(Received for publication, July 21, 1995)
From the
-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)
receptor channels play important roles in plasticity,
neurotransmission, and neurotoxicity in the central nervous system.
AMPA, but not N-methyl-D-aspartate (NMDA), receptor
signaling in rat cortical neurons was found to involve a G-protein
coupled to a protein kinase cascade. While both NMDA and AMPA activated
p
mitogen-activated protein kinase (MAPK) in neurons, only
AMPA-induced MAPK was inhibited by pertussis toxin. AMPA, but not NMDA,
caused an association of a G-protein
subunit with a Ras, Raf
kinase, and MAP/ERK kinase (MEK)-1 complex. The evidence indicates that
AMPA triggers MAPK activation via a novel mechanism in which G-protein
dimers released from G
bind to a Ras protein complex
causing the activation of Ras, Raf kinase, MEK-1, and finally MAPK.
Glutamate is the major excitatory amino acid in the brains of
vertebrates, and its receptors are believed to mediate a wide range of
physiological and pathological processes including neurotransmission,
plasticity, excitotoxicity, and various forms of
neurodegeneration(1) . Many of the actions of glutamate are
coupled to the influx of extracellular Ca mediated
directly or indirectly by NMDA (
)receptors present on
neurons(1, 2) . More recently, it has been determined
that the AMPA glutamate receptor, which normally passes
Na
, is also directly Ca
-permeable (3, 4) and can be an important source of
Ca
influx in some types of
neurons(3, 4, 5) . Transient changes in
intracellular Ca
are known to trigger a plethora of
cellular responses in neurons including the activation of protein
kinases and phosphatases and changes in gene
expression(1, 2) . The NMDA-mediated entry of
Ca
has been shown to activate MAPK(6) , a
serine/threonine kinase known to be important in the transmission of
extracellular signals to the nucleus of
cells(7, 8, 9) . While the exact role and
impact of NMDA-activated MAPK in neuronal cells is unclear, it may be
that MAPK serves as a conduit through which NMDA receptor-mediated
Ca
signals are converted into relevant nuclear
responses. By contrast, it is not known if the entry of Ca
via AMPA glutamate receptors is coupled to signaling
pathways responsible for the activation of MAPK in neurons.
It is
becoming increasingly evident that the influx of Ca through different ion channels can elicit distinct cellular
responses in neurons(2) . For example, the entry of
Ca
through NMDA receptors and L-type
voltage-sensitive calcium channels (VSCCs) have been shown to transmit
signals to the nucleus and to regulate gene expression through two
distinct signaling pathways (10) . It is therefore possible
that mechanisms may be present in neurons by which different
Ca
ion channels can differentially activate MAPK
through distinct signaling pathways. In the present study we
investigated whether Ca
entry through the AMPA
glutamate receptor stimulates MAPK in rat cortical neurons, and if so,
whether the mechanism of MAPK activation by AMPA is different from that
induced by NMDA.
Figure 3:
AMPA, but not NMDA or KCl, causes the
association of a G-protein subunit with Ras complex.
Following the treatments indicated below, Ras immunoprecipitates were
subjected to SDS-10% PAGE, electrotransferred, and probed with an
anti-G-protein
subunit antibody as described (16) . The
blots were then stripped and reprobed with an anti-Ras
antibody(16) . A, untreated controls or cultures
treated with 100 ng/ml PTX for 20 h were stimulated with 100 µM AMPA for 3 min. B, cultures were left unstimulated (laneC) or were stimulated with 100 µM AMPA (laneA), 100 µM NMDA (laneN), or 50 mM KCl (laneK) for 3 min. C, the effect of extracellular
Ca
on the coprecipitation of G-protein
subunit
with Ras. Cultures were incubated in DMEM with or without 2.4 mM Ca
for 15 min and then stimulated with the
indicated doses of AMPA for 3 min.
Figure 4: The association of Raf kinase and MEK-1 with Ras in neurons stimulated by AMPA. Cultures were treated with AMPA as described in the legend to Fig. 3, and the immunocomplex precipitated by the Ras antibody was separated, transferred, and blotted with the following antibodies (16) : A and B, anti-B-Raf or C, anti-MEK-1.
AMPA, NMDA, and KCl-induced depolarization all caused
transient 2.5-fold increases in MAPK activity in primary rat
cortical neurons, which peaked 3 min after treatment (Fig. 1A). However, whereas MAPK activity returned to
near basal levels within 10 min of NMDA or KCl addition,
AMPA-stimulated MAPK declined gradually over a 20-60-min period (Fig. 1A). The mechanism responsible for the prolonged
activation of MAPK by AMPA was further investigated.
Figure 1:
Effects of AMPA, NMDA, and KCl on MAPK
activation. A, cortical neurons (24) were stimulated
with AMPA, NMDA, or KCl and at the indicated times MAPK activity was
determined (25) by the ability of immunocomplexed MAPK to
phosphorylate myelin basic protein (MBP). B, MAPK
activity was determined in neuronal cultures treated with the indicated
concentrations of AMPA for 3 min. C and D, neurons
incubated in medium with or without Ca or
Na
(24) , respectively, were treated with the
indicated agents for 3 min before MAPK activity was determined. E, neurons were treated with 5 µM nifedipine for
15 min and then challenged with the indicated agents for 3 min before
MAPK activity was determined. For A, C, D,
and E the concentrations of AMPA, NMDA, KCl, and epidermal
growth factor (EGF) were 100 µM, 100
µM, 50 mM, and 100 ng/ml, respectively. Each
point is the mean ± S.E. of radioactivity incorporated into
myelin basic protein of three separate experiments in
triplicate.
AMPA induced a
dose-dependent increase of MAPK activity (Fig. 1B). The
stimulation of MAPK by AMPA, NMDA, and KCl, but not epidermal growth
factor, was abolished when cortical neurons were incubated in
Ca-free medium (Fig. 1C). However,
the increase in MAPK activity by AMPA was unperturbed by incubation in
Na
-free medium (Fig. 1D), indicating
that the influx of Na
and/or
Na
-induced depolarization through the AMPA receptor
was not responsible for MAPK activation. Moreover, while the L-type VSCC blocker, nifedipine(15) , abolished the
stimulation of MAPK activity mediated by KCl-induced depolarization, it
had no effect on the activation of MAPK by either AMPA or NMDA (Fig. 1E). This finding is in agreement with a previous
report (6) demonstrating that glutamate and NMDA-stimulated
MAPK activity is a Ca
-dependent, VSCC-independent
process. Thus, L-type VSCCs appear linked to the
depolarization-induced activation of MAPK by KCl, whereas the entry of
Ca
via these channels was not likely a
significant factor in MAPK activation by AMPA. The increase in MAPK
activity by AMPA was blocked by pretreatment of neurons with the
selective AMPA receptor antagonist, CNQX, but not by the NMDA
channel blocker, MK801, or MCPG, a metabotropic receptor blocker.
Furthermore, trans-(1S,3R)-ACPD, a glutamate
metabotropic receptor agonist, did not stimulate MAPK activity (data
not shown). Collectively, these results indicate that the influx of
Ca
directly through the AMPA receptor itself was
likely responsible for the protracted activation of MAPK by AMPA in rat
cortical neurons.
MAP kinase activation arises from a protein kinase cascade, which in some cells involves protein kinase C (PKC)(16, 17) . However, the down-regulation of PKC (18) in neurons by chronic exposure to the phorbol ester, phorbol 12-myristate 13-acetate (PMA), did not alter AMPA-induced activation of MAPK, although it did ablate MAPK stimulation triggered by acute exposure to phorbol 12-myristate 13-acetate (Fig. 2A). Moreover, the potent PKC inhibitor, calphostin C, had no effect on AMPA-induced MAPK (data not shown). Therefore, the activation of MAPK by AMPA was clearly a PKC-independent process.
Figure 2:
AMPA activates Ras and MAPK
through a PTX-dependent but PKC-independent pathway. A,
neurons were treated with 100 nM PMA (TPA) for 20 h
and stimulated with 100 µM AMPA or 100 nM PMA for
3 min. MAPK activity was then determined(25) . Results
represent the mean ± S.E. of three separate determinations in
triplicate. B, untreated controls or cultures treated with 100
ng/ml PTX for 20 h were labeled with
[P]H
PO
for 4 h and then
stimulated with 100 µM AMPA for 2 min. Ras was
immunoprecipitated from cell lysates, and bound guanine nucleotides
were separated by thin layer chromatography(11) . C, densitometric determinations of GTP/(GTP + GDP)
ratios from the autoradiograph presented in B. The
results are representative of six separate determinations. D, untreated controls or cultures treated with 100
ng/ml PTX for 20 h were left unstimulated or were stimulated with 100
µM AMPA, 100 µM NMDA, or 50 mM KCl
for 3 min before MAPK activity was determined. Results are
representative of the mean ± S.E. of three experiments in
triplicate. MBP, myelin basic
protein.
It is well established that the Ras protein occupies a
pivotal position in the cascade activation of
MAPK(19, 20, 21, 22, 23, 24) .
Exposing cortical neurons to AMPA rapidly caused about a 30% increase
of Ras activity (Fig. 2, B and C) measured as
the ratio of the GTP- to GDP-bound form of the protein. Since
Ras-dependent activation of MAPK has been associated with receptors
coupled to G-proteins (25, 26, 27) and
receptors with intrinsic tyrosine kinase(28) , we studied the
effects of PTX on AMPA-induced MAPK activation. Surprisingly,
pretreating cortical neurons with PTX inhibited the increase in MAPK
seen with AMPA but not that observed with either NMDA or KCl (Fig. 2D). Moreover, the increase in Ras activity
effected by AMPA was sensitive to PTX treatment (Fig. 2, B and C), indicating that the activation of MAPK through
the AMPA receptor, but not the NMDA receptor, involved a PTX-sensitive
G-protein (presumably a member of the G family of proteins)
apparently coupled to the AMPA-induced stimulation of Ras in cortical
neurons.
Although most G-protein-mediated processes are modulated by
activated subunits, it is now evident that the
subunits of G-proteins can also regulate the activity of various
effectors(29, 30, 31, 32, 33) .
As shown in Fig. 3A, a G-protein
subunit was
found in the immunocomplex precipitated by an anti-Ras antibody in
AMPA-challenged neurons but not in control cultures. By contrast,
neither NMDA nor KCl stimulation resulted in the coprecipitation of a
subunit with Ras (Fig. 3, B and C). The
AMPA-induced association of the
subunit with the Ras
immunocomplex was dose-dependent (Fig. 3D) and
substantially reduced in cultures subjected to PTX pretreatment (Fig. 3A). As was the case with AMPA-stimulated MAPK,
the coprecipitation of the
subunit with Ras was dependent on
extracellular Ca
(Fig. 3C) but not
Na
(data not shown). These findings strongly suggest
that AMPA, but not NMDA or KCl, induced a dissociation of G
from
dimers and that the subsequent binding of the
free
subunit to Ras protein, probably through its pleckstrin
homology domain(34, 35, 36) , stimulated a
downstream cascade leading to MAPK activation.
If a G-protein
dependent activation of Ras is an early step in the AMPA-induced
stimulation of MAPK, then the downstream involvement of Raf kinase
could be
anticipated(37, 38, 39, 40) . Since B-raf transcripts are found at their highest levels
in brain (41) and B-Raf, rather than Raf-1, is
responsible for nerve growth factor-stimulated MEK-1 activation in PC12
cells(42) , we determined whether stimulating cortical neurons
with AMPA caused the coprecipitation of B-Raf with Ras. As shown in Fig. 4A, while both the p and p
forms of Raf kinase were detected in the immunocomplex
precipitated by the anti-Ras antibody, only the levels of
p
kinase were increased after exposing
neurons to AMPA. Moreover, the p
kinase
that coprecipitated with Ras in AMPA-challenged neurons exhibited a
slow gel mobility shift, an indicator of Raf kinase
activation(38) . As was observed with the
subunit, the
coprecipitation of p
with Ras was
dependent on extracellular Ca
(Fig. 4B). We next determined whether MEK-1 (MAP
kinase/ERK-activating kinase) was present in the complex containing
G-protein
subunit and Raf kinase. MEK acts immediately downstream
of Raf kinase, and upon its phosphorylation and activation, it
phosphorylates and activates MAPK(43, 44) . As
demonstrated in Fig. 4C, while there was no indication
of an increased association of MEK-1 with the Ras complex precipitated
from AMPA-stimulated cells, an AMPA dose-dependent activation of MEK-1
was observed, measured as a slow gel mobility shift of the protein.
Both the binding of MEK-1 to the Ras complex and its subsequent
activation by AMPA were found to be dependent on extracellular
Ca
(data not shown).
The results of this study are
consistent with the hypothesis that the transmembrane influx of
Ca through AMPA receptors induces the dissociation of
G
from a PTX-sensitive G-protein, and that once
released, the G
subunit is responsible for
initiating a cascade of events involving the activation of Ras,
p
kinase, MEK-1, and finally MAPK. This
novel mechanism of MAPK activation in cortical neurons appears distinct
from that responsible for increased MAPK activity effected by NMDA and
KCl. The evidence also indicates that while AMPA, like NMDA and
KCl-mediated depolarization, activates MAPK by processes dependent on
the influx of extracellular Ca
, it is apparent that
different routes of Ca
entry trigger different
mechanisms leading to MAPK activation. This conclusion is in agreement
with a recent report indicating that NMDA and L-type
Ca
channels activate calmodulin II kinase in neurons
by two distinct signaling pathways that are coupled to different routes
of Ca
entry(2) . It is reasonable to suggest
that neurons could use these different paths to MAPK activation to
generate divergence in the downstream signals and cellular responses to
MAPK activity. Elucidating the signaling pathways specifically coupled
to the entry of Ca
through AMPA, NMDA, VSCCs, and
other ionotropic channels will facilitate our understanding of the role
these receptors play in neurons under both physiological and
pathological conditions.