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INTRODUCTION |
Signal transduction elicited by hormones, growth factors, and
several other extracellular stimuli involves a sequential activation of
cytosolic protein kinases, which are collectively known as the
mitogen-activated protein kinase
(MAPK)1 cascades (reviewed in
Refs. 1-4). In growth factor signaling, the key elucidated MAPK
cascade is the one using the extracellular signal-regulated kinase
(ERK, also known as p42/44 MAPK), which is initiated by the small
GTP-binding protein, Ras. Upon stimulation, Ras assumes its active,
GTP-bound form, and recruits the protein kinase Raf-1, which transmits
the signal further via MEK, ERK, and p90RSK to various cytoplasmic and
nuclear target molecules. Other MAPK cascades (reviewed in Refs. 5-7)
are: (i) Jun N-terminal kinase (JNK) cascade, which utilizes MEKK1,
MKK4/7, and JNKs to activate transcription factors such as c-Jun, ATF2,
and Elk1; (ii) p38 MAPK cascade, which uses MEKKs, MKK3/6, and P38
-
to activate Elk, ATF2, CHOP, and MEF; and (iv) big MAPK (BMK,
ERK5) cascade, which uses MEKK3, MEK5, and BMK to transmit EGF and
oxidative stress signals.
The family of receptors coupled to G-proteins (GPCR) is the largest
group of integral membranal receptors involved in signal transduction
(reviewed in Refs. 8-12). Members of this group of receptors transmit
their signals primarily via GTP-binding proteins (G-proteins), which
are heterotrimeric proteins composed of
,
, and
subunits.
The G-proteins are classified according to the subtype of their
subunit into four groups: Gs, Gi,
Gq, and G12. Upon activation, the
heterotrimer dissociates into a GTP-bound, activated,
subunit
(G
), and a dimer of G
. Whereas a small number of common
mechanisms is responsible for receptor Tyr kinase-mediated activation of ERK (13), GPCRs are thought to activate ERK by divers
signaling pathways. All four G
subunits (12), the 
subunits
(14), receptor-interacting proteins such as Src (15),
-arrestin
(16), dynamin (17), and transactivation of receptor Tyr kinases by
GPCRs (18) are capable of initiating several downstream signaling
pathways culminating in MAPK activation. The various MAPK cascades
appear to be important components in the downstream signaling events
initiated by the GPCRs, although different receptors often use
different mechanisms for this purpose (12).
Gonadotropin releasing hormone (GnRH) is a hypothalamic decapeptide,
which serves as a key regulator of the reproductive system. In the
pituitary, the signals of GnRH are transmitted via a specific GPCR
(GnRH receptor; GnRHR), which upon activation interacts with the
heterotrimeric Gq protein (reviewed in Refs. 19, 20). This
interaction then initiates a variety of intracellular signaling events
that culminate in the production and secretion of the leutinizing hormone and follicle-stimulating hormone. In recent studies it was
shown that the JNK, ERK, and p38 MAPK cascades are activated in
response to GnRH stimulation of
T3-1 cells (21-28). The mechanism of JNK activation by GnRH has been shown to involve sequential activation of protein kinase C (PKC), Src, CDC42, and MEKK1 (28); and
the mechanism of p38 MAPK activation by GnRH seems also to be
PKC-dependent (27). The mechanism of ERK activation was
studied by several laboratories over the past few years and was shown to involve PKC, and an unknown protein-tyrosine kinase (PTK) that is
only partially sensitive to the PTK inhibitor genistein (reviewed in
Ref. 12).
Here we show that the activation of ERK by GnRH in the
T3-1 cells
involves two distinct signaling pathways, which converge at the level
of Raf-1. One of these pathways involves a direct activation of Raf-1
by PKC, whereas the other pathway involves activation of Raf-1 by Src
and Ras. Transactivation of EGF receptor, or activation through either
-arrestin or G
, which play a role in the signaling of many
other GPCRs to the ERK cascade, are not involved in the GnRHR to ERK
pathway. On the other hand, dynamin seems to be important for this
pathway, because it is essential for the activation of Ras, in a
PKC-independent manner. Thus, although the GnRHR to ERK signaling is
mainly mediated by Gq-PKC, another pathway involving
dynamin is required for the Src-mediated activation of Ras, which
supports the step of Raf-1 activation by PKC.
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MATERIALS AND METHODS |
Buffers--
Homogenization buffer (buffer H) consisted of 50 mM
-glycerophosphate (pH 7.3), 1.5 mM EGTA,
1 mM EDTA, 1 mM DTT, 0.1 mM sodium
orthovanadate, 1 mM benzamidine, aprotinin (10 µg/ml), leupeptin (10 µg/ml), and pepstatin (2 µg/ml). Buffer A consists of
50 mM
-glycerophosphate (pH 7.3), 1.5 mM
EGTA, 1 mM EDTA, 1 mM DTT, 0.1 mM
sodium orthovanadate. Radioimmune precipitation buffer consisted of 137 mM NaCl, 20 mM Tris, pH 7.4, 10% (v/v) glycerol, 1% Triton X-100 0.5% (w/v) deoxycholate, 0.1% (w/v) SDS,
2.0 mM EDTA, 1.0 mM phenylmethylsulfonyl
fluoride, and 20 µM leupeptin. Ral buffer consisted of 40 mM Tris-HCl, pH 7.5, 200 mM NaCl, 2 mM MgCl2, 1% Nonidet P-40, 1 µg/ml
aprotinin, 1 µg/ml leupeptin, 250 mM phenylmethylsulfonyl
fluoride, 10 mM NaF, and 1 mM sodium orthovanadate.
Stimulants, Inhibitors, Antibodies, and Miscellaneous
Reagents--
[D-Trp6]GnRH, a stable GnRH
analog (GnRH-a), genistein (PTK inhibitor), enolase, protein
A-Sepharose, and protein G-Sepharose were obtained from Sigma Chemical
Co. (St. Louis, MO). GF109203X, PD098059, SB203580, Wortmannin, and
12-O-tetradecanoylphorbol-13-acetate where purchased from
Calbiochem. AG18 and AG1478 and PP1 were also obtained from Calbiochem.
Polyclonal anti-hemagglutinin epitope antibody was from Santa Cruz
Biotechnology. Monoclonal anti-GFP was from Roche Molecular
Biochemicals. Mouse monoclonal anti-active MAPK antibody (DP-ERK
antibody) was from Sigma, Israel (Rehovot, Israel). Antiphosphotyrosine
antibody was from Santa Cruz Biotechnology. Antibody to
-epitope of
PKC was a gift from Dr. Chaya Brodie (Bar-Ilan University, Israel).
Plasmids--
N-terminally truncated FAK (DN-FAK; FRNK) was
cloned in pCDNA1 using BamHI/XhoI sites. HA-ERK2,
N-17 Ras, and L-61 Ras were prepared as previously described (29).
Mammalian expression vectors containing wild-type dynamin, dominant
negative dynamin (K44A-dynamin),
-arrestin2 in pCMV5, and dominant
negative
-arrestin (V54D-
-arrestin2) in pCDNA3 were a gift
from Dr. M. Caron (Duke University, Durham, NC). GFP-Ras was a gift
from Dr. Y. Klug (Tel Aviv University, Israel), and MEK1 was cloned in
pEGFP-N1 in-frame with GFP using an ApaI and
BamHI restriction site. CD8-tagged
-ARK was from Dr. Zvi
Vogel (Weizmann Institute of Science, Israel). PKC
with a
-epitope tag was a gift from Dr. Chaya Brodie (Bar-Ilan University,
Israel). DN-EGF receptor (K721A) in pCDNA3 was provided by Dr Y. Yarden (Weizmann Institute of Science, Israel).
Transfection, Stimulation, and Harvesting of
T3-1
Cells--
Subconfluent
T3-1 cells were transfected with 5 µg of
the examined plasmid. In case of cotransfection, 5 µg of each plasmid were used. The transfection was carried out using the calcium phosphate
technique. The total amount of plasmid was adjusted to 10 µg with
vector DNA in the control experiments. The transfection efficiency was
10-30%, as determined by transfection with a plasmid that contained
-Gal and appropriate staining. Two days after transfection, the
cells were serum-starved for 16 h and incubated for the desired
time intervals with GnRH-a in the presence or absence of various
inhibitors. After stimulation, cells were washed twice with ice-cold
PBS, washed once with buffer A, and subsequently harvested in ice-cold
buffer H. Cell lysates were centrifuged (20,000 × g,
20 min), and the supernatant was assayed for protein content.
Western Blot Analysis--
Cell supernatants, which contained
the cytosolic proteins, were collected, and aliquots from each sample
(20 µg) were separated on 10% SDS-PAGE followed by Western blotting
with the appropriate antibodies. Alternatively, immunoprecipitated
antibodies were boiled in sample buffer and subjected to SDS-PAGE and
Western blotting. The blots were developed with alkaline phosphatase or horseradish peroxidase-conjugated anti-mouse or anti-rabbit Fab antibodies (Jackson Laboratories).
Immunoprecipitation with Anti-HA Antibodies--
For
immunoprecipitation, protein A-Sepharose was mixed with HA antibodies
at 37 °C for 1 h, after which the beads were washed twice with
PBS and twice in buffer H. Cell lysates (300 µg) were added to the
beads and swirled end to end at 4 °C for 2 h. The immunocomplexes were washed once with radioimmune precipitation buffer,
washed twice with 0.5 M LiCl in 100 mM Tris, pH
8.0, and finally washed with buffer A.
Ras Activation Assay--
Cells were stimulated and washed as
described above. Ras activation was assayed as described previously
(30). In brief, cells were lysed in Ral buffer, and lysates (300 µg
of protein) were incubated with 20 µg of GST-Raf (RBD) and were
washed three times in a buffer containing 20 mM Hepes, pH
7.5, 150 mM NaCl, 0.1% Triton X-100, and 10% glycerol.
The amount of Ras pulled-down was then assessed by Western blotting
using anti-Ras antibody. To study Ras activity in transfected cells, a
construct of GFP-Ras was used. After GnRH stimulation, the cells were
lysed in Ral buffer and 300 µg of protein was subjected for further
treatment. The active, GTP-bound form of Ras was precipitated by the
GST-Raf (RBD, 20 µg) and washed as above, and the activated GFP-Ras
was then detected with anti-GFP antibody.
Src Activity--
Cell lysates (400-500 µg of protein in
homogenization buffer containing 1% Triton X-100) were incubated with
anti-c-Src-antibodies precoupled to protein A-Sepharose and swirled end
to end at 4 °C. The immunocomplexes were washed once with
radioimmune precipitation buffer, twice with 0.5 M LiCl in
0.1 M Tris-HCl, pH 8.0, and once with buffer A. The washed
immunoprecipitate were resuspended in a kinase assay buffer (28), and
the c-Src activity was determined using acid-denatured enolase (3 µM) as substrate in the presence of 20 µM
[
-32P]ATP (8000 cpm/pmol). The enzymatic reactions
were terminated by the addition of sample buffer. The samples were then
subjected to SDS-PAGE and autoradiography.
FAK Activity--
Cell lysates (400-500 µg of protein in
buffer H + 1% triton X-100) were incubated with anti-FAK antibody
coupled to protein A-Sepharose and swirled end to end at 4 °C for
2 h. The immunoprecipitates were washed as above and mixed with
sample buffer. Active FAK was determined by Western blot analysis using
anti-phosphotyrosine antibody.
Raf-1 Activity--
The activity of Raf-1 was determined by
immunoprecipitation using an anti-C-terminal Raf-1 antibody and a
subsequent in vitro kinase assay with recombinant MEK. This
was performed in the same reaction mixture described for MEK except
that 2 µg of recombinant MEK1 were used instead of ERK in each
reaction. The reactions were terminated by the addition of sample
buffer, and the samples were subjected to SDS-PAGE analysis. The gels
were blotted onto nitrocellulose membrane, and the phosphorylation of
MEK was assessed by x-ray autoradiography.
MEK Activity--
The activity of transfected GFP-tagged MEK was
assessed by immunoprecipitation with anti-GFP antibody followed by a
subsequent in vitro kinase reaction. In brief,
T3-1 cells
transfected with GFP-MEK were stimulated with GnRH-a and harvested in
buffer H. The cell lysates were incubated with 5 µg/assay of GFP
antibody precoupled to protein G-Agarose and were washed as described
for immunoprecipitation. Immunocomplex kinase reaction was carried out
in a reaction mixture containing 1 µg of recombinant ERK, 10 µM MgCl2, 1.5 µM DTT, 75 mM
-glycerophosphate, pH 7.3, 0.075 µM
sodium vanadate, 3 µM PKI peptide, 1.25 mM
EGTA, 10 µM calmidazolium, and 20 µM
[
-32P]ATP (300 cpm/pmol) for 20 min at 30 °C. The
reactions were terminated by the addition of sample buffer, and the
samples were subjected to SDS-PAGE analysis. The gels were blotted onto
nitrocellulose membrane, and the phosphorylation of ERK was assessed by
x-ray autoradiography.
PKC Activity--
The activity of transfected
-epitope-tagged
PKC
was assessed using cellular fractionation. In brief, transfected
T3-1 cells were stimulated with GnRH-a, homogenized in buffer H, and
centrifuged at 15,000 × g. Pellets containing plasma
membranes were washed twice in buffer H and suspended in buffer H
containing Triton X-100. Translocated PKC in the membranes was
determined by Western blot analysis using antibody to the
PKC
-epitope.
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RESULTS |
Role of PTKs in ERK Activation by GnRH--
Various MAPK cascades
(ERK, JNK, and BMK) are activated in response to GnRH stimulation of
T3-1 cells. We have previously shown that the stimulation of JNK
activity by GnRH is mediated by a unique pathway, which includes
sequential activation of PKC, Src, CDC42, and probably also MEKK1 (28).
PKC was implicated also in the activation of ERK by GnRH (25), but the
other components involved in this pathway remained unclear. In this
study we used anti-doubly phosphorylated ERK (DP-ERK) antibody to
detect its phosphorylation and activation upon GnRH-a treatment of
T3-1 cells. ERK phosphorylation was detected 5 min after GnRH-a
treatment (Fig. 1A), peaked at
15 min, and was slightly reduced 15 min later. No change was detected
in the total amount of ERK as judged by the equal staining with
anti-general ERK antibody (Fig. 1A). These results appear
similar to the trend of ERK activation by GnRH (Ref. 25 and data not
shown), indicating that the anti-DP-ERK antibody can serve as a tool to
study ERK activation in
T3-1 cells.

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Fig. 1.
The effect of PKC and PTK inhibitors on ERK
activation by GnRH. A, sensitivity of ERK activation by
GnRH to various inhibitors: Subconfluent T3-1 cells were pretreated
(15 min) with 200 µM genistein (Gen), 3 µM GF109203X (GF), 25 µM PD98059
(PD), 25 nM wortmannin (Wor), or 20 µM SB203580 (SB) before stimulation or left
untreated as control. GnRH-a (10 7 M) was
added for 0, 5, 15, and 30 min and the activated form of ERK was
determined by Western blot analysis with anti-diphospho ERK antibody
(DP). The total amount of ERK was detected with the 7884 antibody (Ref. 60; total). These results are an average of four
separate experiments. B, sensitivity of ERK activation by
GnRH to inhibitors of PTKs: Subconfluent T3-1 cells were pretreated
with 100 µM AG18, 5 µM AG1478, or 5 µM PP1 for 15 min before stimulation or left untreated as
control. Addition of GnRH-a and analysis of active ERK was carried out
as above. These results are an average of three separate
experiments.
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To study the mechanism of ERK activation we first used inhibitors of
various intracellular signaling cascades. As previously reported (25),
the PTK inhibitor genistein partially (40%) inhibited, and the PKC
inhibitor GF109203X abolished the GnRH-induced ERK activation. The MEK
inhibitor PD98059 inhibited not only GnRH-stimulated ERK-activity but
abolished also its basal activity, whereas the PI3K inhibitor
wortmannin and the p38 MAPK inhibitor SB203580 had no significant
effect on ERK activation. The results suggest that GnRH signaling
toward ERK is mainly transmitted via PKC and to a lesser extent also by
PTKs. Interestingly, the moderate effect of genistein on ERK activation
was achieved under conditions where it completely abolished the GnRH
activation of JNK (28). One possibility for this differential effect is
that the PTK involved in the GnRH to ERK pathway is distinct from Src,
which is the PTK operating in the GnRH-JNK pathway (28), and this
distinct PTK is only mildly sensitive to genistein. To test this
possibility, we examined the effects of additional PTK inhibitors on
ERK activation by GnRH-a. We found (Fig. 1B) that the
specific inhibitor of Src, PP1, which abolished endogenous src
activation (data not shown), inhibited the GnRH-induced ERK activation
to a similar extent as genistein. Similarly, the general PTK inhibitor
AG18 had a small effect (~25%) on the GnRH-induced ERK activation.
However, the EGF receptor inhibitor AG1478, which abolished the
EGF-induced ERK activation in
T3-1 cells (data not shown), had no
effect on ERK activation by GnRH-a. The data suggest that Src is
partially involved in the activation of ERK by GnRH but probably not
via transactivation of EGF or other growth factor receptors as
suggested for other GPCRs (31, 32).
Src, but Not FAK or EGF Receptor, Plays a Role in GnRH to ERK
Signaling--
Signaling by Src is often mediated via the focal
adhesion kinase (FAK), which is usually instrumental in integrin
signaling (33). We found that Src and FAK are activated in response to GnRH in
T3-1 cells, although the onset of Src activation appeared before that of FAK (Refs. 25 and 28 and Fig.
2). A useful tool in the study of GnRH
signaling in
T3-1 cells is the co-overexpression of either
constitutively active or dominant negative forms of upstream components
together with a tagged form of the examined kinase (28). Therefore, we
overexpressed hemagglutinin epitope-tagged ERK (HA-ERK2) in
T3-1
cells, stimulated the cells with GnRH, lysed the cells,
immunoprecipitated the HA-containing proteins with anti-HA antibodies,
and blotted with anti-DP-ERK antibodies. ERK activation measured by
this method was essentially identical to that found for the endogenous
ERK, indicating that overexpression can also serve as a useful tool in
the study of ERK activation by GnRH. To study the role of Src/FAK in
the GnRH to ERK signaling we co-overexpressed a dominant negative form
of FAK (FRNK (34)) with the HA-ERK2 in
T3-1 cells. This FAK
construct, which inhibits the activity of the endogenous FAK in
T3-1
cells (data not shown), had no effect on GnRH-induced ERK activation
(Fig. 2B). This lack of effect, together with the late onset
of FAK activation by GnRH-a stimulation, indicates that FAK is not
involved in GnRH to ERK signaling. On the other hand, the C-terminal
Src kinase (CSK), which acts as a dominant interfering mutant of Src
(28, 35) had a partial inhibitory effect on the GnRH to ERK pathway.
This is demonstrated by a ~35% inhibition in the activation of
ERK by GnRH-a caused by overexpression of CSK in
T3-1 cells
(Fig. 2B). This result, together with the ~40% inhibition
cause by PP1, strongly suggests that Src is partially involved in the
activation of ERK by GnRH in
T3-1 cells.

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Fig. 2.
Activation of Src and FAK by GnRH-a and the
effect of Src and FAK on ERK activation by GnRH-a. A,
activation of Src and FAK by GnRH: Subconfluent T3-1 cells were
treated with GnRH-a (10 7 M) for the indicated
times. Stimulation was terminated by washing the cells with an ice-cold
PBS followed by Src and FAK immunoprecipitation. Activity of Src was
determined using acid-denatured enolase. FAK activity was detected by
anti-phosphotyrosine antibody. B, the effect of CSK, which
inhibits Src activity, and of dominant negative FAK on GnRH-a
stimulation of ERK: Subconfluent T3-1 cells were cotransfected with
CSK and HA-ERK2 for Src experiment and with dominant negative-FAK and
HA-ERK2 for the FAK experiment. Two days after transfection, the cells
were serum-starved for 16 h and then either treated with GnRH-a
(10 7 M) or left untreated. Activated HA-ERK2
was determined with anti-diphospho antibody (DP). The amount
of immunoprecipitated HA-ERK2 was determined by Western blot analysis
with anti-HA antibody. Activation (-fold) (GnRH-stimulated/Basal for
each of the constructs) is shown in the bar graph in the
bottom. The results in the bar graphs are the
average of three experiments.
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It was recently reported that EGF receptor might be involved in GnRH
signaling (36). However, our initial results indicated that there is no
inhibition of GnRH-induced ERK activation by the inhibitor of EGF
receptor AG1478 (Fig. 1B). To further confirm the lack of
participation of EGF receptor in GnRH-ERK signaling, we cotransfected
the dominant negative mutant of EGF receptor (K721A) with HA-ERK2 into
T3-1 cells. The cells were then serum-starved and activated with
either EGF or GnRH-a followed by the determination of ERK activation.
The dominant negative EGF receptor had no influence on ERK activation
by GnRH-a (Fig. 3A) under
conditions where it prevented ERK activation by EGF (Fig.
3B). These results, together with the lack of effect of
AG1478 strongly suggest that EGF receptor is not involved in the
activation of ERK by GnRH.

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Fig. 3.
The effect of EGF receptor on ERK activation
by GnRH-a. Subconfluent T3-1 cell were cotransfected with
dominant negative (DN)-EGF receptor and HA-ERK2. Two days after
transfection, the cells were serum-starved for 16 h and then
either treated with GnRH-a (10 7 M;
A), EGF (50 ng/ml; B), or left untreated (where
indicated). Activated HA-ERK2 was determined with anti-diphospho
antibody. The amount of immunoprecipitated HA-ERK2 was determined by
Western blot analysis with anti-HA antibody. The results in the
bar graph represent the average of three experiments.
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Activation of Raf-1 and Ras by GnRH--
To understand the nature
of the partial involvement of Src in the GnRH to ERK pathway, we
studied the effect of GnRH on the upstream components of the ERK
cascade, including the protein serine/threonine kinase Raf-1. After
treatment of
T3-1 cells with GnRH-a and inhibitors, the cells were
lysed, Raf-1 was immunoprecipitated, and its activity toward
recombinant MEK was measured as described (37). Raf-1 activity was
stimulated within 5 min after GnRH activation (data not shown); its
activity peaked 10 min after stimulation and declined thereafter (Fig.
4). Similar to the inhibition of ERK
activation by GnRH, we found that GF109203X completely inhibited Raf-1
activation by GnRH-a, whereas PP1 and genistein had a partial
inhibitory effect (~30% inhibition) and AG1478 had no significant
influence on Raf-1 activation by GnRH-a. The small GTP-binding protein
Ras, was also transiently activated by GnRH-a. Using a pulldown
and a GTP loading assays, we found that activation of Ras was detected
within 2 min from activation, peaked at 5-10 min, and declined
thereafter (Fig. 5 and data not shown).
However, the mechanism involved in this activation seems to be distinct from that of Raf-1 and ERK as judged from the differential sensitivity to the various inhibitors used. Thus, the Src inhibitors genistein and
PP1 abolished the activation of Ras by GnRH-a, GF109203X had only a
partial effect, and the EGF receptor inhibitor AG1478 had no effect
upon Ras activation. These data indicate that, although both Ras and
Raf-1 are activated in response to GnRH in
T3-1 cells, the upstream
mechanism that leads to this activation is different. Indeed, when a
dominant negative form of Ras (N-17 Ras) was transfected into
the
T3-1 cells, it only partially (30%) inhibited ERK activation by
GnRH-a (Fig. 6), whereas a constitutively active form of Ras (L61-Ras) caused a large elevation of ERK (18- to
22-fold above basal level). Therefore, although Ras is capable of
activating the Raf-1/ERK pathway, GnRH activation of Raf-1/ERK is only
partially Ras-dependent. The most plausible explanation for
these data is that the main pathway operates via direct activation of
Raf-1 by PKC (38), and that this activation requires only a minor
contribution of activated Ras as previously suggested for the
activation of Raf-1 by 12-O-tetradecanoylphorbol-13-acetate (39).

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Fig. 4.
Activation of Raf-1 by GnRH-a.
Subconfluent T3-1 cells were pretreated (15 min) with 200 µM genistein, 3 µM GF109203X
(GF), 5 µM PP1, or 5 µM AG1478
before stimulation or left untreated as control. GnRH-a
(10 7 M) was added for the indicated times.
Cell extracts were immunoprecipitated with anti-Raf-1 C-terminal
antibody, and Raf-1 activity was determined by phosphorylation of
recombinant MEK. The bar graph in the bottom
panel represents an average of three separate experiments.
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Fig. 5.
Activation of Ras by GnRH-a.
Subconfluent T3-1 cells were pretreated (15 min) with 200 µM genistein (Gen), 3 µM
GF109203X (GF), 5 µM PP1, or 5 µM AG 1478 or left untreated as control. GnRH-a
(10 7 M) was added for the indicated times.
Activated Ras in the cell lysate was determined by Ras pulldown assay
using GST-RBD as described under "Material and Methods." The
results in the bar graph are average of three separate
experiments.
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Fig. 6.
Effect of Ras on ERK activation by
GnRH-a. T3-1 cells were cotransfected with HA-ERK2 together
with either N-17 Ras or L61-Ras. Two days after transfection, the cells
were serum-starved for 16 h and then either treated with GnRH-a
(10 7 M) for the indicated times or left
untreated. Activated HA-ERK2 was visualized with anti-diphospho
antibody (DP). The amount of immunoprecipitated HA-ERK2 was
determined by Western blot analysis with anti-HA antibody
(HA). The results in the bar graph are average of
three experiments. Activation (-fold) (GnRH-stimulated/Basal for each
construct) is indicated in the bar graph.
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The Involvement of Dynamin, but Not
-Arrestin or G
, in ERK
Activation by GnRH--
Recently, the 
subunits of G-proteins as
well as
-arrestin and dynamin have been implicated in the
G
-independent GPCR-ERK signaling (reviewed in Ref. 12). For example,
it was shown that Gi-coupled receptor stimulation of
mitogen-activated protein kinase is mediated by G
-induced
activation of Ras (40). To study the possible role of the 
subunit in GnRH to ERK signaling, we used a chimera of CD8 (which
allows anchoring to the membrane) fused to the C terminus of
-adrenergic receptor kinase (ARK-C), which contains a G
binding domain (CD8-ARK-C). It has been previously shown that this
chimera acts as a scavenger of the 
dimer (41, 42). Although this
construct was able to inhibit GPCR signaling toward ERK in COS7 cells,
its overexpression had no significant effect on either the basal or the
GnRH-induced activation of ERK in
T3-1 cells (Fig.
7), indicating that the signaling from
GnRHR to ERK utilizes a G
-independent pathway. Another protein
that was implicated in the signal transmission of GPCRs is
-arrestin, which acts as a mediator of receptor internalization
(43). Recently, it was also shown that
-arrestin can act as a
scaffold protein and transmit the signals of Gq-coupled receptors
toward ERK by forming a complex that contains internalized receptor,
Raf-1, and activated ERK (44). We examined the possible involvement of
-arrestin using either an inactive form of this protein
(V54D-
-arrestin2), which inhibits the activity of all endogenous
-arrestins (43), or by overexpressing wild-type
-arrestin2, which
should increase ERK activation by GPCRs (16, 45). Thus, we coexpressed
these two constructs in
T3-1 cells together with HA-ERK2 and
followed HA-ERK activation using anti-phospho ERK antibodies. As seen
in Fig. 7, neither form of
-arrestin had any effect on ERK
activation by GnRH. Both the G
subunits and
-arrestin do not
seem to participate in the process of ERK activation by GnRH, although
they can influence GPCR signaling in other systems.

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Fig. 7.
Effect of 
subunits and -arrestin on ERK activation
by GnRH-a. T3-1 cells were cotransfected with HA-ERK2 together
with either the scavenger of  -subunits (CD8 fused to  ARK;
CD8), -arrestin2 (Arre), or dominant negative
-arrestin2 (DnArre). Two days after transfection, the
cells were serum-starved for 16 h and then either treated with
GnRH-a (10 7 M) for the indicated times or
left untreated. Activated HA-ERK2 was determined with anti-diphospho
ERK antibody (DP) as described under "Materials and
Methods." The amount of immunoprecipitated HA-ERK2 was determined by
Western blot analysis with anti-HA antibody (HA). The
results in the bottom bar graph are an average of three
experiments. Activation (-fold) (GnRH-stimulated/basal for each
construct) is indicated in the bar graph.
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As well as
-arrestin, dynamin seems to be a key regulator of the
internalization processes of GPCR (46). As such, dynamin has also been
implicated in GPCR signaling, including the activation of MAPKs by
several GPCRs (16). The role of this protein in the
T3-1 cells was
examined, as described for
-arrestin, by co-overexpression of either
wild-type or a dominant negative form of dynamin (K44A-dynamin (16,
47)) together with HA-ERK2. Although the wild-type form of dynamin had
no significant influence on the activation of ERK by GnRH, the dominant
negative form of dynamin partially inhibited (45%) both the basal and
the GnRH-induced ERK activation (Fig. 8).
This inhibition of both basal and GnRH-stimulated activities is similar
to the results obtained with dominant negative Ras (Fig. 6) and to some
extent also to the results with CSK, suggesting that dynamin, Src, and
Ras operate on the same signaling pathway.

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Fig. 8.
Effect of dynamin on ERK activation by
GnRH-a. T3-1 cells were cotransfected with HA-ERK2 together
with either dynamin (Dyn) or dominant negative dynamin
(DN-Dyn). Two days after transfection, the cells were
serum-starved for 16 h and then either treated with GnRH-a
(10 7 M) for the indicated times, with
peroxovanadate (Na3VO4 (100 µM) and H2O2 (200 µM); VOOH), or left untreated. Activated HA-ERK2 was
determined with anti-diphospho ERK antibody (DP). The amount
of immunoprecipitated HA-ERK2 was determined by Western blot analysis
with anti-HA antibody (HA). The results in the bottom
bar graph are an average of three experiments. Activation
(-fold) (GnRH-stimulated/basal for each of the constructs) is indicated
in the bar graph.
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Dynamin, but Not
-Arrestin, Is Involved in Ras Activation by
GnRH--
To confirm that
-arrestin is not involved in GnRH-induced
stimulation of ERK and to further study the role of dynamin in this
process, we undertook to explore the influence on other components of
the GnRH to ERK signaling pathway. First we examined whether
-arrestin has any effect on the GnRH activation of Ras. Thus, either
wild-type or dominant negative forms of
-arrestin2 were cotransfected into
T3-1 cells together with GFP-Ras. After GnRH-a stimulation, the active, GTP-bound form of Ras was precipitated by the
Ras binding domain of Raf-1 (Raf-RBD), and activated GFP-Ras was then
detected with anti-GFP antibody. Similar to the effect on ERK2, no
effect of the
-arrestin constructs on Ras activation could be
detected under the conditions used (Fig. 9). Therefore, as was
predicted by the lack of effect on ERK,
-arrestin does not seem to
play a role also in the GnRH-induced signaling toward Ras. This
observation is in agreement with the lack of
-arrestin involvement
in GnRH-induced internalization of the GnRHR (48), suggesting that
-arrestin does not play a significant role in GnRH signaling.
Unlike
-arrestin, the other internalization mediator, dynamin
appears to be involved in GnRH-induced GnRHR internalization (48) and,
as demonstrated above (Fig. 8), in the activation of ERK by GnRH. We
then undertook to elucidate the possible mechanism by which dynamin
transmits the GnRH signals toward the downstream components of the ERK
cascade. First, the possible involvement of dynamin in Ras activation
by GnRH was examined by cotransfecting either wild-type or dominant
negative (K44A) forms of dynamin together with GFP-Ras. Similar to the
effect on ERK, the wild-type dynamin had no influence on Ras activity
under the conditions examined. However, the dominant negative form of
dynamin completely abrogated the GnRH activation of Ras under the
conditions examined, indicating that dynamin lies upstream of Ras in
the pathway that leads from the GnRHR. Similar to ERK, MEK activity was
only partially inhibited by the dominant negative and to some extent
also by wild-type dynamin (Fig.
9B). The fact that the
inhibition of MEK activation by dominant negative dynamin was very
similar to the inhibition of ERK activity makes it unlikely that
dynamin influences the MEK-ERK level of the cascade as previously
suggested in other systems (49). Moreover, unlike the inhibition of
Ras, ERK, and MEK activities by dominant negative dynamin, there was no
influence of this construct on the GnRH-induced membranal translocation of PKC
(Fig. 9B), indicating that dynamin acts
independently of this PKC. Because PKC
is one of the main PKC
isoforms that participate in GnRH signaling in
T3-1 cells (Ref. 50
and data not shown), the role of dynamin in the activation of Ras seems to be confined to its influence on the Src/Ras step without any significant influence on Raf-1 stimulation by PKC (Fig.
10).

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Fig. 9.
Effect of -arrestin
on GnRH activation of Ras and effect of dynamin on GnRH activation of
Ras, PKC, and MEK. A, effect of -arrestin on GnRH
activation of Ras: T3-1 cells were cotransfected with either
wild-type (WT) or dominant negative (DN) forms of
-arrestin together with GFP-Ras. Two days after transfection, the
cells were serum-starved for 16 h and either treated with GnRH-a
(10 7 M for 10 min,) or left untreated.
Activated GFP-Ras was determined using Ras precipitation with GST-RBD
as described under "Materials and Methods." The results in the
bottom bar graph are an average of two experiments.
Activation (-fold) (GnRH-stimulated/basal for each of the constructs)
is indicated in the bar graph. B, effect of
dynamin on GnRH activation of Ras, PKC, and MEK: T3-1 cells were
cotransfected with either wild type (WT-Dyn) or dominant
negative dynamin (DN-Dyn) together with GFP-Ras, GFP-MEK, or
epitope tagged-PKC . Two days after transfection, the cells were
serum-starved for 16 h and then either treated with GnRH-a
(10 7 M for 10 min for Ras and PKC and 4 min
for MEK) or left untreated. Activated GFP-Ras was determined using a
Western blot with anti-GFP antibody following Ras precipitation using
GST-RBD as described under "Materials and Methods." Activity of
transfected GFP-MEK was measured by immunoprecipitation with anti-GFP
antibody and subsequent in vitro kinase reaction using
recombinant ERK as a substrate (see "Materials and Methods"). To
determine PKC translocation to the membrane, membranal fractions were
collected and membranal (active) PKC was determined by Western blot
analysis using antibody to the PKC epitope tag. The results in the
bar graph represents the average of three experiments and
represent the percentage of -fold activation of GnRH-stimulated enzyme
in cells transfected with dominant negative or wild type dynamin as
compared with activation (-fold) in cells transfected without dynamin
constructs.
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Fig. 10.
Schematic representation of GnRH signaling
toward the MAPK cascades. Broken lines indicate an
indirect activation, and the solid line indicates a direct
activation. Dyn, dynamin.
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DISCUSSION |
GPCRs comprise the largest group of integral membranal proteins,
which transmit their signals primarily via four groups of G-proteins,
Gs, Gi, Gq, and G12 and
also via receptor-interacting molecules and transactivation of growth
factor receptors. The G-proteins and the other components are capable
of transmitting signals from the receptor to MAPK cascades via distinct
pathways, which often form an elaborate network of signaling cascades
(12). However, the exact mechanisms, which are involved in each of
these pathways, are still mostly obscure. Here we studied the receptor for GnRH, which was shown to operate via Gq or
G11 in pituitary cells (51). This system is of particular
interest because, in pituitary cells, GnRH does not promote growth and
differentiation and does not constitute a stress signal and therefore
might utilize a unique signaling pathway to activate the MAPK cascades
(12, 20).
In previous studies we and others have shown that the ERK (21, 25), JNK
(28), and p38MAPK (27) cascade are activated by GnRH in a
PKC-dependent manner. However, although JNK activation seems to be fully dependent on Src and only partially (~70%) on PKC,
ERK activation was shown to be fully dependent on PKC but only
partially (~30%) on PTK. Because PTKs have been shown to play an
important role in the activation of ERK by GPCR (12), the small effect
of PTK inhibitors observed in our studies was surprising. We have ruled
out the possibility that the PTK involved in the GnRH to ERK activation
is only partially sensitive to genistein. This was demonstrated by
showing that none of the general PTK inhibitors used here inhibit ERK
activation by more than 40%. Moreover, the Src inhibitor PP1 and the
inhibitory kinase CSK, which fully inhibit Src activity, also cause
similar inhibitions. Because Src is activated by GnRH and has a major
role in the transmission of signals to the JNK cascade, the most likely
explanation for our results is that the activation of ERK is supported
to some extent by Src, although the main pathway leading to ERK
activation is PTK-independent.
PTK-induced activation of ERK is known to involve the small GTP-binding
protein Ras. Interestingly, although GnRH causes a significant
activation of Ras, a dominant negative form of Ras had only a minor
inhibitory effect on ERK activation. Because the effect was similar to
that exerted by Src, it is likely that Ras acts downstream of Src in a
pathway that partially supports the activation of ERK (Fig. 10).
Indeed, Ras activation by GnRH was abolished by the Src inhibitor, PP1.
Interestingly, it has been previously demonstrated (39) that
stimulation of PKC in COS-7 cells led to activation of Ras and
formation of Ras·Raf-1 complexes, but the activation of Raf-1 by PKC
was not completely blocked by dominant negative Ras. These data
indicate that PKC activates Raf-1 by a mechanism distinct from that
initiated by PTKs and that only a small amount of activated Ras is
needed to allow the activation of Raf-1 by PKC. The fact that ERK
activation is only partially dependent on Src but is fully dependent on
PKC agrees well with this suggested model. Thus, we believe that, upon
GnRH stimulation, Raf-1 is mainly activated by PKC but requires a small
amount of active Ras. This possibility was demonstrated before in other
experimental systems (38, 39) and explains the partial inhibition of
ERK by interfering mutants of Src and Ras (Fig. 10). It has previously
been shown that calcium may be involved in the activation of ERK by
GnRH in
T3-1 cells (25, 52). According to our model this effect is
probably on the Src/dynamin/Ras branch of the pathway and not on the
PKC-Raf-1 step, which was shown to be independent of calcium influx
(52) and not on the Raf-1-MEK-ERK steps, which were shown to be
calcium-independent in several cellular systems (1).
It should be noted that a recent study claimed that the activation of
ERK by GnRH in
T3-1 cells is inhibited equally by PKC and PTK
inhibitors and that Ras activation by GnRH is mediated by EGF receptor
(36). The reason for these slight discrepancies between our results and
the results reported by Grosse et al. (36) is not clear.
However, one possible explanation could be the different lengths of
serum starvation, which modify the content of many signaling components
(53). We found that the minimal time needed for a complete removal of
MAP kinase phosphatases and complete quiescence in
T3-1 cells is
14 h (data not shown). Alternatively, it is also possible that the
T3-1 cells are modified under different growing conditions to form
distinct subpopulations that exhibit different repertoire of signaling
molecules as was shown also in other cell lines such as PC12. These
explanations can be used also for the different results obtained in
regard to the activation of JNK by GnRH in different laboratories
(54).
Another point of interest is the mechanism of Src activation by GnRH.
We have previously shown that Src is activated by GnRH via a mechanism
that is partially (~70%) dependent on PKC (28). Similarly, we found
that the activation of Ras is only partially dependent on PKC, but
fully dependent on Src, supporting the notion that Ras is activated by
Src. Such pathway of sequential activation of Src-Ras-ERK was reported
also for some other GPCRs (12, 55-57). However, the fact that Src and
Ras are only partially dependent on PKC raises the question as to what
might be the other pathway involved in the activation of Src/Ras. An
answer to this question may come from the recent observations that,
upon GPCR stimulation, Src can be activated in a G
-independent
manner and therefore we undertook to study the role of these additional
signaling molecules as outlined below. Many GPCRs were shown to
transmit their signal through transactivation of either EGF receptor-
or cytoskeleton-associated PTKs (FAK and PYK). Our results
indicate that those components are not involved in GnRH signaling to
ERK. Thus, EGF receptor does not seem to be activated in response to
GnRH (25), and the specific inhibitor of EGF receptor (AG1478) or
dominant negative form of the EGF receptor had no effect on
GnRH-induced ERK stimulation. Moreover, FAK does not seem to
participate in GnRH to ERK signaling, because a dominant negative FAK
had no effect on ERK activation by GnRH. Finally, PYK does not
seem to be expressed to any detectable level in
T3-1 cells as judged
by immunoblotting and immunoprecipitation experiments (data not shown),
and therefore is unlikely to participate in the GnRH to ERK pathway.
Although the G
subunits are important transducers of GPCR signaling,
dissociated 
subunits have been implicated in the transmission of
GPCRs signaling as well. Thus, 
dimers can act via PTKs (such as
Src), via a direct activation of Ras or via a direct activation of
either the protein serine/threonine kinase KSR-1 or activation of
phosphatidylinositol 3-kinase (58, 59). In addition, GPCRs can
operate via
-arrestin- and dynamin-mediated internalization (16),
and
-arrestin may serve as a scaffold for additional signaling
molecules and initiate a second wave of G-protein-independent,
heptahelical receptor-mediated signals that activate the MAPK cascades
(12). Interestingly, in the
T3-1 system, we found that neither a
scavenger of 
dimer nor the dominant negative form of
-arrestin affect the GnRH-induced ERK activation. However,
overexpression of dominant negative dynamin reduced the activation of
both basal and GnRH-induced ERK activation indicating that dynamin,
unlike the other upstream components examined, may participate in the
PKC-independent activation of Src/Ras.
Recently it was shown that, in addition to its role in the
internalization of GPCRs, dynamin is also necessary for the direct activation of ERK by MEK (49). This may indicate that the inhibition by
the dominant negative dynamin is downstream of Raf-1 and not upstream
of the Src/Ras pathway. To determine the site of dynamin action, we
examined its role on several components of the cascade. Interestingly,
we found that GnRH-induced Ras activity was significantly inhibited
(~80%) by the dominant negative dynamin, whereas PKC activity was
not affected under the same conditions. Because Src activation is
mostly dependent on PKC (~70%), inhibition by the dominant negative
dynamin upstream of Src at the PKC-independent pathway should have
resulted in only ~30% inhibition of Ras activation, which is much
smaller than the complete inhibition obtained. Moreover, the inhibition
of the GnRH-induced MEK activation was similar to that of ERK
activation. Therefore, we suggest that the dynamin is required for the
process of Ras activation by Src, and the step of Raf-1 activation by
PKC is probably not affected by the dominant negative dynamin under the
conditions used.
In summary, we studied here the mechanism of ERK activation by GnRH in
the pituitary derived
T3-1 cell line. We show that ERK activation is
fully dependent on PKC but only partially dependent on Src, Ras, and
dynamin. Because it has previously been shown that Raf-1 activation by
PKC is only partially dependent on active Ras, our results are best
explained by the involvement of two distinct pathways in the
GnRH-mediated stimulation of Raf-1/ERK. One of these pathways involves
a direct activation of Raf-1 by PKC; the other involves Ras
activation by Src and dynamin.