(Received for publication, April 3, 1995)
From the
Receptors that couple to the heterotrimeric G proteins, G
The ubiquitous mitogen-activated protein kinases (MAPK)
Recently, a
number of receptors that couple to heterotrimeric G proteins have been
shown to stimulate MAPK
activation(16, 17, 18) , including both
receptors that couple to G
Given the diversity of effector molecules
regulated by G protein-coupled receptors, it is possible that MAPK
activation by this class of receptor can proceed by multiple pathways.
Such diversity is suggested by the observation that cellular expression
of G
Activation of either G
Figure 1:
Effect of
Figure 2:
Effect of RasN17 and N
A role for PTKs in G
Figure 3:
Effect of protein-tyrosine kinase
inhibitors on G
Figure 4:
Stimulation of MAPK activation and IP
production by coexpression of various combinations of G
Figure 5:
Differential effects of
Figure 6:
Effect of the protein-tyrosine kinase
inhibitors genistein and herbimycin A on G
Collectively, these results suggest that the release of
G
Previous
studies of the G protein subunit responsible for mediating
G
The effects of PTK inhibitors on G
It is
likely that other signaling pathways capable of mediating MAPK
activation and mitogenesis via G protein-coupled receptors exist. A
distinct signaling mechanism may be mediated by the G protein-coupled
receptor for platelet-activating factor (PAF). PAF stimulates both PI
hydrolysis and MAPK activation, but does not activate p21
We thank Dr. Kazushige Touhara for helpful discussion,
S. Exum for technical assistance, and D. Addison and M. Holben for
excellent secretarial services.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
or G
, can stimulate phosphoinositide (PI) hydrolysis
and mitogen-activated protein kinase (MAPK) activation. PI hydrolysis
produces inositol 1,4,5-trisphosphate and diacylglycerol, leading to
activation of protein kinase C (PKC), which can stimulate increased
MAPK activity. However, the relationship between PI hydrolysis and MAPK
activation in G
and G
signaling has not been
clearly defined and is the subject of this study. The effects of
several signaling inhibitors are assessed including expression of a
peptide derived from the carboxyl terminus of the
adrenergic
receptor kinase 1 (
ARKct), which specifically blocks signaling
mediated by the
subunits of G proteins (G
),
expression of dominant negative mutants of p21
(RasN17) and p74
(N
Raf),
protein-tyrosine kinase (PTK) inhibitors and cellular depletion of PKC.
The G
-coupled
2A adrenergic receptor (AR) stimulates
MAPK activation which is blocked by expression of
ARKct, RasN17,
or N
Raf, or by PTK inhibitors, but unaffected by cellular
depletion of PKC. In contrast, MAPK activation stimulated by the
G
-coupled
1B AR or M1 muscarinic cholinergic receptor
is unaffected by expression of
ARKct or RasN17 expression or by
PTK inhibitors, but is blocked by expression of N
Raf or by PKC
depletion. These data demonstrate that G
- and
G
-coupled receptors stimulate MAPK activation via distinct
signaling pathways. G
is responsible for mediating
G
-coupled receptor-stimulated MAPK activation through a
mechanism utilizing p21
and p74
independent of PKC. In contrast, G
mediates
G
-coupled receptor-stimulated MAPK activation using a
p21
-independent mechanism employing PKC and
p74
. To define the role of G
in
G
-coupled receptor-mediated PI hydrolysis and MAPK
activation, direct stimulation with G
was used. Expression of
G
resulted in MAPK activation that was sensitive to
inhibition by expression of
ARKct, RasN17, or N
Raf or by PTK
inhibitors, but insensitive to PKC depletion. By comparison,
G
-mediated PI hydrolysis was not affected by
ARKct,
RasN17, or N
Raf expression or by PTK inhibitors. Together, these
results demonstrate that G
mediates MAPK activation and PI
hydrolysis via independent signaling pathways.
(
)represent a point of convergence for mitogenic
signals originating from several distinct classes of cell surface
receptor. Activation of these kinases may be achieved via several
pathways. Tyrosine phosphorylation of growth factor receptors (e.g. the epidermal growth factor receptor) leads to the formation of a
membrane-associated multi-protein
complex(1, 2, 3) , which catalyzes GTP
exchange on and activation of the low molecular weight GTP-binding
protein, p21
(4) . Activation of
p21
results in the membrane association and
activation of the serine/threonine protein kinase
p74
, which initiates a protein phosphorylation
cascade culminating in the phosphorylation and activation of
MAPK(5, 6, 7, 8, 9) .
Alternatively, activators of PKC (e.g. phorbol esters) have
been reported to stimulate MAPK activation (10, 11, 12) through both
p21
-dependent and independent
mechanisms(13, 14, 15) .
and to G
. Activation
of G protein-coupled receptors catalyzes exchange of GTP for GDP on the
G protein
subunit (G
) leading to complex dissociation and
release of free G
-GTP and G
subunits (19, 20) , each of which may then interact with
effector molecules. Once released, G
subunits modulate numerous
effectors, including adenylate cyclases, phospholipase C (PLC)
isoforms, and ion channels (21) . G
subunits activate
isoforms of adenylate cyclase(22, 23) and
PLC(24, 25, 26, 27, 28, 29) ,
phospholipase A2(30) , potassium channels(31) , and
phosphatidylinositol 3`-kinase(32, 33) . In addition,
G
subunits mediate translocation of
-adrenergic receptor
kinases (
ARK) from the cytosol to their membrane receptor
substrates(34) .
subunits or constitutively activated G
subunits, but not constitutively activated G
subunits,
leads to MAPK activation(18) . The G
-mediated
pathway may utilize PLC-dependent PKC activation, whereas the mechanism
of G
-mediated p21
and MAPK activation (16, 17, 18) has not been clearly defined.
- or G
-coupled
receptors releases both free G
and G
subunits, either of
which might initiate a sequence of events leading to MAPK activation.
The relative contributions of G
subunit-mediated
p21
activation versus G
- or G
-mediated PLC activation to the
stimulation of MAPK by these receptors remains in question. This study
compares the mechanisms of MAPK activation employed by G
-
and G
-coupled receptors using inhibitors of cellular signal
transduction (e.g. a G
subunit-sequestrant peptide,
dominant negative mutants of p21
and
p74
, protein-tyrosine kinase inhibitors, and
cellular depletion of PKC) in a transfected cell system. We find that
G
-coupled receptor-mediated MAPK activation is
predominantly G
-mediated, PKC-dependent, and
p21
-independent and cannot be dissociated from
the ability of these receptors to activate PLC, whereas the
G
-coupled receptor pathway is G
subunit-mediated,
p21
-dependent, PKC-independent, and dissociable
from PLC activation.
Materials
COS-7 and CHO cells were from the
American Type Culture Collection. Culture media and
LipofectAMINE® were from Gibco-BRL. Fetal bovine serum and
gentamicin were from Life Technologies, Inc. Monoclonal antibody 12CA5
was from Boehringer Mannheim. Protein A-agarose was from Pharmacia
Biotech Inc. Dowex AG1-X8 was from Bio-Rad. Econofluor, myo-[H]inositol, and
[
-
P]ATP were from DuPont NEN. Herbimycin A,
genistein, myelin basic protein (MBP), carbachol,(-)-epinephrine,
and phorbol 12-myristate 13-acetate (PMA) were from Sigma. UK-14304 was
from Pfizer. Dowex AG1-X8 was from Bio-Rad. Pertussis toxin was from
List Biologicals.
DNA Constructs
The cDNAs for the human 2-C10
AR and
ARK1 were cloned in our laboratory(35) . Human M1
AChR cDNA was provided by E. Peralta(36) ; cDNAs encoding
G
1, G
2, G
3, G
4, G
1, G
2, and G
3 were
provided by M. Simon; DNA encoding hemagglutinin (HA)-tagged
p44
(Erk1) was from J. Pouysségur; DNA encoding
the p21
dominant negative mutant
was from D. Altschuler and M. Ostrowski; and DNA encoding the
p74
dominant negative mutant (N
Raf) was
from L. T. Williams. The
ARKct peptide-encoding minigene,
containing cDNA encoding the carboxyl-terminal 195 amino acids of
ARK1, was prepared as described previously(28) .
Cell Culture and Transfection
COS-7 cells were
maintained in Dulbecco's modified Eagle's medium
supplemented with 10% fetal bovine serum (FBS) and 50 µg/ml
gentamicin. CHO cells were maintained in F-12 medium supplemented with
10% FBS and 50 µg/ml gentamicin. For transient transfection using
LipofectAMINE, cells at 80% confluence in six-well culture plates were
washed once with serum-free medium and incubated for 3-5 h at 37
°C in 1.0 ml of serum-free medium containing the indicated amounts
of plasmid DNA and 6.0 µl of LipofectAMINE/µg of DNA. The
transfection mixture was then replaced with 2.0 ml of growth medium and
the cells maintained for 24 h. Prior to assay, transfected cells were
incubated overnight in medium containing 0.5% FBS or medium containing
10% FBS plus 2 µCi/ml [H]inositol for
experiments measuring MAPK activity or IP production, respectively.
Measurement of MAPK Activity
Activity of
epitope-tagged p44 (Erk1) was
determined following immunoprecipitation, using MBP as
substrate(37) . Briefly, serum-starved transfected cells were
stimulated for 5 min with the indicated agonist, lysed in 200 µl of
cold lysis buffer (50 mM Tris-HCl, pH 8, 150 mM NaCl,
1% (v/v) Nonidet P-40, 0.5% (w/v) sodium deoxycholate, 0.1% SDS, 10
mM NaF, 10 mM sodium pyrophosphate, 0.1 mM
phenylmethylsulfonylfluoride) and clarified by centrifugation (15 min,
16000
g). Supernatants were transferred to tubes
containing 6.5 µg of anti-HA 12CA5 antibody and 25 µl of a 50%
slurry of protein A-agarose beads to immunoprecipitate
p44
(rotated 1 h at 4C). Immune
complexes were washed twice with cold lysis buffer and twice with
kinase buffer (20 mM HEPES, pH 7.4, 10 mM
MgCl
, 1 mM dithiothreitol). Phosphorylation of MBP
was performed in 40 µl of kinase buffer containing 250 µg/ml
MBP, 20 µM ATP, and 4 µCi/ml
[
-
P]ATP at room temperature for 30 min.
Reactions were terminated by the addition of 2
Laemmli sample
buffer and
P-labeled MBP resolved by SDS-polyacrylamide
gel electrophoresis. Quantitation of labeled MBP was performed using a
Molecular Dynamics PhosphorImager.
Measurement of IP Production
Transfected cells,
labeled overnight with [H]inositol, were washed
once with phosphate-buffered saline, incubated in phosphate-buffered
saline containing 10 mM LiCl and 1.0 mM CaCl
, and stimulated as indicated for 45-60 min.
Cells were lysed by addition of 0.8 ml of 0.4 M perchloric
acid (1.0 ml/well), and lysates were neutralized by addition of 0.4 ml
of 0.72 M KOH, 0.6 M KHCO
. Total IP
accumulation was determined by Dowex anion exchange chromatography and
liquid scintillation spectroscopy.
Stimulation of IP Production and MAPK Activity by
G
To
determine whether diversity exists in the mechanisms of activation of
MAPK employed by G- and G
-coupled Receptors
- and G
-coupled receptors, we
studied the effects of the G
subunit-sequestrant
ARKct
peptide, dominant negative mutants of p21
and
p74
, cellular depletion of PKC, and inhibitors
of protein-tyrosine kinases on MAPK activation mediated by each class
of receptor. As shown in Fig. 1, both PI hydrolysis and MAPK
activation stimulated by the G
-coupled
2A AR are
sensitive to pertussis toxin (PTX), while PI hydrolysis and MAPK
activity stimulated by the G
-coupled
1B AR and M1AChR
are PTX-insensitive. Since expression of
ARKct selectively
inhibits G
-mediated signal transduction, this peptide can be
utilized to distinguish G
and G
signaling
pathways(28) . Coexpression of
ARKct blocks
2A
AR-stimulated PI hydrolysis and MAPK activation but does not affect
1B AR- or M1AChR-mediated signaling (Fig. 1). These results
suggest that G
mediates both G
-coupled
receptor-stimulated PI hydrolysis and MAPK activation. In contrast,
G
-coupled receptor-mediated PI hydrolysis and MAPK
activation are
ARKct-insensitive, suggesting that these effects
are mediated by the G
subunits.
ARKct peptide expression and PTX treatment on G
- and
G
-coupled receptor-stimulated PI hydrolysis and MAPK
activation. COS-7 cells were transiently transfected with plasmid DNA
encoding
2A AR (0.2 µg/well),
1B AR (0.3 µg/well), or
M1AChR (0.3 µg/well) as described. In experiments in which MAPK
activation was measured, cells were cotransfected with plasmid DNA
encoding p44
(0.1 µg/well). Where indicated, cells
were preincubated overnight with PTX (100 ng/ml) prior to assay. Cells
were stimulated with 1.0 µM UK-14304 (
2A
AR), 100 µM epinephrine (
1B AR), or 1.0
mM carbachol (M1AChR) for 45 min prior to
determination of IP production (A) or for 5 min prior
to determination of MAPK activity (B). Data are
expressed as a percent of total inositol phosphates or MAPK activity
produced by agonist stimulation in control (vehicle pretreated) cells.
-Fold IP production stimulated by agonist for each receptor under
control conditions was 3.0-fold for
2A AR, 3.3-fold for
1B
AR, and 9.6-fold for M1AChR. -Fold MAPK activity stimulated by agonist
for each receptor under control conditions was 7.4-fold for
2A AR,
3.2-fold for
1B AR, and 3.0-fold for M1AChR. Values shown
represent the mean ± S.E. from at least three separate
experiments. Openbar, control; filledbar, PTX;shadedbar,
ARKct.
G-coupled receptor-mediated MAPK activation requires
p21
and p74
activity(38, 39, 40) . In order to
compare the roles of p21
and p74
in G
- and G
-coupled receptor
mediated MAPK activation, the effects of the dominant negative mutants
RasN17 and N
Raf on receptor-stimulated MAPK activation were
assessed. As shown in Fig. 2A, expression of RasN17 or
N
Raf inhibits
2A AR-stimulated MAPK activation. In contrast,
MAPK activation via the G
-coupled M1AChR is not affected by
RasN17 expression but is sensitive to inhibition by N
Raf. Results
similar to the M1AChR were obtained with the G
-coupled
1B AR (data not shown).
Raf expression
or PKC depletion on G
- and G
-coupled
receptor-mediated MAPK activation. COS-7 cells (A) were
cotransfected with plasmid DNA encoding p44
(0.1
µg/well),
2A AR (0.2 µg/well), or M1AChR (0.3 µg/well)
plus either vector plasmid alone (openbars) or
plasmid DNA encoding RasN17 (darkshadedbars) or N
Raf (2.0 µg/well; lightshadedbars). Cells were stimulated with or
without agonist for 5 min and MAPK activity determined as described.
CHO cells (B) were cotransfected with plasmid DNA encoding
p44
(0.1 µg/well) plus either
2A AR (0.2
µg/well),
1B AR (0.3 µg/well), G
1 and G
2
subunits (0.5 µg each/well), or empty plasmid vector. Cells were
pretreated with (filledbar) or without (openbar) 1.0 µM PMA for 24 h prior to
stimulation for 5 min with or without agonist or 1.0 µM PMA and MAPK activity was measured. Data are expressed as -fold
MAPK activation in which the MAPK activity produced in unstimulated
cells was defined as 1.0. Values shown represent mean ± S.E.
from four separate experiments.
G- and
G
-mediated MAPK activation are also distinguished by
differences in dependence upon PKC. Fig. 2B depicts the
effects of cellular PKC depletion in transiently transfected CHO cells.
Following prolonged exposure to PMA, further stimulation with phorbol
ester fails to provoke MAPK activation, demonstrating that the cells
are functionally depleted of PKC activity. The G
-coupled
2A AR fully activates MAPK in PKC-depleted cells, but the
G
-coupled
1B AR response is abolished, as is
M1AChR-stimulated MAPK activation (data not shown). These data suggest
that the signaling pathway utilized by G
-coupled receptors
involves G
, p21
, and p74
and is independent of PKC, whereas the G
-coupled
receptor pathway involves G
and PKC but is independent
of p21
. The two pathways apparently converge
downstream of p21
, since both are inhibited by
expression of N
Raf.
-coupled
receptor-mediated MAPK activation has been
proposed(41, 42) . As shown in Fig. 3A, MAPK activation mediated by the
2A AR,
but not the M1 AChR, is inhibited by pretreatment with the PTK
inhibitors genistein or herbimycin A. By comparison, PI hydrolysis
stimulated by either G
- or G
-coupled receptors
is unaffected by pretreatment with PTK inhibitors. These data suggest a
role for protein-tyrosine phosphorylation in MAPK activation mediated
by G
-coupled receptors, but not by G
-coupled
receptors. These results also suggest that G
-mediated MAPK
activation can be dissociated from PI hydrolysis.
and G
-stimulated PI hydrolysis
and MAPK activation. COS-7 cells were transfected with plasmid DNA
encoding
2A AR (0.2 µg/well), or M1AChR (0.3 µg/well). In
experiments in which MAPK activation was measured, cells were
cotransfected with plasmid DNA encoding p44
(0.1
µg/well). Cells were pretreated for 20 min with vehicle, genistein
(100 µM), or herbimycin A (1.0 µg/ml) and stimulated
for 5 min prior to determination of MAPK activity (A)
or for 45 min prior to measurement of IP production (B). Data
are expressed as the percent of IP production or MAPK activity produced
by agonist stimulation in control (vehicle-pretreated) cells. -Fold
MAPK activity stimulated by agonist for each receptor under control
conditions was 8.5-fold for
2A AR and 3.8-fold for M1AChR. -Fold
IP production stimulated by agonist for each receptor under control
conditions was 3.0-fold for
2A AR and 9.5-fold for M1AChR. Values
shown represent mean ± S.E. from at least three separate
experiments. Openbar, control; filledbar, genistein; shadedbar,
herbimycin A.
Stimulation of PI Hydrolysis and MAPK Activity by
Cellular Expression of G
In order to clarify
the role of G Subunits
subunits in G
-coupled
receptor-mediated PI hydrolysis and MAPK activation, we studied the
effects of direct G
expression on both processes. As shown in Fig. 4, expression of G
1 alone in COS-7 cells provokes a
small increase in MAPK activity, presumably due to an association with
endogenous G
subunits. Coexpression of combinations of
G
1
1, G
1
2, G
1
3, G
2
2, and
G
2
3 subunits each cause a 6-10-fold increase in basal
MAPK activation compared to control cells. Transfection with plasmids
encoding G
2 and G
1 subunits, and all the combinations using
G
3 or G
4 subunits, fails to activate MAPK beyond the basal
level exhibited in cells transfected with G
1 alone. The
combinations of G
and G
that stimulate MAPK activity also
promote a 3-4-fold increase in PI hydrolysis compared to control
cells. Thus, G
and G
subunit combinations known to form
G
complexes when coexpressed in cellular systems (43, 44, 45, 46) (G
1
1,
G
1
2, G
1
3, G
2
2, and G
2
3) all
stimulate both MAPK activation and PI hydrolysis, while those
combinations which are incapable of forming complexes (G
2
1,
G
3
1, and G
3
2) do not produce activity greater than
that observed by transfection of G
1 alone.
and G
subunits. COS-7 cells were transfected with pRK5 vector (Control), or plasmid DNA encoding the indicated combinations
of G
and G
subunits (1.0 µg each/well). For determination
of MAPK activity, cells were cotransfected with plasmid DNA encoding
p44
(0.1 µg/well). The ability of each combination of
G
subunit to stimulate MAPK activation (A) and IP
production (B) was determined as described. Data are presented
as -fold stimulation of MAPK activity or IP production normalized to
the value obtained in cells transfected with vector alone. Values shown
represent the mean ± S.E. from three separate
experiments.
As observed with
G-coupled receptor-mediated signaling,
G
-stimulated MAPK activation and PI hydrolysis appear to be
independent processes. Although
ARKct expression blocks both MAPK
activation and IP accumulation stimulated by G
1
2,
coexpression of either RasN17 or N
Raf abolishes
G
-mediated MAPK activation, without affecting
G
-mediated IP production (Fig. 5). In addition, the
PTK inhibitors genistein and herbimycin A attenuate
G
-stimulated MAPK activation in a dose-dependent manner, with
no effect on G
-stimulated IP production (Fig. 6).
Moreover, as shown in Fig. 2B, G
1
2-mediated
MAPK activation is unaffected by PKC depletion of CHO cells.
ARKct,
RasN17, and N
Raf expression on G
-stimulated MAPK
activation and IP production. COS-7 cells were transfected with plasmid
DNA encoding G
1 and G
2 subunits (1.0 µg/well each), plus
either pRK5 vector,
ARKct, RasN17, or N
Raf (2.0 µg/well).
Where indicated, p44
(0.1 µg/well) was cotransfected
for determination of MAPK activity. The effect of
ARKct, RasN17,
or N
Raf expression on G
-mediated MAPK activation (A) and IP production (B) were determined as
described. Data are presented as -fold stimulation of MAPK activity or
IP production normalized to the value obtained in cells transfected
with vector alone. Values shown represent the mean ± S.E. from
four separate experiments.
-stimulated MAPK
activation and IP production. COS-7 cells were cotransfected with pRK5
vector or plasmid DNA encoding G
1 and G
2 (1.0 µg
each/well). Where indicated, p44
(0.1 µg/well) was
cotransfected for determination of MAPK activity. Cells were pretreated
for 2 h with the indicated concentration of genistein or herbimycin A,
and MAPK activity (left panel) and IP production (right
panel) were determined as described. The data are presented as
percent of G
subunit-stimulated MAPK activity or IP
production in cells not exposed to genistein or herbimycin A. Absolute
values for G
-stimulated signaling under control conditions
were 4.8-fold for MAPK activation and 4.8-fold for IP production. The
values represent the mean ± S.E. from at least four separate
experiments. Open bar, control; bar with thin diagonal
stripes, 10 µM genistein; lightly shaded
bar, 50 µM genistein; dark shaded bar, 100
µM genistein; bar with thick diagonal stripes,
200 µM genistein; solid bar, 1.0 µg/ml
herbimycin.
subunits is responsible for mediation of
G
-coupled receptor-stimulated PI hydrolysis and MAPK
activation. G
likely stimulates increased IP accumulation by
directly activating a G
-sensitive
PLC(24, 25, 26, 27, 28, 29) .
Although G
stimulates both MAPK activation and PI hydrolysis,
the sensitivity of G
-mediated MAPK activation to RasN17,
N
Raf, and PTK inhibitors, as well as its insensitivity to PKC
depletion, indicate that G
-mediated PI hydrolysis and MAPK
activation represent independent signaling pathways.
-coupled receptor-stimulated MAPK activation have produced
contradictory results(16, 17, 18) . One study
found that expression of the G
subunit of transducin (
T),
which like the
ARKct peptide sequesters free G
subunits,
inhibited M1AChR-stimulated MAPK activation(17) , suggesting
that G
-coupled receptor-mediated MAPK activation was
mediated by G
subunits. Another study reported that neither
M1AChR- nor bombesin receptor-stimulated MAPK activation was sensitive
to inhibition by
T subunits(18) . The present results
strongly support the hypothesis that multiple mechanisms exist for G
protein-coupled receptor-mediated MAPK activation. The data demonstrate
that G
- and G
-coupled receptor-mediated MAPK
activation can be distinguished by several characteristics. (i)
Expression of the G
subunit-sequestrant
ARKct peptide
blocks MAPK activation stimulated by receptors coupled to G
but not to G
; (ii) G
-coupled receptor
mediated MAPK activation is abolished by PKC depletion, while
G
-coupled receptor mediated MAPK activation is unaffected;
(iii) expression of the dominant negative mutant RasN17 inhibits
G
- but not G
-mediated MAPK activation; and (iv)
the PTK inhibitors genistein and herbimycin A attenuate
G
-mediated, but not G
-mediated MAPK activation.
-coupled receptor- and
G
subunit-mediated MAPK activation suggest a role for a
protein-tyrosine kinase in the G
-mediated signaling pathway.
We have found that MAPK activation resulting from expression of a
constitutively activated p21
mutant,
p21
(47) , is insensitive
to PTK inhibitors, indicating that the inhibitor-sensitive step lies
upstream of p21
.(
)
Genistein has been reported to inhibit several classes of
protein-tyrosine kinases including epidermal growth factor receptor,
pp60
, and
pp110
(48) , while herbimycin A
demonstrates more selective inhibition of Src family kinases (e.g.Src, Yes, Fbs, Ros, Abl, and ErbB)(49) . Since both genistein and herbimycin A
inhibit G
subunit-mediated MAPK activation, we speculate that
a tyrosine phosphorylation, possibly mediated by a member of the Src family, is required for G
-mediated
p21
activation. If so, the pathways of MAPK
activation utilized by classical tyrosine kinase growth factor
receptors and G
may converge at a very early point.
in CHO cells(50) . PAF-stimulated MAPK activation,
but not PI hydrolysis, is inhibited by PTX, suggesting that the PAF
receptor activates MAPK via a signaling pathway that utilizes a
PTX-sensitive G protein and is independent of both PI hydrolysis and
p21
. It has also been reported that PKC, which
is activated in response to numerous G protein-coupled receptors,
provokes MAPK activation via both p21
-dependent
and independent mechanisms (13, 14, 15) .
These data suggest that considerable heterogeneity exists in the
signaling pathways employed by G proteincoupled receptors leading to
MAPK activation. The mechanism utilized by a given receptor to
stimulate MAPK activation is likely dependent on the class of G protein
to which the receptor can couple and on the signaling machinery
available in a given cell type.
ARK, the
adrenergic
receptor kinase;
ARKct, the carboxyl-terminal peptide of
ARK;
G proteins, GTP-binding proteins; G
and G
, the
and
the
subunits, respectively, of G proteins; PKC,
Ca
-dependent protein kinase; PLC, phospholipase C;
PTK, protein-tyrosine kinase; RasN17, a dominant negative mutant of
p21; N
Raf, a dominant negative mutant of p74; PAF,
platelet-activating factor; PMA, phorbol 12-myristate 13-acetate; MBP,
myelin basic protein; CHO, Chinese hamster ovary; IP, inositol
phosphate; PTX, pertussis toxin; FBS, fetal bovine serum.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.