From the Oral and Pharyngeal Cancer Branch, NIDR, National Institutes of Health, Bethesda, Maryland 20892-4330 and the ¶ Max Planck Research Unit Molecular Cell Biology, Medical Faculty, University of Jena, 07747 Jena, Germany
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ABSTRACT |
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The serine/threonine protein kinase Akt has
recently been shown to be implicated in the pathway leading to cell
survival in response to serum and growth factors in a variety of
cellular systems. However, the existence of a biochemical route
connecting this kinase to the large family of receptors that signal
through heterotrimeric G proteins is yet to be explored. In this study, we set out to investigate whether GTP-binding protein (G
protein)-coupled receptors (GPCRs) can stimulate Akt activity and
survival pathways and, if so, to define the mechanism(s) whereby this
class of cell surface receptors could regulate Akt function. Using
ectopic expression of GPCRs in COS-7 cells as a model, we have observed
that both m1 and m2 muscarinic acetylcholine receptors, representative
of those GPCRs coupled to Gq and Gi
proteins, respectively, can readily activate an epitope-tagged form of
Akt kinase and prevent UV-induced apoptosis. We have also found
that the pathway connecting G proteins to Akt implicates signals
emanating from Gq, G
i, and
dimers, but not from G
s or G
12, in each case
acting through a pathway that involves a phosphatidylinositol-3-OH
kinase activity. Moreover, our findings suggest a role for a novel
-sensitive complex, p101·phosphatidylinositol-3-OH kinase-
,
in the transduction of signals leading to Akt stimulation and cell
survival by GPCRs and open new avenues for research on the function of
the large family of G protein-linked receptors in the regulation of
anti-apoptotic pathways.
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INTRODUCTION |
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Receptors coupled to heterotrimeric GTP-binding proteins (G
proteins)1 are the largest
group of integral membrane receptors involved in the transmission of
signals from the extracellular environment to the cytoplasm (1). A wide
range of external stimuli, including neurotransmitters, growth factors,
hormones, light, odorants, and certain taste ligands, can activate
specific members of this family and promote a conformational change
that is transmitted to the cytoplasmic side of the receptor protein
(1). This leads to a physical interaction between the receptor and the
GDP-bound G protein heterotrimer which causes the dissociation of the
guanine nucleotide and the incorporation of GTP in the G protein subunit, thus releasing the
heterodimer (2). In turn, GTP-bound
G protein
subunits and
complexes initiate, independently, a wide variety of intracellular signaling pathways (3).
Although the G protein-coupled receptor (GPCR) family is involved in many functions performed by fully differentiated cells, these receptors are also expressed in most proliferating cells, and they have been implicated in embryogenesis, tissue regeneration, and growth stimulation (4). The nature of the growth regulatory pathway(s) stimulated by GPCRs has just begun to be elucidated (5). In our laboratory, we have used the ectopic expression of human muscarinic receptors for acetylcholine (mAChRs) in NIH 3T3 cells as a model system for studying mitogenic signaling through G protein-linked receptors. In this biological setting, we have shown that certain mAChR subtypes can effectively transduce mitogenic signals and, when activated persistently, induce the transformed phenotype (6). Interestingly, two recent reports have demonstrated that the activation of endogenously expressed muscarinic receptors is per se sufficient to block apoptosis in neuronal cells (7, 8). These results suggest that in addition to their role in cell growth, GPCRs might also activate yet to be defined survival pathways.
In this regard, the serine-threonine kinase Akt/PKB, which was first identified as the human homolog of a transforming oncogene (9), has been shown recently to control intracellular pathways preventing cell death in response to a variety of extracellular stimuli (10) and in a wide range of cellular systems (11, 12). The mechanism of activation of Akt by tyrosine kinase growth factor receptors has been established recently (13). However, the regulation of this intriguing kinase by GPCRs is still unclear. Initial reports showed that lysophosphatidic acid, acting through Gi-coupled receptors, was unable to stimulate Akt activity in NIH 3T3 cells (14). Furthermore, direct activation of PKC by phorbol esters also failed to activate Akt in this fibroblast cell line (14, 15). On the other hand, recent reports have described Akt stimulation in response to GPCRs in rat epididymal fat cells (16) and human embryonic kidney 293-EBNA cells (17), albeit by a yet to be determined mechanism.
In this study, we set out to investigate whether Akt can be activated
effectively by GPCRs using ectopically expressed receptors and
different G protein subunits in COS-7 cells as a model system. We found
that both Gq- and Gi-coupled GPCRs,
m1 and m2 receptors, respectively, can readily activate an
epitope-tagged form of Akt kinase. We also show that both
signal-transducing molecules generated upon GPCR activation,
complexes and
subunits, can effectively promote Akt activation in a
PI3K-dependent manner. Moreover, our findings suggest a
role for the novel PI3K
and its associated regulatory subunit, p101,
in the transduction of signals leading to Akt stimulation by GPCRs. We
also present evidence that GPCRs can induce survival pathways through
PI3K
acting on Akt.
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EXPERIMENTAL PROCEDURES |
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Cell Lines and Transfection--
COS-7 cells were cultured in
Dulbecco's modified Eagle's medium supplemented with 10% fetal
bovine serum. Cells were transfected by the DEAE-dextran technique,
adjusting the total amount of DNA to 1-4 µg/plate with
pcDNA3--galactosidase DNA, when necessary (18).
Expression Plasmids--
An epitope-tagged Akt (pCEFL-HA-Akt) as
well as the membrane-targeted mutant (pCEFL-myr-HA-Akt) were generated
by inserting the coding region of Akt and myr-Akt (kindly provided by
Dr. P. N. Tsichlis) into the pCEFL expression vector. Expression
plasmids for m1 and m2 mAChRs, for the and
isoforms of the PI3K
and their mutants, as well as expression plasmids for
1,
2, and
2* subunits of heterotrimeric G
proteins were reported previously (18-20). Plasmids expressing the
coding region of PI3K
and the p101 protein fused to the Glu-Glu tag
were kindly provided by L. R. Stephens and are described elsewhere
(21). Plasmids expressing the GTPase-deficient, constitutively
activated forms of representative G protein
subunits
(G
12-Q227L, G
i2-Q205L,
G
q-Q209L, and G
s-R201C) have been
described already (19, 22, 23). An expression plasmid for a chimeric
molecule between the extracellular and transmembrane domain of CD8
fused to the carboxyl-terminal 222 amino acids of
ARK, which
includes both the
-binding domain and the PH domain of
ARK
(pcDNA-CD8-
ARK), has also been described recently (22, 24).
Akt Assay and Western Blots--
Akt activity in lysates from
COS-7 cells transfected with an expression vector for an epitope-tagged
Akt (pCEFL-HA-Akt) was determined upon immunoprecipitation with the
anti-HA-specific monoclonal antibody 12CA5 (Babco) using histone 2B
(Boehringer) as substrate, essentially as described (15). Briefly,
cells grown on 10-cm plates were washed once in cold phosphate-buffered saline and lysed on ice with 1 ml of lysis buffer containing protease and phosphatase inhibitors (1% Triton X-100, 10% glycerol, 137 mM NaCl, 20 mM Tris-HCl, pH 7.5, 1 µg/ml
aprotinin and leupeptin, 1 mM phenylmethylsulfonyl
fluoride, 20 mM NaF, 1 mM disodium
pyrophosphate, and 1 mM Na3VO4).
After preclearing the samples by centrifugation, lysates were
immunoprecipitated with 1 µl of anti-HA monoclonal antibody using
-binding beads (Amersham Pharmacia Biotech) to sediment the
immunocomplexes. After three 1-ml washes with lysis buffer, one 1-ml
wash with water, and one 1-ml wash with kinase buffer (20 mM Hepes, pH 7.4, 10 mM MgCl2, 10 mM MnCl2), reactions were performed for 30 min
at 25 °C under continuous agitation in kinase buffer containing 0.05 mg/ml histone 2B, 5 µM ATP, 1 mM
dithiothreitol, and 10 µCi of [
-32P]ATP. The
products of the kinase reactions were fractionated in 15%
SDS-polyacrylamide gels, transferred to nylon membranes (Immobilon),
and exposed. Resulting autoradiograms were quantified with the use of a
Molecular Dynamics densitometer. When necessary, the same membranes
were analyzed subsequently by Western blot using mouse anti-HA (Babco,
1:500) or goat anti-Akt (C-20, Santa Cruz Biotechnology, 1:250) to
visualize the endogenous protein in PC12 cells. To assess the level of
expression of cotransfected proteins, 50 µl of total lysates were
analyzed by Western blot using goat anti-PI3K
(N-16, Santa Cruz
Biotechnology, 1:250), mouse anti-Glu-Glu (Babco, 1:500), rabbit
anti-G
q (Santa Cruz Biotechnology, 1:250), rabbit
anti-G
s (K-20, Santa Cruz Biotechnology, 1:500), rabbit
anti-G
i (Upstate Biotechnologies, Inc., 1:10,00), rabbit
anti-G
2 (Santa Cruz Biotechnology, 1:500), and specific antisera against G
12 and G
as described previously
(19). Bands were developed by an enhanced chemiluminescence detection
kit (Amersham Pharmacia Biotech) using secondary antibodies coupled to
horseradish peroxidase (Cappel).
Apoptosis Assay--
COS-7 cells were grown on coverslips and
transfected with the indicated plasmids together with an expression
vector for -galactosidase as a marker for transfection, using the
LipofectAMINE-Plus reagent (Life Technologies, Inc.) following the
manufacturer's instructions. Protection from UV-induced apoptosis was
performed essentially as described (12). Briefly, 24 h after
transfection, cells were serum starved overnight in Dulbecco's
modified Eagle's medium containing 10 mM Hepes and
subjected subsequently to UV irradiation (120 mJ, UV-Stratalinker 1800, Stratagene). After the addition of fresh serum-free medium containing
or not 1 mM carbachol, cells were maintained in the
incubator, fixed 8 h later in 4% paraformaldehyde, and
permeabilized with 0.01% Triton X-100. Transfected cells were identified by immunostaining for
-galactosidase expression with a
mouse anti-
-galactosidase antibody (Promega, 1:100) followed by a
rabbit anti-mouse rhodamine-coupled secondary antibody (Sigma, 1:200).
Fragmented DNA was then visualized by the terminal
deoxynucleotidyltransferase-mediated dUTP-FITC nick end labeling
(TUNEL) technique using a kit from Boehringer Manheim, following the
manufacturer's instructions, except that the reaction was carried out
at room temperature instead of at 37 °C. The frequency of apoptosis
was scored by counting several hundred rhodamine-stained (transfected)
cells from at least 20 different fields/coverslip and examining them
for FITC staining (TUNEL-positive) under UV light in an Axioplan2
fluorescence microscope (Zeiss).
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RESULTS |
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Stimulation of muscarinic GPCRs has been reported to induce protection from apoptosis both in cerebellar granule neurons (8) and in the pheochromocytoma cell line PC12 (7), and the Akt kinase has been implicated in survival pathways in many cell types, including PC12 and other neuroectodermal-derived cells (11, 13). Thus, we asked whether activation of endogenous muscarinc receptors would lead to Akt activation in PC12 cells. As shown in Fig. 1A, exposure of PC12 cells to the cholinergic agonist carbachol induced the rapid stimulation of the Akt phosphotransferase activity, to an extent similar to that observed in these cells in response to nerve growth factor acting on its cognate receptors.
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To investigate further the molecular mechanism(s) whereby muscarinic receptors activate Akt and induce protection from cell death, we chose to use a reconstituted system, consisting of the coexpression of m1 and m2 muscarinic receptors together with an epitope-tagged Akt (HA-Akt) in COS-7 cells. Whereas m1 receptors are typical of those coupled through G proteins of the Gq family to phospholipase C activation, m2 is known to couple through Gi to a number of effector pathways, including the inhibition of adenylyl cyclases (25). In cells expressing either muscarinic receptor we observed that carbachol induced an increase in Akt activity, as judged by immune complex kinase reactions using histone 2B as a substrate. When mediated by m1 receptors, induction of histone 2B phosphorylation was evident as early as 1 min after the addition of agonist, showing an early peak at approximately 3 min after stimulation (Fig. 1B). Stimulation of m2 also caused a very rapid activation, which was evident after 1 min and reached a maximum around 15 min (Fig. 1B). Both m1- and m2-induced Akt kinase activity remained elevated for an extended period of time, decreasing to the basal activity as late as 2-3 h after treatment (data not shown).
Recent work has demonstrated that PI3K activity is required for Akt
activation in the majority of the systems described to date (26).
However, PI3K-independent pathways have also been described (17, 27),
including those mediating Akt activation by 3-adrenergic
GPCR in epididymal fat cells (16). In our experimental system,
preincubation of cells with 50 nM wortmannin, a potent PI3K
inhibitor, completely blocked both m1- and m2-mediated stimulation of
Akt (Fig. 1C). Thus both Gq- and
Gi-coupled receptors appear to activate Akt irrespective of
their G protein coupling specificity, utilizing a
PI3K-dependent pathway.
As an approach to investigate which G protein(s) mediates Akt
activation, we used the expression of GTPase-deficient mutationally activated forms of G protein subunits, which can activate effector pathways by obviating the need for receptor stimulation (2). Thus, we
coexpressed the epitope-tagged Akt together with GTPase-deficient mutants for G
s, G
i2, G
q,
and G
12, representing each of the four known
subunits (3). As shown in Fig.
2A, the activated mutant of
G
q was able to trigger Akt activation similar to that caused by Ras when used as control (15, 28). Activated
G
i2 also stimulated Akt, although less efficiently than
G
q, and the activated mutants of G
s and
G
12 had no demonstrable effect under our experimental
conditions. These data indicate that G
q, and to a lesser
extent G
i, can mediate Akt activation by G
protein-linked receptors.
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When activated, GPCRs catalyze the replacement of GDP by GTP bound to
the subunit and induce the dissociation of
-GTP from
dimers. Although the
subunits were thought to be the sole responsible molecules for coupling receptors to second
messenger-generating systems, recent work has established a critical
role for
dimers in signal transduction (3, 19). These data
prompted us to explore whether
dimers might also participate in
signaling to Akt. We observed that when cotransfected,
1
2 subunits induce a remarkable increase
in the phosphorylating activity of the epitope-tagged Akt, although
expression of the HA-Akt was similar for each transfected cell
population (Fig. 2B). In contrast, Akt was activated poorly when coexpressed with
1 or
2 alone or
when coexpressed with
1 and an altered form of
2 subunit, designated
2*, which lacks the
-isoprenylation signal and therefore fails to associate to the
plasma membrane (19) (Fig. 2B). Based upon these results, we
conclude that membrane-bound forms of
subunits of heterotrimeric G proteins can potently stimulate Akt activity in COS-7 cells. In view
of these results, we next sought to explore the relative contributions
of
and
proteins in Akt stimulation by mAChRs. To approach
this question, we employed a chimeric molecule combining the
extracellular and transmembrane domain of CD8 fused to the carboxyl-terminal domain of
ARK which includes the high affinity
binding region of this kinase as described (22). This chimeric molecule targets the COOH-terminal part of
ARK to the plasma membrane where it is expected to bind and sequester free
complexes when dissociated from G
subunits upon receptor
stimulation, thus blocking
-dependent pathways (22).
As shown in Fig. 2C, coexpression of CD8-
ARK with the m2
mAChR nearly abolished the activation of Akt in response to carbachol,
whereas m1-mediated Akt stimulation was only partially impaired by
overexpression of this
-sequestering molecule. In contrast, Akt
activation by other effectors such as Ras was unaffected by CD8-
ARK.
Taken together, these findings strongly suggest that signaling from m2
mAChR to Akt is mediated primarily by the
subunits of
heterotrimeric G proteins, whereas m1-mediated activation is achieved
via G
-dependent and -independent pathways.
m1 receptors and activated Gq efficiently stimulate
phospholipase C
causing the hydrolysis of phosphoinositides (29). This results in the generation of two second messengers: inositol trisphosphate, which leads to the elevation of intracellular
[Ca2+], and diacylglycerol, which activates PKC (25).
However, several lines of evidence suggested that PKC does not
participate in signaling from m1 and G
q to Akt; direct
activation of PKC by phorbol 12-myristate-13-acetate (100 ng/ml for 15 min) provoked no change in Akt activity, nor did specific inhibitors of
PKC such as bisindolylmaleimide (10 µM for 30 min) affect
m1 or Gq-induced Akt activation (data not shown). On the
other hand, wortmannin treatment clearly blocked the stimulation of Akt
by m1 (see above, Fig. 1C) and, similarly, prevented Akt
activation by G
q-QL and
coexpression (data not shown). These data implicated a function for PI3K rather than for PKC
in the pathway connecting G protein-initiated signals to Akt. In this
regard, whereas many cell surface receptors activate PI3K
and
through the tyrosine phosphorylation of their p85 subunit, GPCRs were
shown recently to activate a novel PI3K isoform, PI3K
, which does
not interact with p85 (21). To explore whether this novel PI3K isoform
participates in signaling from G proteins to Akt, we cotransfected a
dominant negative form PI3K
together with activated
G
q, G
i2,
, and activated Ras. As
shown in Fig. 3, cotransfection of a
dominant negative PI3K
did not affect Ras-induced stimulation of
Akt, suggesting that in COS-7 cells Ras might act mainly via PI3K
,
as proposed previously (28). However, this PI3K
mutant nearly
abolished Akt activation by each heterotrimeric G protein subunit,
G
q-QL, G
i2-QL, and
complexes (Fig.
3), as well as by m1 and m2 receptors (data not shown), thus suggesting
that a PI3K
or a PI3K
-like kinase is required for both
- and
-mediated pathways elicited by GPCRs.
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To assess further the involvement of a PI3K activity downstream of GPCR
in the signaling pathway to Akt, we investigated whether the
overexpression of different isoforms of PI3K was per se
sufficient to induce Akt activation. As observed in Fig.
4, expression of both and
isoforms of PI3K triggered Akt activation poorly, although a
constitutively active form of PI3K
(myr-PI3K
) (19) revealed a
greater ability to stimulate Akt than each of the wild type forms.
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For PI3K, a novel noncatalytic subunit unrelated to p85 has been
identified recently (21) and named p101. It has been shown recently
that coexpression of this molecule with PI3K
enhances its basal
activity and potentiates PI3K activation by
dimers (21). We
therefore asked whether the expression of this noncatalytic PI3K
subunit p101 could potentiate the effect of PI3K
on Akt. As shown in
Fig. 4, Akt activity increased dramatically when both proteins were
expressed together. Interestingly, however, we observed that expression
of p101 was able per se to induce an increase in Akt
activity, to an extent comparable to that elicited by PI3K
alone
(Fig. 4). These data suggest that p101 may activate an endogenous PI3K
or a PI3K
-like protein thereby stimulating Akt
phosphorylating activity.
To assess further the biological consequences of Akt activation by
GPCRs, we set out to investigate whether m1 and m2 receptors could
induce survival pathways in COS-7 cells. For that, we took advantage of
the recent observation that transfected COS-7 cells undergo apoptosis
upon UV irradiation (12) and that Akt activation can protect cells from
death in this cellular setting (12). In preliminary experiments, the
ED50 for the apoptotic effect of UV was found to be
approximately 120 mJ, a dose of UV which was utilized for the
subsequent assays. As shown in Fig. 5,
under control conditions less than 10% of the transfected cells
displayed an apoptotic phenotype and were labeled by the TUNEL
reaction. However, when irradiated by UV a fraction of the
untransfected and transfected cells underwent apoptosis, the former
visualized as TUNEL-positive cells (FITC-stained) and the latter as
both anti--galactosidase and TUNEL-positive cells (rhodamine- and FITC-stained, respectively), as depicted in Fig. 5B. Of
interest, the addition of carbachol protected m1- and m2-transfected
cells from UV-induced apoptosis, to an extent similar to that caused by
transfection of an activated form of Akt, myr-Akt (Fig. 5A). In contrast, carbachol treatment produced no apparent consequences in
mock-transfected or myr-Akt-transfected cells. Thus, in COS-7 cells m1
and m2 GPCRs can effectively activate survival mechanisms that are able
to counteract apoptotic insults, most likely through Akt.
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Based on our previous results, we wanted to study further the role of
PI3K in the survival pathway stimulated by GPCRs. Initial experiments exploring the effect of wortmannin on the protective activity of m1 and m2 were inconclusive, as treatment of serum-starved cells with wortmannin caused per se a noticeable apoptotic
effect (data not shown). Accordingly, we next examined the effect of overexpression of the kinase-deficient mutant of PI3K
on the antiapoptotic activity elicited by m1 and m2 receptors. As shown in
Fig. 6, expression of the
kinase-deficient mutant of PI3K
prevented the protective effect of
m1 and m2 stimulation without affecting the basal UV-induced apoptosis
or the protection conferred by expression of an activated form of Akt,
thus suggesting a role for PI3K
in the survival pathway elicited by
GPCRs.
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DISCUSSION |
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The multiplicity of intracellular signaling pathways activated by
the large family of GPCRs has just begun to be appreciated. In
particular, recent work has provided a wealth of evidence that this
family of cell surface receptors can trigger biochemical routes
communicating the membrane to the nucleus, thereby controlling key
molecules involved in gene expression regulation (4, 5). Furthermore,
these receptors can affect normal and aberrant cell growth as well as
regulate antiapoptotic pathways (7, 8). In this regard, it has been
shown that activation of muscarinic GPCRs in PC12 cells can induce cell
survival (7). Here, we show that stimulation of PC12 cells with the
cholinergic agonist carbachol can activate Akt to an extent comparable
to that observed in response to nerve growth factor, a natural
survival-promoting factor for these cells (30), thus suggesting that
the Akt kinase might participate in the survival pathway(s) elicited by
GPCRs. These cells, as many neuroectodermal derived cells, express a mixed population of muscarinic receptor subtypes (31), each exhibiting
a distinct coupling selectivity, thus limiting the ability to
characterize the pathway linking these receptors to Akt. However, when
ectopically expressed in COS-7 cells both m1 and m2, Gq-
and Gi-coupled receptors, respectively, were able to
activate Akt efficiently, thus providing an experimental model where
the activation of Akt by these GPCRs could be examined in a molecularly
defined reconstituted system. In these cells, we found that whereas
activated Gi2 can induce Akt activity poorly, both
activated G
q and
complexes were potent
stimulators of this serine/threonine kinase. In line with this
observation, a
-sequestrant, CD8-
ARK, nearly abolished Akt
stimulation in response to m2 activation but had a more limited effect
on m1-induced Akt activation. Collectively, these data indicate that
whereas Gi-coupled receptors signal primarily to Akt
through
dimers, Gq-coupled receptors utilize both
-dependent and -independent mechanisms, the latter
likely acting through G
q.
Of interest, PKC did not appear to link Gq to Akt, but
pharmacological and biochemical evidence supported a role for a PI3K downstream from GPCRs and heterotrimeric G protein subunits in the
pathway leading to Akt, and we obtained data to suggest that the
PI3K
·p101 dimer is a likely candidate to communicate G proteins to
Akt. This finding can help explain the seemingly contradictory results
on Akt activation by GPCRs (14-17; see above) because PI3K
is
highly expressed in hematopoietic cells but poorly expressed in other
tissues. In COS-7 cells, we can detect limited expression of PI3K
(not shown) (20) which is consistent with the observation that
p101 expression alone is sufficient to activate Akt, albeit to a
limited extent compared with that achieved upon coexpression of PI3K
and p101. Thus, in tissues and cell lines lacking PI3K
, either GPCR
would fail to activate Akt (14, 15) or other PI3K isoforms, such as
PI3K
(32), might link heterotrimeric G proteins to Akt.
To examine the biological consequences of activating GPCRs in COS-7
cells, we investigated the ability of m1 and m2 receptors to protect
COS-7 cells from UV-induced apoptosis. In this reconstituted system, we
found that both GPCRs were able to activate cell survival pathways
effectively, most likely through Akt. Furthermore, we observed that the
protective effect elicied by m1 and m2 was nearly abolished by
expression of a dominant-negative mutant form of PI3K, suggesting
that the GPCR-PI3K
pathway is biologically relevant in this cellular
setting.
In summary, our findings raise the possibility of the existence of a
novel pathway activating Akt and preventing apoptosis by cell
surface receptors. This pathway involves extracellular ligands
acting on GPCRs and the consequent activation of Gi or Gq proteins, depending on the coupling selectivity. In
turn, these G proteins will release subunits, and
dimers and activated G
, particularly G
q, will then
stimulate PI3K
·p101 complexes or other yet to be identified PI3K
isoforms, leading to Akt activation and promoting cell survival.
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ACKNOWLEDGEMENTS |
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We thank Dr. Stevens for providing the p101
and PI3K expression vectors and Drs. Tsichlis and Franke for the
kind gift of Akt expression plasmids.
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FOOTNOTES |
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* 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.
Supported by a postdoctoral fellowship from the Spanish Ministerio
de Educación y Cultura.
§ Supported by the Fundación Argentina para el Desarrollo Infantil.
To whom correspondence should be addressed: Oral and
Pharyngeal Cancer Branch, NIDR, National Institutes of Health, 9000 Rockville Pike, Bldg. 30, Rm. 211, Bethesda, MD 20892-4330. Fax:
301-402-0823.
1
The abbreviations used are: G protein,
GTP-binding protein; GPCR, G protein-coupled receptor; mAChR,
muscarinic acetylcholine receptor; PKC, protein kinase C; PI3K,
phosphatidylinositol-3-OH kinase; HA, hemagglutinin; ARK,
-adrenergic receptor kinase; FITC, fluorescein isothiocyanate;
TUNEL, terminal deoxynucleotidyltransferase-mediated dUTP-FITC nick end
labeling.
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REFERENCES |
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