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INTRODUCTION |
Glutamate is the major excitatory neurotransmitter in the
central nervous system. Ionotropic glutamate receptors are divided into
three groups according to their pharmacological and
electrophysiological characteristics:
-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA)1 receptors, kainate
receptors, and N-methyl-D-aspartate receptors (reviewed in Ref. 1). AMPA-type glutamate receptors are oligomeric structures, formed by the assembly of four subunits (GluR1-4; Refs.
2-4), and their diversity is further increased by post-transcriptional modifications such as alternative splicing and RNA editing (reviewed in
Ref. 1).
GluR4-containing AMPA receptors are specifically expressed in neurons
and glia in several regions of the central nervous system (5-10),
where they are responsible for signal transmission at high rates (11).
In the rat hippocampus, GluR4 subunits are expressed early in
development, and specifically mediate synaptic delivery of AMPA
receptors at early stages (10). GluR4-containing AMPA receptors are
delivered to hippocampal synapses by spontaneous activity, a mechanism
that seems to be subunit specific (10). In fact, targeting of AMPA
receptors to the post-synaptic membrane was described to be
specifically mediated by the subunit composition of the receptors
(12).
Targeting of AMPA receptors to the postsynaptic membrane of excitatory
synapses is thought to be mediated through interaction of the C termini
of AMPA receptor subunits with scaffolding proteins. The AMPA
receptor-interacting proteins include PDZ (for PSD-95, Disc Large, and Z0-1) domain-containing
proteins, like glutamate receptor-interacting protein (GRIP) or
AMPA-binding protein, protein interacting with protein kinase C 1 (PICK1), syntenin and synapse-associated protein 97 (SAP97), and
proteins lacking a PDZ domain, like stargazin and neuronal
activity-regulated pentraxin (Narp; reviewed in Ref. 13). GluR4 AMPA
receptor subunit was reported to associate with stargazin, GRIP, and
syntenin (14, 15). The plasma membrane protein stargazin is believed to
mediate AMPA receptor targeting to the membrane surface (14), whereas
GRIP was proposed to play a role in receptor stabilization at synapses
(16). The role of syntenin, which was also described to interact with
syndecans (17), in addition to GluR1-3 and GluR2c, is not known. None of the interactions described for GluR4 so far can account for its
specific delivery to hippocampal synapses following spontaneous synaptic activity.
AMPA receptors are known to be regulated by protein phosphorylation
(reviewed in Ref. 18). AMPA receptor phosphorylation modulates channel
conductance (19), peak open probability of the receptor (20),
interaction with PDZ domain-containing proteins (21, 22), clustering
(23), and synaptic delivery of the receptors (24). Several
phosphorylation sites have been mapped in the C termini of AMPA
receptor subunits, including GluR4. GluR4 can be phosphorylated on
Ser-482 by PKA, PKC, and calcium/calmodulin-dependent protein kinase II and on Thr-830 by PKC (25). In hippocampal slices,
PKA activation by spontaneous activity is necessary and sufficient for
delivery of GluR4-containing receptors to postsynaptic sites (26).
Additionally, PKC activation increases Ca2+ influx through
activated AMPA receptor channels in cultured chick retinal neurons
(27), where GluR4 is the main AMPA receptor subunit expressed (28).
PKC has been implicated in a variety of neuronal functions, including
modulation of ion channel activity and synaptic transmission (reviewed
in Ref. 29). PKC
expression is developmentally regulated, and PKC
is expressed postnatally in the rat brain (30), playing a role in
both long term potentiation and in learning and memory (31-33).
Because GluR4 AMPA receptor subunit is phosphorylated and its
phosphorylation may mediate synaptic delivery (26), early in
development, we studied the biochemical interaction of GluR4 with
PKC
. Our work shows that PKC
interacts with GluR4 AMPA receptor
subunit, both in rat brain and in chick retina cultured neurons, and
that bound PKC
is able to preferentially phosphorylate GluR4 on
Ser-482, relatively to other substrates. Furthermore, co-transfection
of PKC
with GluR4 in human embryonic kidney (HEK) cells increases
GluR4 subunit surface expression. Together these results indicate that
the association between PKC
and GluR4 plays a role in regulating the
function of GluR4-containing AMPA receptors.
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EXPERIMENTAL PROCEDURES |
Materials--
Trypsin was purchased from Invitrogen
(Paisley, Scotland), and basal Eagle's medium, penicillin, and
streptomycin were obtained from Sigma (Madrid, Spain). PGEX4T-2 and
pGEX-1
T vectors, glutathione-Sepharose 4B, protein A-Sepharose
CL-4B, anti-GST antibody, alkaline phosphatase-conjugated goat
anti-mouse and goat anti-rabbit secondary antibodies, and [
-32P]ATP, were all obtained from Amersham Biosciences
(Uppsala, Sweden). Rabbit anti-goat alkaline phosphatase-conjugated
antibody was from Zymed Laboratories Inc. (San
Francisco, CA). Fetal calf serum was from Biochrom KG (Berlin,
Germany), the pET23b vector was from Novagen (Madison, WI), the pBK-CMV
vector was from Stratagene (Amsterdam, Netherlands), and
nickel-nitrilotriacetic acid-agarose was from Qiagen (Hilden, Germany).
Isopropyl-1-thio-
-galactopyranoside was from Promega (Madison, WI).
Histone H1, phorbol 12-myristate 13-acetate (PMA), and the PKC
pseudosubstrate peptide 19-31 were from Calbiochem (San Diego, CA).
Complete Mini protease inhibitor mixture and microporous polyvinylidene
difluoride membranes were obtained from Roche Diagnostics GmbH
(Switzerland, Basel). UltraLinkTM Plus immobilized
streptavidin gel and EZ-LinkTM Sulfo-NHS-SS-Biotin were
from Pierce. The rabbit polyclonal anti-GluR4 antibody was purchased
from Chemicon (Temecula, CA), and the mouse monoclonal anti-PKC
,
anti-PKC
, and anti-PKAc
antibodies were from Transduction
Laboratories (Lexington, KY). The anti-rabbit secondary antibody
conjugated to Alexa 488 and the Texas Red-conjugated anti-mouse
antibody were from Molecular Probes (Leiden, Netherlands).
Chick Retinal Cultures--
Monolayer primary cultures of chick
retina amacrine-like cells were prepared from 8-day-old chick embryos
as previously described (34, 35). After treatment with trypsin (0.1%,
15 min, 37 °C) in Hanks' balanced salt solution, the cells were
plated on tissue culture dishes (at a density of 1.35 × 106 cells/cm2) for cell extract preparation, or
on glass coverslips (at a density of 0.75 × 106
cells/cm2) for immunocytochemistry. Chick retina
amacrine-like neurons were maintained in basal Eagle's medium,
buffered with 25 mM HEPES and 10 mM
NaHCO3, pH 7.4, and supplemented with 5% heat-inactivated fetal calf serum, penicillin (100 units/ml), and streptomycin (100 µg/ml). The cells were kept at 37 °C in a humidified incubator of
5% CO2 and 95% air, for 5 days.
Recombinant Proteins--
cDNA fragments coding for the
intracellular C-terminal domain of GluR4, or fractions of the
C-terminal domain, were amplified by PCR, using GluR4 cDNA as a
template. PCR products were subcloned into the pGEX4T-2 vector (via
BamHI and EcoRI sites for the whole C-terminal
length; via BamHI and SalI sites for the
truncated constructs) or into the pGEX-1
T vector (BamHI
and EcoRI sites, for the constructs coding amino acids
815-828 and amino acids 829-882 of GluR4). The entire coding sequence
of PKC
was amplified by reverse transcription-PCR from total RNA
isolated from rat brain cortex, using the specific primers
5'-agcagctagcatggcgggtct-3' and 5'-ccgctcgagcatgacgggcacag-3', which
include the restriction sites for NheI and XhoI,
respectively. PKC
cDNA was subcloned into pBK-CMV vector
restriction endonuclease sites NheI and XhoI (for
HEK 293T cell transfection) or a His tag was added to the C terminus of
PKC
by subcloning PKC
cDNA in frame into restriction endonuclease sites NheI and XhoI in the pET23b
vector. Recombinant proteins were expressed in BL21 Escherichia
coli transformed with the constructs described above. Bacteria
grown to A600 = 0.8 were induced with
isopropyl-1-thio-
-galactopyranoside (100 µM) for 2 h, and then lysed with phosphate-buffered saline (PBS)
containing 1% Triton X-100 and a protease inhibitor mixture. The cells
were sonicated and shaken for 30 min at 4 °C, and the insoluble
fraction was then removed by centrifugation at 12,000 × g, for 10 min at 4 °C. GST fusion proteins were purified
by glutathione-Sepharose affinity chromatography, and His-tagged PKC
was purified using nickel-nitrilotriacetic acid-agarose according to
the protocol of the manufacturers. Recombinant proteins were dialyzed
overnight against Tris-buffered saline (TBS).
Immunocytochemistry--
Chick retinal cells were kept in
culture for 5 days and were then fixed with 2% paraformaldehyde for 30 min at room temperature. Cells were washed in PBS after fixation,
permeabilized with 0.3% Triton X-100 for 10 min at 4 °C, and
blocked in 0.2% bovine gelatin in PBS for 1 h at room
temperature. Cells were incubated with a rabbit polyclonal anti-GluR4
antibody (10 µg/ml) and a mouse monoclonal anti-PKC
(5 µg/ml)
antibody for 1 h, at room temperature. Neurons were washed with
0.1% bovine gelatin in PBS with 0.1% Tween 20 and incubated with
anti-rabbit secondary antibody conjugated with Alexa 488 and Texas
Red-conjugated anti-mouse antibody. Images were obtained with a Zeiss
confocal microscope.
Extract Preparation and Immunoprecipitation--
Cultured chick
retinal neurons were washed with ice-cold PBS, scraped with
lysis buffer (20 mM Tris, 2 mM EGTA, 2 mM EDTA, and a protease inhibitor mixture) containing 1%
Triton X-100, sonicated, and centrifuged at 1000 × g,
for 10 min at 4 °C. The supernatants containing soluble proteins
were used to immunoprecipitate the GluR4 AMPA receptor subunit. Rat
cerebellum membranes were prepared according to the procedure described
by Luo and colleagues (36). Membrane proteins were then diluted 1:1
with lysis buffer containing 1% Triton X-100 (for Triton X-100
extracts), or with lysis buffer containing 1% Triton X-100 and 2%
SDS, and boiled for 30 s (for SDS extracts), to disassemble
oligomeric receptor complexes. These samples were diluted 7 times with
lysis buffer containing 1% Triton X-100 before immunoprecipitation.
GluR4 and GluR1 immunoprecipitates were obtained by incubation of 2.5 µg of anti-GluR4 polyclonal antibody with chick retina extracts, or
membranes obtained from rat cerebellum, and by incubation of 1 µl of
GluR1 polyclonal antibody (a kind gift from Dr. Richard Huganir) with
rat cerebellum membranes, overnight at 4 °C. These samples were then
incubated for 90 min with 100 µl of protein A-Sepharose beads (50%)
and extensively washed (twice with TBS containing 1% Triton X-100,
twice with TBS containing 1% Triton X-100 and 0.5 M NaCl,
and twice more with TBS). The immunoprecipitated proteins were eluted
by boiling in 1× Laemmli sample buffer and were separated by
SDS-PAGE.
GST Binding Assays--
Extracts of cultured chick retina cells
were prepared as described above. Whole rat brain extracts were
prepared by homogenizing the tissue in lysis buffer with 1% Triton
X-100 followed by centrifugation at 1000 × g, for 10 min at 4 °C. The resulting supernatant was re-centrifuged at
1000 × g, for 10 min at 4 °C, and the soluble brain
proteins were used for binding studies. Ten µg of fusion protein was
incubated with chick retinal extracts, rat brain homogenates, or
recombinant His-tagged PKC
overnight at 4 °C. The mixture was
incubated with 50% glutathione-Sepharose beads for 30 min at 4 °C.
Beads were washed extensively with TBS containing 1% Triton X-100 and
0.5 M NaCl, and binding proteins were eluted with 1×
Laemmli sample buffer. Samples were analyzed by Western blotting.
Gel Electrophoresis and Immunoblotting--
Samples were
resolved by SDS-PAGE in 12% acrylamide gels. For immunoblot analysis,
proteins were transferred onto a polyvinylidene difluoride membrane by
electroblotting (40 V, overnight, at 10 °C). The membranes were
blocked for 45 min with 5% (w/v) nonfat dry milk plus 0.1% (v/v)
Tween 20 in TBS, and probed during 1 h at room temperature with
anti-PKC
(0.05 µg/ml), anti-PKC
(0.05 µg/ml), anti-GluR4 (0.5 µg/ml), anti-GluR1 (1:5000), anti-GST (1:2000), or anti-PKAc
(0.25 µg/ml) antibodies. Following several washes, the membranes were
incubated for 1 h, at room temperature, with alkaline
phosphatase-conjugated goat anti-mouse secondary antibody for
anti-PKC
, anti-PKC
, and anti-PKAc staining, with alkaline
phosphatase-conjugated goat anti-rabbit secondary antibody for
anti-GluR1 and anti-GluR4 antibodies, or with alkaline
phosphatase-conjugated rabbit anti-goat secondary antibody for anti-GST
staining. The blots were washed again, and immunostaining was
visualized by the enhanced chemifluorescence method on a Storm 860 gel
and blot imaging system (Amersham Biosciences).
Phosphorylation Assays--
GST pull-down assays were performed
using GluR4 C-terminal fragments fused to GST and whole rat brain
extracts. GST fusion proteins and interacting proteins were pulled down
with glutathione-Sepharose beads, which were washed extensively with
TBS containing 1% Triton X-100 and 0.5 M NaCl, and
incubated in a phosphorylation buffer containing 100 mM
Hepes, 20 mM MgCl2, 250 µM ATP, 5 µCi of [
-32P]ATP, 200 µM
CaCl2, and phosphatidylserine/diacylglycerol (50 µg/ml/5 µg/ml) for 30 min, at 30 °C. The bound proteins were
eluted from beads with 1× Laemmli sample buffer and separated by
SDS-PAGE in 12% acrylamide gels. Polyacrylamide gels were stained with Coomassie Blue R, destained, and then extensively washed with ultrapure
H2O and 10% glycerol. Gels were then dried for 45 min, at
80 °C, exposed to a storage Phosphor Screen (Eastman Kodak Co.) and
analyzed on a Storm 860 gel and blot imaging system.
Histone Phosphorylation Assays--
Histone H1 (20 µg) was
incubated with purified protein kinase C from rat brain, in a
phosphorylation buffer containing 100 mM Hepes, 20 mM MgCl2, 250 µM ATP, 5 µCi of
[
-32P]ATP, 200 µM CaCl2, and
phosphatidylserine/diacylglycerol (50 µg/ml/5 µg/ml), in the
presence of equimolar amounts of GST (8.0 µg), GST fused to GluR4
C-terminal fragments corresponding to amino acids 815-828 (8.6 µg)
or to amino acids 815-838 (9.0 µg), or in the presence of the PKC
pseudosubstrate peptide 19-31 (RFARKGALRQKNV; 0.62 µg), for 30 min,
at 30 °C. Reactions were stopped with Laemmli sample buffer, and the
samples were separated by SDS-PAGE followed by autoradiography (as
described above). Histone H1 phosphorylated bands were quantified using
ImageQuant (Amersham Biosciences) software and plotted.
Transfection of Cultured HEK 293T Cells--
HEK 293T cells
maintained at 37 °C in a humidified incubator of 5%
CO2, 95% air were transiently transfected with 10 µg of cDNA (pBK-CMV- PKC
and/or pGW1-GluR4), using the calcium
phosphate coprecipitation method, as previously described (25).
Receptor Surface Expression--
Cultured HEK 293T cells were
stimulated with 200 nM PMA, for 10 min, 48 h after
transfection. After stimulation the cells were washed with culture
medium and then incubated at 37 °C in a humidified incubator of 5%
CO2, 95% air for 3 h. Cells were washed twice with
PBS/Ca2+/Mg2+ (PBS supplemented with 0.5 mM MgCl2 and 1 mM
CaCl2) and incubated with 1 mg/ml EZ-LinkTM
Sulfo-NHS-SS-Biotin in PBS/Ca2+/Mg2+ for 30 min
at 4 °C. Cells were then washed twice with
PBS/Ca2+/Mg2+ supplemented with 0.1% bovine
serum albumin, and once with PBS/Ca2+/Mg2+ and
scraped with lysis buffer containing 1% Triton X-100 and 0.1%
SDS. The cells were then sonicated and centrifuged at 1000 × g, for 10 min at 4 °C. Aliquots of the supernatants
containing soluble proteins (4% of total) were used to determine
expression of total GluR4 and the remaining samples incubated with
UltraLinkTM Plus immobilized streptavidin gel for 2 h
at 4 °C (according to the protocol of the manufacturers).
Streptavidin beads were extensively washed, twice with TBS containing
1% Triton X-100, once with TBS containing 1% Triton X-100 and 0.5 M NaCl, and once more with TBS. The biotinylated proteins
were eluted by boiling in 1× Laemmli sample buffer and were analyzed
by immunoblotting. The digital images were quantified using ImageQuant
software (Amersham Biosciences). Surface receptor expression was
determined from the surface biotinylated/total receptor ratio.
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RESULTS |
PKC
Associates with GluR4 in Vivo--
To examine whether
PKC
is associated with GluR4 in vivo, the interaction of
PKC
with GluR4 was tested in rat cerebellum and in primary chick
retina cultures, where GluR4 is highly expressed (11, 28, 37).
Immunoprecipitation of GluR4 from rat cerebellum membrane extracts or
from cultured chick retina cells resulted in specific
co-immunoprecipitation of PKC
(Fig.
1). Pre-absorption of the GluR4 antibody
with its antigen blocked the co-immunoprecipitation of PKC
in the
retina cell extracts (Fig. 1B), confirming the specificity
of the association. In contrast, PKC
, which is also expressed in
this preparation (28), did not co-immunoprecipitate with GluR4 AMPA
receptor subunit (Fig. 1B). Rat cerebellum membrane extracts
prepared in 1% SDS and boiled, to ensure disassembly of tetrameric
AMPA receptor complexes (2-4), were also tested. Immunoprecipitation
of either GluR4 or GluR1 from rat cerebellum membranes, solubilized in
SDS, also specifically co-immunoprecipitated PKC
(Fig.
1A). Immunocytochemistry experiments using an antibody that
specifically recognizes GluR4 showed a punctuate distribution of
GluR4-containing AMPA receptors, along the dendrites, in cultured chick
retina cells (Fig. 2). Double labeling
with anti-GluR4 and anti-PKC
antibodies revealed that some of the
GluR4 punctuate distribution is co-localized with PKC
(Fig.
2).

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Fig. 1.
PKC associates with
GluR4 in vivo. A, rat cerebellum
membrane preparation was solubilized with 0.5% Triton X-100 (Triton
X-100 extracts) or with 0.5% Triton X-100 and 1% SDS (SDS extracts),
and the extracts were used to immunoprecipitate GluR4 and GluR1. The
immunoprecipitates were then analyzed for the presence of GluR1, GluR4,
and PKC . B, total homogenates of cultured chick retina
neurons were solubilized with 1% Triton X-100 and were
immunoprecipitated either with anti-GluR4 antibody or with the same
antibody with antigen pre-absorption. The immunoprecipitated complex
was resolved by SDS-PAGE and probed with anti-GluR4, anti-PKC , and
anti-PKC antibodies. Whole rat brain lysates were used as a positive
control in immunoblot staining (A and B).
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Fig. 2.
Co-localization of GluR4-containing AMPA
receptors with PKC in cultured neurons.
Cultured chick retina cells were double-labeled with rabbit anti-GluR4
and mouse anti-PKC antibodies, followed by Alexa 488-conjugated
anti-rabbit IgG and Texas Red-conjugated anti-mouse IgG. Merged images
show a partial co-localization of GluR4 punctuate staining and PKC
immunoreactivity. Arrowheads indicate areas of
co-localization, and the scale bar represents 5 µm.
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Analysis of the PKC
Binding Domain in GluR4--
To map the
amino acid segment in GluR4 AMPA receptor subunit that binds PKC
,
GST fusion proteins with the C-terminal domain of GluR4 were produced,
because this region is the main intracellular domain of the receptor
subunit (see Fig. 3D; Refs. 1,
38, and 39). GST fused to full-length GluR4 C terminus or to partial segments of GluR4 C terminus were used to pull down interacting proteins, present either in Triton X-100 solubilized rat brain homogenates (Fig. 3A) or cultured chick embryo retinal
amacrine-like neurons (Fig. 3B). We found that PKC
present both in the rat brain (Fig. 3A) and in the chick
retina culture extracts (Fig. 3B) binds full-length GluR4 C
terminus and the C-terminal fragments corresponding to amino acids
815-852 and 815-838. GST fused to the protein segment corresponding
to amino acids 815-828 of GluR4 also bound PKC
in GST pull-down
assays using rat brain homogenates (Fig. 3A). However, the
fusion protein lacking amino acids 815-828 was unable to bind PKC
(Fig. 3A). This result suggests that the membrane-proximal
region between amino acids 815 and 828 in the C terminus of GluR4 is
important for the interaction with PKC
.

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Fig. 3.
PKC interacts with
GluR4 C-terminal sequence. A rat brain extract (A), an
extract of cultured chick retina neurons (B), or purified
recombinant PKC (C) was incubated with GST fused to GluR4
C-terminal peptides (GST-full-length GluR4 C terminus or GST fused to
GluR4 C-terminal amino acids 815-852 (GST-GluR4 (815-852)), 815-838
(GST-GluR4 (815-838)), 815-828 (GST-GluR4 (815-828)), or 829-882
(GST-GluR4( 815-828)), as indicated. Glutathione-Sepharose beads
were used to pull down GST fusion proteins (detected with anti-GST
antibody; Fig. 1A). Analysis of the pull-down samples with
an anti-PKC antibody showed that PKC was present when the
extracts were incubated with GST fusion proteins containing GluR4
C-terminal amino acids 815-828 (A and B).
Experiments with recombinant PKC show direct interaction of
recombinant PKC with GluR4 C-terminal fragment (C).
D, topology and C-terminal sequence of GluR4 AMPA receptor
subunit. Whole rat brain lysates were used as a positive control in
immunoblot staining (A-C).
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Because there are several scaffolding proteins described to interact
with glutamate receptors (reviewed in Ref. 40) and, in some cases,
known to mediate the interaction of AMPA receptors with kinases and
phosphatases (41), we investigated whether PKC
can directly bind to
the C terminus of GluR4 AMPA receptor subunit or whether an adaptor
protein is required. Purified recombinant PKC
with a C-terminal His
tag was incubated with GST fused to the GluR4 C-terminal segment
containing amino acids 815-838. Again, glutathione-Sepharose was used
to pull down GST fusion proteins and the presence of PKC
in these
samples was analyzed by immunoblotting with a monoclonal anti-PKC
antibody. As shown in Fig. 3C, recombinant PKC
was
detected in pull-down samples, indicating that it directly binds the
C-terminal domain of GluR4.
Phosphorylation within GluR4-PKC
Complexes--
Previous work
(25) showed that the GluR4 AMPA receptor subunit is phosphorylated by
PKC at the C-terminal domain, mainly on Ser-482. Our results
demonstrating that PKC
binds directly to the GluR4 AMPA receptor
subunit suggest that bound PKC
may facilitate GluR4 AMPA receptor
phosphorylation. To test this hypothesis, we performed GST pull-down
assays from rat brain extracts and, after extensively washing beads,
incubated immobilized GST fused to GluR4 C-terminal segments and
interacting proteins, including bound PKC
(Fig.
4A, middle
blot), in the presence of [
-32P]ATP. Eluted
samples were then analyzed by SDS-PAGE, and autoradiography. GluR4
C-terminal fusion protein was phosphorylated by bound kinases (Fig.
4A, upper panel). The S842A GluR4
C-terminal fusion protein, which lacks the major GluR4 phosphorylation
site, was not phosphorylated by bound kinases, but binds PKC
(Fig.
4A). T830A GluR4-GST protein, lacking a minor PKC
phosphorylation site on GluR4, was still phosphorylated in pull-down
samples. Deletion of GluR4 C terminus after amino acid 828 did not
disrupt its binding to PKC
, but eliminated the phosphorylation sites
(Thr-830 and Ser-482) on GluR4 and therefore prevented its
phosphorylation (Fig. 4A). Our results suggest that PKC
bound to GluR4 phosphorylates Ser-482 in GluR4.

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Fig. 4.
Phosphorylation within
PKC -GluR4 complexes. A rat brain extract
was incubated with GST-GluR4 C-terminal proteins, and the fusion
proteins, together with interacting proteins, were pulled down with
glutathione-Sepharose beads. A, pull-down samples were
incubated with [ -32P]ATP for 30 min at 30 °C in PKC
phosphorylation buffer. Phosphorylated GST-GluR4 proteins are shown in
the upper panel. PKC present in the pull-down
samples was detected by Western blotting, and total GST fusion protein
was labeled with an anti-GST antibody (middle
panels). Western blotting analysis using a monoclonal
antibody that recognizes the catalytic domain of PKA (PKAc ) showed
that PKAc was not present in GluR4 C-terminal complexes
(lower panel). Whole rat brain lysates were used
as a positive control. B, sequence alignment of PKC
pseudosubstrate domain and GluR4 C-terminal amino acids 815-828.
Homologous amino acids (box) and conserved pseudosubstrate
amino acids (*) are indicated. C, histone H1 was
incubated with [ -32P]ATP and purified protein kinase
C, for 30 min at 30 °C, in the absence or presence of GST-GluR4
C-terminal proteins or the PKC pseudosubstrate peptide 19-31, as
indicated. Samples were subjected to SDS-PAGE, and the gels were
stained with Coomassie Blue R (middle panel) and
autoradiographed (top panel), to detect histone
phosphorylation. Histone H1 phosphorylated bands were quantified using
ImageQuant software (Amersham Biosciences) and plotted
(bottom graphic). Results are presented as
means ± S.E. of three independent experiments. Statistical
significance was determined by analysis of variance followed by
Dunnett's test.
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PKA was previously described to phosphorylate GluR4 on Ser-482 (25),
and it has been reported to interact with GluR1, through A kinase
anchor protein, via SAP97 (41). Therefore, to exclude the
hypothesis of GluR4 phosphorylation by PKA eventually present in GluR4
complexes, we looked for the presence of PKA in pull-down samples. As
shown in Fig. 4A, no PKA immunoreactivity was detected in
GluR4 C-terminal complexes using an antibody against the catalytic fragment of PKA
(an ubiquitously expressed PKA isoform).
The GluR4 C-terminal segment that interacts with PKC
is homologous
to PKC
pseudosubstrate sequence (Fig. 4B), and its
interaction with PKC
may mimic the pseudosubstrate interaction. In
the activated state of the kinase, the pseudosubstrate is displaced
from the catalytic groove that enables it to interact with substrates. GluR4, through its C-terminal membrane-proximal segment, which presents
homology to the pseudosubstrate, might preferentially occupy the
vacated catalytic site. To test this hypothesis, we tested the effect
of PKC interaction with GluR4 C terminus on the kinase activity on
another substrate. GluR4 C-terminal amino acids 815-828 and 815-838,
which were shown to bind PKC
, inhibited histone H1 phosphorylation
by purified protein kinase C from rat brain to the same extent as the
PKC pseudosubstrate peptide (Fig. 4C). This result supports
the hypothesis that PKC
bound to GluR4 C terminus preferentially
phosphorylates GluR4, to the detriment of other substrates.
PKC
Activation Increases GluR4 Surface Expression--
It was
recently shown that, early in development, rat hippocampal GluR4
Ser-482 phosphorylation by PKA is necessary and sufficient for
GluR4-containing AMPA receptors delivery to synapses (26). Additionally, stimulation of GluR4-expressing HEK cells with PMA resulted in Ser-482 phosphorylation (25). To evaluate the contribution of GluR4 Ser-482 phosphorylation by PKC
to the expression of GluR4-containing AMPA receptor at the plasma membrane, we transfected HEK 293T cells with GluR4, or co-transfected cells with GluR4 and
PKC
. Cells were then stimulated with 200 nM PMA for 10 min. Three hours after the stimulus, cells were incubated with a
biotinylation reagent that reacts with the extracellular domains of
membrane proteins. Proteins extracted from HEK 293T cells were then
incubated with streptavidin gel, which allowed the purification of
biotinylated proteins. Surface and total GluR4 were compared using
quantitative immunoblotting. Co-transfection of PKC
with GluR4
resulted in increased GluR4 subunit surface expression following
stimulation with PMA (Fig. 5), indicating
that phosphorylation by PKC targets GluR4 to the plasma membrane.

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Fig. 5.
GluR4 surface expression in transfected HEK
293T cells. Cultured HEK cells were transfected with GluR4 or
co-transfected with GluR4 and PKC , as indicated. Cells were
biotinylated following stimulation with 200 nM PMA, for 10 min, and surface versus total GluR4 bands were quantified
using the ImageQuant software (Amersham Biosciences) and plotted.
Results are presented as means ± S.E. of three independent
experiments. Statistical significance was determined by the
t test.
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 |
DISCUSSION |
Phosphorylation of AMPA receptors is critical in the control of
synaptic function and plasticity (reviewed in Ref. 18). In this study
we found that PKC
associates with GluR4 AMPA receptor subunit
in vivo and in vitro. Our results further suggest
that this interaction localizes the kinase in close proximity to GluR4, facilitating receptor phosphorylation, and that phosphorylation by PKC
targets the receptor to the plasma membrane.
Co-immunoprecipitation experiments showed that the AMPA receptor
subunit GluR4 associates with the
conventional isoform of PKC, both
in rat cerebellum and in chick retinal cultures (Fig. 1, A
and B). Additionally, PKC
and GluR4 showed partial
overlapping distributions in chick embryo retina cultures (Fig. 2).
Recombinant and native PKC
were retained by GST-GluR4 C terminus,
and deletion analysis showed that the membrane-proximal region of GluR4
C terminus (conserved among AMPA receptor subunits), from amino acids
815-828, is crucial for the binding of GluR4 to PKC
(Fig. 3,
A-C). The corresponding sequence in GluR1 was described to
interact with the protein 4.1N, the neuronal homologue of the
erythrocyte membrane cytoskeletal protein 4.1 (42). PKC
binding to
the GluR4 C-terminal sequence may bring close together the kinase and
its substrate, thereby influencing receptor phosphorylation.
In contrast to the direct interaction between GluR4 and PKC
,
reported here, AMPA receptors have been shown to associate with protein
kinases mainly through adaptor proteins (reviewed in Ref. 18). PICK1,
which interacts with PKC
, is now known to associate with GluR2/3 and
GluR4c (43). PICK1 is co-localized with PKC
and AMPA receptors at
excitatory synapses and was described to homo-oligomerize through its
PDZ domain. In an heterologous expression system, PICK1 was shown to
induce AMPA receptor clustering (44). PICK1 dimers may target PKC
to
AMPA receptors, thus providing a mechanism for selective
phosphorylation of AMPA receptors. GluR2 was determined to be
phosphorylated on Ser-880 by PKC (22), and activation of this kinase
increases phosphorylation of GluR2 Ser-880 and induces long term
depression in the cerebellum (21, 45). Moreover, long term depression
induction in cultured Purkinje cells resulted in Ser-880
phosphorylation and in a long lasting disruption of GluR2 clusters
(23). PICK1 was also reported to form a complex with the mGluR7a
metabotropic glutamate receptors and PKC
. In this case, PICK1 was
shown to play an inhibitory role on PKC
phosphorylation of mGluR7a
(46).
SAP97 binds GluR1 AMPA receptor subunit and was reported to be
important for recruitment of PKA, PKC, and protein phosphatase PP2B
through AKAP79/150. This protein forms a complex with SAP97 that
directs PKA (41) or protein phosphatase PP2B (47) to GluR1,
facilitating GluR1 Ser-845 phosphorylation or dephosphorylation. However, it has recently been reported that the interaction between GluR1 and SAP97 occurs predominantly in the biosynthetic and secretory pathway (48), raising the question of whether the kinase and phosphatase in the complex can regulate receptor activity at synapses.
Having established that GluR4 directly assembles with PKC
, we
searched for functional consequences of this interaction. Previous work
showed that GluR4 is phosphorylated on Ser-482 by PKA, PKC, and
calcium/calmodulin-dependent protein kinase II (25). Our results showed that GST fused to GluR4 C terminus pulled down PKC
,
and that there was phosphorylation of GluR4 Ser-482 within those
complexes (Fig. 4A). In addition, incubation of the GluR4 C-terminal domain, which binds PKC
, with histone H1 (1:3 molar ratio) and purified PKC inhibited histone phosphorylation (Fig. 4C). Sequence alignment of PKC
pseudosubstrate domain and
GluR4 C-terminal amino acids 815-828, the crucial peptide for PKC
binding, shows sequence homology (Fig. 4B). This suggests
that interaction of PKC with the GluR4 C-terminal region, through the
catalytic domain of the kinase, may prevent histone H1 phosphorylation. PKC pseudosubstrate domain contains several basic residues, and it was
suggested that this domain binds to an acidic sequence of the PKC
catalytic domain (49, 50), distinct from the ATP-binding core and the
phosphate transfer region. GluR4 may bind this region in PKC through
its membrane-proximal segment, analogous to the pseudosubstrate,
thereby positioning the phosphorylation site for preferential
phosphorylation by PKC.
We have previously found that PKC up-regulates AMPA receptor activity
in chick embryo retinal cultures, where GluR4 is the main AMPA receptor
subunit expressed (27, 28). Recent work showed that in the rat
hippocampus GluR4 Ser-482 phosphorylation by PKA, activated by
spontaneous activity early in development, is necessary and sufficient
for GluR4-containing AMPA receptors delivery to synapses (26). PKC also
phosphorylates GluR4 Ser-482 in transfected HEK 293T (25). Our results
show that PKC
expression in GluR4 transfected HEK 293T cells
increase GluR4 surface expression upon stimulation with PMA, when
compared with PKC
-deficient cells (Fig. 5). Our results argue for a
role for anchored PKC
in GluR4 receptor subunit phosphorylation and
targeting to the plasma membrane.