(Received for publication, September 18, 1995; and in revised form, November 13, 1995)
From the
Mitogen-activated protein kinase (MAPK) is activated in response
to both receptor tyrosine kinases and G-protein-coupled receptors.
Recently, G-coupled receptors, such as the
adrenergic receptor, were shown to mediate Ras-dependent MAPK
activation via a pathway requiring G-protein
subunits
(G
) and many of the same intermediates involved in
receptor tyrosine kinase signaling. In contrast, G
-coupled
receptors, such as the M
muscarinic acetylcholine receptor
(M
AChR), activate MAPK via a pathway that is
Ras-independent but requires the activity of protein kinase C (PKC).
Here we show that, in Chinese hamster ovary cells, the
M
AChR and platelet-activating factor receptor (PAFR)
mediate MAPK activation via the
-subunit of the G
protein. G
-mediated MAPK activation was sensitive to
treatment with pertussis toxin but insensitive to inhibition by a
G
-sequestering peptide (
ARK1ct).
M
AChR and PAFR catalyzed G
-subunit GTP
exchange, and MAPK activation could be partially rescued by a pertussis
toxin-insensitive mutant of G
but not by similar
mutants of G
. G
-mediated MAPK activation was
insensitive to inhibition by a dominant negative mutant of Ras (N17Ras)
but was completely blocked by cellular depletion of PKC. Thus,
M
AChR and PAFR, which have previously been shown to couple
to G
, are also coupled to G
to activate a novel
PKC-dependent mitogenic signaling pathway.
Mitogen-activated protein kinase (MAPK) ()can be
activated by a variety of extracellular stimuli, including those
mediated by receptor tyrosine kinases (RTKs) and G-protein-coupled
receptors (GPCRs)(1, 2, 3) . The mitogenic
signaling pathway mediated by the epidermal growth factor RTK involves
a cascade of protein-protein interactions, leading to Ras-dependent
MAPK activation(4, 5) . Agonist binding to the
epidermal growth factor RTK leads to receptor dimerization and
autophosphorylation, resulting in a phosphotyrosine-dependent
association with Shc. The subsequent interaction between
Tyr(P)-phosphorylated Shc and the Grb2 adaptor protein causes a
translocation of the Grb2-SOS complex to the membrane, where SOS
mediates guanine nucleotide exchange on Ras (6) .
Recently,
subunits derived from PTX-sensitive heterotrimeric
G-proteins were also shown to mediate Ras-dependent MAPK
activation(7, 8, 9, 10) . Release of
G
promotes the tyrosine phosphorylation of Shc and
its subsequent association with Grb2-SOS. Both RTK- and
G
-mediated MAPK activation are completely blocked
by the expression of dominant negative mutants of mSOS1 and Ras,
demonstrating that RTKs and G
activate MAPK via a
common signaling pathway involving Shc, Grb2, SOS, and Ras(7) .
MAPK activation via G-coupled receptors, such as
AR and the lysophosphatidic acid receptor, is
sensitive to inhibition by the C-terminal fragment of
ARK1
(
ARK1ct), a competitive inhibitor of
G
-mediated signals(10) . However, not all
GPCRs mediate MAPK activation exclusively via receptor-catalyzed
release of
subunits. For example, in COS-7 cells, MAPK
activation via receptors coupled to members of the PTX-insensitive
G
family, such as M
AChR and the
adrenergic receptor (
AR), is insensitive to the
G
-sequestrant
ARK1ct peptide(11) .
Instead, MAPK activation occurs predominantly via a PKC-dependent
pathway. The GTP-bound
-subunit of the G
protein
activates phosphoinositide hydrolysis (12) and protein kinase C
(PKC). Once activated, PKC stimulates MAPK activity via a poorly
understood mechanism involving the activation of Raf
kinase(13, 14) .
MAPK activation in CHO cells
stably transfected with PAFR cDNA has been reported to be sensitive to
PTX and independent of Ras(15) . We have studied MAPK
activation by GPCRs in COS-7 and CHO cells and find that the mechanism
of MAChR- and PAFR-mediated MAPK activation varies between
cell types. Our data demonstrate the existence of a novel PKC-dependent
mitogenic signaling pathway, which is mediated by the
-subunit of
the PTX-sensitive G
-protein and which is independent of Ras
activation.
Figure 1:
Comparison of the effects of PTX
treatment and ARK1ct peptide on MAPK activation in COS-7 and CHO
cells. A, COS-7 cells were transiently co-transfected with
p44
plus the indicated cDNAs with or without
the
ARK1ct peptide cDNA. The effects of PTX treatment (100 ng/ml,
20 h) and
ARK1ct peptide expression on basal and
agonist-stimulated MAPK activity, assessed as phosphorylation of MBP by
immunoprecipitated p44
, was determined
following a 5-min exposure to epinephrine (100 µM),
carbachol (1 mM), PAF (100 nM), or UK14304 (10
µM). For G
, cells were transfected
with G
and G
cDNAs (stimulated) either
with or without
ARK1ct. B, CHO cells were transiently
co-transfected with p44
plus the indicated cDNAs with
or without the
ARK1ct peptide cDNA. MAPK activity was determined
as described above. Data shown represent the mean ± S.D. for
duplicate samples from a representative experiment, which was
replicated 3 times with comparable results. White column, basal; shaded column, stimulated; black column, stimulated (PTX treated); hatched column, stimulated plus
ARK1ct.
In CHO cells, three patterns emerged.
First, the PTX-insensitive AR-mediated signal remained
PTX-insensitive, as found in COS-7 cells. Similarly, the
G
-dependent signals, mediated by either
AR or by transfected G
, remained
sensitive to
ARK1ct. In contrast, stimulation of PAFR-transfected
CHO cells mediated a 5-fold increase in MAPK activity. Moreover, MAPK
activation via M
AChR and PAFR was abolished by PTX
treatment but remained insensitive to G
sequestration by
ARK1ct expression (Fig. 1B). Thus, M
AChR can activate MAPK
via two distinct pathways, one sensitive (CHO cells) and one
insensitive (COS-7 cells) to PTX, while neither pathway appears to be
mediated by G-protein
subunits. Like the M
AChR,
PAFR can mediate PTX-sensitive,
ARK1ct-insensitive MAPK activation
in CHO cells. Interestingly, M1AChR and PAFR mediated PTX-insensitive
phosphoinositide hydrolysis in both COS-7 and CHO cells (data not
shown).
Figure 2:
Activation of G-protein
-subunit by M
AChR and PAFR in CHO cells. A,
CHO whole cell lysates were immunoblotted using the indicated
anti-G
subunit polyclonal antibody and visualized by
enzyme-linked chemiluminescence. B, CHO cells were transiently
transfected with M
AChR or PAFR cDNAs. G
GTP exchange was measured, after a 10-min stimulation with the
indicated agonist (100 nM PAF or 1 mM carbachol (Carb)), by GTP azidoanilide labeling and visualized by
autoradiography(36) . Control cells were transfected with empty
pRK5 vector (V).
PAFR has previously been shown to
couple to G in NCB-20 cells(20) , whereas
M
AChR, like
AR, is known to couple only to
members of the G
family(21, 22) . To
determine whether these receptors were capable of coupling to G
in CHO cells, we measured G
GTP exchange in
permeabilized cell preparations. As shown in Fig. 2B,
agonist stimulation of either PAFR or M
AChR mediated a
2-3-fold increase in the incorporation of the photoactivatable
GTP analog into G
in immunoprecipitates from CHO cell
lysates, indicating that both receptors are capable of coupling to and
activating G
. The specificity of the anti-G
antibody was confirmed by its inability to detect G
subunits in immunoblotting assays of whole cell lysates or
partially purified membrane preparations (data not shown).
Agonist-stimulated MAPK activity was measured in CHO cells
co-transfected with MAChR plus the PTX-insensitive mutants
of G
, G
, G
, and
G
(G
PT, G
PT,
G
PT, and G
PT, respectively). As
shown in Fig. 3, MAPK activation by M
AChR was almost
completely inhibited by PTX in control cells and in cells transfected
with the PTX-insensitive mutants of G
. In contrast,
M
AChR-mediated MAPK activation was rescued by
G
PT in cells treated with PTX, suggesting that the
G
protein is able to mediate MAPK activation by
M
AChR in CHO cells. G
PT was unable to
rescue
AR-mediated MAPK activation from PTX
inhibition (data not shown).
Figure 3:
Effect of co-expression of PTX-insensitive
G-protein -subunits on M
AChR-mediated MAPK activation.
CHO cells were transiently co-transfected with
p44
, M
AChR, and the indicated
PTX-insensitive mutant G-protein
-subunit. Cells were incubated
overnight in serum-free medium in the presence of 100 ng/ml PTX prior
to the determination of carbachol-induced MAPK activation. Data are
presented as the percent of carbachol-stimulated MAPK activity measured
in the absence of PTX. Data shown represent the mean ± S.D. for
duplicate samples from a representative experiment, which was
replicated 3 times with comparable results. Control cells were
transfected with empty pRK5 vector (V).
Figure 4:
Effects of PKC depletion and dominant
negative N17Ras expression on MAPK activation in CHO cells. CHO cells
were transiently co-transfected with the indicated receptor cDNA (or
G) with or without the N17Ras cDNA. Where
indicated, cells were incubated overnight in serum-free medium in the
presence or absence of PMA (1 µM) to deplete endogenous
PKC activity prior to the determination of MAPK activity. PKC depletion
was confirmed by unresponsiveness to further PMA stimulation (data not
shown). Data shown represent the mean ± S.D. for duplicate
samples from a representative experiment, which was replicated 3 times
with comparable results.
To determine the role of PKC in the
PTX-sensitive activation of MAPK by MAChR and PAFR, we
pretreated cells overnight with phorbol ester to deplete endogenous
PKC. As shown in Fig. 4, MAPK activation by
AR,
M
AChR, and PAFR was completely blocked by PKC depletion,
whereas G
and G
-coupled
AR were unaffected. Thus, in CHO cells,
M
AChR and PAFR couple to G
to activate MAPK via
a signaling pathway that is independent of Ras but dependent on the
activity of PKC.
We have characterized the mitogenic signaling pathways
mediated by several G protein-coupled receptors in COS-7 and CHO cells.
The data demonstrate the existence of a novel mitogenic signaling
pathway mediated via the -subunit of the G
protein. In
CHO cells, activation of endogenous G
mediates
PKC-dependent MAPK activation. Although it is not clear that PAFR and
M
AChR activate G
under physiological
conditions, we have shown that these receptors, when transiently
expressed in CHO cells, activate MAPK via the
-subunits of
G
.
The G protein is the least well
characterized of the known PTX-sensitive G proteins. G
is
localized primarily to the growth cones in the mammalian brain (26) and may be involved in neuronal development and
differentiation. G
is known to mediate a variety of
intracellular effects, including inhibition of adenylyl
cyclase(27) , inhibition of voltage-dependent Ca
channels(28, 29) , and stimulation of
phosphoinositide hydrolysis(30) . Intracellular injection of a
constitutively active mutant of G
(Q205LG
-
) mediates a PKC-dependent resumption of
the Xenopus oocyte cell cycle(31) . Expression of
Q205LG
-
stimulates mitogenesis in NIH 3T3 cells, and
prolonged expression leads to cellular transformation(32) .
In addition to G, the PAF receptor has been reported to
couple to the PTX-insensitive G
and the PTX-sensitive
G
and G
(20) . PAF mediates a variety
of physiological effects, including increased expression of the
c-fos and c-jun protooncogenes(33) ,
increased neurite outgrowth in PC12 pheochromocytoma
cells(34) , elevation of intracellular
Ca
(35) , and increased phosphoinositide
hydrolysis(15) . M
AChR has previously been shown to
couple primarily to G
(21, 22) , whereas
our data demonstrate a coupling with G
to mediate mitogenic
signaling. In contrast,
AR, which is also coupled to
G
in COS-7 cells, is unable to couple to G
in CHO cells, demonstrating that the interaction between
M
AChR and G
is specific.
The activation of
MAPK via a PTX-sensitive, but Ras-independent, pathway is inconsistent
with the known mechanism of G-mediated mitogenic signaling,
which requires G-protein
subunits and the activation of Shc,
Grb2, SOS, and Ras(7, 10) (Fig. 5). Our data
show that the PTX-sensitive activation of MAPK was insensitive to the
G
sequestering
ARK1ct peptide and, moreover,
was specifically rescued by a PTX-insensitive mutant of
G
, demonstrating the direct involvement of the
-subunit of G
in mitogenic signaling.
Figure 5:
Model of G-protein-mediated mitogenic
signaling. The convergent pathways of GPCR- and RTK-mediated mitogenic
signaling are shown. Signals mediated by RTKs and G-coupled
receptors converge at, or before, Shc to mediate Ras-dependent MAPK
activation. In contrast, receptors coupled to PTX-sensitive G
or PTX-insensitive G
activate PKC which, in turn,
can mediate Ras-independent MAPK activation. Dotted arrows indicate multiple or uncharacterized steps in the pathway. Jagged lines indicate lipid modifications of proteins. MEK, MAPK/extracellular regulated kinase
kinase.
It has been
suggested that PKC stimulation is capable of mediating MAPK activation
via direct phosphorylation of Raf(13) . Consistent with this
observation, G-mediated MAPK activation was unaffected by
the N17Ras dominant negative mutant and required the activity of PKC.
These data corroborate the observation that PAFR, when stably expressed
in CHO cells, is unable to mediate an increase in the GTP-bound form of
Ras(15) . Interestingly, transfection of COS-7 cells with
wild-type G
cDNA did not introduce a PTX-sensitive
component to the M
AChR-mediated signal, (
)suggesting that additional downstream components, absent
from COS-7 cells, may be required for G
to mediate a
mitogenic signal.
A model of the known mitogenic signaling pathways
mediated by GPCRs (Fig. 5) shows how RTKs and
G-coupled receptors activate MAPK in a Ras-dependent
manner, whereas receptors coupled to G
and G
activate MAPK via a pathway that requires PKC. The mechanism by
which G
activates PKC and subsequently MAPK remains unknown
and is the subject of further investigation.