From the Ernest Gallo Clinic and Research Center and the
Department of Neurology, University of California San
Francisco, San Francisco, California 94110-3518
Received for publication, September 6, 2002, and in revised form, December 23, 2002
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ABSTRACT |
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We recently identified a novel mechanism
for modulation of the phosphorylation state and function of the
N-methyl-D-aspartate (NMDA) receptor via the
scaffolding protein RACK1. We found that RACK1 binds both the NR2B
subunit of the NMDA receptor and the nonreceptor protein-tyrosine
kinase, Fyn. RACK1 inhibits Fyn phosphorylation of NR2B and decreases
NMDA receptor-mediated currents in CA1 hippocampal slices (Yaka, R.,
Thornton, C., Vagts, A. J., Phamluong, K., Bonci, A., and Ron, D. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 5710-5715). Here, we identified the signaling cascade by which RACK1 is released from the NMDA receptor complex and identified the consequences of the
dissociation. We found that activation of the cAMP/protein kinase A
pathway in hippocampal slices induced the release of RACK1 from NR2B
and Fyn. This resulted in the induction of NR2B phosphorylation and the
enhancement of NMDA receptor-mediated activity via Fyn. We identified
the neuropeptide, pituitary adenylate cyclase activating polypeptide
(PACAP(1-38)), as a ligand that induced phosphorylation of NR2B and
enhanced NMDA receptor potentials. Finally, we found that activation of
the cAMP/protein kinase A pathway induced the movement of RACK1 to the
nuclear compartment in dissociated hippocampal neurons. Nuclear RACK1
in turn was found to regulate the expression of brain-derived
neurotrophic factor induced by PACAP(1-38). Taken together our results
suggest that activation of adenylate cyclase by PACAP(1-38) results in the release of RACK1 from the NMDA receptor and Fyn. This in turn leads
to NMDA receptor phosphorylation, enhanced activity mediated by Fyn,
and to the induction of brain-derived neurotrophic factor expression by RACK1.
The ionotropic glutamate receptor subtype,
N-methyl-D-aspartate
(NMDA),1 plays an essential
role in neuronal development, excitotoxicity, synaptic plasticity, and
learning and memory (2). The ligand-gated NMDA receptor channel is a
heteromer comprised of NR1 and at least one of four NR2 subunits (A-D)
(3). The cytoplasmic tail of the NR2B subunit is phosphorylated on
tyrosine residues (4) and is the most abundant tyrosine-phosphorylated
protein in the postsynaptic density (5). Phosphorylation of NMDA
receptor subunits regulates the activity of the channel (6).
Specifically, application of a tyrosine kinase inhibitor causes a
progressive decrease in NMDA receptor-mediated currents, and
conversely, inhibition of protein-tyrosine phosphatases results in an
increase in NMDA receptor-mediated currents (7). Subsequent studies
have identified the Src family of protein-tyrosine kinases (PTKs) as
enzymes that phosphorylate the NR2 subunits, regulating NMDA receptor
activities (6-8). Hence, modulation of NMDA receptor phosphorylation
by Src family protein-tyrosine kinases is likely to play an important role in modulating glutamate-mediated pathways.
In the recent years it has become increasingly apparent that the
formation of localized signaling complexes, which include receptors,
kinases, phosphatases and their substrates, are highly important for
the regulation of signal transduction cascades (9, 10). Scaffolding
proteins play a major role in the assembly of such signaling complexes
(11). Recently, we identified the scaffolding protein RACK1 as a novel
regulator of the phosphorylation state and function of the NMDA
receptor. We found that RACK1 interacts with both the cytoplasmic tail
of the NR2B subunit and Fyn and inhibits the ability of Fyn to
phosphorylate the NR2B subunit and consequently inhibits NMDA
receptor-mediated excitatory postsynaptic currents (EPSCs) in CA1
hippocampal neurons (1). Peptides that inhibit the interactions between
RACK1 and Fyn and RACK1 and NR2B restored the ability of Fyn to
phosphorylate NR2B in the presence of RACK1, and enhanced NMDA
receptor-mediated EPSCs via activation of the Src family
protein-tyrosine kinase Fyn (1). Based on these results we propose a
model wherein RACK1 localizes Fyn in close proximity with its substrate
the NR2B subunit but prevents the phosphorylation of the subunit until
the appropriate signal occurs. This signal should cause the
dissociation of RACK1 from the NMDA receptor complex, allowing Fyn to
phosphorylate the channel, resulting in enhanced channel activity.
Although it is well established that activation of Src family
protein-tyrosine kinases such as Fyn are required for the
phosphorylation of NMDA receptor subunits and the regulation of channel
activity, the signaling cascades that lead to kinase activation and the subsequent phosphorylation of the NMDA receptor subunits are not fully
understood. Here we identify a signaling pathway that induces the
release of RACK1 from the NMDA receptor complex and the functional consequences of RACK1 dissociation.
Reagents
The polyclonal anti-Fyn, anti-NR2B, anti-NR2A, and anti-CREB
antibodies were purchased from Santa Cruz Biotechnologies. Monoclonal anti-phosphotyrosine, anti-RACK1, and anti-NR2B antibodies were purchased from Transduction Laboratories. The polyclonal
anti-(pY1336)NR2B, anti-(pY1252)NR2B and anti-(pY1472)NR2B antibodies
were a generous gift from M. Greenberg, Harvard University, and are
described elsewhere (12). Forskolin, ifenprodil, and picrotoxin were
purchased from Sigma. Ovine PACAP(1-38), 1,9-dideoxyforskolin, and
protein phosphatase 2 were purchased from Calbiochem. Phorbol
12-myristate 13-acetate (PMA) was purchased from Alexis Corporation.
NBQX and D-AP5 were purchased from Tocris. Sodium
orthovanadate was purchased from Sigma and prepared according to
manufacturer's instructions.
Animals
Fyn Recombinant Proteins
Full-length RACK1 amino acids 1-317 (Tat-RACK1), N-terminal
fragment of RACK1 amino acids 1-180 (RACK1 Preparation of Slice Homogenates
Transverse hippocampal slices (250-300 µm) were prepared from
3-4-week-old male rats or Fyn Immunoprecipitation
The membranal fraction from brain homogenates was precleared by
incubation with protein G-agarose (Invitrogen). The samples were
centrifuged, and the protein quantity was determined using bicinchoninic (BCA) reagent (Pierce). Immunoprecipitation was performed
with 5 µg of the appropriate antibodies, with ~0.5 mg of protein
diluted in 1× immunoprecipitation buffer (1% Triton X-100, 150 mM NaCl, 10 mM Tris HCl, pH 7.4, 1 mM EDTA, 1 mM EGTA, and protease and
phosphatase inhibitor mixtures). After overnight incubation at 4 °C,
protein G-agarose was added, and the mixture was incubated at 4 °C
for 2 h. The agarose resin was washed extensively and samples
resolved by SDS-PAGE on a 10% gel. Membranes were probed with the
appropriate antibodies, and immunoreactivity was detected with ECL and
processed using the STORM system.
Primary Hippocampal Neurons
Newborn rats at P0 were decapitated, and hippocampi were
dissected bilaterally. Cells were dissociated by enzyme digestion with
papain followed by brief mechanical trituration and plated on
poly-D-lysine- (Sigma) treated chamber slides
(Nalgene). Cells were plated (1 × 105 cells/chamber)
and maintained in Neurobasal medium (Invitrogen) supplemented with B27,
penicillin, streptomycin, and Glutamax-1 (Invitrogen) and maintained in
culture for 3 weeks.
Immunocytochemistry
After treatment, RACK1 immunofluorescence was performed as
described previously (14, 15). Briefly, cells were washed in cold
phosphate-buffered saline containing 0.3% Triton X-100, fixed in cold
methanol, and blocked in phosphate-buffered saline containing 0.3%
Triton X-100 and 3% normal goat serum. Immunofluorescence was
performed with monoclonal IgM anti-RACK1 (1:100). Staining was detected
with secondary antibodies conjugated to Texas Red (1:250). Slides were
viewed with a laser scanning confocal microscope (Bio-Rad MRC-1024). Z
field images were processed by obtaining the middle Z field sections
using NIH Image 1.61 (National Institutes of Health) and Adobe
Photoshop (Adobe Systems Inc.).
Nuclear Extraction
Hippocampal slices were homogenized, and nuclear and non-nuclear
extracts were prepared using an NE-PER kit in accordance with the
manufacturer's protocol (Pierce).
Electrophysiology
Transverse hippocampal slices (400-450 µm) were prepared from
3-5-week-old male Fyn+/+, Fyn RT-PCR
Total RNAs were isolated using Trizol reagent (Invitrogen) and
reverse transcribed using a Reverse Transcription System kit (Promega)
at 42 °C for 30 min. BDNF and control GPDH
expression were analyzed by PCR with temperature cycling parameters
consisting of initial denaturation at 94 °C for 2 min followed by 35 cycles of denaturation at 94 °C for 30 s, annealing at 52 °C
for 30 s, extension at 72 °C for 1 min, and a final incubation
at 72 °C for 7 min. The BDNF primers were based on a
coding frame of the rat BDNF gene: upstream, 5'-TTG AGC ACG TGA TCG AAG
AGC-3', and downstream, 5'-GTT CGG CAT TGC GAG TTC CAG-3'. The
GPDH primers were based on the rat GPDH gene:
upstream, 5'-TGA AGG TCG GTG TCA ACG GAT TTG GC-3', and downstream,
5'-CAT GTA GGC CAT GAG GTC CAC CAC-3'. The PCR products were separated
by 1.8% agarose gel and photographed by Eagle Eye II (Stratagene).
Data Analysis
NR2 Phosphorylation--
Scanned images of the bands
corresponding to NMDA receptor subunits were quantitatively analyzed by
densitometry with the NIH Image 1.61 program providing peak areas;
values were expressed as a ratio of phosphorylated to total amount of
NMDA receptor subunits. At the conditions used, all blots were within
the linear range. Statistical analysis was performed using Student's
t test for significant differences.
BDNF mRNA Expression--
Scanned images of the RT-PCR
products were analyzed quantitatively by densitometry with the NIH
Image 1.61 program. The intensities of BDNF and the corresponding GPDH
controls were measured, and the BDNF:GPDH ratio was calculated. The
results are presented as the mean ratio ± S.D. Titration of the
GPDH RT-PCR was performed using 5-cycle increments from 20 to 60 cycles. At the conditions used (35 cycles), GPDH amplification was
observed to be within the linear range.
Identification of a Signal Transduction Cascade That Induces the
Dissociation of RACK1 from the NMDA Receptor Complex--
Several
signal transduction cascades such as PKA (13, 14) and PKC (15-17) have
been shown to affect the localization and/or function of RACK1. We
found previously that the adenylate cyclase activator, forskolin,
induces the translocation of RACK1 to the nucleus in glioma and
neuroblastoma cell lines (14). We therefore hypothesized that
activation of the cAMP signaling cascade which results in the nuclear
compartmentalization of RACK1 could release RACK1 from the NMDA
receptor complex to enable Fyn to phosphorylate the NR2B subunit.
Accordingly, hippocampal slices were treated with vehicle or 10 µM forskolin, and the integrity of the complex was
determined by immunoprecipitation. Anti-RACK1 and anti-NR2B antibodies
were used to immunoprecipitate RACK1 and NR2B, respectively, and
Western blot analysis with anti-RACK1, anti-Fyn, and anti-NR2B antibodies was used to detect coimmunoprecipitation of the
corresponding proteins. As shown in Fig.
1, in mouse hippocampal slices treated with vehicle, anti-RACK1 coimmunoprecipitated Fyn and NR2B, and anti-NR2B antibodies coimmunoprecipitated Fyn and RACK1. These results
are in line with our initial observation of complex formation in rat
hippocampal slices (1). However, incubation of hippocampal slices with
forskolin, but not its inactive analog dideoxyforskolin, resulted in
the dissociation of Fyn and NR2B from RACK1 (Fig. 1, left
panel) and caused Fyn and RACK1 to dissociate from the NR2B
subunit (Fig. 1, right panel). These results suggest that activation of the cAMP signaling pathway initiates the release of RACK1
from the NMDA receptor complex.
Forskolin and PACAP(1-38) Induce Specific Phosphorylation of
NR2B--
If forskolin-induced release of RACK1 from the NMDA receptor
complex is mediating Fyn phosphorylation of the NR2B subunit, then
forskolin should induce the phosphorylation of NR2B in a Fyn-dependent manner. To test the hypothesis, we determined
whether forskolin induced the phosphorylation of the NR2B subunit in
hippocampal slices from wild type (Fyn+/+) mice and from
mice in which the Fyn gene was deleted (Fyn
Next, we assessed whether the activation of hippocampal
Gs-coupled receptors that activate adenylate cyclase would,
like forskolin, enhance tyrosine phosphorylation of NR2B.
Interestingly, we identified the neuropeptide PACAP(1-38) as a ligand
that caused an enhancement of tyrosine phosphorylation of NR2B (Fig.
3a, top two panels) but not NR2A (Fig. 3a, bottom two panels) in rat
hippocampal slices. Next we determined which tyrosines were
phosphorylated on NR2B upon PACAP(1-38) treatment, using
phospho-specific anti-NR2B antibodies raised against phospho-tyrosine
residues, previously identified to be phosphorylated by Fyn (18). At
concentrations as low as 10 nM, PACAP(1-38) induced an
increase in phosphorylation of tyrosines 1336 and 1252 (Fig.
3b) and 1472 (data not shown) on NR2B. We found previously
that RACK1 interaction with Fyn and NR2B prevents the ability of Fyn to
phosphorylate the subunit (1). Thus, if PACAP-induced phosphorylation
of NR2B is indeed mediated via the release of RACK1 from the NMDA
receptor complex, then increasing RACK1 intracellular levels should
inhibit PACAP(1-38)-induced phosphorylation by the interaction of
exogenous RACK1 with Fyn and NR2B. To test the hypothesis, we used the
Tat protein transduction method (13, 19) to transduce RACK1 into
hippocampal neurons (Fig. 3c) and monitored
PACAP(1-38)-induced NR2B phosphorylation. Preincubation of slices with
1 µM Tat-RACK1 inhibited PACAP(1-38)-induced NR2B
phosphorylation of tyrosines 1336 and 1252 (Fig. 3d and data not shown), and there was no significant effect of Tat-RACK1 alone on
basal levels of NR2B phosphorylation. In addition, Tat-RACK1
The PACAP(1-38) receptors are positively coupled to the cAMP/PKA
pathway via G Forskolin and PACAP(1-38) Enhance NMDA Receptor-mediated fEPSPs in
Hippocampal Slices of Wild Type but Not Fyn
Next we verified that PACAP(1-38)-induced enhancement of NMDA receptor
function occurred in hippocampal slices from rat. Bath application of
10 nM PACAP(1-38) increased NMDA receptor-mediated fEPSPs
in rat slices (Fig. 5c) as was observed in the mouse. The concentration of PACAP(1-38) which produced the enhancement in rat
hippocampal slices was 10-fold higher than in slices from mice;
however, this higher concentration was also required for NR2B
phosphorylation in rat slices and therefore is likely to be the result
of a species difference in PACAP(1-38) sensitivity. To examine whether
PACAP(1-38)- or forskolin-induced enhancement of NMDA
receptor-mediated fEPSPs was specific for the NR2B-containing NMDA
receptor channels, fEPSPs were recorded in the presence of the NMDA
receptor antagonist ifenprodil. At low concentrations, ifenprodil is
selective for NR2B-containing receptors (27). Therefore we determined
the IC50 for ifenprodil inhibition on NMDA
receptor-mediated fEPSPs in our rat hippocampal slice preparation. We
found that 5 µM inhibited 50% of NMDA receptor-mediated
fEPSPs. Next, we bath applied 5 µM ifenprodil, and 20 min
later 10 µM forskolin or 10 nM PACAP were
added to the bath. As predicted, 5 µM ifenprodil
completely abolished the PACAP(1-38)- or forskolin-induced increase of
NMDA receptor-mediated fEPSPs (Fig. 5c, top panel traces, and data not shown). If the PACAP(1-38)-induced increase of fEPSPs is mediated by Fyn phosphorylation of NR2B then inhibition of
Fyn kinase activity should abolish this increase. Therefore, fEPSPs
were recorded in the presence or absence of a specific inhibitor for
the Src family of protein-tyrosine kinases, protein phosphatase 2. As
shown in Fig. 5c, PACAP(1-38)-induced enhancement of fEPSPs
was abolished when PACAP(1-38) was coapplied with 25 nM of
protein phosphatase 2. Taken together, these results suggest that
activation of the cAMP/PKA pathway results in the dissociation of RACK1
from the NMDA receptor complex, phosphorylation of NR2B subunit of the
NMDA receptor by Fyn, and the consequent enhancement of NMDA receptor
channel activity in a Fyn-dependent manner.
Forskolin and PACAP(1-38) Induce the Translocation of RACK1 to the
Nuclear Compartment in Hippocampal Neurons--
The intracellular
compartmentalization of RACK1 is not fixed, and different stimuli can
induce its movement to distinct intracellular compartments (13-15).
Therefore, we hypothesized that the release of RACK1 from the NMDA
receptor by activation of the cAMP/PKA pathway would lead to its
redistribution to a different site. We have shown previously that
forskolin induces the nuclear compartmentalization of RACK1 in glioma
and neuroblastoma cell lines (15). To assess whether forskolin could
enhance the nuclear compartmentalization of RACK1 in the hippocampus,
dissociated hippocampal neurons were treated with vehicle or forskolin,
and RACK1 nuclear compartmentalization was determined by
immunohistochemistry and confocal microscopy. RACK1 was found in
processes and cell bodies but not in nuclei in control cells (Fig.
6a, left panel).
Forskolin treatment resulted in a decrease of RACK1 in the processes
and a marked increase in RACK1 staining in the nuclei (Fig.
6a, right panel), suggesting that forskolin
induced the redistribution of a portion of RACK1 into the nucleus. We
also performed a similar experiment in rat hippocampal slices using
subcellular fractionation and isolating nuclear and non-nuclear
fractions. Forskolin treatment increased RACK1 in the nuclear fraction
and decreased RACK1 in the non-nuclear fraction (Fig. 6b).
Next, we hypothesized that if the activation of cAMP/PKA pathway by
forskolin induces the translocation of RACK1 to the nucleus, then this
effect will also be detected with PACAP(1-38) treatment. Rat
hippocampal slices were incubated with PACAP(1-38), and the presence
of nuclear RACK1 was assessed by subcellular fractionation. As shown in
Fig. 6c, the same concentration of PACAP(1-38) (10 nM) which increased NR2B phosphorylation and enhanced NMDA
receptor function (Figs. 3 and 5) also increased RACK1 in the nuclear
fraction. These results suggest that the dissociation of RACK1 from the
NMDA receptor complex may be caused by the translocation of RACK1 to
the nuclear compartment induced by forskolin or PACAP(1-38).
PACAP(1-38) Induces BDNF mRNA Expression via Nuclear
RACK1--
Finally, we analyzed the functional consequences of
PACAP-38-induced RACK1 nuclear compartmentalization. Recently we found that in C6 glioma cells nuclear RACK1 mediates the induction of the
immediate early gene c-fos (13). In cerebellar granule
cells, PACAP(1-38) induces c-fos expression via the
cAMP/PKA pathway (28). PACAP(1-38) has been also shown to increase the
expression of the brain-derived neurotrophic factor (BDNF) in cortical
neurons and in astrocytes (29). We therefore assessed whether
PACAP(1-38) induced expression of BDNF in the hippocampus. As shown in
Fig. 7a, incubation of primary
hippocampal neurons with 10-100 nM PACAP(1-38) resulted
in a dose-dependent increase in mRNA of BDNF. To
determine whether PACAP(1-38) induction of BDNF is mediated via
nuclear RACK1, we incubated hippocampal neurons with PACAP(1-38) and
in the presence and absence of a dominant negative fragment of RACK1 (Tat-RACK1 Compartmentalization of signaling proteins in close proximity to
the NMDA receptor is highly important for the regulation of
phosphorylation of the NMDA receptor subunits and the regulation of
function of NMDA receptor activity. Recently, we identified RACK1 as a
scaffolding protein for Fyn kinase and the NR2B subunit of the NMDA
receptor and revealed a novel molecular mechanism by which RACK1
modulates the function of Fyn and the NMDA receptor (1). RACK1 binds
directly to both Fyn and the cytoplasmic tail of NR2B, inhibits the
phosphorylation of NR2B by Fyn, and decreases NMDA receptor-mediated
EPSCs in CA1 hippocampal neurons (1). Here, we identified the cAMP/PKA
pathway as an important signaling mechanism responsible for 1) the
release of RACK1 from the NMDA receptor complex, 2) the subsequent
tyrosine phosphorylation of the NR2B subunit, 3) the increase in NMDA
receptor-mediated fEPSPs in hippocampal slices in a
Fyn-dependent manner, and 4) the increase in BDNF
expression via RACK1. Based on our results we propose the following
model (Fig. 8). RACK1 binds the
cytoplasmic tail of NR2B and Fyn. This allows Fyn to be localized in
close proximity to its substrate. However, when RACK1 is present in the
complex, Fyn cannot phosphorylate NR2B (Fig. 8a). Activation
of adenylate cyclase by forskolin or activation of
Gs-coupled PACAP(1-38) receptors that activate
the cAMP/PKA pathway releases RACK1 from NR2B and Fyn and induces its
nuclear compartmentalization (Fig. 8b). Once RACK1 is
removed from the NMDA receptor, Fyn is then free to phosphorylate NR2B,
which in turn results in an increase in the activity of the NMDA
receptor channel. Lastly, nuclear RACK1 will induce the expression of
BDNF (Fig. 8b).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/
mice (129SvImJ/C57BL.6J hybrids) were
purchased from Jackson Laboratories. Mice were mated in-house with
129/SVJ wild type mice to generate Fyn+/
mice.
Fyn+/
mice were then mated to generate
Fyn
/
and Fyn+/+ littermate controls, which
were used for biochemical and electrophysiological experiments. The
genotyping of mice was determined by RT-PCR analysis of products
derived from tail mRNA. Only male mice were used in the studies.
The age of the individuals ranged from 3 to 5 weeks. For each colony,
mice were randomly (with respect to the genotype) housed in groups of
five individuals in Plexiglas cages. Food and water were available
ad libitum. Animals were kept in conditions of constant
temperature (23 °C), humidity (50%), and light-dark cycle (light on
from 6:00 am to 6:00 pm). 3-4-week-old male Sprague-Dawley rats were
purchased from Simonsen and kept under the same conditions.
C), or C-terminal
fragment of RACK1 amino acids 138-317 (RACK1
N) were subcloned into
pTAT-HA and expressed in and purified from Escherichia coli
as described previously (13).
/
and Fyn+/+
mice. Slices were maintained for at least 2 h in artificial
cerebrospinal fluid that contained 126 mM NaCl, 1.2 mM KCl, 1.2 mM NaH2PO4, 0.01 mM MgCl2, 2.4 mM
CaCl2, 18 mM NaHCO3, and 11 mM glucose, saturated with 95%O2 and
5%CO2 at 25 °C. After a 1-2-h recovery, slices were
treated and prepared as 10% homogenates in ice-cold 320 mM
sucrose, 10 mM Tris-HCl, pH 7.4, 10 mM EDTA, 10 mM EGTA, protease inhibitor mixture, and phosphatase
inhibitor mixture, with a glass/Teflon homogenizer (12 strokes). After
a short centrifugation (1000 × g, 2 min, 4 °C), to
remove nuclei and large debris, the supernatant was centrifuged at
10,000 × g for 30 min at 4 °C to obtain crude
synaptosomal fraction. The membranal pellet was resuspended in
solubilization buffer (1% deoxycholate, 10 mM Tris-HCl, pH 7.4, 10 mM EDTA, 10 mM EGTA, and protease and
phosphatase inhibitor mixture).
/
mice or
3-4-week-old male Sprague-Dawley rats. The slices were allowed to
recover for at least 1-2 h in artificial cerebrospinal fluid perfusion
medium as described above. After recovery, slices were submerged and
superfused continuously with artificial cerebrospinal fluid at
25 °C. Field excitatory postsynaptic potentials (fEPSPs) were
recorded from stratum-radiatum of CA1 region with glass microelectrodes filled with 2 M NaCl. To obtain NMDA receptor-mediated
fEPSPs, 100 µM picrotoxin and 10 µM NBQX
were added to the bath solution to block
-aminobutyric acid type A
receptor- and (AMPA)
-amino, 3-hydroxy, 5-methyl,
4-isoxazolepropionic acid receptor-mediated IPSPs and EPSPs,
respectively. To evoke fEPSPs, Schaffer collateral/commissural afferents were stimulated with 0.1-Hz pulses using steel bipolar microelectrodes at intensities adjusted to produce an evoked response that was 50% of the maximum-recorded fEPSP for each recording. The
maximal rate of change in fEPSP within a time window selected around
the rising phase was calculated. Data were collected using an
Axopatch-1D amplifier (Axon instruments, Foster City, CA), filtered at
2 kHz, and digitized at 5-10 kHz. To obtain AMPA receptor-mediated fEPSPs, 100 µM picrotoxin and 50 µM D-AP5
were added to the bath solution to block
-aminobutyric acid type
A receptor- and NMDA receptor-mediated IPSPs and EPSPs, respectively.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Forskolin induces release of NR2B and Fyn
from RACK1 in hippocampal slices. Hippocampal slices from wild
type mice were treated with vehicle (Control) 10 µM forskolin, or 10 µM
1-9-dideoxyforskolin (dd Forskolin) for 15 min. Slices were
homogenized, and immunoprecipitations (IP) were performed
using anti-RACK1 (5 µg, left panel) and anti-NR2B (5 µg,
right panel) antibodies. Control immunoprecipitation with
anti-mouse IgM was also included (right panel). The presence
of proteins in hippocampal homogenates was verified by Western blot
(Input, left panel). Samples were separated by
SDS-PAGE on a 10% gel and probed with anti-NR2B (top
panels), anti-Fyn (middle panels), and anti-RACK1
antibodies (bottom panels). Results shown are representative
of three experiments.
/
).
Hippocampal slices were treated with vehicle or forskolin, the NR2B
subunit was immunoprecipitated, and phosphorylation was determined with
anti-phosphotyrosine antibodies. As shown in Fig. 2, forskolin caused a significant
increase in the phosphorylation of the NR2B subunit in
Fyn+/+ mice (Fig. 2, top two panels).
Importantly, the tyrosine phosphorylation of the NR2B subunit was not
detected in Fyn
/
(Fig. 2, middle two
panels), suggesting that as predicted, Fyn is the kinase that
phosphorylates the NR2B subunit of the NMDA receptor upon cAMP/PKA
stimulation. Because RACK1 localizes Fyn specifically to the NR2B
subunit but not NR2A (1, and data not shown), we hypothesized that the
release of RACK1 from the NMDA receptor complex would not alter the
phosphorylation state of the NR2A subunit. Indeed, there was no
detectable phosphorylation of the NR2A subunit in hippocampal slices
from Fyn+/+ mice treated with forskolin (Fig. 2,
bottom two panels).
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Fig. 2.
Forskolin induces tyrosine phosphorylation of
NR2B from Fyn+/+ but not from
Fyn /
mice. Hippocampal slices from Fyn+/+ (top
two and bottom two panels) or Fyn
/
(middle two panels) mice were preincubated for 15 min with
10 µM Na2VO4 and then incubated
for 15 min in the absence (control) or presence of 10 µM
forskolin. Immunoprecipitations (IP) were performed using
anti-NR2A or anti-NR2B antibodies, and the controls were mouse IgG
(top four panels) and goat IgG (bottom two
panels). Control anti-NR2A and anti-NR2B antibodies were included.
Membranes were probed with anti-phosphotyrosine (pY),
anti-NR2A, or anti-NR2B antibodies, as indicated. Results shown are
representative of four experiments.
N that
lacks the binding sites for Fyn and NR2B (1) was inactive (Fig.
3e), suggesting that Tat-RACK1 inhibition of NR2B
phosphorylation is specific. Taken together, these results suggest that
NR2B phosphorylation is mediated by the release of RACK1 from NMDA
receptor, enabling Fyn to phosphorylate the subunit.
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Fig. 3.
PACAP(1-38) induces tyrosine phosphorylation
of NR2B which is inhibited by RACK1. a, 300-µm rat
hippocampal slices were incubated with 10 µM
Na2VO4 for 30 min followed by bath application
of 100 nM PACAP(1-38) for 10 min. Immunoprecipitations
were performed using anti-NR2B or anti-NR2A antibodies. Membranes were
probed with anti-phosphotyrosine (pY; first and
third panels), anti-NR2B (second panel), or
anti-NR2A antibodies (bottom panel). Results were quantified
using NIH Image. The histogram depicts levels of tyrosine
phosphorylation normalized to total NR2A and NR2B and presented as
percent of control ± S.D. of three experiments (**significantly
greater than NR2A p < 0.01, t test).
b, rat hippocampal slices were preincubated with 10 µM Na2VO4 for 30 min. Slices were
then treated with 10 nM PACAP(1-38) for 15 min. Membranes
were probed with the anti-phospho-NR2B-specific antibodies
anti-[pTyr1252]NR2B and anti-[pTyr1336]NR2B. Results shown are
representative of three experiments. c, to ensure
transduction of the Tat fusion protein, rat hippocampal slices were
incubated with 1 µM Tat-RACK1 for 2 h. Slices were
then washed, homogenized, and the deoxycholate-soluble fraction was
isolated. Immunoprecipitation (IP) with anti-HA antibodies
was performed, and proteins were resolved by SDS-PAGE. Membranes were
probed with anti-HA (left panel) and anti-RACK1 antibodies
(right panel). Results shown are representative of three
experiments. d, rat hippocampal slices were incubated with
phosphate-buffered saline (Control, lanes 1 and
2) or with 1 µM Tat-RACK1 (lanes 3 and 4) for 2 h followed by a 30-min incubation with 10 µM Na2VO4. Slices were then
treated without (lanes 1 and 4) or with 10 nM PACAP(1-38) (lanes 2 and 3) for
15 min. Membranes were probed with the anti-[pTyr1336]NR2B. Anti-NR2B
antibodies were used to verify equal loading. The histogram depicts
levels of tyrosine phosphorylation normalized to total NR2B and
presented as percent of control ± S.D. of three experiments.
**significantly lower than PACAP(1-38) alone, p < 0.01, t test. e, rat hippocampal slices were
incubated with phosphate-buffered saline (control) or with 1 µM Tat-RACK1 N for 2 h followed by a 30-min
incubation with 10 µM Na2VO4.
Slices were then treated without (control) or with 10 nM
PACAP(1-38) for 15 min. Membranes were probed with the
anti-phospho-NR2B-specific antibodies anti-[pTyr1336]NR2B. Anti-NR2B
antibodies were used to verify equal loading. Results shown are
representative of three experiments.
s, and PACAP(1-38) is the most potent
endogenous activator of the cAMP signaling pathway (20). However,
PACAP(1-38) receptors have also been linked to the activation of the
PKC pathway via G
q (20). In addition, PKC activation has
been shown to increase the function of the NMDA receptor channel via
Src (21, 22). We therefore also tested the ability of the PKC activator PMA to release RACK1 from Fyn and NR2B and to induce NR2B
phosphorylation in rat hippocampal slices. Slices were incubated with
vehicle or 500 nM PMA, and the presence of the
Fyn, RACK1, and NR2B complex and the phosphorylation state of NR2B were
determined. PMA treatment did not result in the dissociation of RACK1
from Fyn and NR2B (Fig. 4a),
nor did PMA induce the phosphorylation of NR2B (Fig. 4b),
suggesting that activation of PKC is not involved in the release of
RACK1 from the NMDA receptor complex which results in NR2B
phosphorylation. Taken together, our results suggest that activation of
the cAMP/PKA pathway by PACAP(1-38) releases RACK1 from the NMDA
receptor complex to induce Fyn phosphorylation of NR2B.
View larger version (23K):
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Fig. 4.
PKC activation does not release Fyn and NR2B
from RACK1 and does not induce tyrosine phosphorylation of NMDA-NR2B in
hippocampal slices. a, rat hippocampal slices were
preincubated for 30 min with 10 µM
Na2VO4 and then incubated with 500 nM PMA or vehicle for 30 min. Immunoprecipitations
(IP) were performed using anti-RACK1 antibodies. Samples
were separated by SDS-PAGE on a 10% gel and probed with anti-NR2B,
anti-Fyn, and anti-RACK1 antibodies. Results shown are representative
of three experiments. b, rat hippocampal slices were treated
as described in a, and immunoprecipitations were performed
using mouse monoclonal anti-NR2B antibodies. Samples were separated by
SDS-PAGE on a 10% gel and probed with anti-phosphotyrosine
(pY) antibodies and polyclonal anti-NR2B. Results shown are
representative of three experiments.
/
Mice--
Tyrosine phosphorylation has been shown previously to
increase the activity of the NMDA receptor channel (7, 8, 23). PACAP(1-38) was found to enhance NMDA receptor function in chick cortical neurons (24), in sympathetic preganglionic neurons (25), and
in the CA1 region of the hippocampus (26). However, the molecular
mechanism that mediates PACAP induction of NMDA receptor activity is
unknown. We proposed that activation of the cAMP/PKA pathway by
forskolin or PACAP(1-38) resulting in the phosphorylation of the NR2B
by Fyn caused the enhancement of NMDA receptor-mediated activities. To
examine this possibility, hippocampal slices from Fyn+/+
and Fyn
/
mice were treated with 10 µM
forskolin or 1 nM PACAP(1-38), and NMDA receptor-mediated
fEPSPs were recorded from hippocampal CA1 region. A robust increase in
fEPSPs was measured in hippocampal slices from Fyn+/+ mice
when forskolin, but not dideoxyforskolin was bath applied (Fig.
5a). The same increase was
observed upon bath application of PACAP(1-38) (Fig. 5b).
Importantly, there was no change in fEPSPs in hippocampal slices from
Fyn
/
mice treated with either forskolin (Fig.
5a) or PACAP(1-38) (Fig. 5b), suggesting that
Fyn is required for forskolin- or PACAP(1-38)-induced potentiation of
NMDA receptor-mediated fEPSPs. To rule out the possibility that
PACAP(1-38)-induced enhancement of NMDA receptor-mediated potentials
was cause by enhanced presynaptic glutamate release, AMPA
receptor-mediated fEPSPs were recorded in the CA1 region. As shown in
Fig. 5b, there was no change in AMPA receptor-mediated fEPSPs after bath application of PACAP(1-38), suggesting that PACAP(1-38)-induced enhancement of NMDA fEPSPs occurred
postsynaptically.
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Fig. 5.
PACAP(1-38) or forskolin enhances NMDA
receptor-mediated fEPSPs in CA1 in a Fyn-dependent manner.
a, NMDA receptor-mediated fEPSPs slope plotted for
recordings made from CA1 hippocampal slices. For all recordings, 10 µM Na2VO4 was bath applied 30 min
before the application of drugs. fEPSPs were recorded from
Fyn+/+ mice in the presence of 10 µM
forskolin ( , n = 6) or 10 µM
dideoxyforskolin (dd-Forskolin, the inactive analog of
forskolin (
, n = 5) and from Fyn
/
in
the presence of 10 µM forskolin (
, n = 7), as indicated by the horizontal bar. Data are presented
as the mean percent of base line ± S.E. b, NMDA
receptor-mediated fEPSPs were recorded in the presence of 1 nM PACAP(1-38) in Fyn+/+ mice (
,
n = 6) and in Fyn
/
mice (
,
n = 5). The AMPA receptor-mediated fEPSP slope was
recorded from CA1 hippocampal slices of Fyn+/+ mice (
,
n = 5) before and during bath application of 1 nM PACAP(1-38). The horizontal bar indicates
the period of PACAP(1-38) application. Data are presented as the mean
percent of base line ± S.E. c, NMDA receptor-mediated
fEPSPs were recorded in the presence of 10 nM PACAP(1-38)
(
, n = 5), 25 nM protein phosphatase 2 (PP2,
, n = 5), or PACAP + protein
phosphatase 2 (
, n = 5). The horizontal
bar indicates the period of drug application. Data are presented
as the mean percent of base line ± S.E. The traces on
the left inset represent NMDA receptor synaptic responses in the CA1 field (average of six single sweeps) taken
during base-line recordings and after 10 min of PACAP application. The
traces on the right inset represent NMDA receptor
synaptic responses (average of six single sweeps) taken during
base-line recordings after 10 min of 5 µM ifenprodil
application and during 10 min coapplication of ifenprodil and PACAP as
indicated.
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Fig. 6.
Forskolin induces RACK1 nuclear
compartmentalization in hippocampal neurons. a, dissociated
hippocampal neurons were treated with vehicle (Control) or
10 µM forskolin for 15 min at 37 °C. Cells were then
fixed and stained with anti-RACK1 antibodies and viewed at ×60
magnification using a confocal microscope. Images shown are the
individual middle sections of a projected Z series. Results are
representative of three experiments. b, rat hippocampal
slices were incubated with vehicle (Control) or 10 µM forskolin for 15 min. Slices were homogenized and
nuclear and non-nuclear fractions prepared. Nuclear (50 µg,
lanes 1 and 2) and non-nuclear (50 µg,
lanes 3 and 4) extracts were separated by
SDS-PAGE, transferred to nitrocellulose membrane, and probed with
anti-RACK1 or anti-CREB antibodies. Results are representative of two
experiments. c, rat hippocampal slices were incubated with
vehicle (Control) or 10 nM PACAP(1-38) for 5, 15, and 30 min. After treatment, slices were homogenized and the
nuclear fraction prepared. Nuclear extracts (50 µg/lane)
were separated by SDS-PAGE, transferred to nitrocellulose, and probed
with anti-RACK1 or anti-CREB antibodies. The histogram depicts
quantification of RACK1 levels in the nuclear fraction normalized to
CREB and presented as mean percent of control ± S.D.
(n = 3).
C) which inhibits RACK1 nuclear compartmentalization and
function (13). Preincubation of hippocampal neurons with 1 µM Tat-RACK1
C inhibited PACAP(1-38)-induced BDNF
expression at 10 nM (not shown) or even 100 nM
PACAP(1-38) (Fig. 7b). However, Tat-RACK1
C alone did not
affect the basal levels of BDNF mRNA (Fig. 7b).
Therefore, our results suggest that the release of RACK1 from the NMDA
receptor complex results not only in the enhancement of NMDA receptor
activity by Fyn but also in an increase in the mRNA levels of BDNF
via RACK1 itself.
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Fig. 7.
PACAP(1-38) increases BDNF expression via
nuclear RACK1. a, dissociated hippocampal neurons were
incubated with vehicle (lane 1) or increasing concentrations
of PACAP(1-38) (lanes 2-4) for 30 min at 37 °C. Total
RNAs were used to analyze the expression of BDNF and
GPDH by RT-PCR. PCR products were separated on an agarose
gel and photographed by Eagle Eye II. The histogram depicts the mean
BDNF:GPDH ratio ± S.D., n = 3. b, dissociated hippocampal neurons were incubated with
vehicle (lane 1), with 100 nM PACAP(1-38) for
30 min (lane 2), preincubated with 1 µM
Tat-RACK1 C for 30 min (lane 3), or preincubated with 1 µM Tat-RACK1
C for 30 min and then treated with 100 nM PACAP(1-38) for an additional 30 min (lane
4). RNA isolation and RT-PCR were performed as described in
a. The histogram depicts the mean
BDNF:GPDH ratio of ± S.D.,
n = 3.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (12K):
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Fig. 8.
RACK1 forms a complex with Fyn and
NR2B and is released upon adenylate cyclase stimulation. a,
RACK1 binds the cytoplasmic tail of NR2B and Fyn. This allows Fyn to be
localized in close proximity to its substrate. However, when RACK1 is
present in the complex, Fyn cannot phosphorylate NR2B. b,
activation of adenylate cyclase by forskolin or activation of
Gs-coupled PACAP(1-38) receptor PAC1 that activates the
cAMP/PKA pathway releases RACK1 from NR2B and Fyn and induces its
nuclear compartmentalization. Once RACK1 is removed from the NMDA
receptor, Fyn is free to phosphorylate the NR2B subunit, which in turn
results in an increase in the activity of the NMDA receptor channel. As
a result of RACK1 nuclear translocation, BDNF expression is
enhanced.
We found that the activation of the cAMP/PKA signaling pathway resulted
in the release of RACK1 from the NMDA receptor complex. Several reports
suggest a link among RACK1, cAMP signaling, and NMDA receptors. Yarwood
et al. (30) identified a direct interaction between RACK1
and the cAMP-specific phosphodiesterase 4 isoform phosphodiesterase 5 (30), and recently phosphodiesterase 4 was found to regulate the
function of the NMDA receptor and thus is likely to be part of the
postsynaptic density complex as well (31). Moreover, several reports
suggest a link between cAMP/PKA signaling and the phosphorylation state
and activity of the NMDA receptor channel. In vitro, PKA
phosphorylates the NR1 subunit of the NMDA receptor (32, 33). Forskolin
enhances NMDA receptor activity in spinal cord dorsal horn neurons and
in hippocampal neurons in culture (34, 35), and the NMDA
receptor-associated protein, Yotiao (36), interacts with PKA holoenzyme
and type 1 protein phosphatase (37). Taken together, these findings
strongly suggest that the cAMP machinery is compartmentalized within
close proximity to RACK1 and the NMDA receptor, allowing the rapid
dissociation of RACK1 upon activation of a Gs-coupled
receptor, and the rapid regulation of the NMDA receptor function.
We identified the neuropeptide PACAP(1-38) as a ligand responsible for
the phosphorylation of the NR2B subunit and the enhancement of NMDA
receptor-mediated activities via Fyn. PACAP(1-38) mediates its
activities via two receptors, type 1 (PAC1), which binds PACAP(1-38) in high affinity, and type 2 (PAC2), which binds PACAP(1-38) and vasoactive intestinal peptide at equal affinities (38). The PAC1
receptor is highly expressed in several regions of the central nervous
system including the hippocampus (38). The hippocampus mainly expresses
the PAC1 but not PAC2 receptors (39), and electron microscope studies
revealed that the PAC1 receptors are found in postsynaptic densities of
synaptic junctions in brain regions including the hippocampus (40).
Therefore, it is likely that the increase in tyrosine phosphorylation
of NR2B occurs via activation of the PAC1 receptors by PACAP(1-38).
PAC1 receptors are coupled mainly to the cAMP/PKA pathway via
Gs, but also to the phospholipase C/PKC pathway via
activation of G
q-coupled receptors (19). However, it is
unlikely that PACAP(1-38) is mediating the phosphorylation of NR2B and
the enhancement of NMDA receptor activities via the PLC/PKC pathway.
First, we could not detect dissociation of the NR2B·RACK1·Fyn
complex upon activation of PKC with the PKC activator PMA. Second,
activation of the phospholipase C/PKC pathway requires higher
concentrations of PACAP(1-38) (41) compared with the concentrations
used here, which enhanced the phosphorylation and function of the NMDA
receptor. Interestingly, PACAP(1-38) has been implicated in learning
and memory. PAC1 deletion mutant mice show deficits in contextual fear
conditioning, which is a hippocampus-dependent associative
learning, and in a passive avoidance learning (42), and thet also show
impairment of hippocampal mossy fiber long term potentiation (43).
Thus, it is possible that the enhancement of NMDA receptor function by
PACAP(1-38) is important for synaptic plasticity, a process that
underlies several forms of learning and memory.
In addition to PACAP(1-38) activities on NMDA receptor, we found that the peptide enhanced the expression of BDNF via nuclear RACK1. Recently, we found that in C6 glioma cells nuclear RACK1 mediates the induction of expression of the immediate early gene c-fos in a cAMP/PKA-dependent manner (13). In cerebellar granule cells, PACAP(1-38) was found to induce c-fos expression via the cAMP/PKA pathway (28). Because the promoter region of BDNF contains several AP1 sites (44), it is possible that PACAP(1-38) induces BDNF expression via c-fos. Interestingly, BDNF has also been strongly implicated in the mechanism leading to enhancement of synaptic strength (45). Presynaptically, BDNF enhances glutamate release in hippocampal neurons (46-48). Postsynaptically, BDNF enhances the activity of NMDA receptors (49, 50), at least in part, by inducing the phosphorylation of NR1 and NR2B but not NR2A subunits (51). BDNF is essential for the formation of long-term potentiation, and BDNF knockout mice have impaired long-term potentiation that is restored with viral infection of BDNF (52-57). In addition, in excised membrane patches from cultured hippocampal neurons, acute exposure to BDNF increases NMDA single channel open probability, and this effect is dependent on the presence of the NR2B subunit (58). These findings raise the possibility that the increase in BDNF levels via the nuclear compartmentalization of RACK1 initiates a positive feedback loop for further enhancing the function of hippocampal NMDA receptors.
In summary, we have identified cAMP/PKA as an important regulatory
signaling pathway for the localization and function of the scaffolding
protein RACK1. The movement of other scaffolding proteins has been
shown to be important for proper transduction of signaling cascades and
regulation of gene expression. For example, the scaffolding protein
STE5 moves to the nucleus to induce transcription (59). The dual
function of RACK1 in response to cAMP/PKA activation, i.e.
an enhancement in NMDA receptor phosphorylation and activity as well as
an increase in expression of BDNF, leads to the interesting possibility
that the dynamic compartmentalization of scaffolding proteins adds yet
another dimension to signaling events. Our results also suggest
that the dynamic compartmentalization of RACK1 is important for the
regulation of processes leading to synaptic plasticity.
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ACKNOWLEDGEMENTS |
---|
We thank S. Dowdy, University of California San Diego, for providing the pTAT-HA plasmid and M. Greenberg, Harvard University, for providing the phospho-NR2B antibodies. We thank our colleagues Fredrick (Woody) Hopf, Patricia Janak, Claire Thornton, and Jennifer Whistler for critical reading of the manuscript.
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FOOTNOTES |
---|
* This work was supported by funds provided by the state of California for medical research on alcohol and substance abuse through the University of California San Francisco (to D. R.) and NIAAA (R01AA/MH13438-O1A1) (to D. R.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ To whom correspondence should be addressed: Ernest Gallo Clinic and Research Center, 5858 Horton St., Suite 200, Emeryville, CA 94608. Tel.: 510-985-3150; Fax: 510-985-3101; E-mail: dorit@itsa.ucsf.edu.
Published, JBC Papers in Press, January 10, 2003, DOI 10.1074/jbc.M209141200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: NMDA, N-methyl-D-aspartate; BDNF, brain-derived neurotrophic factor; CREB, cAMP-response element-binding protein; EPSC(s), excitatory postsynaptic current(s); EPSP(s), excitatory postsynaptic potential(s); fEPSP(s), field excitatory postsynaptic potential(s); GPDH, glycerol-3-phosphate dehydrogenase; HA, hemagglutinin; IPSP(s), inhibitory postsynaptic potential(s); PACAP, pituitary adenylate cyclase-activating polypeptide; PKA, protein kinase A; PKC, protein kinase C; PMA, phorbol 12-myristate 13-acetate; RT, reverse transcription.
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