDL, fused to LexA (this peptide was an artifactual sequence
generated aberrantly during PCR construction of another bait). The
two-hybrid screen yielded multiple isolates of two distinct clones
(clones 5 and 14). Clone 5 was a cDNA fragment of GRIP1, encoding amino acids 363-1112 (containing PDZ4 through the C terminus). Clone 14 encoded amino acids 556-1002 of ABP-L/GRIP2 (beginning within PDZ5 and
extending through the C terminus). Additional GRIP1 and ABP-L/GRIP2
cDNA sequences were obtained by hybridization screening of  ZAP II
rat cortical and hippocampal cDNA libraries (Stratagene, La Jolla, CA)
using as probes the GRIP1 and ABP-L/GRIP2 cDNA fragments described
above. The 5' ends of GRIP1 and ABP-L/GRIP2 were obtained by 5' rapid
amplification of cDNA ends using a Marathon-Ready rat brain cDNA
library (Clontech). DNA sequences were obtained by automated
sequencing. The nucleotide sequence of ABP-L/GRIP2 has been deposited
in GenBank under accession number AF112182
Northern blots. A rat poly-A mRNA multi-tissue Northern blot
(Clontech) was probed with 32P-labeled ABP-L/GRIP2 cDNA
fragments (corresponding to amino acids 556-1002) under
high-stringency conditions using ExpressHyb (Clontech) and exposed at
 80°C on XAR-5 film (Eastman Kodak, Rochester, NY) for 50 hr.
Antibodies. GRIP antibodies (termed "1756" and
"C8399") were raised by immunizing two different rabbits with a
hexahistidine (H6)-tagged fusion protein
incorporating GRIP1 amino acids 664-1112 (includes PDZ6 and extends to
the C terminus of the protein); 1756 antibodies were then
affinity-purified on a Sulfolink column (Pierce, Rockford, IL) coupled
to a soluble H6-tagged fusion protein of GRIP1 containing
PDZ4-PDZ6 (residues 463-761) and have been described (Wyszynski et
al., 1998). C8399-I antibodies were affinity-purified on a Sulfolink
column coupled to a soluble glutathione S-transferase fusion protein incorporating GRIP1 amino acids 664-1112. The rabbit anti-PSD-95 antibody (CSK) has been previously described (Kim and Sheng, 1996). The following antibodies were obtained from commercial sources: polyclonal anti-GluR2/3 and monoclonal anti-GluR2-N antibodies (Chemicon, Temecula, CA); anti-NR1 monoclonal antibody 54.1 (PharMingen, San Diego, CA); anti-glutamic acid decarboxylase (GAD)
monoclonal antibodies (Boehringer Mannheim, Indianapolis, IN);
anti-GAD-6 monoclonal antibodies (Developmental Studies Hybridoma Bank,
University of Iowa, Iowa City, IA); and nonimmune purified rabbit IgG
and anti-synaptophysin monoclonal antibodies (Sigma, St. Louis, MO).
The rabbit and guinea pig anti-GluR1 antibodies were gifts from Richard
L. Huganir (Johns Hopkins University School of Medicine, Baltimore, MD).
Immunohistochemistry on brain and cultured neurons. Brain
immunohistochemistry was performed on Vibratome-cut 50 µm floating brain sections from Sprague Dawley rats (~6 weeks of age) perfused transcardiacally with 4% paraformaldehyde and treated with proteinase K as described (Lin et al., 1998). Primary anti-GAD monoclonal antibodies were used at 1 µg/ml. Hippocampal neuron cultures were prepared from hippocampi dissected from embryonic day 18 rat embryos by
the methods of Banker and Cowan (1977) and Goslin and Banker (1991).
Neurons were fixed at 3 weeks in culture with 4% paraformaldehyde and
4% sucrose in phosphate buffer, permeabilized with 0.25% Triton X-100, and immunolabeled using mouse anti-GluR1, mouse anti-GluR2-N, mouse anti-synaptophysin, or mouse anti-GAD-6 antibodies. All double
labeling was performed with rabbit anti-GRIP antibody 1756 antibodies
(1 µg/ml). Staining was visualized using Cy3- or FITC-conjugated secondary antibodies (Jackson ImmunoResearch, West Grove PA).
Immunoelectron microscopy. For electron microscopy, five
Sprague Dawley rats were perfusion-fixed with a mixture of 2-4%
paraformaldehyde and 2% glutaraldehyde; brains were post-fixed for
2-4 hr. Fifty-micrometer-thick transverse sections were cut on a
Vibratome and processed for osmium-free embedment (for details, see
Phend et al., 1995). Thin sections were cut and collected on 300-mesh
uncoated nickel grids and stained for GRIP using anti-GRIP1 antibodies
C8399-I and 1756. After treatment with 4% paraphenylenediamine in
Tris-buffered saline containing detergent (TBS/T; 0.1 M
Tris, pH 7.6, with 0.005% Tergitol NP-10) for 1 hr, grids were rinsed
and incubated overnight at 37°C in primary antibody (1:100), rinsed
in TBS/T, pH 7.6, transferred to TBS/T, pH 8.2, and incubated for 1 hr
in the secondary antibody [goat anti-rabbit IgG conjugated to 10 nm
(Amersham, Arlington Heights, IL) or 18 nm (E-Y Labs, San Mateo, CA)
gold particles]. Grids were then rinsed and counterstained with uranyl acetate and Sato's lead. In control experiments, in the absence of
primary rabbit serum, virtually no gold particles were detected; after
exposure to nonimmune rabbit serum, sparse gold particles were seen
that showed no obvious pattern. Grids were examined at 80 kV on a JEOL
(Tokyo, Japan) 200CX electron microscope. For quantitation, micrographs
at 29,000-40,000× original magnification were digitized on a flat-bed
scanner. Locations of gold particles were measured using NIH Image
software running on a Macintosh platform. For each grid studied, the
first 10 synapses with clearly visible synaptic structures, labeled
with at least one gold particle within 100 nm on either side of the
postsynaptic membrane, were considered. To define "axodendritic"
position, we measured the distance between the center of each gold
particle and the outer leaflet of the postsynaptic membrane. To define
"lateral" synaptic position, we measured the distance from each end
of the active zone to a line drawn perpendicular to the synapse running
through the center of the particle.
Immunoprecipitation. Coimmunoprecipitation of GRIP, GluR2/3,
and GluR1 complexes from rat cortex was done on sodium deoxycholate (DOC)-extracted rat brain membranes as described (Luo et al., 1997). In
brief, DOC extracts were prepared by resuspending rat brain cortex P2
fraction in a suitable volume (1 ml/gm of tissue) of ice-cold Tris-EDTA
buffer (10 mM Tris-HCl and 5 mM EDTA, pH 7.4)
and sonicated briefly. One-tenth volume of ice-cold DOC buffer (10%
sodium deoxycholate and 500 mM Tris-HCl, pH 9.0) was added to the sonicate, followed by incubation at 36°C for 30 min. After the
addition of 0.1 volume of Triton X-100 buffer (1% Triton X-100 and 500 mM Tris-HCl, pH 9.0), the samples were dialyzed against ice-cold binding buffer (50 mM Tris-HCl, pH 7.4, and 0.1%
Triton X-100). Unextractable material was pelleted at 37,000 × g for 40 min, and the supernatant was used for immunoprecipitation.
For immunoprecipitation, GRIP (C8399-I), GluR2/3, GluR2-N, or rabbit
IgG antibodies were prebound to protein A-Sepharose beads by incubating
the antibodies with protein A-Sepharose beads (Sigma) at a ratio of 20 µg of antibodies/40 µl of protein A-Sepharose beads in 200 mM Na-borate buffer, pH 8.0, at 4°C for 12 hr. The protein A-Sepharose/antibody-bound beads were then washed with 200 mM Na-borate buffer, pH 9.0, added to 100 µg of
DOC-extracted protein (see above), and allowed to incubate at 4°C for
2-3 hr. The immunoprecipitation reaction was then centrifuged at high speed in a microcentrifuge; the supernatant was saved, and the immunoprecipitate pellet was washed three times with binding buffer.
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RESULTS |
Cloning of ABP-L/GRIP2, a splice variant of ABP containing seven
PDZ domains
By yeast two-hybrid screening (see Materials and Methods), we
obtained not only multiple isolates of a cDNA fragment (clone 5) from
the original GRIP gene (Dong et al., 1997; GRIP1) but also a cDNA
fragment (clone 14) that we subsequently found to be virtually
identical to ABP within the region of overlap (Srivastava et al.,
1998). We obtained the full-length coding sequence of this ABP-related
gene, which showed interesting differences from the full-length ABP
sequence published by Srivastava et al. (1998). Compared with ABP, our
open reading frame contained an N-terminal extension of 52 amino acids,
an internal deletion of 41 amino acids between PDZ3 and PDZ4, and a
C-terminal extension that contains an additional seventh PDZ domain
(PDZ7; Fig. 1). Otherwise, the two
proteins are essentially identical, suggesting that the extensions and
deletions noted in our clone arise from differential splicing. We
called the novel protein encoded by our cDNA "ABP-L/GRIP2," because
it is a longer splice variant of ABP, and because it is clearly a close
relative of GRIP1 (Dong et al., 1997), both in terms of primary
sequence and domain organization. Because ABP is essentially contained
within ABP-L/GRIP2, we presume that ABP and ABP-L/GRIP2 are splice
variants of the same gene. In this paper, we use the name GRIP1 for the
original GRIP described by Dong et al. (1997) and ABP-L/GRIP2 for the
long splice variant of ABP. The generic term GRIP will be used to refer
to GRIP1 and ABP-L/GRIP2 proteins as a set.
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Figure 1.
Top, Amino acid sequence alignment
of GRIP1 and ABP-L/GRIP2. The third line
(Cons.) shows the consensus amino acids that are common
between GRIP1 and ABP-L/GRIP2. The seven PDZ domains are
underlined. Bottom, Rat poly-A mRNA
Northern blot showing the tissue distribution of ABP-L/GRIP2 mRNAs.
Positions of RNA molecular size markers are shown in kilobases.
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Over its entire length, ABP-L/GRIP2 is 56% identical and 68% similar
to GRIP1 at the amino acid level (Fig. 1). The greatest similarity
between GRIP1 and ABP-L/GRIP2 occurs over the respective PDZ domains
1-7, with highest identity for PDZ6 (93%) and lowest identity for
PDZ3 (62%). There is also significant homology between GRIP1 and
ABP-L/GRIP2 in the intervening sequences between each PDZ domain.
During the cloning and sequencing of overlapping cDNAs, presumptive
alternative splice forms of both GRIP1 and ABP-L/GRIP2 were noted that
contained small insertions or deletions at certain sites in both cDNA
sequences (data not shown). The particular splice variant of
ABP-L/GRIP2 protein shown in Figure 1 is 1002 amino acids long and has
a predicted Mr of 107,733.
By Northern analysis, ABP-L/GRIP2 mRNA is expressed predominantly in
the brain, in the form of a major ~6 kb transcript and a minor ~2.8
kb transcript (Fig. 1, inset). Similar-size mRNAs were
present in the heart, but less abundantly. ABP-L/GRIP2 mRNAs of smaller
size were detected in liver (~2.4 kb) and in testis (~1 kb). Thus
ABP-L/GRIP2 mRNA shows a tissue expression pattern distinct from GRIP1,
which is predominantly expressed in brain and testis and expressed at a
low level in kidney (Wyszynski et al., 1998).
Ultrastructural localization of GRIP protein in rat brain
Two different polyclonal rabbit antisera were raised against
fusion proteins containing PDZ6 through the C terminus of GRIP1 and
affinity-purified (Wyszynski et al., 1998). The affinity-purified antibodies (termed 1756 and C8399-I; see Materials and Methods) specifically and effectively recognize both GRIP1 and ABP-L/GRIP2, as
assessed by immunoblotting of recombinant bacterial fusion proteins and
rat brain extracts (Wyszynski et al., 1998; data not shown). The
cross-reactivity of 1756 and C8399 antibodies is not surprising,
because the PDZ6 and PDZ7 domains are highly conserved between GRIP1
and ABP-L/GRIP2 (Fig. 1). Although unable to distinguish between GRIP1
and ABP-L/GRIP2, these antibodies are nevertheless specific for GRIP
and hence useful for probing the set of GRIP1 and ABP/GRIP2 proteins in
the brain. Using these "pan-GRIP" antibodies, we have recently
described the immunohistochemical localization of GRIP in various
regions of rat brain (Wyszynski et al., 1998). At the light microscopy
(LM) level, GRIP is widely distributed within brain neurons, showing a
punctate staining pattern on top of a diffuse intracellular labeling
(Wyszynski et al., 1998).
To obtain higher resolution, we studied the subcellular localization of
GRIP by postembedding immunogold electron microscopy (EM) of adult rat
neocortex (Fig. 2). A similar labeling
pattern was seen using two GRIP antibodies prepared from different
rabbits and purified in different ways (C8399-I and 1756), supporting the specificity of immunogold labeling. In all blocks, the highest density of gold particles coding for GRIP was associated with asymmetric synapses (Fig. 2A,D,E).
Labeled symmetric synapses were rare; of 114 labeled synapses studied
in three rats, only 2 were symmetric. Labeling (at a lower level
density) could also be seen over nonsynaptic plasma membranes (Fig.
2B,C). Considerable intracellular
labeling was present within spines and within dendritic shafts (Fig.
2C,F); in dendritic shafts particles were
typically associated with microtubules (Fig. 2F). In
some cases, GRIP immunoreactivity was seen in axons, axon terminals,
and somatic endoplasmic reticulum (data not shown). Thus based on
immuno-EM, GRIP is present at excitatory synapses but additionally
associated with nonsynaptic membranes and intracellular
compartments.
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Figure 2.
EM photomicrographs of immunogold labeling (18 nm gold particles) for GRIP in layers II/III of rat somatic sensory
cortex. A, Gold particles (arrowheads)
label two asymmetric synapses made by the same terminal onto spines.
B, GRIP immunogold labeling (arrowheads)
at nonsynaptic sites of the plasma membrane of two spines.
C, Gold particles labeling the postsynaptic membrane of
a perforated synapse with additional intracellular labeling within the
dendritic spine (arrow). D, Gold particle
labeling encroaches into the presynaptic cytoplasm
(arrow); the postsynaptic labeling
(arrowhead) coincides with the "inner" margin of the
PSD. E, Immunogold labeling (arrowhead)
at an asymmetric synapse extends into the postsynaptic cytoplasm.
F, Large dendritic profile immunopositive for GRIP;
arrows point to labeling adjacent to microtubules.
Sp, Spine; asterisks mark presynaptic
terminals. Scale bars: A-D, 0.25 µm; E,
F, 0.5 µm. G, H, Quantitative
analysis of the distribution of GRIP immunogold particles at the
synapse. G, Distribution of gold particles in the
axodendritic axis. One hundred sixty particles lying within 175 nm of
the postsynaptic membrane were measured in 70 random electron
micrographic fields, selected to reveal clear synaptic membranes; data
were pooled from three animals. Abscissa, Distance from
the center of each gold particle to the postsynaptic membrane (in
nanometers); ordinate, labeling density (arbitrary
units); zero density corresponds to the x-axis.
H, Lateral distribution of gold particles along the
synapse (only those within 100 nm of the postsynaptic membrane were
considered). Lateral position is normalized; center of
the active zone corresponds to 0; and edge corresponds
to 1.0.
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The synaptic labeling for GRIP was concentrated over the postsynaptic
membrane and the postsynaptic density and was generally sparser over
the presynaptic profile. Approximately one-third (87 of 266) of
asymmetric synapses were labeled for GRIP. This is probably an
underestimate, considering the limited sensitivity of the method and
our use of single, rather than serial, sections to assess each synapse.
To quantify the staining pattern at synapses, we measured the
distributions of gold particles associated with synapses in 70 randomly
selected fields from three animals. Along the axodendritic axis,
particle density was highest at the postsynaptic membrane with some
spread (up to 150 nm) toward the presynaptic and postsynaptic
compartments (Fig. 2G). The labeling density appeared higher
at the center of the active zone, declining toward the edge of the PSD
(Fig. 2H).
Localization of GRIP in cultured hippocampal neurons
We also assessed the cellular and subcellular localization of GRIP
by immunocytochemistry of hippocampal neurons in low-density culture
(Fig. 3A-D). A
similar labeling pattern was seen using anti-GRIP antibodies 1756 and
C8399-I. GRIP showed fine punctate and diffuse staining in cell bodies
and dendrites (Fig. 3A-D). In addition, GRIP
appeared to be concentrated in sparse puncta of larger diameter (0.5-2
µm) along the somata and dendrites of many neurons (Fig.
3B,C). Double-label
immunocytochemistry with GRIP 1756 and synaptophysin antibodies
revealed GRIP localization to a subset of synaptic sites (Fig.
3A). A large fraction of the GRIP puncta, however, appeared
nonsynaptic.
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Figure 3.
Immunocytochemical localization of GRIP in
cultured hippocampal neurons and in rat brain.
A-E, Double-label immunocytochemistry of
hippocampal neurons (3 weeks in low-density culture) with antibodies to
GRIP (1756, A-D) and synaptophysin
(Syp, A), GluR2
(B), or glutamic acid decarboxylase
(GAD, C, D). GRIP is
visualized by Cy3 secary antibody (red), and
synaptophysin, GluR2, and GAD are visualized by FITC secondary antibody
(green). Colocalization is shown in
yellow. A subset of GRIP-immunoreactive puncta showed
colocalization with synaptophysin (A) and GluR2
(B). Inset, Enlargement of the
boxed region in B, showing examples of
GRIP/GluR2 colocalization in dendritic spine-like structures
(arrowheads). There is also extensive overlap of GRIP
and GluR2 staining in the cell body (B). The
larger GRIP puncta colocalized with a subset of GAD-labeled inhibitory
terminals (C). GABAergic neurons also had higher
levels of diffuse somatodendritic GRIP immunoreactivity than did
pyramidal neurons (D). E, Confocal
image showing partial colocalization of GRIP and GAD in area CA1 of
hippocampus. Note colocalized immunoreactive puncta on pyramidal cell
bodies and adjacent neuropil. p, Stratum pyramidale;
so, stratum oriens. Scale bars: A,
D, 10 µm (for A-D); E,
40 µm.
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Double labeling with 1756 and anti-GluR2 antibodies showed extensive
overlap of GRIP and AMPA receptor staining in cell bodies and major
dendritic shafts but only limited colocalization of GRIP and GluR2 in
dendritic puncta [~10% of GluR2 puncta colocalized with GRIP along
dendrites (Fig. 3B, arrowheads)]. On the other hand, double labeling with 1756 antibodies and an antibody against GAD,
to reveal inhibitory presynaptic sites, showed extensive colocalization
of GRIP and GAD in the larger puncta, although colocalization was not
complete (Fig. 3C). Interestingly, diffuse cytoplasmic GRIP
immunoreactivity was also much higher in GABAergic neuronal somata and
their processes than in pyramidal neurons (Fig. 3D).
Association of GRIP with inhibitory neurons and synapses has also been
observed with independent GRIP antibodies (R. L. Huganir, personal
communication). Overall, the pattern of immunostaining in
embryonic hippocampal cultures indicated that GRIP is highly expressed
in GABAergic neurons and enriched in inhibitory nerve terminals. The
nature of the nonsynaptic GRIP staining is unclear, but it is
consistent with the GRIP labeling of nonsynaptic membranes and
intracellular compartments seen by EM (Fig. 2). Earlier biochemical fractionation had also suggested a widespread subcellular distribution of GRIP (Wyszynski et al., 1998).
Immunocolocalization of GRIP and GAD was confirmed in vivo
by double labeling of rat brain (for detailed information on the pattern of cellular labeling, see Burette et al., 1999). Punctate GRIP
staining in the neuropil of rat brain is greatly enhanced by light
protease treatment of the sections. In hippocampus a subset of these
GRIP immunoreactive puncta colocalized with GAD-positive puncta on cell
bodies and proximal dendrites of pyramidal neurons (Fig.
3E), consistent with the localization of GRIP in inhibitory synapses. Unfortunately, the protease digestion was incompatible with
synaptophysin staining; thus we were unable to perform double labeling
to test whether the GRIP puncta in general were synaptic in brain
sections (data not shown).
Association of GRIP and AMPA receptors in vivo
Although our immuno-EM data revealed the presence of GRIP at
excitatory synapses in rat brain, it does not prove that GRIP is
biochemically associated with AMPA receptors at these sites. To test
whether GRIP and AMPA receptors exist in a protein complex, we
optimized a coimmunoprecipitation assay that allowed us to immunoprecipitate GRIP or AMPA receptor subunits virtually
quantitatively from detergent extracts prepared from rat brain
membranes (Fig. 4A).
Because our antibodies recognize both GRIP1 and ABP/GRIP2, we can
assess AMPA receptor association with the entire known family of GRIP
proteins.
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Figure 4.
Coimmunoprecipitation of native complexes
containing GRIP and AMPA receptor subunits from rat brain.
A, Detergent solubility of GRIP and AMPA receptor
subunits. Crude synaptosomal membrane fraction from rat cortex was
extracted with sodium deoxycholate (DOC); equal
percentages of the resulting extract (Sol) and
pellet fractions were immunoblotted for GRIP, GluR2/3, GluR1, NR1, and
PSD-95. B, Membrane proteins from adult rat cortex were
detergent-solubilized as described in Materials and Methods. Parallel
samples of brain extract were immunoprecipitated (IP)
with nonspecific rabbit IgGs or affinity-purified antibodies to GRIP or
GluR2/3, as indicated. In lanes 4-6, the GRIP
antibodies were heat-inactivated by boiling before use. Each
immunoprecipitation experiment is shown in three lanes:
I, input to the immunoprecipitation reaction;
P, immunoprecipitated pellet; and S,
supernatant remaining after the immunoprecipitation. Equal fractions of
each sample (~10%) were loaded, thus allowing direct estimate of the
fraction of each protein immunoprecipitated (the amount of protein in
I = P + S). The
I, P, and S fractions of
the various immunoprecipitation reactions were immunoblotted
(IB) for GRIP, GluR2/3, GluR1, NR1, and PSD-95, as
indicated to the right of each row.
Rows are also labeled
(A-E) for reader orientation (see
Results). C, D, Same as for
B, except that GluR2-N antibodies were used for
immunoprecipitation in C, and solubilization and
immunoprecipitation was performed in the presence of either GluR2/3 or
NR2B C-terminal competitor peptide (0.5 mM final
concentration) in D.
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Our extraction conditions using deoxycholate detergent solubilized the
large majority of GRIP and AMPA receptors (typically >80%; Fig.
4A). Using GRIP antibodies for immunoprecipitation, close to all of the extracted GRIP could be pelleted with the protein
A-Sepharose beads, leaving little or undetectable amounts of GRIP in
the supernatant (Fig. 4B, compare row
A, lanes 8, 9). This GRIP
immunoprecipitate contained most of the GluR2/3 in the extract, leaving
behind a minor fraction of GluR2/3 in the supernatant (Fig.
4B, row B, lanes 8, 9).
In multiple experiments, the fraction of GluR2/3 that was
coimmunoprecipitated with GRIP ranged from ~40 to ~80%.
Furthermore, approximately half of the extracted GluR1 (an AMPA
receptor subunit that does not bind directly to GRIP) was
coimmunoprecipitated with GRIP (Fig. 4B, row C,
lanes 8, 9). Control experiments showed that the NMDA receptor
subunit NR1 and PSD-95 were not coimmunoprecipitated with GRIP
antibodies (Fig. 4B, rows D, E, lane 8).
Because PSD-95 can be efficiently coimmunoprecipitated with NR1 under
the same conditions (data not shown), this result indicates that
GRIP-GluR2/3-containing complexes are distinct from complexes
containing NR1 and PSD-95.
In the converse reaction, GluR2/3 antibodies were able to
quantitatively immunoprecipitate GluR2/3 from the extract and to coimmunoprecipitate approximately half of the extracted GRIP protein (Fig. 4B, rows A, B, lanes 11, 12; data
not shown). In multiple experiments, the fraction of GRIP that was
coimmunoprecipitated with GluR2/3 ranged from ~30 to ~50%. The
same immunoprecipitates contained a majority of the GluR1 (Fig.
4B, row C, lanes 11, 12). Again, the
GRIP-GluR2/3 coimmunoprecipitation is specific, because no detectable
NR1 and PSD-95 was pelleted with GluR2/3 (Fig. 4B, rows D, E, lane 11). In further negative controls, equal
amounts of nonspecific rabbit immunoglobulins or heat-inactivated GRIP antibodies were unable to precipitate GRIP, GluR2/3, GluR1, NR1, or
PSD-95 (Fig. 4B, rows A-E, lanes 2, 5).
Similar results were reproduced in several experiments.
We also used an independent antibody (GluR2-N) directed against the
extracellular N-terminal region of GluR2 to perform the immunoprecipitation (Fig. 4C). GluR2-N immunoprecipitated
approximately half of the GluR2/3 and GluR1 immunoreactivity from the
brain extract, consistent with the association of these subunits in heteromeric AMPA receptors (Fig. 4C, rows B, C, lanes
2, 3). A significant fraction of GRIP was specifically
coimmunoprecipitated with GluR2-N antibodies (Fig. 4C,
row A, lanes 2, 3). However, no detectable NR1 and PSD-95
was pelleted with GluR2-N (Fig. 4C, rows D, E, lane
2). Thus using multiple antibodies we could show substantial
association of AMPA receptors and GRIP in adult rat brain.
If the immunoprecipitation of GRIP-AMPA receptor subunit complexes from
brain detergent extracts was attributable to artifactual association of
these proteins occurring after solubilization, a synthetic peptide
comprising the last 10 residues of the GluR2/3 C terminus should
compete for PDZ domain binding and hence should reduce or abolish the
coimmunoprecipitation of GRIP and GluR2/3 by competing for binding to
the GRIP PDZ domain. However, even in the presence of a 0.5 mM concentration of GluR2/3 C-terminal peptide, GluR2/3
showed equally substantial coimmunoprecipitation with GRIP (Fig.
4D). These results indicate that AMPA receptor subunits are largely associated with GRIP in native complexes in rat
brain, but that a significant fraction of GRIP is not associated with
AMPA receptors.
GRIP and GluR2/3 were also coimmunoprecipitated from juvenile rat brain
(postnatal day 7) with similar efficiency to adult, indicating that
GRIP and AMPA receptors are highly associated in immature brain.
However, in neuronal culture (3 weeks in culture), we found poor
association (<10%) of GRIP and AMPA receptor subunits by
coimmunoprecipitation (data not shown). This poor coimmunoprecipitation in cultured neurons correlates with the weak colocalization of GRIP and
GluR2 seen by immunocytochemistry (Fig. 3).
| |
DISCUSSION |
ABP and ABP-L/GRIP2 are splice variants of the same gene
ABP, a GluR2/3-binding protein with six PDZ domains, has been
recently reported (Srivastava et al., 1998). Based on sequence comparisons with the ABP-L/GRIP2 protein described here, ABP and ABP-L/GRIP2 are almost certainly alternative splice variants of the
same gene. ABP is a splice form that has been truncated at the
C-terminal region, thereby losing the seventh PDZ domain. The reported
size of ABP on SDS-PAGE matches the minor 97 kDa band that we detect
consistently with our pan-GRIP antibodies (Wyszynski et al., 1998);
furthermore, Srivastava et al. (1998) also reported higher molecular
weight bands on their ABP Western blots that probably correspond, at
least in part, to "full-length" ABP-L/GRIP2 polypeptides. It will
be of interest to identify the binding partners of PDZ7 in GRIP,
because its absence in ABP implies that the function of PDZ7 can be
uncoupled from that of the other six PDZ domains. The significance of
the alternative splicing of ABP/ABP-L/GRIP2 remains to be determined.
A major fraction of GluR2/3 is associated with GRIP
The binding of GluR2/3 to GRIP has been previously studied only
in vitro and in heterologous expression systems (Dong et
al., 1997; Srivastava et al., 1998). We show here that AMPA receptor subunits are associated with GRIP in vivo. Indeed, a major
fraction of GluR2/3 appears to be complexed with GRIP in the brain
under optimal conditions for immunoprecipitation. Approximately half of
the extractable GluR1 is also associated with GRIP, presumably via
heteromultimerization with GluR2/3, because GluR1 cannot bind directly
to the PDZ domains of GRIP. That a lesser fraction of GluR1 than of
GluR2/3 is associated with GRIP in rat brain cortex may be accounted
for in part by the existence of AMPA receptors that contain GluR1 but
not GluR2/3 (Martin et al., 1993; Kharazia et al., 1996; Wenthold et
al., 1996; Kondo et al., 1997). When it is not in a heteromeric
configuration with GluR2/3, GluR1 may be associated with scaffold
proteins other than GRIP. It is intriguing, given that much of GluR1
appears not to be complexed with GRIP, that a recent report showed
GluR1 to be coimmunoprecipitated with SAP97, a PSD-95-related protein
(Leonard et al., 1998).
Although most of the extractable GluR2/3 is complexed with GRIP, a
substantial proportion of GRIP appears not to be associated with
GluR2/3. The simplest interpretation of this observation is that AMPA
receptors interact with only a subset of GRIP in the brain. The
remaining fraction of GRIP may be involved in the assembly of protein
complexes that do not include AMPA receptor subunits.
Several findings support the idea that GRIP may play additional roles
unrelated to the anchoring of AMPA receptors at synapses. First, GRIP
is only modestly enriched in synaptic or PSD fractions when compared
with PSD-95, the prototypical postsynaptic scaffold protein (Wyszynski
et al., 1998). Second, significant amounts of GRIP are present in
subcellular fractions that are depleted of synaptic membranes, such as
the cytosolic fraction S3 and the synaptic vesicle-enriched fraction
LP2 (Wyszynski et al., 1998). Third, although immunogold EM shows a
concentration of GRIP at asymmetric synapses in adult brain neurons, it
also reveals GRIP immunoreactivity at nonsynaptic membranes and in
intracellular compartments of dendritic spines and shafts. Fourth,
immunocytochemistry of cultured embryonic neurons from hippocampus
reveals that a prominent signal for GRIP is in GABAergic neurons and
their nerve terminals, and a large fraction of GRIP immunoreactivity is
nonsynaptic. Finally, GRIP expression is high very early during
cortical development and does not correlate temporally with AMPA
receptor expression or synaptogenesis (Wyszynski et al., 1998). Taken
together, these data argue that a large proportion of GRIP is not
associated with excitatory synapses, where AMPA receptors are presumed
to be concentrated. Perhaps this nonsynaptic fraction of GRIP is
represented by the subset of GRIP that does not coimmunoprecipitate
with GluR2/3. Based on the precedent of PSD-95, it is quite likely that
there are multiple binding partners for each individual PDZ domain of GRIP. Thus at synaptic sites, GRIP may be complexed with AMPA receptors, whereas at nonsynaptic sites, GRIP may be complexed with a
different set of interacting proteins. It is also possible that the
GRIP-AMPA receptor interaction occurs only after both proteins reach
the synapse, in which case the nonsynaptic GRIP may be in an unbound
(AMPA receptor-free) form in transit to its final location.
The above discussion is prejudiced toward the idea that AMPA receptors
are highly concentrated in excitatory postsynaptic membranes, and that
the site of GluR2/3-GRIP interaction is primarily at synaptic sites.
However, to reach their synaptic destination, AMPA receptor proteins
themselves have to be sorted via specific vesicle trafficking pathways
emanating from the cell body. Furthermore, recent evidence points to a
regulated trafficking of AMPA receptors at postsynaptic sites and the
existence of intracellular and subsynaptic pools of AMPA receptor
subunits (Molnár et al., 1993; Baude et al., 1995; Nusser et al.,
1998; Petralia et al., 1999). These considerations imply the existence
of AMPA receptor proteins residing in intracellular membranous
compartments on the way to, and in the vicinity of, the synapse.
Consistent with this, we note that the staining pattern of AMPA
receptors in neurons is typically more diffuse than that of NMDA
receptors, suggesting a more widespread subcellular distribution for
AMPA receptors (Rao et al., 1998). If GluR2/3 subunits are as highly
associated with GRIP as the coimmunoprecipitations would suggest, then
it is possible that some of the nonsynaptic GRIP that we observe is
complexed with intracellular pools of AMPA receptors. Thus GRIP could
play a role in AMPA receptor trafficking in addition to its function in
synaptic anchoring of AMPA receptors. In this regard, it will be
important to determine at what stage of the secretory pathway the
GRIP-GluR2/3 interaction takes place.
Localization of GRIP in GABAergic neurons and terminals
A surprising finding was that in hippocampal neuron cultures GRIP
immunoreactivity colocalized more prominently with GAD, a marker of
GABAergic neuronal somata and terminals, than with AMPA receptors. The
association of GRIP with GABAergic neurons in culture is consistent
with immunohistochemistry of rat brain sections, which showed the most
striking expression of GRIP protein in interneurons of hippocampus and
cortex (Wyszynski et al., 1998). The immunostaining pattern in cultured
neurons indicates the enrichment of GRIP in GABAergic somata and
dendrites and nerve terminals. A partial colocalization of GRIP- and
GAD-immunoreactive puncta was also seen in brain by confocal
microscopy, supporting the idea that GRIP is concentrated in GABAergic
presynaptic terminals in vivo. The particularly abundant
expression of GRIP in GABAergic neurons is of interest. GABAergic
interneurons contain AMPA receptor clusters that are more insoluble in
Triton X-100 than AMPA receptors in pyramidal cells (Allison et al.,
1998). Moreover, relative to pyramidal neurons, GABAergic neurons show
a particularly high content and low variability of AMPA receptors at
excitatory postsynaptic sites (Nusser et al., 1998). Whether these
postsynaptic characteristics are related to the high levels of GRIP in
these neurons remains to be determined.
Synthesis of LM and EM findings: multiple pools of GRIP
In contrast to LM immunocytochemistry of hippocampal cultures, we
did not observe a concentration of GRIP signal over nerve terminals at
symmetric (presumed inhibitory) synapses by immuno-EM of neocortex.
Instead, the highest density of GRIP labeling occurred over asymmetric
synapses, as might be expected for an AMPA receptor-binding protein. We
are confident of the specificity of each staining pattern because they
were consistently obtained with multiple independent GRIP antibodies in
appropriately controlled experiments. The apparently different results
in hippocampal culture and adult rat neocortex may reflect different
developmental stages of these preparations and/or the differential
strengths and weakness of LM versus postembedding EM
immunolocalization. The postembedding immunogold EM technique is
especially sensitive to the density of the antigen and may miss
antigens that are relatively soluble and easily lost during the
processing of the thin sections (such as may occur for GRIP in
GABAergic nerve terminals). The accessibility or "visibility" of
the antigen may also vary with the different methods. Proteins in the
PSD may be less accessible to antibodies and often are more difficult
to stain by LM immunohistochemistry than the same proteins in
cytoplasmic compartments (e.g., see Watanabe et al., 1998). In support
of this, we found that punctate GRIP staining in the brain at the LM
level is dramatically revealed by protease treatment of brain sections.
On the other hand, the thin sectioning of postembedding immuno-EM can
directly reveal antigens in hard-to-access places like the PSD.
Alternatively, it is possible that real differences exist between the
subcellular distribution of GRIP in hippocampal culture and in the
intact brain. In particular, we note that in contrast to the brain,
GRIP and AMPA receptor subunits are poorly associated in neuron
cultures, as assessed by coimmunoprecipitation. This correlates with
the limited colocalization of GRIP and AMPA receptor subunits at
synaptic puncta in cultured neurons. In this sense, cultured
hippocampal neurons may represent an imperfect model for GRIP-AMPA
receptor interactions.
Because of the apparent discrepancies discussed above, it is important
to synthesize the results from multiple immunohistochemical approaches
and to combine them with more quantitative biochemical fractionation
studies. By doing this, we conclude that multiple pools of GRIP exist
in neurons spread among several subcellular locations. GRIP is not only
present at the excitatory postsynaptic membrane; it is also at
inhibitory synapses and in intracellular compartments close to and
distant from the synapse. Moreover, only approximately half of GRIP is
associated with AMPA receptors. The future challenge will be to define
the molecular roles played by these various different subsets of GRIP
and to relate these functions to the trafficking and function of AMPA receptors.
| |
FOOTNOTES |
Received March 1, 1999; revised May 5, 1999; accepted May 18, 1999.
This work was supported by National Institutes of Health Grants NS35050
(to M.S.), CA66268 (to M.W.), NS35527 (to R.W.), and NS34361 (to
D.G.S.) and a grant from the Korea Research Foundation (to E.K.). M.S.
is Assistant Investigator of the Howard Hughes Medical Institute. We
thank Carlo Sala, Fu-chia Yang, and Sheila Rudolph-Correia for
experimental help. We are especially grateful to Anuradha Rao and Ann
Marie Craig for double-label immunocytochemistry of hippocampal neurons
with antibodies to GRIP and glutamic acid decarboxylase.
Correspondence should be addressed to Dr. Morgan Sheng, Howard Hughes
Medical Institute, Wellman 423, Massachusetts General Hospital, 50 Blossom Street, Boston, MA 02114.
| |
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Surface Targeting of the Dopamine Transporter Involves Discrete Epitopes in the Distal C Terminus But Does Not Require Canonical PDZ Domain Interactions
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L. E. Swan, C. Wichmann, U. Prange, A. Schmid, M. Schmidt, T. Schwarz, E. Ponimaskin, F. Madeo, G. Vorbruggen, and S. J. Sigrist
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K. J. Seidenman, J. P. Steinberg, R. Huganir, and R. Malinow
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Interaction of the Postsynaptic Density-95/Guanylate Kinase Domain-Associated Protein Complex with a Light Chain of Myosin-V and Dynein
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TOP
ABSTRACT
INTRODUCTION
