(Received for publication, July 16, 1995; and in revised form, August 17, 1995)
From the
The enzymatic activity of mitogen-activated protein kinases (MAP
kinases) increases in response to agents acting on a variety of cell
surface receptors, including receptors linked to heterotrimeric G
proteins of the G and G
family. Recently, it
has been shown that stimulation of
-adrenergic receptors, which
are typical of those that act through G
to activate
adenylyl cyclases, potently activates MAP kinases in the heart,
resulting in the hypertrophy of the cardiac muscle (Lazou, A.,
Bogoyevitch, M. A., Clerk, A., Fuller, S. J., Marshall, C. J., and
Sudgen, P. H.(1994) Circ. Res. 75, 938-941). We have
observed that exposure of COS-7 cells to a
-adrenergic agonist,
isoproterenol, raises intracellular levels of cAMP and effectively
activates protein kinase A (PKA) and an epitope-tagged MAP kinase.
However, MAP kinase stimulation by isoproterenol was neither mimicked
by expression of an activated mutant of G
, nor by
treatment with PKA-stimulating agents. Moreover, pretreatment of COS-7
with a permeable cAMP analog, 8-Br-cAMP, markedly decreased MAP kinase
activation by either isoproterenol or epidermal growth factor. Thus, in
COS-7 cells cAMP and PKA do not appear to mediate MAP kinase activation
by
-adrenergic receptors. Signaling from
-adrenergic
receptors to MAP kinase was inhibited by transfection of a chimeric
molecule consisting of the CD8 receptor and the carboxyl terminus of
the
-adrenergic receptor kinase, which includes the
-binding domain. MAP kinase activation by isoproterenol was
not affected by depletion of protein kinase C, but it was completely
abolished by expression of Ras-inhibiting molecules. We conclude that
signaling from
-adrenergic receptors to MAP kinase involves an
activating signal mediated by
subunits acting on a
Ras-dependent pathway and a G
-induced inhibitory
signal mediated by cAMP and PKA. The balance between these two opposing
mechanisms of regulation would be expected to control the MAP kinase
response to
-adrenergic agonists as well as to other biologically
active agents known to act on G
coupled receptors,
including a number of hormones, neurotransmitters, and lipid mediators.
Mitogen-activated protein kinases (MAP kinases) ()appear to play a central role in mitogenic signaling
pathways stimulated by growth-promoting factors acting on a variety of
cell surface receptors (1, 2) . These kinases actively
participate in converting extracellular stimuli to intracellular
signals affecting the expression of genes necessary for a number of
biological functions, including cell growth and
differentiation(2) . The pathway linking cell surface receptors
to MAP kinases has just begun to be elucidated. The tyrosine kinase
class of receptors signals to MAP kinase by a multistep process. In the
case of ligand-activated EGF receptors, it involves binding to the
adaptor protein GRB2 which causes the recruitment to the membrane of
SOS, a guanine nucleotide exchange factor for p21
(3, 4) and the consequent exchange of GDP
for GTP bound to p21
. This initiates the
activation of a linear cascade of protein kinases including c-Raf (5) and MEKK(6) , and MEK1 and MEK2(7) , which
ultimately phosphorylate MAP kinases on both threonine and tyrosine
residues, resulting in a dramatic increase in their enzymatic activity (7) . In turn, MAP kinases phosphorylate and modulate the
function of key enzymes and nuclear transcription factors(8) .
The pathway linking G protein-coupled receptors to MAP kinases is
still poorly understood. Recent reports indicate that MAP kinases can
be activated by a number of receptors linked to G. For
example, triggering
-adrenergic(9) , m2
muscarinic (10, 11) , and D2 dopaminergic (11) receptors, as well as receptors for lysophosphatidic acid (12) can all potently activate MAP kinase in a pertussis
toxin-sensitive manner. The enzymatic activity of MAP kinases can also
be induced upon stimulation of receptors coupled to G
, such
as m1 muscarinic (10, 13) and bombesin (11) receptors, in this case through a pathway only partially
dependent on protein kinase C(10, 13) . Activation of
MAP kinase appears not to be exclusively linked to cell proliferation,
as only a few of these receptors can signal cell proliferation. In this
regard, accumulating evidence suggests that persistent activation of
the MAP kinase might lead to differentiation or hypertrophy in a cell
type-specific manner (14) . For example, stimulation of nerve
growth factor receptors in PC12 rat pheochromocytoma cells or insulin
receptors in L1 murine preadipocytic cell line provokes a marked and
prolonged activation of MAP kinases and induces cells to acquire a
fully differentiated phenotype(15, 16) . In addition,
hormonal stimulation of a number of G protein-coupled receptors
naturally expressed in the heart elevates the enzymatic activity of MAP
kinases and causes the hypertrophy of muscle cells(17) .
Interestingly, MAP kinase activation and the hypertrophic response was
shown to be elicited by agonist acting on receptors coupled to either
G
or G
, such as
and
-adrenergic receptors, respectively(17) . The latter
represents one of the few examples of receptors coupled to G
activating MAP kinase so far reported(17) . This is
particularly interesting because
-adrenergic receptors couple to
adenylyl cyclases to raise intracellular levels of cAMP(18) ,
and recently available data indicate that elevated cAMP can block MAP
kinase activation by oncogenic Ras proteins(19) , tyrosine
kinase receptors(20, 21, 22) , and G
protein-coupled receptors(23, 24) . Thus, receptors
coupled to G
would be expected to diminish rather than to
stimulate MAP kinase activity.
In this study, we have used the
expression of an epitope-tagged MAP kinase in COS-7 cells as an
experimental model to study the signaling pathway connecting
endogenously expressed -adrenergic receptors to MAP kinase. We
have found that signaling from these G
-coupled receptors to
MAP kinase has two distinctive components: an activating pathway
mediated by
subunits acting through Ras, and a
G
-induced inhibitory signal mediated by cAMP and PKA.
A DNA fragment encoding the
carboxyl-terminal 222 amino acids of human bARK1(26) , a region
that includes the -binding domain(27) , was amplified
with the oligonucleotides 5`-CCGGATCCACCATGggaatcaagttactggac-3` and
5`-CCGAATTCgaggccgttggcactgcc-3`, and subcloned as a BamHI-EcoRI fragment in a modified pGEX4T3 (Pharmacia
Biotech Inc.) bacterial expression plasmid containing a short
oligonucleotide encoding a COOH-terminal Myc epitope, (
)and
then transferred as a BamHI/NotI fragment to
pcDNA-CD8. The resulting DNA construct, designated
pcDNA-CD8-
ARK-C, was expected to express the extracellular and
transmembrane domains of CD8 fused to an intracellular domain
containing the
binding portion of human
ARK and a
COOH-terminal Myc epitope.
To determine whether stimulation of endogenously expressed
-adrenergic receptors induces MAP kinase activation, COS-7 cells
were transfected with an expression plasmid carrying the cDNA for an
epitope-tagged MAP kinase, serum starved, and then treated with
increasing concentrations of isoproterenol for 5 min. As seen in Fig. 1A, the
-adrenergic agonist effectively
induced MAP kinase activation in a dosedependent fashion, reaching a
maximum at concentrations of isoproterenol above 10 µM.
MAP kinase activation was also time dependent, reaching its maximum
between 3-5 min (Fig. 1B). Pretreatment of cells
with the
-adrenergic antagonist propranolol (4 µM)
completely abolished MAP kinase activation by isoproterenol but not by
EGF (Fig. 1B), thus confirming that stimulation of MAP
kinase in response to isoproterenol is mediated by
-adrenergic
receptors.
Figure 1: MAP kinase activation by isoproterenol. A, dose-response curve. Serum starved COS-7 cells were treated with increasing concentrations of isoproterenol for 5 min. Cell lysates were processed as described under ``Experimental Procedures.'' Data represent the average ± S.E. of triplicate samples and are expressed as fold increase in radioactivity incorporated into MBP with respect to control, untreated cells. Under these experimental conditions, radioactivity incorporated into MBP for control cells was 5377 ± 810 counts/min. B, time course activation of MAP kinase by isoproterenol and its blockade by propranolol. Serum-starved cells were treated for the indicated times with 10 mM isoproterenol, and control cells or cells pretreated with 4 µM propranolol for 20 min were stimulated for 5 min with either isoproterenol (10 mM) or EGF (100 ng/ml). MAP kinase activity was determined in immunoprecipitates using MBP as substrate as described under ``Experimental Procedures.''
-Adrenergic receptors are typical of those coupled
through G
to the stimulation of adenylyl
cyclase(18) . Thus, addition of isoproterenol would be expected
to promote an increase in cAMP levels and consequently to activate
PKA(33) . To study whether this second messenger-generating
system was responsible for activating MAP kinase, we treated cells with
a number of agents known to raise cAMP levels and/or to activate PKA.
As shown in Fig. 2, treatment for 5 min with 100 µM isoproterenol, 10 µM forskolin, or expression of a
constitutively activated mutant of G
, G
QL (34) induced a remarkable increase in cAMP levels as
compared to control cells. In contrast, neither EGF nor serum elicited
any demonstrable effect on intracellular cAMP. Consistent with these
results, PKA activity was also greatly enhanced in cells treated with
isoproterenol, forskolin, or in cells transfected with a constitutively
activated mutant of G
(Table 1). Furthermore,
treatment of COS-7 cells with the cell-permeable analog of cAMP,
8-Br-cAMP (1 mM), elicited a remarkable increase in PKA
activity (Table 1). However, whereas isoproterenol induced a
10-fold increase in MAP kinase activity, neither 8 Br-cAMP nor
G
QL were able to activate MAP kinase to any
significant extent (Table 1). Thus, the cAMP and PKA response to
isoproterenol might not be sufficient to explain its activating effect
on MAP kinase. On the other hand, the fact that forskolin can also
elevate MAP kinase activity is more likely to result from some of the
pleiotropic effects of this drug (35) rather than as a
consequence of stimulating the cAMP/PKA pathway.
Figure 2:
Effect of cAMP raising agents on
intracellular levels of cAMP. COS-7 cells transfected with insertless
expression vector (control) or transfected with G-QL
were grown to 90% confluence in 24-well plates. Medium was replaced by
Eagle's medium containing 1 mM 3-isobutyl-methylxantine,
and cells were stimulated with 10% serum, 10 µM
isoproterenol, 10 µM forskolin, or 100 ng/ml EGF for 5 min
and processed as described under ``Experimental Procedures.''
Results represent the average ± S.E. of three independent
experiments.
Recently, it has
been shown that in certain cell types cAMP-raising agents can potently
inhibit the activation of MAP kinase in response to a variety of
mitogens(19, 20, 21, 22, 23, 24) .
Thus, we examined whether increased cAMP levels and PKA activity would
affect -adrenergic receptor-induced MAP kinase activation. To that
end, COS-7 cells were transfected with the activated form of
G
or were pretreated with 8-Br-cAMP (1 mM)
for 20 min prior to stimulation with isoproterenol or EGF. Both
cotransfection with G
QL (not shown) and pretreatment
with 8-Br-cAMP markedly decreased MAP kinase activation by either
isoproterenol or EGF, as shown in Fig. 3. This effect of
8-Br-cAMP was blocked when added together with a PKA-specific
inhibitor, H-8 (5 µM)(36) . As shown in Fig. 4A, under these conditions H-8 restored MAP kinase
activation by isoproterenol and EGF almost to the levels found in
control, unpretreated cells (Fig. 4A). Furthermore,
treatment of cells with the PKA inhibitor potentiated MAP kinase
activation by isoproterenol, up to 2-fold after 5 min of stimulation,
when MAP kinase activation is at its peak (Fig. 4B). Taken
together, these findings demonstrate that the cAMP-PKA pathway does not
mediate activation of MAP kinases in response to the
-adrenergic
agonist. On the contrary, this biochemical route negatively regulates
the MAP kinase signaling pathway in COS-7 cells.
Figure 3: Effect of pretreatment with 8-Br-cAMP on MAP kinase activation. Serum-starved COS-7 cells transfected with pcDNA-HA-MAP kinase were either left untreated or pretreated for 20 min with 1 mM 8-Br-cAMP prior to stimulation with 10 µM isoproterenol or 100 ng/ml EGF for 5 min. MAP kinase activity was determined in anti-HA immunoprecipitates using MBP as substrate. Results represent average ± S.E. of triplicate samples from a representative assay. Similar results were obtained in three independent experiments.
Figure 4: Effects of pretreatment with the PKA inhibitor H-8 on MAP kinase activation. A, pretreatment with H-8 reverts the inhibiting effect of 8-Br-cAMP on MAP kinase activation. COS-7 cells transfected with the epitope-tagged MAP kinase were serum starved and pretreated with 5 µM H-8, 1 mM 8-Br-cAMP, or the combination of both for 20 min before stimulation with 10 µM isoproterenol or 100 ng/ml EGF for 5 min. MAP kinase activity was determined in immunoprecipitates using MBP as substrate. Results represent average ± S.E. of three independent experiments. B, time course activation of MAP kinase by 10 µM isoproterenol in COS-7 cells transfected with the HA-MAP kinase expression plasmid treated or untreated (control) with 5 µM H-8 for 20 min. MAP kinase activity was determined as above. Results represent average ± S.E. of triplicate samples from a representative experiment.
Agonist-dependent
activation of G protein-coupled receptors induces the replacement of
GDP by GTP bound to the subunit and causes the dissociation of
-GTP from
subunits(37) . Although the GTP-bound
subunit was thought to be alone responsible for activating
effector molecules, accumulating evidence supports an active role for
the G
dimers in signal transmission(38) .
To explore whether
complexes participate in MAP kinase
stimulation by
-adrenergic receptors, we took advantage of the
observation that overexpression of the
subunit of transducin
(G
) or the carboxyl-terminal domain of
-adrenergic
receptor kinase (
ARK) can block
-dependent pathways,
probably by binding and sequestering free
dimers(39) . Thus, we engineered a chimeric molecule between
the extracellular and transmembrane domain of CD8 (25) fused to
the COOH-terminal domain of
ARK, which would be expected to
express CD8 antigen at the cell surface, and to localize the
ARK
COOH-terminal domain to the inner face of the plasma membrane.
Immunofluorescence analysis of intact cells transfected with this
expression construct revealed that both CD8 and CD8-
ARK-C chimera
were efficiently expressed (Fig. 5). No fluorescence was
observed in cells transfected with the vector control or if the primary
antibody was omitted (not shown). As shown in Fig. 6,
coexpression of G
(Fig. 6A) or
CD8-
ARK-C (Fig. 6B) nearly abolished activation of
MAP kinase in response to isoproterenol. In contrast, CD8 alone had no
demonstrable effect (Fig. 6B). MAP kinase activation by
EGF was not affected by any these constructs, thus further
demonstrating the specificity of this approach(10) . Thus,
taken together these data strongly suggest that signaling from
-adrenergic receptors to MAP kinase is mediated by
subunits rather than by the
subunit of G
.
Figure 5:
Detection of membrane-targeted ARK by
immunofluorescence. COS-7 cells transfected with pcDNA-CD8 (A)
or pcDNA-CD8-
ARK-C (B) were immunostained with anti CD8
(1:100) antibody and goat anti-mouse fluorescein
isothiocyanate-conjugated secondary antibody (1:100) as described under
``Experimental Procedures.''
Figure 6:
Effect of scavenging proteins
on MAP kinase activation. A, effect of G
.
Expression plasmids containing G
or the pcDNA vector (1
µg each) were transfected into COS-7 cells together with
pcDNA-HA-MAP kinase. Cells were then serum starved and subsequently
stimulated with 10 µM isoproterenol or 100 ng/ml of EGF
for 5 min. MAP kinase activity was determined in immunoprecipitates
using MBP as substrate. Results represent average ± S.E. of
three independent experiments, expressed as percentage of response with
respect to vector-transfected cells. Under these experimental
conditions, radioactivity incorporated into MBP for vector-transfected
cells untreated, or treated with isoproterenol or EGF were 5,377
± 810, 27,458 ± 2,016, and 111,360 ± 8,945
counts/min, respectively. B, membrane-targeted
ARK-C
blocks MAP kinase activation by isoproterenol. Plasmids containing the
CD8-
ARK chimera CD8 or the pcDNA vector (1 µg each) were
cotransfected into COS-7 cells together with pcDNA-HA-MAP kinase. Cells
were then processed as above. MAP kinase activity was determined in
immunoprecipitates using MBP as substrate, run in a 12%
SDS-polyacrylamide gel electrophoresis gel, and subsequently
autoradiographed. Similar results were obtained in three independent
experiments. &cjs2117;, vector;
,
G
.
Addition
of isoproterenol to COS-7 cells did not induce any demonstrable
hydrolysis of phosphatidylinositol (data not shown). However,
triggering -adrenergic receptors in other cells results in the
stimulation of PKC(40) . Thus, we asked whether PKC plays a
significant role in signaling MAP kinase activation by
-adrenergic
receptors by depleting cells of PKC by prolonged treatment with high
concentrations of phorbol esters(41) . As shown in Fig. 7, this procedure abolished MAP kinase activation by a
subsequent challenge with PKC-activating concentrations of TPA. In
contrast, PKC depletion did not affect MAP kinase activation by
isoproterenol or EGF, demonstrating that signaling from
-adrenergic receptors to MAP kinase involves a PKC-independent
pathway.
Figure 7: Effects of protein kinase C down-regulation on MAP kinase activation by isoproterenol. Serum-starved cells were treated with either 10 µM isoproterenol, 100 ng/ml EGF, or 100 ng/ml TPA for 5 min with or without a 12-h pretreatment with 1 µg/ml TPA. MAP kinase activity was determined in immunoprecipitates using MBP as substrate as described under ``Experimental Procedures.'' Results represent average ± S.E. of triplicate samples from a representative experiment.
We next explored a role for Ras in MAP kinase activation by
-adrenergic receptors by transfecting cells with expression
plasmids carrying Ras-inhibitory molecules, such as the dominant
inhibitory mutant ras N17 (42) and
Rap-1a(43) . As shown in Fig. 8, cotransfection of
either ras-inhibiting construct nearly abolished MAP kinase
activation by isoproterenol. In contrast, expression of Ras-blocking
molecules failed to affect MAP kinase stimulation by a constitutively
activated form of Raf, Raf BXB (44) . Thus, these data strongly
suggest that signaling from
-adrenergic receptors to MAP kinase
involves a Ras-dependent pathway.
Figure 8: Effect of ras-inhibitory proteins on MAP kinase activation by isoproterenol. Plasmids containing ras-N17, rap-1a, Raf-BXB, or the pcDNA vector (1 µg each) were transfected into COS-7 cells together with the epitope-tagged MAP kinase cDNA. Serum-starved cells were subsequently stimulated with 10 µM isoproterenol for 5 min. MAP kinase activity was determined in immunoprecipitates using MBP as substrate. Results represent average ± S.E. of triplicate samples from a representative experiment. Similar results were obatined in four independent experiments.
In the present study we have set out to investigate the
signaling pathway linking -adrenergic receptors endogenously
expressed in COS-7 cells to a transfected, epitope-tagged MAP kinase.
We have found that triggering COS-7 cells with the
-adrenergic
agonist isoproterenol raises intracellular levels of cAMP, potently
stimulates PKA, and induces a time- and dose-dependent activation of
MAP kinase. However, we observed that the enhanced MAP kinase activity
elicited by isoproterenol was not mimicked by expression of a
constitutively activated form of G
or by treating
cells with a permeable cAMP-analog, 8-Br-cAMP, although both potently
stimulated PKA activity. Taken together, these findings strongly
suggest that the cAMP-PKA pathway does not mediate MAP kinase elevation
in response to isoproterenol. In fact, the only cAMP raising agent
capable of eliciting a MAP kinase response was forskolin. Thus, this
effect of forskolin, which was previously reported by
others(11) , is likely to represent a nonspecific effect of
this drug rather than being induced by increased PKA activity.
Previous studies have shown that pretreatment with cAMP-raising
agents strongly inhibits the MAP kinase activation elicited in response
to a variety of mitogens(21, 22, 23) . In
line with these observations, MAP kinase activation in response to
isoproterenol or EGF was also markedly decreased by pretreatment with
8-Br-cAMP or by overexpression of an activated form of
G. It has been proposed that elevation of cAMP blocks
signaling to MAP kinase by a mechanism involving the phosphorylation of
Raf-1 by PKA(21, 23) . Whether this is also the case
in COS-7 cells is under current investigation. In this regard, we have
observed that pretreatment of COS-7 cells with the PKA inhibitor H-8 (36) prevents the blocking effect of 8-Br-cAMP, thus supporting
the existence of a PKA-dependent pathway inhibiting MAP kinase in these
cells. Furthermore, exposure of cells to H-8 potentiated MAP kinase
activation by isoproterenol, further demonstrating that PKA is not
necessary to elevate MAP kinase activity in response to
-adrenergic receptor stimulation. Furthermore, this observation
raises the possibility that G
-coupled receptors might send,
simultaneously, both activating and inhibitory signals to MAP kinase,
the latter mediated by cAMP acting on PKA.
Recent studies from our
laboratory have provided evidence that MAP kinase activation by
muscarinic acetylcholine receptors is mediated by the
subunits of the heterotrimeric G
and G
proteins(10) . As discussed above, expression of
constitutively activated G
failed to mimic the
activating effect of isoproterenol on MAP kinase. Thus, we explored
whether G
complexes released upon activation of
G
by
-adrenergic receptors mediate in MAP kinase
stimulation. We initially studied the ability of overexpressing G
to affect the MAP kinase response to isoproterenol. G
is highly expressed in the retina and participates in linking
light-induced changes in rhodopsin to a cGMP
phosphodiesterase(45) . In COS-7 cells, expression of G
is expected to associate to free
subunits released
during G protein stimulation, thus preventing
-dependent
pathways(45) . We observed that coexpression of G
did not have any demonstrable effect on MAP kinase activation by
EGF, but nearly abolished MAP kinase stimulation mediated by
-adrenergic receptors thus suggesting that G
subunits are involved in linking
-adrenergic receptors to
the MAP kinase pathway. As an alternative approach, we took advantage
of the recent observation that the carboxyl-terminal domain of the
ARK protein binds efficiently to
complexes(46) . In this case, we anchored the
-binding domain of
ARK to the plasma membrane by fusing
it to the intracellular domain of the CD8 lymphocyte cell surface
receptor. Immunofluorescence analysis of intact, transfected COS-7
cells revealed that this construct was effectively expressed and
localized to the plasma membrane. This CD8-
ARK-C chimera abolished
signaling from
-adrenergic receptors to MAP kinase, without
affecting the EGF-induced response. Thus, we conclude that
G
complexes released upon activation of receptors
coupled to G
are responsible for signaling MAP kinase
activation.
Because dimers have been shown to activate
certain subtypes of phospholipase C(47) , which might implicate
PKC, we next explored a role for PKC in the MAP kinase response to
isoproterenol. For that, cells were challenged with this
-adrenergic agonist upon depletion of functional PKC by prolonged
treatment with phorbol esters. Under these conditions, the MAP kinase
response to a subsequent stimulation of PKCs with phorbol esters was
completely abolished, but MAP kinase activation elicited by
isoproterenol was identical to that of unpretreated cells. Thus, PKC
does not appear to play a significant role in MAP kinase activation by
-adrenergic receptors. On the other hand, it has been recently
shown that receptors coupled to G
and G
activate MAP kinase in a ras-dependent
fashion(10, 48, 49, 50) . In this
study, we show that ras-blocking proteins such as ras N17 (42) and rap-1a (43) completely block
isoproterenol-induced MAP kinase activation, thus strongly suggesting
that Ras also participates in signaling from G
-coupled
receptors to MAP kinase. Taken together, these findings strongly
suggest that
complexes released from G
,
G
, or G
can each effectively couple to effector
molecules acting on the Ras-MAP kinase pathway. The identity of
molecules linking
subunits of these heterotrimeric G
proteins to Ras is under current investigation.
Our present findings
demonstrate that G-coupled receptors such as
-adrenergic receptors signal to MAP kinase in a unique and complex
manner, which involves two counteracting pathways: an activating route
mediated by
subunits of G proteins acting on a ras-dependent pathway and an inhibitory route involving
, elevated intracellular cAMP levels, and PKA
activation. The balance between these two mechanisms would be expected
to determine the outcome of the signal sent to MAP kinases, and a
number of cell type-specific factors are likely to regulate this
balance. For example, whereas in COS-7 cells isoproterenol triggers a
marked activation of MAP kinase, addition of this
-adrenergic
agonist to adipocytes not only fails to stimulate MAP kinase, but
potently blocks insulin action(22) . The identity of those
tissue-specific factors involved in balancing these opposing signals
acting simultaneously on MAP kinases are still not known and warrant
further investigation. On the other hand, a large number of natural
agonists such as hormones, neurotransmitters, and lipid mediators are
known to stimulate G
-coupled receptors and, therefore, they
would be expected to exert a similar dual effect on MAP kinases. Thus,
our study raises the possibility that the
-Ras-MAP kinase
pathway might play an unexpected role determining the nature of the
biological responses elicited in vivo by each of these natural
agonists.