From the Department of Physiology, Cornell University Medical College, New York, New York 10021
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ABSTRACT |
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Heterotrimeric G protein-coupled receptors can
activate the mitogen-activated protein kinase (MAPK) cascade. Recent
studies using pharmacological inhibitors or dominant-negative mutants of signaling molecules have advanced our understanding of the pathways
from G protein-coupled receptors to MAPK. However, molecular genetic
analysis of these pathways is inadequate in mammalian cells. Here,
using the well characterized Gs- and protein
kinase A-deficient S49 mouse lymphoma cells, we provide the molecular genetic evidence that Gs
is responsible for transducing
the
-adrenergic receptor signal to MAPK in a protein kinase
A-dependent pathway involving Rap1 and Raf (but not Ras)
molecules.
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INTRODUCTION |
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G proteins serve their physiological roles by transducing signals from a broad class of cell-surface receptors to specific effector proteins (1-4). A variety of intracellular signal transduction pathways are regulated by G proteins, including the mitogen-activated protein kinase (MAPK)1 pathway (5). Although the activation mechanism of the MAPK cascade by receptors with intrinsic tyrosine kinase activity has been well studied, the route from G proteins to the MAPK cascade in mammalian cells is less understood (6, 7).
Recent studies in cultured cell lines with pharmacological inhibitors and dominant-negative mutants of certain signaling molecules have revealed the participation of some molecular components in the regulation of MAPK by G protein-coupled receptors (for review, see Ref. 5). Although the detailed biochemical steps are far from clear, these studies have shown that G protein-coupled receptors use pathways very similar to those utilized by receptor tyrosine kinases to activate the prototype Raf/MEK/MAPK cascade. Gq- and Gi-coupled receptors transmit the signals to MAPK through a pathway involving tyrosine kinase, adapter proteins Shc and Grb2, guanine nucleotide exchange factor Sos, Ras, and Raf in most cases (for review, see Ref. 5). Phosphatidylinositol 3-kinase has been implicated to act upstream of tyrosine kinases in the Gi/MAPK pathway in some cells (8-12).
For receptors coupled to Gs, overexpressing G or
Gs
subunits in COS-7 cells has shown that whereas
G
subunits have the capacity to stimulate MAPK, the ability of
Gs
to stimulate MAPK is controversial (13, 14). It is
also unclear if cAMP and protein kinase A (PKA) participate in the
Gs-coupled receptor/MAPK pathway. Whereas one group
reported that cAMP, forskolin, and Gs-coupled receptors can
stimulate MAPK in COS-7 cells (13), another reported that cAMP and PKA
do not mediate activation of MAPK by Gs-coupled receptors
in COS-7 cells (14). It was proposed that the Gs-coupled
-adrenergic receptor used the G
subunit to activate the MAPK
pathway through Ras and used the Gs
subunit to inhibit
MAPK activation through cAMP and PKA (14). These contradictory results
regarding whether the
-subunit or the
-subunits of
Gs protein mediate the receptor stimulation of MAPK and
whether PKA is involved in the Gs/MAPK pathway in mammalian
cells prompted us to address this question genetically. For the most
part, signaling by heterotrimeric G proteins has not been studied
genetically in mammalian cells.
S49 mouse lymphoma cells have played an important historical role in G
protein research (15). A variant of S49 cells lacking Gs
was instrumental in defining the function of and characterizing Gs
(15). Elevation of intracellular cAMP levels results
in growth arrest in the G1 phase of the cell cycle and
later (after several days) in cell death (16, 17). Mutants have been
selected that are resistant to cytolysis. These mutants include
cyc
(which lacks Gs
) (18),
UNC (which has a mutation of arginine at position
372 of Gs
and thus uncouples the interaction of
Gs
with the receptors) (19), and
kin
(which lacks protein kinase A activity)
(20).
These Gs and PKA mutant S49 cells should be very useful
in a molecular genetic study to understand the role of
Gs
and PKA in the
-adrenergic receptor/MAPK signaling
system. In this study, using these mutant S49 cells, we demonstrate
that Gs
transduces the
-adrenergic receptor signal to
MAPK in a protein kinase A-dependent pathway involving Rap1
and Raf (but not Ras) molecules.
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EXPERIMENTAL PROCEDURES |
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S49 Mouse Lymphoma Cells-- S49 mouse lymphoma T cells were obtained from the Cell Culture Facility at the University of California at San Francisco and were grown in Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated horse serum as described (16, 17, 21). Transient transfection was done with 2 µg of plasmid DNA and LipofectAMINE (Life Technologies, Inc.) in six-well culture plates as described previously (22-25). Transfection efficiency was ~20%.
Immunoprecipitation and Immunoblot Analysis--
S49 whole cell
extracts were prepared as follows. Cells were harvested from 10-cm
plates and washed twice with cold phosphate-buffered saline, and
pellets were resuspended in 0.8 m1 of extraction buffer (150 mM NaCl, 10 mM Tris, pH 7.4, l mM
EDTA, l mM EGTA, 0.1% SDS, 1% sodium deoxycholate, 1%
Triton X-100, 0.5% Nonidet P-40, 0.2 mM
phenylmethylsulfonyl fluoride, 0.2 mM sodium orthovanadate, 0.02 mg/m1 tosylphenylalanyl chloromethyl ketone, and 0.03 mg/m1 leupeptin). Resuspended pellets were passed five times through a
26-gauge needle and centrifuged at 5000 rpm for 5 min at 4 °C to
remove insoluble material, and the supernatant was saved as the whole
cell extract. For immunoprecipitation, 10 µl of protein G-agarose was
added to the whole cell lysate to preclear. Then, 5 µl of primary
antibody was added and continuously incubated at 4 °C for 30 min.
After another 2-h incubation with 30 µl of protein G-agarose beads,
the immunocomplex was washed three times with extraction buffer and
three times with wash buffer (10 mM Tris, pH 7.4, and 1 mM EDTA). The immunocomplex was then subjected to
SDS-polyacrylamide gel electrophoresis. Western blotting with anti-ERK-1, anti-Gs, anti-G
, and
anti-Gi
was done as described (22-25). Anti-G protein
antibodies were from Santa Cruz Biotechnology. Membrane filters were
incubated in 1× Tris-buffered saline/5% milk for 1 h and then
incubated in primary antibody for 2 h at room temperature. Blots
were washed three times with Tris-buffered saline/Tween-20 and one time
with Tris-buffered saline and then incubated with secondary antibody
for 2 h at room temperature. Blots were washed again, and signal
was detected with ECL (NEN Life Science Products).
MAPK Assay--
For treatments, cells were stimulated with 100 µM isoproterenol or 1 µM somatostatin for 5 min. This brief treatment (5 min) with isoproterenol did not cause cell
death. Whole cell lysate was prepared, and immunocomplex MAPK assay was
performed as described previously using myelin basic protein (10 µg)
as substrate (23, 24). ERK-1 immunoprecipitation was done with a
monoclonal antibody to ERK-1 (Transduction Laboratories). Kinase assay
buffer contained 30 mM Tris-HCl, pH 8, 20 mM
MgC12, 2 mM MnC12, and 10 µM ATP. The mixture was preincubated for 3 min before 10 µCi of [-32P]ATP was added. After 10 min at
30 °C, samples were separated by 12% SDS-polyacrylamide gel
electrophoresis. Gels were transferred to nitrocellulose membrane
filters and exposed for autoradiography. Quantitation was performed
using a Molecular Dynamics PhosphorImager.
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RESULTS |
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Gs Is Required for Transmitting the
-Adrenergic
Receptor Signal to MAPK--
In wild-type S49 mouse lymphoma cells,
the agonist isoproterenol activates the endogenous
Gs-coupled
-adrenergic receptor, leading to the
stimulation of MAPK activity (Fig.
1A). Isoproterenol-induced increase in MAPK activity was not sensitive to pertussis toxin and
could be blocked by the
-adrenergic receptor-specific antagonist propranolol (data not shown). To genetically determine whether Gs
or G
subunits transduce the receptor signal to
MAPK, we have taken advantage of the availability of mutant S49 cells
that have genetic defects in
-receptor signaling.
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Gs Is the Signal Transducer in the
-Adrenergic
Receptor/MAPK Pathway--
The requirement for Gs
may
be due to its signaling role or to its requirement in maintaining the
structural integrity of trimeric G proteins or both. To distinguish
between the signaling versus structural role, we introduced
into cyc
cells (a null Gs
mutant background) a Gs
mutant that still complexes with
the G
subunit and is still able to couple to the
-adrenergic
receptor (that is, the structural role is still fulfilled), but is
unable to stimulate its downstream target adenylyl cyclase (that is,
the signaling role is impaired). If such a mutant is unable to rescue
the cyc
mutant response to
-adrenergic
receptor stimulation of MAPK, then Gs
is very likely the
signal transducer. If such a mutant is able to rescue the
cyc
response to
-adrenergic receptor
stimulation of MAPK, Gs
is probably needed for
structural integrity. Therefore, we tested two Gs
mutants with amino acid changes in the effector contact region of
Gs
, previously described to be defective in stimulating adenylyl cyclase, but still interacting with G
and the
-adrenergic receptor (27, 28). As shown in Fig.
2, wild-type Gs
rescues the cyc
cell response to
-adrenergic
stimulation of MAPK, whereas neither of the two mutants could rescue
the response. Furthermore, expression of a constitutively activated
Gs
mutant (
sQ227L, with
Gln227 changed to Leu) (29) leads to stimulation of MAPK,
indicating that Gs
is not only required but also
sufficient to activate the MAPK pathway. Thus, we conclude that
Gs
is the signal transducer in the
-adrenergic
receptor/MAPK pathway.
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PKA Is Required for Transmitting the -Adrenergic Receptor Signal
to MAPK--
Binding of isoproterenol to the
-adrenergic receptor
results in the activation of Gs
, leading to stimulation
of adenylyl cyclase and elevation of the intracellular levels of cAMP.
Most cAMP-mediated intracellular responses are mediated through protein kinase A in mammalian cells (30). As shown in Fig.
3, stimulation of MAPK by the
-adrenergic receptor is blocked in kin
mutant S49 cells that lack protein kinase A activity (20), whereas
stimulation of MAPK by the Gi-coupled somatostatin receptor is normal in kin
cells. This result further
demonstrates that the Gs
/adenylyl cyclase/cAMP/protein
kinase A cascade links the
-adrenergic receptor to MAPK in S49 mouse
lymphoma cells.
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Downstream Components of the PKA/MAPK Pathway in S49
Cells--
Activation of MAPK by PKA in some cells has been proposed
to act through a Ras-independent but B-Raf- and
Rap1-dependent signaling pathway (31). To examine the role
of Ras, Raf, and Rap1 in the PKA/MAPK pathway in S49 cells, we tested
the effects of dominant-negative mutants of Ras, Raf, and Rap1 (Fig.
4). Expression of a dominant-negative Ras
mutant (RasN17) (32) in S49 cells had no effect on the stimulation of
MAPK by Gs-coupled -receptors (Fig. 4A),
whereas it inhibited the MAPK stimulation by Gi-coupled
somatostatin receptors (Fig. 4B). Transfection of a
dominant-negative Raf mutant (a truncated Raf mutant with the conserved
region 1, which interferes with the activation of endogenous Raf
including B-Raf) (33-36) into S49 cells blocked the MAPK stimulation
by both Gs- and Gi-coupled receptors (Fig. 4).
While there was no effect on the stimulation of MAPK by the
Gi-coupled somatostatin receptor, transfection of a
dominant-negative Rap1 mutant (Rap1N17) (31) blocked the MAPK
stimulation by the Gs-coupled
-receptor (Fig.
4C). These data suggest that in S49 cells, as in some other
mammalian cells, the PKA/MAPK pathway requires Rap1 and Raf, but not
Ras (31, 37).
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DISCUSSION |
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In summary, using various mutant S49 mouse lymphoma cells, we have
provided the first genetic evidence for Gs and protein kinase A transducing the
-adrenergic receptor signal to MAPK. In
cyc
mutant S49 cells that lack
Gs
proteins, stimulation of the
-adrenergic receptor
failed to activate MAPK. In UNC mutant S49 cells that Gs
is unable to couple to the
-adrenergic receptor,
MAPK could not be stimulated by the
-receptor. Two Gs
mutants that can complex with the G
subunit and
-receptor, but
are unable to stimulate adenylyl cyclase, failed to rescue the
cyc
mutant response to
-receptor
stimulation of MAPK. Wild-type Gs
rescued the
cyc
cell response. Furthermore, in
kin
mutant S49 cells that lack protein kinase
A activity, stimulation of MAPK by the
-receptor was blocked.
Moreover, dominant-negative mutants of Rap1 or Raf, but not Ras,
suppressed the
-receptor-induced MAPK stimulation. These data
collectively demonstrate that the Gs-coupled adrenergic
receptor uses Gs
, transducing the signal to a PKA-,
Rap1-, and Raf-dependent, but Ras-independent, pathway leading to MAPK activation in S49 mouse lymphoma cells.
Previously, Faure et al. (13) have shown that overexpressing
G or constitutively activated Gs
could lead to
activation of MAPK and that cAMP, forskolin, and Gs-coupled
receptors could stimulate MAPK in COS-7 cells. On the other hand,
Crespo et al. (14) reported that only overexpression of
G
subunits, but not the activated Gs
subunit,
could increase MAPK activity in COS-7 cells. It was proposed that in
COS-7 cells, whereas G
transduces a positive signal to increase
MAPK activity, Gs
, through protein kinase A, inhibits
the MAPK stimulation (14). The reason for this discrepancy is not
clear. The suggestion that G
mediates the
-adrenergic receptor
signal to MAPK is based on two types of experiments (14). One is that,
as mentioned above, overexpression of G
could lead to increased
MAPK activity. Another is that overexpression of a G
-binding
fragment from the
-adrenergic receptor kinase protein or
Gt
could attenuate the stimulation of MAPK by
-adrenergic receptors.
We were unable to perform a genetic analysis of the role of G
subunits due to the lack of null mutants of G
or G
subunits in
S49 cells. G
is likely required in a structural role for the
integrity of G protein function, but is unlikely to play a major
signaling role for the following reasons. First, in
cyc
mutant cells, the basal activity of MAPK
is similar to that in wild-type cells. If G
is the major signal
transducer, as in Saccharomyces cerevisiae, then
in cyc
cells, MAPK could be constitutively
active, as in G
null mutant cells in S. cerevisiae (38,
39). Second, two mutant Gs
(
s89 and
s389) subunits did not rescue the
cyc
cell response despite being able to
release G
upon receptor stimulation. Third, in S49 cells, the
isoforms of adenylyl cyclases can be stimulated by Gs
,
but not by G
or calcium/calmodulin (40). These data suggest that
if G
is needed, it is for structural reasons only, not for
activating downstream targets. Therefore, in S49 mouse lymphoma cells,
Gs
, not G
, transduces the receptor signal to
MAPK.
In the fission yeast Schizosaccharomyces pombe,
the -subunit of G protein carries the signal to the MAPK pathway
(41). In the budding yeast S. cerevisiae, the
-subunit
of G protein couples the receptor to the MAPK cascade (38, 39). Given
that the
-subunit of Gs transduces
Gs-coupled receptor signal to MAPK in S49 cells and that
the
-subunit of Gi likely conveys the message from
Gi-coupled receptors to MAPK (13, 42, 43), these different
usages of the
and
subunits might be reminiscent of S. pombe versus S. cerevisiae MAPK signaling
pathways. Thus, mammalian cells have both yeast pathways that are
utilized by different families of G proteins.
The cAMP and protein kinase A effect on the MAPK pathway depends on
cell type: in some cells, they are stimulatory to the MAPK pathway,
whereas in other cells, they are inhibitory (44, 45). A recent study
has determined that these stimulatory or inhibitory effects are
dictated by the expression of B-Raf (31). Protein kinase A directly
activates the small G protein Rap1, which in turn, selectively and
directly activates B-Raf, leading to the activation of MAPK. We found
that B-Raf is expressed in S49 cells and that isoproterenol could
stimulate B-Raf activity in S49 cells. Also, we have examined the
activation of MEK, an upstream activator of MAPK, and obtained results
similar to those for MAPK activation. Thus, we propose the activation
sequence as -adrenergic receptor/Gs
/adenylyl
cyclase/cAMP/PKA/Rap1/B-Raf/MEK/MAPK. This Gs
/MAPK
pathway represents the first complete biochemical pathway for G
protein/MAPK signaling.
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ACKNOWLEDGEMENTS |
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We thank Drs. T. Kozasa, A. Gilman, and P. Stork for providing plasmids. We are grateful to Drs. R. Iyengar, L. Levin, T. Maack, and the members of our laboratory for reading the manuscript.
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FOOTNOTES |
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* This work was supported by grants from NIH, the National Science Foundation, and the American Heart Association.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.
Beatrice F. Parvin Investigator of the American Heart Association,
New York City Affiliate. To whom correspondence should be addressed:
Dept. of Physiology, Cornell University Medical College, 1300 York
Ave., New York, NY 10021. Tel.: 212-746-6362; Fax: 212-746-8690;
E-mail: xyhuang{at}mail.med.cornell.edu.
1 The abbreviations used are: MAPK, mitogen-activated protein kinase; MEK, mitogen-activated protein kinase/extracellular signal-regulated protein kinase kinase; PKA, protein kinase A.
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REFERENCES |
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