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The Journal of Neuroscience, June 15, 1999, 19(12):4748-4754
Characterization of Phosphorylation Sites on the Glutamate
Receptor 4 Subunit of the AMPA Receptors
Ana Luísa
Carvalho1,
Kimihiko
Kameyama2, and
Richard L.
Huganir2
1 Center for Neuroscience of Coimbra, Department of
Biochemistry, University of Coimbra, 3000 Coimbra, Portugal, and
2 Howard Hughes Medical Institute, Department of
Neuroscience, The Johns Hopkins University School of Medicine,
Baltimore, Maryland 21205
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ABSTRACT |
Recent studies have suggested that protein phosphorylation of
glutamate receptors may play an important role in synaptic
transmission. Specifically, the phosphorylation of AMPA
receptors has been implicated in cellular models of synaptic
plasticity. The phosphorylation of the glutamate receptor 1 (GluR1)
subunit of AMPA receptors by protein kinase A (PKA), protein kinase C
(PKC), and Ca2+/calmodulin-dependent protein kinase
II (CaMKII) has been characterized extensively. Phosphorylation
of this subunit occurs exclusively on the intracellular C-terminal
domain. However, the GluR1 subunit C terminus shows low homology to the
other AMPA receptor subunits. In this paper we characterized the
phosphorylation of AMPA receptor subunit GluR4, using site-specific
mutagenesis and biochemical techniques. We found that GluR4 is
phosphorylated on serine 842 within the C-terminal domain in
vitro and in vivo. Serine 842 is phosphorylated
by PKA, PKC, and CaMKII in vitro and is phosphorylated in transfected cells by PKA. Two-dimensional phosphopeptide
analysis indicates that serine 842 is the major phosphorylation site on GluR4. In addition, we identified threonine 830 as a potential PKC
phosphorylation site. These results suggest that GluR4, which is the
most rapidly desensitizing AMPA receptor subunit, may be modulated by phosphorylation.
Key words:
glutamate; AMPA receptors; GluR4; phosphorylation; PKA; PKC
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INTRODUCTION |
Rapid excitatory neurotransmission
in the CNS is mediated mostly by ionotropic glutamate receptors,
which are ligand-gated ion channels that play a role in synaptic
plasticity (Bliss and Collingridge, 1993 ; Linden, 1994), neuronal cell
death (Choi, 1988), and in some forms of neuronal degeneration (Zuo et
al., 1997). Ionotropic glutamate receptors can be divided according to
their molecular structure, sensitivity to agonists, and physiological properties into AMPA, kainate, or NMDA receptors. Molecular
cloning studies identified receptor subunits for these receptor classes (Hollmann and Heinemann, 1994): glutamate receptors 1-4 (GluR1-4) are
AMPA receptor subunits, GluR5-7 and KA1 and KA2 comprise kainate receptors, whereas NMDA receptors are formed by assembly of the NR1
subunit with NR2A-NR2D subunits. Initially, glutamate receptors were
viewed as members of the family of ligand-gated ion channels, typified
by the nicotinic acetylcholine receptors, and thus were assumed to be
pentamers; each subunit was thought to have four transmembrane domains
and extracellular N and C termini. However, it now has been
demonstrated that the topology profile for glutamate receptors consists
of three transmembrane domains, a channel-lining reentrant membrane
loop, an extracellular N-terminal domain, and an intracellular
C-terminal domain (Hollmann et al., 1994; Bennett and Dingledine, 1995;
Wo and Oswald, 1995). Moreover, some recent evidence suggests that
ionotropic glutamate receptors may share a tetrameric structure with
the voltage-gated potassium channels (Mano and Teichberg, 1998;
Rosenmund et al., 1998).
GluR4 has a limited expression in the brain, but it is highly expressed
in the thalamus and in the cerebellum (Keinänen et al., 1990;
Monyer et al., 1991). GluR4 is particularly prominent in cerebellar
granule cells, where, along with GluR2, it probably accounts for most
of the non-NMDA receptor channels (Monyer et al., 1991; Mosbacher et
al., 1994). Swanson and coworkers have shown that the
GluR4flip channels expressed in human embryonic kidney
(HEK) 293 cells have agonist-dependent conductance properties (Swanson
et al., 1997) resembling those of native high-conductance channels in
cerebellar granule cells (Wyllie et al., 1993). Alternative splicing of
the "flip-flop" region of AMPA receptor subunits regulates the
desensitization properties of AMPA receptors and has the largest effect
on GluR4 desensitization. Homomeric AMPA GluR4flop
receptors expressed in oocytes have desensitization time constants of
~1 msec, whereas the GluR4flip homomeric receptors
desensitize approximately four times more slowly (Mosbacher et al.,
1994), suggesting that rapid desensitization of AMPA receptors can be
regulated by the expression and alternative splicing of GluR4. Indeed,
fast synaptic transmission and submillisecond desensitization have been
detected in the cerebellum granule cells by postnatal days 11-17
(Silver et al., 1992), when GluR4flop is expressed (Monyer
et al., 1991). GluR4flop also is expressed in auditory
cells of the cochlear nucleus, where the fast kinetics of the AMPA
receptor are physiologically important in transmitting the signals
necessary for sound localization (Raman et al., 1994) and selective
targeting of GluR4 to auditory nerve synapses has been shown (Rubio and
Wenthold, 1997). Preferential expression of GluR4 may be a common
feature of AMPA receptors mediating fast neurotransmission in the brainstem.
Another example of preferential expression of GluR4 is in the spinal
cord motor neurons (Tomiyama et al., 1996), where rapid neurotransmission has been recorded (Smith et al., 1991) and where calcium-permeable AMPA receptors may be involved in excitotoxicity (Carriedo et al., 1996). In fact, both the reticular nucleus of the
thalamus, which exclusively expresses GluR4 (Keinänen et al.,
1990), and basal forebrain cholinergic neurons, which preferentially express GluR4 (Page and Everitt, 1995), are highly susceptible to
AMPA-induced neurotoxicity (Page and Everitt, 1995), suggesting that
GluR4 may be important in AMPA receptor-mediated excitotoxicity.
Biochemical and physiology studies have demonstrated that the function
of AMPA receptors is modulated by protein phosphorylation in
heterologous cells expressing recombinant receptor subunits, in primary
neuronal cultures, and in hippocampal slices. GluR1 has been shown to
be phosphorylated by PKA on Ser-845 in transfected HEK 293 cells (Roche
et al., 1996), and treatment of hippocampal slices with forskolin
increased the phosphorylation of GluR1 Ser-845 (Mammen et al., 1997).
Furthermore, phosphorylation by PKA of Ser-845 on GluR1 potentiates its
response to glutamate (Roche et al., 1996). These results are in
agreement with earlier studies that described an increase in the
amplitude of AMPA receptor responses on PKA activation, both in
cultured neurons (Liman et al., 1989; Greengard et al., 1991; Wang et
al., 1991) and in oocytes expressing GluR1/GluR3 subunits (Keller et
al., 1992). GluR1 also is phosphorylated on Ser-831 by PKC and CaMKII
both in transfected HEK 293 cells and in hippocampal slices (Roche et
al., 1996; Barria et al., 1997a; Mammen et al., 1997). Recent studies
have shown that GluR1 phosphorylation is modulated during cellular
models of synaptic plasticity, including long-term potentiation and
long-term depression (Barria et al., 1997b; Kameyama et al., 1998; Lee
at al., 1998).
In the present study, we characterized the phosphorylation of GluR4
in vitro and in situ, using site-specific
mutagenesis. We demonstrate that GluR4 is phosphorylated in
vitro on Ser-842 by PKA, PKC, and CaMKII, whereas Thr-830 is
phosphorylated in vitro by PKC. In addition, Ser-842 is
phosphorylated basally in HEK 293T cells in situ, and this
phosphorylation is highly regulated by the PKA activator forskolin.
These results indicate that GluR4 is phosphorylated directly and that
phosphorylation of GluR4-containing receptors may regulate rapid
excitatory synaptic transmission.
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MATERIALS AND METHODS |
Materials. GluR4 cDNA was a kind gift of Dr. Steve
Heinemann (Salk Institute, San Diego, CA). PKC and the catalytic
subunit of PKA were purified as previously described (Reimann and
Behman, 1983; Woodgett and Hunter, 1987). Radioisotopes were purchased from DuPont New England Nuclear (Boston, MA), and cellulose thin layer
chromatography (TLC) plates were from Kodak (Rochester, NY). Forskolin,
phorbol 12-myristate 13-acetate (PMA), and CaMKII were obtained from
Calbiochem (La Jolla, CA). Restriction enzymes were purchased from New
England Biolabs (Beverly, MA), and polyvinylidene difluoride membrane
was from Millipore (Bedford, MA).
Cell culture, transfection, and metabolic labeling. HEK 293T
cells maintained at 37°C and 5% CO2 were transiently
transfected with 10 µg of cDNA, using the calcium phosphate
coprecipitation method, as previously described (Tingley et al., 1997;
Mammen et al., 1999). Cells were used 48 hr after transfection. For
metabolic labeling the cells were incubated with 1 mCi/ml
[32P]orthophosphate in phosphate-free minimal
essential medium (Life Technologies, Gaithersburg, MD) for 4 hr,
and treated for 15 min with drugs, as indicated. Cells were scraped in
150 µl of 1% SDS in IP buffer containing 10 mM sodium
phosphate, pH 7.0, 100 mM NaCl, and protease and
phosphatase inhibitors; the cells were diluted by adding 750 µl of
1% Triton X-100 in ice-cold IP buffer and then sonicated. The residual
insoluble fraction was removed by centrifugation at 14,000 × g for 10 min at 4°C. The GluR4 protein or GluR4 mutant
proteins were isolated by immunoprecipitation, using anti-GluR4
antibodies, as described previously for other AMPA receptor subunits
(Roche et al., 1996). Samples were analyzed on a 7.5% polyacrylamide
gel and visualized by autoradiography.
Fusion proteins. Fusion proteins containing the C-terminal
region of GluR4 (amino acids 815-882) or truncated segments of the
C-terminal region (polypeptides corresponding to amino acids 815-838
and to amino acids 815-852 of GluR4) were constructed by PCR
amplification of GluR4 cDNA, by subcloning into restriction endonuclease sites (BamHI and EcoRI for the
whole-length C terminal; BamHI and SalI for the
truncated segments) in the pGEX4T-2 vector (Pharmacia, Piscataway, NJ),
and by expression in BL21 Escherichia coli. Transformed
bacteria were grown in 200 ml cultures, induced with
isopropyl-1-thio- -galactopyranoside (100 µM) for 2 hr,
and then lysed in PBS containing 1% Triton X-100 and protease
inhibitors. The cells were sonicated and shaken at 4°C for 30 min;
the insoluble fraction was removed by centrifugation at 12,000 × g for 10 min at 4°C. Fusion proteins were purified by
glutathione-Sepharose affinity chromatography, according to the
protocol of the manufacturer (Pharmacia), and were dialyzed overnight
against TBS.
GluR4 fusion protein phosphorylation. Phosphorylation of 1 µg of purified fusion proteins was performed by incubation with 300 ng of purified kinases at 30°C for 30 min in a 100 µl total volume.
Phosphorylation reaction buffer for PKC and CaMKII reactions contained
100 mM HEPES, pH 7.4, 20 mM
MgCl2, 200 µM
CaCl2, and 250 µM ATP. For PKC
reactions, 50 µg/ml phosphatidylserine and 5 µg/ml diacylglycerol
were added. Phosphorylation of fusion proteins by PKA was performed in
20 mM HEPES, pH 7.0, 10 mM
MgCl2, and 250 µM ATP. Reactions
included 5 µCi of [ -32P] ATP and were stopped with
SDS-PAGE sample buffer; the phosphorylated fusion proteins were
resolved by SDS-PAGE and visualized by autoradiography. The
phosphorylated proteins were excised from gels for phosphopeptide mapping.
Phosphopeptide mapping and phosphoamino acid analysis.
Phosphopeptide mapping of phosphorylated proteins was performed
essentially as previously described (Blackstone et al., 1994). Briefly,
the polyacrylamide gel fragments containing the phosphorylated GluR4 or
GluR4 fusion proteins were excised from the gel, digested with 0.3 mg/ml trypsin, and spotted onto TLC plates. The tryptic digests were
separated by electrophoresis (400 V) in acetic
acid/pyridine/H2O (19:1:89, v/v) in the first dimension and
by ascending chromatography in pyridine/butanol/acetic
acid/H2O (15:10:3:12, v/v) in the second dimension. The TLC
plates were exposed in PhosphoImager cassettes and visualized with a
Molecular Dynamics PhosphoImager (Sunnyvale, CA).
For phosphoamino acid analysis, peptides resulting from trypsin
digestion were hydrolyzed in 6 M HCl and spotted onto TLC plates, along with phosphoserine, phosphothreonine, and phosphotyrosine standards. The phosphoamino acids were separated by electrophoresis (400 V) in pH 1.9 buffer (formic acid/acetic acid/H2O;
1:10:89, v/v) for 5 cm and then in pH 3.4 buffer (acetic
acid/pyridine/H2O; 19:1:89, v/v) for 9 cm more. The
phosphoamino acid standards were visualized with ninhydrin, and the TLC
plate was exposed to film to visualize the 32P-labeled
amino acids.
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RESULTS |
To examine the phosphorylation of the AMPA receptor GluR4 subunit,
we transfected HEK 293T cells with GluR4 cDNA and labeled them
with [32P] orthophosphate. GluR4 was
immunoprecipitated via an anti-GluR4 antibody. GluR4 migrated on
SDS-PAGE as a protein of ~105 kDa that showed basal phosphorylation,
which was increased on cell treatment with forskolin (Fig.
1A). Phorbol esters had
little effect on the phosphorylation of GluR4 (Fig.
1A). GluR4 protein was excised from the gel, and
two-dimensional phosphopeptide mapping and phosphoamino acid analysis
were performed. Phosphorylation of recombinant GluR4 occurred
predominantly on serine residues (Fig. 1B), but some
phosphothreonine signal was detected. The increase in phosphorylation
in response to forskolin was on serine residues. Tryptic phosphopeptide
mapping of GluR4 revealed two main clusters of phosphopeptides (Fig.
1C). Treatment of the 293T cells with forskolin or with
phorbol esters did not change the phosphopeptide map patterns
significantly (Fig. 1D,E). No novel major
phosphopeptides appeared on cell stimulation with forskolin, although
forskolin greatly increased GluR4 phosphorylation (Fig. 1A), indicating that the site phosphorylated by PKA
is phosphorylated basally in 293 cells.
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Figure 1.
Phosphorylation of GluR4 AMPA receptor subunit
transiently expressed in HEK 293T cells. A, GluR4 was
immunoprecipitated from labeled cells, resolved by SDS-PAGE, and
visualized by autoradiography. B, After digestion with
trypsin, GluR4 was hydrolyzed with 6N HCl, and the resulting amino
acids were separated by electrophoresis. The circles
indicate the migration of ninhydrin-stained phosphoamino acid
standards. GluR4 phosphoamino acids were visualized by autoradiography.
C-E, Phosphorylated GluR4 was excised from the gel and
digested with trypsin; the resulting phosphopeptides were spotted onto
chromatography plates and resolved in two dimensions. HEK 293T cells
expressing GluR4 were labeled with 1 mCi/ml [32P]
orthophosphate and treated for 15 min with control solution
(C), with 10 µM forskolin
(D), or with 200 nM PMA
(E).
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To identify kinases that directly phosphorylate GluR4 and to narrow
down the regions likely to include the phosphorylation sites, we
constructed glutathione S-transferase fusion proteins containing the C-terminal domain of GluR4 or truncated segments of the
C terminus (Fig. 2). Purified bacterial
fusion proteins were phosphorylated in vitro with purified
kinases in the presence of [-32P] ATP (Fig.
3). PKA phosphorylated the C terminal of
GluR4 as well as the peptide corresponding to amino acids 815-852 of
GluR4 (Fig. 3A). However, PKA phosphorylation was eliminated
in the fusion protein containing GluR4 amino acids 815-838, suggesting that major phosphorylation sites for PKA are in the region between amino acids 838 and 852. Indeed, the tryptic phosphopeptide map of the
PKA phosphorylated GluR4 C-terminal fusion protein (Fig. 3C)
was identical to that of 852 truncated C-terminal fusion protein (data
not shown). In both cases the phosphopeptide maps were identical to
those generated by GluR4 isolated from transfected HEK 293T cells (see
Fig. 1D). Very low phosphorylation of the 838 truncated C-terminal fusion protein was observed, and phosphopeptide
mapping revealed only a minor phosphopeptide for this fusion protein
(data not shown). These results demonstrate that the majority of
C-terminal GluR4 phosphorylation by PKA occurs on residues between 838 and 852 of GluR4. PKA phosphorylated the fusion protein on serine residues only (Fig. 3B).
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Figure 2.
GluR4 C terminal. A, Schematic
diagram of the C-terminal region of GluR4, including the fourth
transmembrane domain (TMD IV). The
expanded region shows the amino acid sequence of the C
terminal, and the residues subjected to site-directed mutagenesis are
indicated by their residue numbers. The
arrows indicate the ending of the truncated C-terminal
bacterial fusion proteins. B, Schematic diagram of the
fusion proteins containing segments of GluR4 C terminal.
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Figure 3.
Phosphorylation of GluR4 fusion proteins.
A, Bacterial fusion proteins containing GluR4 C terminal
or partial segments of GluR4 C terminal (amino acids 815-838 or
815-852) were incubated with purified kinases, as indicated, in the
presence of [-32P] ATP, resolved by SDS-PAGE, and
analyzed by autoradiography. B, Phosphoamino acid
analysis of phosphorylated GluR4 fusion proteins. Phosphoamino acid
analysis was performed as described in Figure 1B.
C-E, The phosphorylated GluR4 C-terminal fusion
proteins were excised from the gel and digested with trypsin; the
phosphopeptides were resolved in two dimensions in TLC plates and
visualized by autoradiography. In every case the phosphopeptide map for
the fusion protein containing amino acids 815-852 of GluR4 protein was
identical to that for the full-length C-terminal GluR4 fusion
protein.
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To characterize PKC phosphorylation of GluR4, we phosphorylated GluR4
C-terminal fusion proteins in vitro by purified PKC, and the
resulting tryptic phosphopeptides were resolved (Fig. 3A,D).
PKC phosphorylated the full-length GluR4 C-terminal fusion protein as
well as the 852 truncated fusion protein (Fig. 3A). However,
as with PKA, the majority of PKC phosphorylation was eliminated in the
fusion protein containing amino acids 815-838, suggesting that
the major phosphorylation sites for PKC are in the region between amino
acids 838 and 852. The tryptic phosphopeptide map of the PKC
phosphorylated GluR4 C-terminal fusion protein (Fig. 3D) was
identical to that of the 852 truncated C-terminal fusion protein (data
not shown). In contrast, the major phosphopeptides were absent in the
838 truncated C-terminal fusion protein. The PKC phosphorylated
wild-type and 852 truncated C-terminal fusion proteins contained the
same phosphopeptide clusters (phosphopeptides 1 and 2) that were
observed for recombinant GluR4 isolated from HEK 293T cells; however,
several additional phosphopeptides (phosphopeptides 3 and 4) were
present in the fusion protein. These phosphopeptides were still
phosphorylated by PKC in the 838 truncated C-terminal fusion protein.
The majority of this phosphorylation occurred on phosphothreonine
residues (Fig. 3B).
CaMKII phosphorylation of the fusion proteins produced phosphopeptides
identical to those produced by PKA and PKC phosphorylation. Basically,
the same two clusters of phosphopeptides (1, 2) were observed, both for
the full-length C-terminal GluR4 fusion protein (Fig. 3E)
and for the fusion protein including amino acids 815 to 852 (data not
shown). CaMKII phosphorylated exclusively serine residues (Fig.
3B). The fusion protein containing the shorter GluR4
C-terminal fragment was not phosphorylated by CaMKII (Fig. 3A).
Taking into account that the major phosphorylation sites on the GluR4
C-terminal domain are in the region between amino acids 838 and 852, we
used site-directed mutagenesis to identify which residues are
phosphorylated. Conversion of serine residue 842 to alanine, in the
GluR4 C-terminal fusion protein, resulted in tryptic phosphopeptide
maps of PKA and PKC phosphorylated fusion proteins that lacked the
major phosphopeptides (phosphopeptides 2) (Figs.
4B, 5B).
Similarly, phosphopeptides 2 were missing in the phosphopeptide map for
the GluR4 S842A mutant C-terminal GST fusion protein phosphorylated
in vitro by purified CaMKII (data not shown). Moreover,
mutation of serine 842 to alanine in full-length recombinant GluR4
eliminated the majority of the GluR4 phosphorylation seen in HEK 293T
cells. Phosphopeptide maps of the mutant GluR4 lacked phosphopeptides 2 in unstimulated cells as well as in cells stimulated with forskolin
(Fig. 4D) or with phorbol esters (Fig. 5E). These results suggest
that S842 is the major phosphorylation site in HEK cells.
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Figure 4.
Identification of a PKA phosphorylation site on
the GluR4 subunit. A, B, Purified fusion proteins
containing the C terminal of GluR4 (A) or of
GluR4 S842A mutant (B) were phosphorylated
in vitro with purified PKA. The phosphorylated fusion
proteins were subjected to phosphopeptide mapping. C, D,
HEK 293T cells expressing GluR4 or GluR4 S842A mutant were labeled with
[32P] orthophosphate and stimulated with
forskolin. Phosphorylated wild-type GluR4 (C) or
the S842A GluR4 mutant (D) were
immunoprecipitated, resolved in SDS-PAGE, and analyzed by tryptic
phosphopeptide mapping.
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Figure 5.
Identification of PKC phosphorylation sites on the
GluR4 subunit. A-C, PKC phosphorylation of wild-type
GluR4 (A), S842A GluR4 (B),
and T830A GluR4 (C) C-terminal GST fusion
proteins. Fusion proteins were incubated with purified PKC in the
presence of [-32P] ATP, resolved by SDS-PAGE, excised
from the gel, and digested with trypsin for phosphopeptide mapping
(A-C) or for phosphoamino acid analysis
(G). For phosphoamino acid analysis the tryptic
phosphopeptides were hydrolyzed and resolved by electrophoresis on a
TLC plate, along with phosphoamino acid standards (migration is
indicated by the circles). D-F,
Wild-type GluR4 (D), the S842A GluR4 mutant
(E), or the T830A GluR4 mutant
(F) were expressed in HEK 293T cells, which were
labeled with [32P] orthophosphate before
stimulation with phorbol esters. Wild-type GluR4 and its mutants were
isolated by immunoprecipitation and analyzed by phosphopeptide
mapping.
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Although S842 is the major site of phosphorylation, GluR4 expressed in
HEK 293T cells also was phosphorylated on threonine residues (see Fig.
1B). Interestingly, PKC phosphorylated the C-terminal
GST fusion proteins in vitro on threonine residues. Threonine phosphorylation was observed on all C-terminal fusion proteins, including the fusion protein containing the shortest C-terminal fragment (amino acids 815-838), indicating that GluR4 C
terminus is phosphorylated by PKC on threonine residues in the region
between amino acids 815 and 838. Because Thr-830 is the only threonine
residue in this region and because it is a consensus site (KXT; Pearson
and Kemp, 1991) for PKC phosphorylation, we mutated Thr-830 to alanine
in the GluR4 C-terminal fusion protein and in the full-length GluR4
subunit. In vitro phosphorylation by PKC of the T830A mutant
GluR4 C-terminal fusion protein produced a phosphopeptide map lacking
phosphopeptide 4 (Fig. 5C), and phosphothreonine was no
longer detected (Fig. 5G). However, we have not been able to
detect phosphopeptide 4 in situ for wild-type GluR4 (see
Fig. 1C-E), and the map for the T830A GluR4 mutant
expressed in HEK 293T cells (Fig. 5F) did not reveal
any differences relative to the wild type.
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DISCUSSION |
Protein phosphorylation plays a critical role in the regulation of
glutamate receptor function and is an important mechanism of synaptic
plasticity (Roche et al., 1994; Moss and Smart, 1996). It has been
demonstrated that AMPA receptor function is regulated by the
phosphorylation of Ser-845 by PKA (Roche et al., 1996) and the
phosphorylation of Ser-831 of GluR1 by both PKC (Roche et al., 1996)
and CaMKII (Barria et al., 1997a; Mammen et al., 1997). However, the
phosphorylation sites described for GluR1 are not conserved in other
AMPA receptor subunits, and the phosphorylation of the GluR2-4
subunits has not been well characterized. Previous studies, using
phosphorylation site-specific antibodies, suggested that Ser-696 in
GluR2 and the corresponding site in other AMPA receptor subunits is
phosphorylated on AMPA receptor activation with agonists (Nakazawa et
al., 1995). However, according to the currently accepted model of the
transmembrane topology of glutamate receptors, this region of GluR2 is
extracellular and therefore inaccessible to intracellular kinases.
In this study we characterized the phosphorylation of the GluR4 subunit
in vitro and in transfected cells, using site-specific mutagenesis and phosphopeptide mapping techniques. We demonstrated that
PKA, PKC, and CaMKII directly phosphorylate GluR4 C-terminal fusion
proteins in vitro and that Ser-842 is phosphorylated basally in GluR4 in transfected cells. Forskolin treatment of the transfected cells dramatically increased the phosphorylation of Ser-842, suggesting that PKA phosphorylates this site in situ. In addition, we
showed that PKC phosphorylates Thr-830 in vitro, but we were
not able to detect the phosphopeptide (phosphopeptide 4) containing
Thr-830 in situ (see Fig. 5A). However,
interestingly, the Thr-830 mutant GluR4 C-terminal fusion protein did
not show any phosphothreonine signal when it was phosphorylated by PKC,
indicating that Thr-830 is the major threonine that is phosphorylated
by PKC in GluR4. Because PKA and CaMKII did not phosphorylate GluR4
fusion proteins on threonine residues, phosphorylation of Thr-830 may
account for most of the phosphothreonine signal, both in GluR4 fusion proteins and in recombinant GluR4 expressed in HEK 293T cells.
GluR4 has high homology in the C-terminal with GluR2c, an alternatively
spliced form of GluR2, which contains a longer C-terminal domain
(Köhler et al., 1994), and both Ser-842 and Thr-830, described here as phosphorylation sites for GluR4, are conserved in GluR2c. However, only a minor fraction of the GluR2 transcripts encodes the
longer C terminus, which could not be detected in the rat brain by
in situ hybridization with a specific probe (Köhler et
al., 1994).
Fast synaptic transmission and submillisecond desensitization have been
detected in several parts of the brain, where they have important
physiological function, for instance as specializations for temporal
coding in the auditory neurons of the cochlear nucleus (Raman et al.,
1994; Rubio and Wenthold, 1997). In all cases, rapid kinetics seems to
be associated with heavy GluR4 expression (Monyer et al., 1991; Smith
et al., 1991; Silver et al., 1992; Wyllie et al., 1993; Mosbacher et
al., 1994; Raman et al., 1994; Tomiyama et al., 1996; Rubio and
Wenthold, 1997; Swanson et al., 1997). We have described Ser-842 as a
major phosphorylation site in GluR4 and identified Thr-830 as a
potential phosphorylation site for PKC. These results suggest that,
similar to other glutamate receptor subunits, the phosphorylation of
GluR4 may be an important mechanism in regulating its functional
properties and in modulating excitatory synaptic transmission.
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FOOTNOTES |
Received Dec. 7, 1998; revised April 1, 1999; accepted April 1, 1999.
A.L.C. was supported by a fellowship from the Portuguese Research
Council (PRAXIS XXI).
Correspondence should be addressed to Dr. Richard L. Huganir, Howard
Hughes Medical Institute, Johns Hopkins University, Department of
Neuroscience, 904A PCTB, 725 North Wolfe Street, Baltimore, MD 21205.
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