 |
INTRODUCTION |
Extracellular signals such as growth factors and hormones regulate
cell proliferation. In general, growth factors activate receptor
tyrosine kinases (RTKs),1
whereas most hormones activate G protein-coupled receptors (GPCRs). Intracellular cascades such as the mitogen-activated protein (MAP) kinase, also called the extracellular signal-regulated kinase (ERK),
cascade, are mainly activated as a consequence of agonist stimulation
of RTKs. ERK1/2 are serine/threonine protein kinases activated upon
dual phosphorylation by the MAP kinase kinase MEK (1), which is
phosphorylated and activated by MAP kinase kinase kinases of the Raf
family (2). In mammals, all three Raf isoforms expressed (A-Raf, B-Raf,
and Raf-1) are cytosolic in resting cells and become activated upon
recruitment to the plasma membrane by activated, small G proteins of
the Ras family (H-Ras, K-Ras, N-Ras, and Rap1). Ras, in its active,
GTP-bound conformation, may activate all isoforms of Raf. The levels of
GTP-bound Ras are controlled by the actions of guanine nucleotide
exchange factors and GTPase-activating proteins (GAPs). The two major
Ras-specific guanine nucleotide exchange factors are Son of sevenless
(SOS) (3) and Ras-GRF1 (CDC25Mm) (4). SOS is mainly thought
to activate Ras as a consequence of RTK stimulation, whereas Ras-GRF1
activates Ras in response to Ca2+ signaling and
GPCR-mediated signals (5-8).
Serotonin (5-hydroxytryptamine (5-HT)) mediates its diverse
physiological effects through at least 14 different receptors, of which
13 are GPCRs or so-called seven transmembrane-spanning receptors (9).
The 5-HT receptors, each encoded by a separate gene, have been grouped
into families called 5-HT1 to 5-HT7, with some
of the families containing several members. The human serotonin receptors 5-HT4 and 5-HT7 couple to the
heterotrimeric G protein Gs and exist in multiple splice
variants (10, 11). Stimulation of these receptors leads to activation
of adenylyl cyclase and a rapid increase in the formation of the
intracellular second messenger cAMP. Elevated levels of cAMP have
several intracellular effects, e.g. activation of
cAMP-dependent protein kinase (PKA) and exchange proteins
directly activated by cAMP (Epacs), guanine nucleotide exchange factors
specific for Rap. Both mechanisms have been claimed to be involved in
cAMP-dependent activation of Rap1 (12-14).
The main target for cAMP is PKA, which has cell type-specific
effects on the ERK1/2 cascade. In several cell types,
Ras-dependent ERK1/2 activation is antagonized by PKA
(15-18). In other cell types, PKA appears to activate the ERK1/2
cascade via Rap1 and B-Raf (12, 19). In human embryonic kidney HEK293
cells, isoproterenol stimulation of the endogenous
Gs-coupled
2-adrenergic receptor (
2-AR) activates Ras and Rap1, but only Rap1 is able to
mediate activation of ERK1/2 by recruiting B-Raf to the membrane and
thereafter activate MEK (20). However, at overexpressed levels of
2-ARs, ERK1/2 can be activated through the G
subunits of pertussis toxin-sensitive G proteins, interpreted as a
switch in coupling from Gs to Gi due to
phosphorylation of the
2-AR by PKA (21).
Upon stimulation of Gi- (22) and
Gq-coupled (23) receptors, activation of MAP kinases is
induced by G
subunits that mediate Ras activation through
proteins such as the tyrosine kinase c-Src, Grb2, and SOS (24). Both
phosphoinositide 3-kinase
(25) and the calcium-sensitive kinase
PYK2 (26) have been implicated in the activation of c-Src. The
G
i and G
q subunits may also mediate
ERK1/2 activation by binding to Rap1GAP1, a selective Rap1
GTPase-activating protein (27, 28). In these cases, active Rap1
antagonizes the function of Ras, and inhibition of Rap1 by Rap1GAP1
allows Ras to signal to ERK1/2. Furthermore, in several cell types,
GPCRs coupling to Gi and Gq have been reported
to transactivate the epidermal growth factor receptor (EGFR) and thereby lead to activation of ERK1/2 (29-31).
A Gs-coupled 5-HT receptor has been implicated in ERK
activation and LTP induction in Aplysia (32, 33). In mouse
cortical neurons, several monoamines including 5-HT, as well as
forskolin, activated ERK1/2 and increased the level of GTP-bound Ras,
but the 5-HT receptor involved was not characterized (34). In cultured rat hippocampal neurons, 5-HT activated ERK1/2 through
5-HT7 receptors, but the mechanism was not characterized
(35). In mammalian brain, both 5-HT4 and 5-HT7
receptors are found in the hippocampus (36, 37), and these receptors
are both believed to be important in learning and memory (38). Thus,
there is a need to determine whether 5-HT4 and
5-HT7 receptors can activate ERK, as well as the mechanism
for such activation. We have previously characterized activation of
adenylyl cyclase by different splice variants of the human
Gs-coupled 5-HT4 (39) and 5-HT7
(40) receptors. We now demonstrate that both the human
Gs-coupled serotonin receptors 5-HT4(b) and
5-HT7(a), when transiently expressed in HEK293 and COS-7
cells, activate ERK1/2 through a mechanism dependent on Ras and
independent of Rap1.
 |
EXPERIMENTAL PROCEDURES |
Materials--
HEK293 and COS-7 cells were from American Type
Culture Collection (Manassas, VA). Mouse monoclonal anti-phospho-ERK1/2
was from Cell Signaling Technology (Beverly, MA), sheep polyclonal anti-mouse Ig-horseradish peroxidase and sheep anti-rabbit
IgG-horseradish peroxidase were from Amersham Biosciences, rabbit
polyclonal anti-ERK1/2 was from Upstate Biotechnology (Lake Placid,
NY), mouse monoclonal anti-Ras (IgG1) was from Transduction
Laboratories (BD Biosciences), and rabbit polyclonal anti-Raf-1 and
rabbit polyclonal anti-B-Raf were from Santa Cruz Biotechnology (Santa
Cruz, CA). 5-HT hydrochloride (serotonin),
N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide dihydrobromide (H89), epidermal growth factor (EGF), Dulbecco's modified Eagle's medium, and diethylaminoethyl-dextran hydrochloride (DEAE-dextran) were from Sigma. PD153035 was from Tocris Cookson Ltd.
(Avonmouth, UK), and PD98059 was from Calbiochem. Glutathione-Sepharose 4B and Hybond-P (polyvinylidene difluoride) membrane were from Amersham
Biosciences. LipofectAMINETM reagent and penicillin-streptomycin were
from Invitrogen. UltraCULTURETM general purpose serum-free medium and
L-glutamine were from BioWhittaker Europe (Vervierse, Belgium). Bio West extended duration chemiluminescent substrate was
from UVP (Upland, CA), and the BC assay protein quantitation kit was
from Uptima (Monticon, France).
Plasmids--
The pcDNA3.1(
) vectors encoding the human
5-HT4(b) and 5-HT7(a) receptors were described
previously (39, 40). The pcDNA3.1 vector encoding the dominant
negative Rap1, RapN17, was provided by Dr. Philip J. S. Stork
(Oregon). Rap1GAP1, GST-Raf1-RBD, and GST-RalGDS-RBD constructs were
provided by Dr. Johannes L. Bos (Utrecht, The Netherlands) (41, 43).
The pCMV vectors encoding the dominant negative Ras, RasN17,
constitutively active Ras, RasV12, dominant negative Raf1, RafS621A,
and constitutively active Raf1, Raf-CAAX, were from
Clontech. The pcDNA3.1 vectors encoding the
dominant negative B-Raf, N-B-Raf, and constitutively active B-Raf,
CAAX-B-Raf, were provided by Dr. Roser Buscà (Nice, France) and
Dr. Alain Eychene (Orsay, France) (42).
Cell Culture and Transfection--
COS-7 cells were cultured in
Dulbecco's modified Eagle's medium supplemented with 10% fetal calf
serum and 50 mg/liter gentamicin at 37 °C in a humidified atmosphere
of 5% CO2 in air and transfected at 60-70% confluence
with the 5-HT4(b) or 5-HT7(a) receptors using DEAE-dextran according to the manufacturer's protocol. HEK293 cells
were cultured in UltraCULTURETM general purpose serum-free medium with
2 mM L-glutamine and 100 units/ml
penicillin-streptomycin at 37 °C in a humidified atmosphere of 5%
CO2 in air and transfected at 60-70% confluence with the
indicated cDNA(s) using LipofectAMINE according to the
manufacturer's protocol. When necessary, empty vector
(pcDNA3.1(
); Invitrogen) was included to assure that each dish
received the same amount of DNA (1.0 and 2.9 µg of plasmid DNA per
35-mm and 60-mm dish, respectively). If not otherwise indicated, cells
were kept in serum-free medium as described until treatment and lysis,
~48 h after transfection. When indicated, cells were preincubated
with 20 µM H89 for 25 min, 5 µM PD98059 for
20 min, or 1 µM PD153035 for 18 h. Cells were
stimulated with agonist (10 µM 5-HT or 5 nM
EGF) for 5 min, if not otherwise indicated. All experiments were
carried out in doublets with at least three replicates, if not
otherwise indicated.
Western Blotting--
Equal amounts of cell lysate proteins were
separated by SDS-PAGE and electroblotted onto polyvinylidene difluoride
membranes. The membranes were incubated with primary antibodies
(anti-phospho-ERK1/2, 1:2,000; anti-Ras, 1:1,000; anti-Rap1, 1:500) in
5% nonfat dry milk in phosphate-buffered saline-0.05% Tween and
thereafter incubated with corresponding horseradish
peroxidase-conjugated secondary antibodies. The immobilized horseradish
peroxidase-conjugated secondary antibodies were visualized with Bio
West chemiluminescent substrate and analyzed with a UVP BioChemie
system. After detection of phospho-ERK1/2, membranes were stripped by
treatment with 0.5 M NaOH for 5 min at room temperature and
extensive washing with distilled H2O and reprobed with
anti-ERK1/2 antibodies (1:10,000) to confirm equal loading.
Phospho-ERK1/2 Assay--
The cells were cultured in
35-mm dishes, transfected and stimulated with agonist as described,
lysed in ice-cold cell lysis buffer (1% SDS, 1 mM
Na3VO4, 50 mM Tris-HCl, pH 7.4 at
room temperature), scraped with a rubber policeman, sheared
through a 25-gauge syringe, and immediately snap frozen in liquid
N2. The thawed cell lysates were cleared at 13,000 × g at 4 °C, and the protein concentrations in the
supernatants were quantified using the BC assay protein quantitation
kit using BSA as standard. Equal amounts of protein were prepared for
separation by SDS-PAGE.
Pull-Down Experiments in HEK293 Cells--
The cells were
assayed as described by van Triest et al. (43). Briefly,
HEK293 cells were maintained in 60-mm dishes, transfected, and
stimulated as described. The cells were lysed in ice-cold pull-down
lysis buffer (10% glycerol, 1% Nonidet P-40, 50 mM
Tris-HCl, pH 7.5 at room temperature, 200 mM NaCl, 2 mM MgCl2, 1 mM phenylmethylsulfonyl fluoride, 2 µg/ml aprotinin, 1 µg/ml leupeptin, and 10 µg/ml
trypsin inhibitor). Equal amounts of cleared cell lysates were
incubated at 4 °C on a tumbler for 1 h with GST-Raf1-RBD or
GST-RalGDS-RBD constructs precoupled to glutathione-Sepharose beads for
Ras or Rap1 pull-down experiments, respectively. The beads were
pelleted and washed four times with ice-cold pull-down lysis buffer.
The proteins were eluted from the beads by boiling for 5 min in 1× loading buffer. The protein samples were loaded and resolved on 12.5%
SDS-polyacrylamide gels. Western blotting was performed as described.
 |
RESULTS |
ERK1/2 Activation via 5-HT4(b) and
5-HT7(a) Receptors in COS-7 and HEK293 Cells--
Initial
experiments indicated that treatment of COS-7 cells transiently
transfected with Gs-coupled human 5-HT4(b) or
5-HT7(a) receptors with 10 µM 5-HT induced
dual phosphorylation and thus activation of ERK1/2. Similar results
were obtained with HEK293 cells. Because HEK293 cells do not express
endogenous 5-HT receptors and had a lower background of ERK1/2
phosphorylation, these cells were selected for further study. Based on
the potency of 5-HT to stimulate adenylyl cyclase activity through
human 5-HT4(b) (39) or 5-HT7(a) (40) receptors
and 5-HT concentration-response experiments with ERK1/2 phosphorylation
(data not shown), a concentration of 10 µM 5-HT was
chosen for the remaining experiments. A high resolution time course
showed that the maximal 5-HT-induced phosphorylation of ERK1/2 through
5-HT4(b) receptors occurred after 5 min (Fig. 1A). Time courses with more
extended periods of stimulation showed that 5-HT-induced activation of
ERK1/2 through 5-HT4(b) (data not shown) and
5-HT7(a) (Fig. 1B) receptors peaked after ~5
min and then decreased. There was no second peak or sustained
activation of ERK1/2.

View larger version (43K):
[in this window]
[in a new window]
|
Fig. 1.
Activation of ERK1/2 through
5-HT4(b) and 5-HT7(a) receptors.
Time courses of activation of ERK1/2 after 5-HT stimulation of
HEK293 cells transiently transfected with the human
5-HT4(b) (A) or 5-HT7(a)
(B) receptor. The cells were stimulated for the indicated
periods of time and assayed for detection of phospho-ERK1/2 as
described under "Experimental Procedures." Proteins were separated
on 10% SDS-PAGE and electroblotted over to polyvinylidene difluoride
membranes before incubation with antibodies. A and
B show representative Western blots probed with
phospho-specific ERK1/2 antibodies (ppERK1/2; top
panels) and subsequently probed with anti-ERK1/2 antibodies
(ERK1/2; bottom panels) to confirm equal
loading.
|
|
Activation of ERK1/2 through 5-HT4(b) and
5-HT7(a) Receptors Is PKA- and
MEK-dependent--
All splice variants of the human
serotonin receptors 5-HT4 and 5-HT7 are
Gs-coupled receptors that upon agonist stimulation induce a
rapid increase in the formation of cAMP, leading to activation of PKA
or Epac. To examine the role of PKA in the signaling pathway, HEK293
cells transiently transfected with either 5-HT4(b) or
5-HT7(a) were preincubated with the PKA inhibitor H89.
Pretreatment with H89 led to a partial inhibition of 5-HT-induced
activation of ERK1/2, as shown in Fig.
2A for the
5-HT4(b) receptor. However, a more efficient inhibition was
observed when the transiently transfected cells were maintained in
serum-free Dulbecco's modified Eagle's medium for 16-18 h before
treatment with 5-HT, as shown in Fig. 2B for the
5-HT7(a) receptor. MEK plays a central role in the
classical signaling pathway from cell surface receptors to ERK1/2.
Pretreatment with the selective MEK inhibitor PD98059, as described
under "Experimental Procedures," blocked 5-HT-induced activation of
ERK1/2 in cells transfected with 5-HT4(b) receptors (data
not shown) or 5-HT7(a) (Fig. 2C). Taken
together, the above data show that 5-HT-induced activation of
ERK1/2 by human 5-HT4(b) and 5-HT7(a)
receptors is mediated through a pathway dependent on MEK and at least
partially dependent on PKA.

View larger version (34K):
[in this window]
[in a new window]
|
Fig. 2.
Activation of ERK1/2 is partly blocked by a
PKA inhibitor and dependent on MEK. A and B,
HEK293 cells transiently transfected with 5-HT4(b)
(A) or 5-HT7(a) (B) were treated with
5-HT or vehicle in the absence or presence of the PKA inhibitor H89 (20 µM), as indicated. The cells were harvested, and Western
blotting was performed as described under "Experimental
Procedures." C, HEK293 cells transiently transfected with
5-HT7(a) were pretreated with the MEK inhibitor PD98059 (5 µM) or vehicle before treatment with 5-HT or vehicle.
Representative Western blots shown in A-C were probed as
described in Fig. 1.
|
|
Activation of ERK1/2 through 5-HT4(b) and
5-HT7(a) Receptors Does Not Require EGFR
Phosphorylation--
Several studies have shown that
Gs-coupled receptors may transactivate RTKs (29-31).
Transiently transfected HEK293 cells were preincubated with the EGFR
kinase inhibitor PD153035 to determine possible involvement of EGFR
transactivation. The presence of PD153035 did not influence the
5-HT-induced activation of ERK1/2 through 5-HT4(b) (data
not shown) or 5-HT7(a) receptors (Fig. 3A). As a positive control,
the presence of PD153035 was shown to completely eliminate activation
of ERK1/2 by EGF in nontransfected HEK293 cells (Fig. 3B).
The above data suggest that activation of ERK1/2 through
5-HT4(b) and 5-HT7(a) receptors does not
require transactivation of EGFRs.

View larger version (38K):
[in this window]
[in a new window]
|
Fig. 3.
Activation of ERK1/2 through
5-HT7(a) receptors does not require EGFR
phosphorylation. A, HEK293 cells transiently
transfected with 5-HT7(a) receptors were treated with 5-HT
(10 µM) or vehicle in the absence or presence of PD153035
(1 µM) as indicated. B, nontransfected HEK293
cells were treated with EGF (5 nM) or vehicle in the
absence or presence of PD153035. Representative Western blots probed as
described in Fig. 1 are shown in A and B.
|
|
5-HT-induced Activation of ERK1/2 Is Independent of
Rap1--
Activation of ERK1/2 by endogenous
2-ARs in
HEK293 cells has been reported to be PKA-dependent and
mediated through a pathway independent of Ras. Although both Ras and
Rap1 were activated by isoproterenol treatment, only Rap1 was capable
of coupling to a Raf isoform and activating ERK1/2 (20). The Rap1
interfering mutant, Rap1N17, and the Rap1-specific GTPase-activating
protein, Rap1GAP1, were used to investigate the role of Rap1 in the
activation of ERK1/2 through 5-HT4(b) and
5-HT7(a) receptors in co-transfection experiments. The
presence of Rap1N17 (data not shown) or Rap1GAP1 (Fig.
4A) did not influence the
5-HT-induced activation of ERK1/2, although activation of Rap1 was
abolished, as evidenced by pull-down of activated Rap1 (Rap1-GTP) with
the RBD of RalGDS (Fig. 4B).

View larger version (45K):
[in this window]
[in a new window]
|
Fig. 4.
Activation of ERK1/2 through
5-HT4(b) and 5-HT7(a) receptors is independent
of Rap1. HEK293 cells were transiently co-transfected with
5-HT4(b) (A, top panels) or
5-HT7(a) (A, bottom panels, and
B) and Rap1GAP1 or empty vector (pcDNA3.1) and treated
with 10 µM 5-HT or vehicle for 5 min. A,
representative Western blots, probed as described in Fig. 1, are shown.
B, the Ras-binding domain of RalGDS was used to pull down
activated Rap1 (Rap1-GTP) from cell lysates, as described under
"Experimental Procedures." Representative Western blots probed with
anti-Rap1 are shown to illustrate activated (Rap1-GTP,
top panel) and total (Rap1, bottom
panel) Rap1. Note that the presence of Rap1GAP1 completely
eliminated activation of Rap1.
|
|
5-HT Induces Ras Activation through 5-HT4(b) and
5-HT7(a) Receptors--
GPCRs may activate the small
GTPase Ras, which plays a central role in the activation of ERK1/2
through several different types of receptors. Therefore, the RBD of
Raf1, which only binds activated (GTP-bound) Ras, was used in pull-down
experiments to test whether 5-HT could induce activation of Ras. Such
experiments documented activation of Ras by 5-HT through both
5-HT4(b) receptors (data not shown) and
5-HT7(a) receptors (Fig.
5A). Nontransfected HEK293
cells were treated with EGF as a positive control for Ras activation
(Fig. 5A).

View larger version (34K):
[in this window]
[in a new window]
|
Fig. 5.
Activation of ERK1/2 through
5-HT4(b) and 5-HT7(a) receptors is dependent on
Ras. A, Ras pull-down experiments were performed after
stimulating 5-HT7(a) receptor-transfected HEK293 cells with
vehicle or 5-HT (10 µM) or nontransfected HEK293 cells
with vehicle or EGF (5 nM). Pull-down experiments were
carried out in single-dish experiments and repeated at least three
times. A representative Western blot probed with anti-Ras antibodies is
shown. B, HEK293 cells co-transfected with
5-HT4(b) (top panels) or 5-HT7(a)
(bottom panels) and empty vector, RasN17, or RasV12 were
stimulated with vehicle or 5-HT (10 µM) for 5 min. For
each receptor, the panels in B show
representative Western blots probed as described in Fig. 1.
|
|
Activation of ERK1/2 through 5-HT4(b) and
5-HT7(a) Receptors Is
Ras-dependent--
Dominant negative and constitutively
active mutants of Ras were used to further investigate the role of Ras
in the 5-HT-induced activation of ERK1/2. Activation of ERK1/2 through
5-HT4(b) and 5-HT7(a) receptors was highly
reduced in the presence of the dominant negative variant of Ras, RasN17
(Fig. 5B). The data in Figs. 4 and 5 suggest that the
5-HT-induced activation of ERK1/2 is dependent on Ras, but independent
of Rap1. When cells co-transfected with 5-HT4(b) receptors
and the constitutively active variant of Ras, RasV12, were treated with
5-HT, an additional activation of ERK1/2 was observed compared with the
constitutive activation of ERK1/2 in these cells.
Raf Interfering Mutants Inhibit 5-HT-induced Activation of
ERK1/2--
HEK293 cells express several effectors of
activated Ras, e.g. different isoforms of Raf. HEK293 cells
were co-transfected with a point-mutated variant of Raf1, RafS621A,
which interferes with Raf1-mediated signaling, or with the
constitutively active variant RafCAAX to investigate the role of Raf1
in the activation of ERK1/2 through 5-HT4(b) and
5-HT7(a) receptors. The cells were incubated for 16-18 h
in serum-free Dulbecco's modified Eagle's medium before stimulation
with 5-HT, as described under "Experimental Procedures." Expression
of RafS621A suppressed the activation of ERK1/2 mediated through
5-HT4(b) (Fig. 6A,
top panels) and 5-HT7(a) (Fig. 6A,
bottom panels) receptors. When cells co-transfected with
RafCAAX and 5-HT4(b) (data not shown) or
5-HT7(a) (Fig. 6B) receptors were treated with
5-HT, increased phosphorylation of ERK1/2 was observed compared with
the constitutive ERK1/2 phosphorylation in these cells.

View larger version (36K):
[in this window]
[in a new window]
|
Fig. 6.
Raf-1 interfering mutants reduce the
5-HT-stimulated activation of ERK1/2. A, HEK293 cells
co-transfected with 5-HT4(b) (top panels) or
5-HT7(a) (bottom panels) receptor and the
interfering Raf1 mutant RafS621A or empty pcDNA3.1 vector as
control were stimulated and harvested for phospho-ERK1/2 assay as
described under "Experimental Procedures." B, HEK293
cells were transiently co-transfected with the 5-HT7(a)
receptor and constitutively active Raf1 (RafCAAX) or empty vector as
control and stimulated with vehicle or 5-HT (10 µM) for 5 min. The Western blots shown in A and B were
probed as described in Fig. 1. C, overexpression of RafS621A
in HEK293 cells co-transfected with 5-HT7(a) receptors and
RafS621A was confirmed by incubating Western blots with anti-Raf-1. The
interfering mutant RafS621A is clearly overexpressed compared with
endogenous levels of Raf-1 in cells transfected without RasS621A.
|
|
To determine the role of B-Raf, HEK293 cells were co-transfected with
5-HT7(a) receptors and dominant negative (N-B-Raf) or constitutively active (CAAX-B-Raf) B-Raf. The dominant negative mutant
of B-Raf corresponds to the N-terminal region of quail B-Raf (amino
acids 1-443) (42). In several experiments, the presence of N-B-Raf had
only a weak inhibitory effect on 5-HT-induced activation of ERK1/2
(Fig. 7A). In general, the
inhibitory effect of N-B-Raf was weaker than that of RafS621A.
Additional activation of ERK1/2 was induced by 5-HT in the presence of
CAAX-B-Raf, as was the case with RafCAAX (Fig. 7A).
Overexpression of RafS621A and N-B-Raf was verified by Western blot
analysis using anti-Raf1 (Fig. 6C) and anti-B-Raf (Fig.
7B) antibodies, respectively.

View larger version (25K):
[in this window]
[in a new window]
|
Fig. 7.
Dominant negative B-Raf has little effect on
5-HT-stimulated activation of ERK1/2. HEK293 cells co-transfected
with 5-HT7(a) receptor and the dominant negative B-Raf
construct N-B-Raf or the constitutively active B-Raf construct
CAAX-B-Raf or empty vector as control were stimulated and harvested for
phospho-ERK1/2 assay as described under "Experimental Procedures."
A, the presence of N-B-Raf barely reduced the 5-HT-mediated
activation of ERK1/2. Treatment of HEK293 cells co-transfected with
5-HT7(a) receptor and CAAX-B-Raf with 5-HT caused
additional phosphorylation of ERK1/2 compared with the constitutive
level of activation of ERK1/2 in these cells. B,
overexpression of N-B-Raf (N-terminal amino acids 1-443) was confirmed
by incubating Western blots with anti-B-Raf.
|
|
 |
DISCUSSION |
In this paper, we report the activation of ERK1/2 by 5-HT through
the human Gs-coupled serotonin receptors
5-HT4(b) and 5-HT7(a) transiently expressed in
COS-7 and HEK293 cells. We also demonstrate that the pathway in HEK293
cells involves Ras but not Rap1. Pretreatment of transiently
transfected HEK293 cells with H89 suppressed 5-HT-induced dual
phosphorylation of ERK1/2, indicating a role for PKA.
Intracellular signals from the large family of G protein-coupled
receptors are conveyed by
subunits (G
) or
and
subunits (G
) of heterotrimeric G proteins. Of the four main classes of heterotrimeric G proteins (Gs, Gi,
Gq, and G12/13), Gq- and
Gi-coupled receptors have been shown to mediate activation
of ERK1/2 through free G
subunits (44, 45). G
subunits
released upon stimulation of Gs-coupled receptors have not
been shown to mediate activation of ERK1/2, possibly because cells
contain far less Gs than Gi or Gq.
However, G
s activates adenylyl cyclase and thereby PKA, which has been proposed to play an essential role in the ERK1/2 cascade
(12, 20, 34). In line with this, we found that ERK1/2 activation
through the Gs-coupled receptors 5-HT4(b) and
5-HT7(a) was inhibited in cells pretreated with the PKA
inhibitor H89.
In addition to activation of PKA, cAMP may lead to activation of Rap1
through the Rap1-selective exchange factors Epac1 or Epac2 (13, 14,
46). The small GTPase Rap1 is a Ras analogue, which upon isoproterenol
stimulation of HEK293 cells has been reported to recruit B-Raf to the
membrane and thereby activate the ERK1/2 cascade (20). We determined
the role of Rap1 in the observed 5-HT-induced activation of ERK1/2 by
co-transfecting HEK293 cells with either Rap1GAP1 or Rap1N17 and 5-HT
receptors. Even though Rap1N17 is not a true dominant negative (47,
48), it has been used to inhibit Rap1 activity in several studies (12, 20, 49). Therefore, we used Rap1N17 to support the data obtained with
Rap1GAP1. The observed 5-HT-induced activation of ERK1/2 in HEK293
cells was not influenced by co-expression of either Rap1GAP1 or
Rap1N17, although Rap1 activation was eliminated. Thus, the
Gs-coupled 5-HT4(b) and 5-HT7(a)
receptors mediate activation of ERK1/2 through a Rap1-independent
pathway, in contrast to what was reported for
2-ARs
(20).
The small GTPase Ras is the best characterized and serves
as the main activator of the ERK1/2 cascade by linking RTKs and all isoforms of Raf. RTK-mediated activation of Ras involves a series
of SH2- and SH3-dependent protein-protein interactions between tyrosine-phosphorylated receptor, the adapter proteins Shc and
Grb2, and the Ras-guanine nucleotide exchange factor SOS (50, 51).
Certain GPCRs coupling to Gi and Gq mediate
activation of Ras through free G
subunits, e.g. by
stimulation of Shc (44, 45, 52). Isoproterenol stimulation of
endogenous
2-ARs in HEK293 cells was reported to
activate both Rap1 and Ras, but only Rap1 was implicated in the
activation of ERK1/2 (20). The mechanism of Ras activation was not
addressed. Stimulation of HEK293 cells transiently transfected with
5-HT4(b) or 5-HT7(a) receptors also increased
the amount of GTP-bound Ras, and, in addition, 5-HT-induced activation
of ERK1/2 was inhibited by expression of the dominant negative mutant
of Ras, RasN17. Thus, activation of ERK1/2 through Gs-coupled 5-HT4(b) and 5-HT7(a)
receptors in HEK293 cells is Ras-dependent.
Since the original manuscript was submitted for publication, two papers
have been published that support the finding of Rap1-independent activation of ERK1/2 through Gs-coupled receptors (53, 54). Furthermore, in accordance with our finding in HEK293 cells of a
Ras-dependent mechanism for activation of ERK1/2, Enserink
et al. (54) report a Ras-dependent activation of
ERK1/2 in Chinese hamster ovary cells subsequent to elevation of
intracellular cAMP.
Transactivation of RTKs has been proposed as an alternative mechanism
for activation of Ras through GPCRs (55). The signal will pass the
membrane three times, and activation of Ras and the ERK1/2 cascade will
subsequently take place as by direct stimulation of RTKs,
e.g. EGFR. Activation of ERK1/2 through 5-HT4(b)
and 5-HT7(a) receptors was not inhibited in the presence of
the EGFR kinase inhibitor PD153035, indicating that EGFR
transactivation is not required.
The effects of cAMP on the ERK1/2 cascade are cell type-specific. In
some cell types, PKA was found to inhibit RTK-mediated activation of
ERK1/2 (15-18). Raf is the main target for cAMP-mediated regulation of
the ERK1/2 cascade. The serine residues 43, 259, and 621 have all been
suggested to be involved in the regulation of Raf1 activity (56).
Phosphorylation of serine 43 does not inhibit the catalytic activity of
Raf1, but it has been proposed to disrupt the normal Ras-Raf
association and Raf1 activation (15). Furthermore, even though B-Raf
does not have a phosphorylation site analogous to serine 43 in Raf1,
elevation of intracellular cAMP levels by forskolin inhibited the
EGF-dependent activation of B-Raf (57). The serine 621 residue of Raf1 has been suggested as PKA phosphorylation site. Raf1
phosphorylated at this site exhibited reduced affinity for activated
Ras as well as impaired catalytic activity (58). Furthermore, mutation
of serine 621 to alanine resulted in loss of catalytic activity. Thus,
a serine 621 to alanine mutant, RafS621A, has been used as a
"dominant negative" variant of Raf1. However, recent reports have
shown that the list of residues involved in the
PKA-dependent regulation of Raf1 activity may also include
serine residues 233 and 338 (59, 60). Based on the literature
concerning cAMP-mediated regulation of Raf1, RafS621A cannot be
considered a true dominant negative. Still, we found that the
Ras-dependent, 5-HT-induced activation of ERK1/2 was partly
inhibited by co-expression of the Raf1 interfering mutant RafS621A,
implicating Raf1 in the pathway. In melanocytes, dominant negative
B-Raf has been used to show that Ras and B-Raf were involved in
cAMP-mediated activation of ERK1/2 (42). We found only little or no
effect of co-expression of dominant negative B-Raf under conditions
where RafS621A caused more pronounced reduction of ERK1/2 activation by
5-HT. Whether RafS621A is a true dominant negative or only a partial
inhibitor of Raf1 activation, these data suggest that one or more
isoforms of Raf are involved in the activation of ERK1/2 through
5-HT4(b) and 5-HT7(a) receptors in HEK293 cells.
The immediate upstream activator of ERK1/2 is MEK (61), which is
involved in most pathways described for activation of ERK1/2. The
presence of the MEK-specific inhibitor PD98059 inhibited 5-HT-induced activation of ERK1/2 through 5-HT4(b) and
5-HT7(a) receptors. These results suggest that the
5-HT-induced activation of ERK1/2 is MEK-dependent.
The high number of publications concerning activation of MAP kinases
via Gs-coupled receptors illustrates the complexity of this
field. Several publications have shown that activation of Ras may occur
after stimulation of Gs-coupled receptors. However, little
is known about how these receptors or agents that elevate intracellular
levels of cAMP mediate activation of Ras. Src or a Src-like kinase has
been suggested to be involved in the activation of ERK1/2 subsequent to
stimulation of the Gs-coupled A2A-adenosine receptor (53). However, this may not account for the 5-HT-induced Ras-dependent activation of ERK1/2 in HEK293 cells because
the Src-dependent mechanism was observed in Chinese hamster
ovary, PC12, and NIH3T3 cells, but not in HEK293 cells. To our
knowledge, a Ras-specific exchange factor activated by cAMP, analogous
to Epac for Rap1, has not been published. This does not mean that a
Ras-specific exchange factor directly activated by cAMP or PKA phosphorylation does not exist. Alternatively, there may be more indirect actions of PKA via known or unknown proteins that mediate activation of Ras.
The biological significance of the observed activation of ERK1/2 by
5-HT4 and 5-HT7 receptors is unknown. The
biological effects of 5-HT are mediated by at least 14 different
receptors, and there is hardly a target cell for 5-HT that expresses
only one type of 5-HT receptor. Based on current knowledge about signal
transduction mechanisms and ERK activation, other 5-HT receptors than
5-HT4 and 5-HT7 would be expected to play a
more important role in ERK activation. However, examples of biological
systems exist where ERK activation by Gs-coupled serotonin
receptors may be important. In Aplysia, activation of
mitogen-activated protein kinases by serotonin is implicated in memory
formation and occurs through a Gs-coupled 5-HT receptor
(32, 33). In mammalian brain, 5-HT4 and 5-HT7
receptors are both found in the hippocampus (36, 37), and
5-HT7 receptors were recently shown to activate ERKs in
cultured hippocampal neurons (35). The importance of ERK activation for the involvement of these receptors in learning and memory (38) remains
to be determined.
In conclusion, we demonstrate activation of ERK1/2 by 5-HT through the
human Gs-coupled 5-HT4(b) and
5-HT7(a) receptors in COS-7 and HEK293 cells. In HEK293
cells, the observed ERK1/2 activation was Ras-dependent but
Rap1-independent and involved one or more Raf isoforms. Therefore, we
suggest the following pathway for activation of ERK1/2 by 5-HT through
5-HT4(b) and 5-HT7(a) receptors: Gs-coupled 5-HT receptor
cAMP
PKA
Ras
Raf
MEK
ERK1/2. The unknown pathway from cAMP/PKA to Ras
activation remains a challenge for future research.