Dependence of Gonadotropin-releasing Hormone-induced Neuronal
MAPK Signaling on Epidermal Growth Factor Receptor
Transactivation*
Bukhtiar H.
Shah
,
Jae-Won
Soh§, and
Kevin J.
Catt
¶
From the
Endocrinology and Reproduction Research
Branch, NICHD, National Institutes of Health, Bethesda, Maryland
20892 and § Herbert Irving Comprehensive Cancer Center,
College of Physicians and Surgeons, Columbia University,
New York, New York 10032
Received for publication, August 27, 2002, and in revised form, November 13, 2002
 |
ABSTRACT |
The hypothalamic decapeptide,
gonadotropin-releasing hormone (GnRH), utilizes multiple signaling
pathways to activate extracellularly regulated mitogen-activated
protein kinases (ERK1/2) in normal and immortalized pituitary
gonadotrophs and transfected cells expressing the GnRH receptor. In
immortalized hypothalamic GnRH neurons (GT1-7 cells), which also
express GnRH receptors, GnRH, epidermal growth factor (EGF), and
phorbol 12-myristate 13-acetate (PMA) caused marked phosphorylation of
ERK1/2. This action of GnRH and PMA, but not that of EGF, was
primarily dependent on activation of protein kinase C (PKC), and the
ERK1/2 responses to all three agents were abolished by the selective
EGF receptor kinase inhibitor, AG1478. Consistent with this, both GnRH
and EGF increased tyrosine phosphorylation of the EGF receptor. GnRH and PMA, but not EGF, caused rapid phosphorylation of the proline-rich tyrosine kinase, Pyk2, at Tyr402. This was reduced by
Ca2+ chelation and inhibition of PKC, but not by AG1478.
GnRH stimulation caused translocation of PKC
and -
to the cell
membrane and enhanced the association of Src with PKC
and PKC
,
Pyk2, and the EGF receptor. The Src inhibitor, PP2, the C-terminal Src
kinase (Csk), and dominant-negative Pyk2 attenuated ERK1/2 activation
by GnRH and PMA but not by EGF. These findings indicate that Src and
Pyk2 act upstream of the EGF receptor to mediate its transactivation,
which is essential for GnRH-induced ERK1/2 phosphorylation in
hypothalamic GnRH neurons.
 |
INTRODUCTION |
The hypothalamic decapeptide, gonadotropin releasing hormone
(GnRH),1 is a primary
regulatory factor in the neuroendocrine control of reproduction and is
released in an episodic manner from the hypothalamic GnRH neurons. The
pulsatile delivery of GnRH to the anterior pituitary gland is essential
to maintain the circulating gonadotropin profiles that are necessary
for normal reproductive function. In addition to regulating
pituitary gonadotropin release, GnRH has extrapituitary actions in
neural and nonneural tissues and in several types of tumor cells
(1). Immortalized GnRH-producing neurons (GT1-7 neurons) express
several G protein-coupled receptors (GPCRs), including those for GnRH
and luteinizing hormone/human chorionic gonadotropin (2, 3), as well as
- and
-adrenergic (4), muscarinic (5), and serotonergic receptors
(6). These cells retain many of the characteristics of the native GnRH neurons, including the ability to maintain pulsatile GnRH release (1,
3). Recent evidence suggests that the autocrine action of GnRH on
hypothalamic GnRH neurons is involved in the mechanism of pulsatile
GnRH secretion (3).
Agonist activation of specific GPCRs and the resulting dissociation of
their cognate G proteins releases
- and 
-subunits that
regulate phospholipase C-
, adenylyl cyclase, and ion channels, which
in turn control the intracellular levels of inositol phosphates, Ca2+, cAMP, and other second messengers (7, 8). The major
signal transduction pathways in cells expressing GnRH receptors are
initiated by activation of phospholipase C. The consequent calcium
(Ca2+) mobilization and activation of protein kinase C
(PKC) by GnRH are key elements in the hypothalamic control of
gonadotropin secretion from the anterior pituitary gland (1, 3, 5).
Activation of PKC and Ca2+ mobilization during GnRH
receptor stimulation are also responsible for mediating downstream
signals leading to activation of extracellularly regulated
mitogen-activated protein kinases (ERK1/2 MAPKs) that transmit signals
from the cell surface to the nucleus to regulate transcriptional and
other processes (7-13). However, the specific PKC isoforms that are
involved in GnRH-induced ERK1/2 activation in GT1-7 cells are not known.
Mitogenic signaling by GPCRs can also occur through activation of
tyrosine kinases of the Src family, focal adhesion kinases (FAKs), and
receptor tyrosine kinases (RTKs). The RTKs involved in GPCR-mediated
activation of ERK1/2 MAPKs include the EGF-R, platelet-derived growth
factor receptor, and insulin-like growth factor receptor (14-16). The
GPCRs mediating EGF-R transactivation during agonist stimulation
include the AT1 angiotensin receptor (17), the
-adrenoreceptor (18), the P2Y2 purinoceptor (19), and receptors for
endothelin-1, thrombin, lysophosphatidic acid, and bradykinin (20, 21).
GPCR-mediated transactivation of the EGF-R initiates the ERK1/2 MAPK
cascade through recruitment of adaptor proteins, such as the
Shc-Grb2-Sos complex, that activate the small G protein, Ras
(14, 22). Depending on the GPCR agonist and cell type,
Ca2+, PKC, G protein 
subunits, and nonreceptor
tyrosine kinases including Src and Pyk2, have been implicated in
GPCR-induced EGF-R transactivation (14, 22). Endogenous EGF-Rs are
expressed in several model systems, including
T3-1 gonadotrophs,
COS-7 cells, and HEK-293 cells, that have been used in studies on GnRH signaling. However, the role of EGF-R transactivation in GnRH-induced ERK activation has been a subject of controversy and is not clearly defined (9, 10, 23). Also, the signaling molecules involved in
cross-talk between the neuronal GnRH-R and the EGF-R have not been identified.
Depending upon the cell type, GPCRs mediate both Ras-independent ERK1/2
activation via stimulation of PKC and Ras-dependent ERK
activation by receptor and nonreceptor tyrosine kinases (7, 14). GnRH
has been found to activate ERK1/2 MAPKs in
T3-1 gonadotrophs and in
COS-7 cells (8-10, 12-13) and GH3 cells transfected with the GnRH
receptor (11). It also stimulates Jun N-terminal kinase in
T3-1
cells (24) and p38-MAPK in L
T2 gonadotrophs (25). Activation of
these MAPKs by other GPCRs, such as angiotensin II (26, 27), endothelin
(28), adrenomedullin (29), and acetylcholine (30), is mediated through
the proline-rich protein tyrosine kinase, Pyk2. In general, Pyk2
activation in conjunction with Src kinase appears to be a key element
in GPCR-mediated transactivation of the EGF-R (31). However, no
information is available on the role of Pyk2 and the nature of its
interaction with Src and EGF-R during receptor stimulation by GnRH. The
present studies have identified a signaling cascade that mediates
GnRH-induced ERK1/2 phosphorylation in immortalized GnRH neurons
(GT1-7 cells) and is dependent on receptor-mediated activation of PKC,
Src, Pyk2, and the EGF-R.
 |
EXPERIMENTAL PROCEDURES |
Materials--
GnRH was obtained from Peninsula Laboratories,
Inc. (Belmont, CA), EGF was from Invitrogen, and pertussis toxin
was from List Biological Laboratories. Protein assay kits were from
Pierce. ERK1/2 and anti-phospho-ERK1/2
(Thr202/Tyr204) antibodies were from New
England Biolabs, and secondary antibodies conjugated to horseradish
peroxidase were from KPL. Antibodies against Src, EGF-R, phospho-EGF-R
(Tyr1173), and phosphotyrosine (PY20) were from Santa Cruz
Biotechnology, Inc. (Santa Cruz, CA). Anti-phospho-Pyk2
(Tyr402) was from either Calbiochem or
BIOSOURCE International, and anti-phospho-EGF-R (Tyr1068) was from BIOSOURCE
International. Mouse monoclonal hemagglutinin tag antibody was from
Covance Babco (Berkeley, CA). AG1478, Go6983, Ro318220, PP2,
BAPTA, PMA, and wortmannin were from Calbiochem, and antibodies against
PKC isoforms and Pyk2 were from Transduction Laboratories.
LipofectAMINE was from Invitrogen. The Pyk2, dominant negative Pyk2,
constitutively active Src, and Csk constructs were provided by Dr. Zvi
Naor (University of Tel Aviv). PKC isoform-specific dominant negative
and constitutively active constructs tagged with the hemagglutinin
epitope were prepared as previously described (32). Western blotting
reagents and ECL were obtained from Amersham Biosciences or Pierce.
Cell Culture and Transfections--
GT1-7 neurons donated by
Dr. Richard Weiner (University of California, San Francisco) were
grown in culture medium consisting of 500 ml of Dulbecco's modified
Eagle's medium containing 0.584 g/liter L-glutamate and
4.5 g/liter glucose, mixed with 500 ml of F-12 medium containing 0.146 g/liter L-glutamate, 1.8 g/liter glucose, 100 µg/ml
gentamicin, 2.5 g/liter sodium carbonate, and 10% heat-inactivated
fetal calf serum. DNA transfections were performed with LipofectAMINE
according to the manufacturer's instructions.
Inositol Phosphate Measurements--
Cells were labeled for
24 h in inositol-free Dulbecco's modified Eagle's medium
containing 20 µCi/ml [3H]inositol as previously
described (5) and then washed twice with inositol-free M199 medium and
stimulated at 37 °C in the presence of 10 mM LiCl. The
reactions were stopped with perchloric acid, inositol phosphates were
extracted, and radioactivity was measured by liquid scintillation
-spectrometry.
Subcellular Fractionation--
Serum-starved GT1-7 cells were
treated with either PMA or GnRH for the times indicated and then washed
twice with ice-cold PBS and collected in homogenization buffer
containing 25 mM Tris·HCl, pH 7.4, 2 mM EDTA,
10 mM
-mercaptoethanol, 10% glycerol, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml each of aprotinin and leupeptin. After they were kept on ice for 10 min, cells were homogenized with 25 strokes of a Dounce homogenizer. Nuclei and unbroken cells were pelleted by centrifugation at 500 g for 5 min,
and the supernatant was centrifuged at 100,000 × g for
30 min. The high speed supernatant constituted the cytosolic fraction. The pellet was washed three times and extracted in ice-cold
homogenization buffer containing 1% Triton X-100 for 30 min. The
Triton-soluble component (membrane fraction) was separated from the
insoluble material (cytoskeletal fraction) by centrifugation at
100,000 × g for 20 min.
Immunoprecipitation--
After treatment with inhibitors and
drugs, cells were placed on ice, washed twice with ice-cold PBS, lysed
in lysis buffer (50 mM Tris, pH 8.0, 150 mM
NaCl, 1 mM NaF, 0.25% sodium deoxycholate, 1 mM EDTA, 1% Nonidet P-40, 10 µg/ml aprotonin, 10 µg/ml
leupeptin, 10 µg/ml soybean trypsin inhibitor, 10 µg/ml pepstain,
and 1 mM 4-(2-aminoethyl)benzensulfonyl fluoride), and
probe-sonicated (Sonifier cell disruptor). Solubilized lysates were
clarified by centrifugation at 8000 × g for 10 min,
precleared with agarose, and then incubated with specific antibodies
and protein A- or G-agarose. The immunoprecipitates were collected,
washed four times with lysis buffer, and dissolved in Laemmli buffer.
After heating at 95 °C for 5 min, the samples were centrifuged
briefly, and the supernatants were analyzed by SDS-PAGE on 8-16%
gradient gels.
Immunoblot Analysis--
Cells grown in six-well plates
and at 60-70% confluence were serum-starved for 24 h before
treatment at 37 °C with selected agents. The media were then
aspirated, and the cells were washed twice with ice-cold PBS and lysed
in 100 µl of Laemmli sample buffer. The samples were briefly
sonicated, heated at 95 °C for 5 min, and centrifuged for 5 min. The
supernatant was electrophoresed on SDS-PAGE (8-16%) gradient gels and
transferred to polyvinylidene difluoride membranes. Blots were
incubated overnight at 4 °C with primary antibodies and washed three
times with TBST before probing with horseradish peroxidase-conjugated
secondary antibodies for 1 h at room temperature. Blots were then
visualized with enhanced chemiluminescence reagent (Amersham
Biosciences or Pierce) and quantitated with a laser-scanning
densitometer. In some cases, blots were stripped and reprobed with
other antibodies.
 |
RESULTS |
GnRH treatment of GT1-7 cells caused transient stimulation of
ERK1/2 that reached a peak at 5 min and declined thereafter toward the
basal level over 30 min (Fig.
1A). GnRH-induced ERK1/2 activation was concentration-dependent over the 0.2-200
nM range and was abolished by the GnRH receptor antagonist,
[D-pGlu1,D-Phe,D-Trp(3,6)]GnRH (Fig. 1, B and C). GnRH receptors are primarily
coupled to Gq/11 proteins, but some of the physiological
actions of GnRH are known to occur through activation of Gs
or Gi proteins (5, 33). In GT1-7 neurons, nanomolar GnRH
concentrations cause marked elevation of inositol phosphate production
through Gq-mediated activation of phospholipase C and also
stimulate cAMP production. Higher concentrations of GnRH (0.1-1
µM) reduce intracellular cAMP levels in a pertussis
toxin-sensitive manner, suggesting that GnRH activates a Gi
protein in GT1-7 cells (34). Consistent with this, pertussis toxin had
a modest inhibitory effect (~30%) on GnRH-induced ERK activation,
suggesting partial involvement of Gi protein(s) in MAPK
signaling in GT1-7 cells (Fig. 1D).

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Fig. 1.
GnRH stimulation increases phosphorylation of
ERK1/2 in a concentration-dependent manner. A,
time course of the effect of GnRH (200 nM) on ERK1/2 (p44
and p42) phosphorylation at Thr202/Tyr204
(ERK1/2-P) in GT1-7 cells. Cells were grown to 70% confluence and
serum-starved for 20-24 h. After GnRH treatment for selected times,
cells were washed twice with ice-cold PBS and lysed in Laemmli sample
buffer. Cells were sonicated, heated to 95 °C for 5 min, and
centrifuged before loading onto 8-16% gradient gels for SDS-PAGE
analysis. ERK1/2 phosphorylation (ERK1/2-P) was measured by
immunoblotting with phospho-ERK1/2 antibodies. B,
concentration-dependent effects of GnRH on ERK1/2
activation. Cells were treated with various concentrations of GnRH for
5 min. Blots were stripped and reprobed with ERK1/2 antibody as shown
in the lower panels. C, the selective
GnRH receptor antagonist
[D-pGlu1,D-Phe,D-Trp(3,6)]GnRH
(DPGlu) blocks the effect of GnRH on ERK1/2 activation. The
antagonist (10 µM) was added 20 min before the addition
of GnRH (200 nM) for 5 min. D, effect of
pertussis toxin (PTX) on ERK1/2 activation by GnRH (200 nM for 5 min). Serum-starved cells were treated with PTX
(50 ng/ml) for 24 h, and ERK1/2 activation was measured following
stimulation with GnRH (200 nM for 5 min). Con,
unstimulated control cells. All blots are representative of three or
four experiments with similar results.
|
|
The roles of PKC and Ca2+ in agonist-stimulated activation
of ERK1/2 were evaluated in studies with PKC inhibitors and the
Ca2+ chelators BAPTA-2AM and EGTA. GnRH-induced
ERK1/2 activation was found to be highly PKC-dependent and
was abolished by the PKC inhibitors Ro318220 and Go6983 (Fig.
2A). These inhibitors had no
effect on ERK1/2 activation induced by basic fibroblast growth factor
(bFGF) (Fig. 2B) or isoproterenol, a
2-adrenoreceptor agonist (data not shown). Consistent
with its critical role in GnRH action, depletion of PKC by prolonged
PMA treatment (1 µM for 16 h) abolished
agonist-induced ERK1/2 activation (Fig. 2C). However, ERK1/2
activation by GnRH was less sensitive to Ca2+ chelation by
EGTA and BAPTA (Fig. 2D). Consistent with this, the PKC
activator, PMA, was much more effective than the Ca2+
ionophore, ionomycin, in eliciting ERK1/2 activation (Fig.
2E). These findings suggest that GnRH receptor-mediated
ERK1/2 activation in GT1-7 cells is predominantly dependent on
PKC.

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Fig. 2.
Role of PKC and calcium in GnRH-mediated
ERK1/2 activation. A, effects of PKC inhibitors on
ERK1/2 activation by GnRH or bFGF in GT1-7 cells. A and
B, cells were pretreated with Ro318220
(Ro31; 1 µM) and Go6983 (Go69; 1 µM) before stimulation with GnRH (200 nM for
5 min) or bFGF (10 ng/ml for 5 min). C, PKC depletion by
overnight (O/N) PMA treatment (2 µM, 16 h) abolishes ERK1/2 activation by GnRH.
D, effects of Ca2+ chelation on GnRH-induced
ERK1/2 activation. Cells were treated with BAPTA (2 and 20 µM) or EGTA (0.5 and 2.5 mM) followed by
stimulation with GnRH (200 nM) for 5 min. E,
relative effects of GnRH (200 nM), ionomycin (10 µM), and PMA (100 nM) on ERK1/2 activation in
GT1-7 cells. Lower panels, total ERK1/2 levels.
Con, unstimulated control cells. All blots are
representative of three experiments with similar results.
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A major role of PKC in GnRH-induced ERK activation has also been
reported in other cell types (8-10), but little information is
available about the involvement of specific PKC isoforms in this
cascade. GT1-7 cells were found to contain several immunoreactive PKC
isoforms, including
,
,
, and
. Overnight PMA
stimulation (2 µM) caused down-regulation of PKC
,
-
, and -
but not PKC
(Fig.
3A). Among the PMA-sensitive
PKC isoforms, only PKC
and -
were translocated from cytosol to
the cell membrane during treatment with GnRH and PMA (Fig.
3B). These effects of GnRH and PMA were specific, since no
changes in the levels of ERK1/2 and Na+/K+-ATPase were found in cytosol and
membranes, respectively (Fig. 3C). The predominant role of
PKC
in GnRH-induced ERK activation was confirmed in studies with
constitutively active and dominant negative mutants of PKC
. These
results showed that GnRH-induced ERK1/2 phosphorylation was attenuated
by dominant negative PKC
(dnPKC
; Fig.
4) and was increased with transfection of
constitutively active PKC
(data not shown).

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Fig. 3.
GnRH and PMA cause translocation of
PKC and - from
cytosol to the membrane. A, GT1-7 cells express
PMA-sensitive and -insensitive PKC isoforms. Cells were treated with
PMA (2 µM) overnight (O/N),
and cell lysates were analyzed by Western blotting (IB) for
various PKC isoforms. Lower panel, the ERK1/2
levels in control and PMA-treated cells. B and C,
serum-starved cells were stimulated with GnRH (200 nM) and
PMA (200 nM) for the time periods indicated. After washing
with ice-cold PBS, cells were collected and homogenized. The cytosol
and membranes were obtained as described under "Experimental
Procedures." Equal amounts of proteins from control and stimulated
cells were analyzed by SDS-PAGE and detected for PKC isoforms in
cytosol and membranes. As controls, ERK1/2 and
Na+/K+-ATPase were probed in cytosol and
membranes, respectively. All data are representative of two or three
experiments.
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Fig. 4.
The effects of dominant negative
PKC on ERK1/2 activation by GnRH.
A, expression of native and transfected dominant negative
PKC (dnPKC ) in GT1-7 cells transfected with plasmid
DNA (2 and 5 µg) encoding dominant negative PKC . Cells were washed
twice with ice-cold PBS and lysed in Laemmli sample buffer before
loading onto 8-16% gradient gels for SDS-PAGE analysis. The
expression of PKC constructs was detected using antibody against the
hemagglutinin epitope with which these mutant proteins are tagged.
Whereas conventional antibody against PKC detects both native and
exogenous dominant negative PKC proteins, the hemagglutinin antibody
detects only the product of transfected dominant negative PKC with no
immunoreactivity in the nontransfected (NT) cells.
B, effects of overexpression of dominant negative PKC on
GnRH-induced ERK1/2 phosphorylation
(ERK1/2-P). Serum-starved cells were
stimulated with GnRH (200 nM for 5 min) and then washed
twice with ice-cold PBS and lysed in Laemmli sample buffer before
loading onto 8-16% gradient gels for SDS-PAGE analysis. The
quantitated data are shown in the lower panel
(n = 4).
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It is well established that transactivation of receptor tyrosine
kinases such as the EGF-R contributes to GPCR-mediated ERK1/2 activation in certain cell types (14, 22). However, studies on the role
of the EGF-R in GnRH action have not given consistent results (9, 10).
Thus, whereas Grosse et al. (10) implicated transactivation
of the EGF-R in GnRH-induced stimulation of ERK1/2 phosphorylation in
T3-1 pituitary gonadotrophs, Benard et al. (9)
subsequently reported that the major pathway of ERK1/2 activation was
through PKC and activation of Raf-1 and did not involve the EGF-R.
Since GT1-7 cells express receptors for both EGF and GnRH, we
evaluated the role of the EGF-R in GnRH-induced MAPK signaling. In this
cell type, EGF, like GnRH, caused transient activation of ERK1/2 (Fig.
5A). As expected, the
selective EGF-R kinase inhibitor, AG1478, blocked the ERK1/2 activation
induced by EGF (Fig. 5B). EGF stimulation caused rapid and
marked phosphorylation of the EGF-R at Tyr1173 in a time-
and concentration-dependent manner (Fig. 5, C and D). Our data suggest a potential role of PKC in GnRH-induced
ERK1/2 activation. To determine whether PKC acts upstream or downstream of the EGF-R, we examined the effect of PKC inhibition on EGF-induced ERK1/2 activation. Whereas PKC depletion by prolonged PMA treatment or
PKC inhibitors abolished the effects of PMA and GnRH, it had no effect
on EGF responses (Fig. 5, E and F). These data
indicate that EGF-induced ERK1/2 activation is PKC-independent and that PKC acts upstream of the EGF-R during GnRH signaling in GT1-7 cells.

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Fig. 5.
EGF causes marked phosphorylation of ERK1/2
and EGF-R in GT1-7 cells. A, cells were treated with EGF
(50 ng/ml) for the time periods indicated, and ERK1/2 phosphorylation
was determined as described under "Experimental Procedures."
B, the EGF receptor kinase inhibitor, AG1478 (100 nM) completely inhibits ERK1/2 activation by EGF (50 ng/ml). C, time course effect of EGF (50 ng/ml) on EGF-R
phosphorylation (EGF-R-P) at Tyr1173. D,
concentration-dependent effects of EGF (4-min stimulation)
on EGF-R phosphorylation at Tyr1173. Serum-starved
GT1-7 cells treated with EGF were collected in Laemmli sample buffer
and analyzed for immunoblotting with anti-phosphotyrosine EGF-R
antibody at Tyr1173. E,
concentration-dependent inhibitory effect of AG1478 on
EGF-induced phosphorylation of the EGF-R at Tyr1173.
F, PKC depletion by PMA treatment (2 µM)
overnight (O/N) abolishes ERK1/2 activation
induced by GnRH (200 nM for 5 min) and PMA (100 nM for 8 min) but not by EGF (50 ng/ml). G, lack
of effect of PKC inhibitors, Ro318220 (Ro31; 1 µM) and Go6983 (Go69; 1 µM) on
EGF-induced ERK1/2 activation. GT1-7 cells were pretreated with
inhibitors for 20 min and stimulated with EGF (50 ng/ml) for 5 min.
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|
To examine the involvement of EGF-R in GnRH-induced ERK1/2
activation in GT1-7 cells, cells were pretreated with AG1478 and stimulated with GnRH (200 nM for 5 min). As shown in Fig.
6A, GnRH-stimulated ERK1/2
phosphorylation was also abolished by AG1478, indicating its absolute
dependence on transactivation of the EGF-R. The inhibitory action of
AG1478 on GnRH-induced ERK1/2 activation was selective and did not
affect bFGF-stimulated ERK1/2 phosphorylation (Fig. 6B).
Consistent with the role of EGF-R in GnRH signaling, GnRH also
stimulated phosphorylation of the EGF-R as measured with
anti-phosphopeptide antibodies that recognize the phosphorylated molecule at Tyr1173 or Tyr1168 (Fig.
6C), the major sites of Src kinase phosphorylation (35) and
Grb2 binding (36), respectively. These data demonstrate that
transactivation and phosphorylation of the EGF-R are essential for GnRH
signaling through ERK1/2 in GT1-7 cells.

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Fig. 6.
GnRH-induced ERK1/2 activation is dependent
on EGF-R transactivation in GT1-7 cells. A, the EGF
receptor kinase inhibitor, AG1478 (100 nM), inhibits ERK1/2
activation by GnRH (100 nM, 5 min). B, lack of
inhibitory effect of AG1478 on ERK1/2 activation induced by bFGF (10 ng/ml for 5 min). C, time course effects of GnRH on
phosphorylation of the EGF-receptor. Serum-starved cells were
stimulated with GnRH (200 nM), collected in Laemmli sample
buffer, and analyzed for immunoblotting with anti-phosphotyrosine EGF-R
antibody at Tyr1173 or Tyr1068. The
lower panel shows total EGF-R protein.
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Since GnRH-induced ERK1/2 activation is primarily dependent on PKC and
GnRH causes PKC activation through generation of diacylglycerol (8-10), we investigated the effects of PMA on this cascade. The results revealed that PMA caused marked ERK1/2 activation that was
abolished by prior PKC depletion (as shown above in Fig. 5D) and by PKC inhibitors, Ro318220 and Go6983 (Fig.
7, A and B). To
determine whether PMA mimics the effects of GnRH with respect to EGF-R
transactivation, GT1-7 cells were treated with AG1478 and stimulated
with PMA. As shown in Fig. 7C, PMA-induced ERK1/2 activation
was extinguished in a dose-dependent manner by the EGF-R
kinase inhibitor, AG1478, indicating that GnRH-induced ERK1/2 activation occurs through EGF-R transactivation in a
PKC-dependent manner.

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Fig. 7.
The involvement of EGF-R transactivation in
PMA-induced ERK1/2 activation. A, time course effect of
PMA (100 nM) on ERK1/2 activation. B, effects of
PKC inhibitors, Ro318220 (Ro31; 1 µM) and
Go6983 (Go69; 1 µM) on PMA-induced ERK1/2
activation. C, effect of dominant negative PKC mutant (2 µg of DNA) on PMA-induced ERK1/2 activation. Serum-starved GT1-7
cells were pretreated with inhibitors for 15 min before stimulation
with PMA (100 nM) for 8 min. The blots were reprobed with
nonphosphorylated ERK1/2, and data are shown in the lower
panels (n = 3-4).
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Since there is no consensus on the types of intermediate
proteins involved during GPCR-induced transactivation of the EGF-R (14,
36, 37), we examined the roles of Src and Pyk2 in GnRH-induced EGF-R
phosphorylation and ERK activation. In GT1-7 cells, the highly
selective Src kinase inhibitor, PP2, and the Src negative regulatory
kinase, Csk, attenuated the activation of ERK1/2 by GnRH (Fig.
8, A and B).
Similarly, Src inhibition abolished the effect of PMA on ERK1/2
activation (Fig. 8C). In contrast, Src inhibition and Csk
had no effect on EGF-induced ERK1/2 responses (Fig. 8D).
These data suggest that Src has a critical role in GnRH-induced
activation of the EGF-R and ERK1/2. Since our data show that both PKC
and Src act upstream of EGF-R, we examined the interaction between PKC
and Src. As shown in Fig. 8E, GnRH stimulation increased the
association of PKC
and -
with Src.

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Fig. 8.
Role of Src in GnRH-induced ERK1/2
activation. A, concentration-dependent effect of
the Src kinase inhibitor, PP2, on ERK1/2 activation induced by GnRH.
Serum-starved GT1-7 cells were incubated with PP2 (0.01-1
µM) for 20 min and then stimulated with GnRH (200 nM) for 5 min. B, effect of Src negative
regulatory kinase (Csk) on ERK1/2 activation induced by GnRH. Csk DNA
(1 µg) was transfected in GT1-7 cells, and ERK1/2 activation was
measured following stimulation with GnRH (100 nM for 5 min). Blots were reprobed with Src antibody. C,
concentration-dependent effects of Src kinase inhibitor,
PP2, on PMA-induced ERK1/2 activation. Serum-starved cells were treated
with PP2 for 20 min before stimulation with PMA (100 nM for
8 min). D, lack of effects of PP2 (5 µM) and
Csk overexpression on EGF-induced ERK1/2 phosphorylation.
E, GnRH stimulation increases association of PKC and -
with Src. Serum-starved cells were stimulated with GnRH (200 nM) for 5 min, washed with ice-cold PBS, collected in lysis
buffer, immunoprecipitated (IP) with Src antibody, and
immunobloted (IB) with antibodies against PKC and -
(n = 3).
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A role for the nonreceptor proline-rich tyrosine kinase, Pyk2, in
ERK1/2 activation by some GPCRs has been shown (26-30). However, nothing is known about the role of Pyk2 in GnRH signaling. GnRH stimulation of GT1-7 cells caused a marked increase in Pyk2 tyrosine phosphorylation at residue 402 that commenced within 1 min and was
sustained for up to 30 min (Fig.
9A). Like ERK1/2 activation, the stimulatory effect of GnRH on Pyk2 activation was sensitive to both
PKC inhibition and Ca2+ chelation (Fig. 9B).
Consistent with the potential involvement of PKC in this cascade, PMA
also caused phosphorylation of Pyk2 at Tyr402, and this
effect was attenuated by PKC inhibition but not by AG1478 (Fig.
9C). These data suggest that GnRH-induced Pyk2 activation is
primarily PKC-dependent in GT1-7 cells.

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Fig. 9.
GnRH and PMA cause Pyk2 phosphorylation in
GT1-7 cells. A, serum-starved GT1-7 cells were
stimulated with GnRH (200 nM) for the time periods
indicated, and cell lysates were analyzed for Pyk2 activation (Pyk2-P)
using phosphotyrosine antibody to Pyk2 (Tyr402).
B, effects of PKC inhibitors and Ca2+ chelation
on Pyk2 activation by GnRH. Serum-starved GT1-7 cells were treated
with Ro318220 (Ro31; 1 µM), Go6983
(Go69; 1 µM), and BAPTA (BAP; 10 µM) for 20 min before stimulation with GnRH (200 nM, 5 min). C, effects of AG1478 (100 nM), calcium chelator, BAPTA (BAP; 10 µM), and PKC inhibitor, Ro318220 (Ro31; 1 µM) on PMA-induced phosphorylation of Pyk2
(Pyk2-P). GT1-7 cells were incubated with inhibitors for 20 min and stimulated with PMA (100 nM) for 8 min. The blot
was reprobed with ERK1/2 antibody to show the equal loadings of the
protein in each lane. Data are representative of three independent
experiments.
|
|
The dependence of GnRH-mediated ERK1/2 activation on Pyk2 was evaluated
in studies with dominant negative Pyk2 mutants (PKMs). Overexpression
of PKM attenuated the stimulatory effects of GnRH and PMA on ERK1/2
activation, and Pyk2 overexpression enhanced the effect of GnRH on
ERK1/2 activation (Fig.
10A). These data show that
Pyk2 has an important role in GnRH-induced ERK1/2 activation in GT1-7
cells. Previous studies have shown that, depending on the cell types,
GPCR stimulation leads to interaction of Src with Pyk2 and also that
these proteins can cause activation of one another (20, 37). Whether
such an interaction occurs following GnRH stimulation is not known. An
analysis of the cell lysates immunoprecipitated with anti-Src antibody
and immunoblotted with Pyk2 antibody revealed that GnRH increased the
association of Src with Pyk2. Furthermore, Src also
co-immunoprecipitated with the EGF-R in GT1-7 cells (Fig.
10B). Taken together, these results indicate that ERK1/2
activation by GnRH leads to recruitment of a multicomponent signaling
complex that includes PKC
/
, Src, Pyk2, and the EGF-R.

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Fig. 10.
Role of Pyk2 in GnRH-induced ERK activation.
A, effects of overexpression of Pyk2 dominant negative
mutant, PKM, and Pyk2 (1 µg DNA) on ERK1/2 activation induced by PMA
(100 nM for 8 min) and GnRH (200 nM for 5 min).
Blots were stripped and reprobed with ERK1/2 and Pyk2 antibodies. The
data from three experiments were quantitated. B, effects of
GnRH (200 nM) and PMA (100 nM) on the
association of Src with Pyk2 and EGF-R. Agonist-stimulated cells were
collected in lysis buffer, immunoprecipitated (IP) with Src
antibody or preimmune serum (PIS), and immunoblotted
(IB) with either Pyk2 or EGF-R antibody. Data are
representative of two experiments.
|
|
In contrast, although EGF caused marked activation of ERK1/2, it had no
effect on Pyk2 phosphorylation (Fig.
11A). Moreover, Pyk2
activation by GnRH was not prevented by AG1478 (Fig. 11B), indicating that GnRH-induced Pyk2 activation precedes that of the EGF-R
transactivation. Whereas PKM decreased ERK1/2 activation by GnRH and
PMA, it had no effect on EGF-induced ERK1/2 activation and EGF-R
phosphorylation (Fig. 11, C and D). Because both
Pyk2 and FAK show high sequence similarity, cellular localization, and
signaling characteristics (38, 39), we determined whether GnRH-induced
ERK1/2 activation also involves FAK activation. In contrast to its
marked effect on Pyk2 activation, GnRH had little effect on FAK
phosphorylation (data not shown). These data suggest that GnRH causes
selective activation of Pyk2 through its specific receptors in GT1-7
cells.

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Fig. 11.
Pyk2 acts upstream of the EGF-R during GnRH
stimulation. A, effect of EGF (50 ng/ml for 5 min) on Pyk2
phosphorylation. B, lack of effect of AG1478 (100 nM) on GnRH-induced Pyk2 activation. Serum-starved GT1-7
cells were pretreated with inhibitors for 20 min before stimulation
with agonists. Cells were collected in Laemmli sample buffer, and
immunoblot analysis was done as described under "Experimental
Procedures." C and D, lack of effects of
dominant negative Pyk2 (PKM) and Pyk2 overexpression on EGF-induced
phosphorylation of ERK1/2 and EGF-R at Tyr1068.
|
|
 |
DISCUSSION |
The activity of the mammalian pituitary-gonadal axis
is dependent on the pulsatile secretion of GnRH from hypothalamic GnRH neurons. Many of the genomic effects of GnRH in its neuroendocrine target cells are believed to be mediated by the activation of MAPKs,
which convey GnRH signaling from the cell surface to the nucleus for
regulation of genes controlling the functions of GnRH neurons and
pituitary gonadotropes (1, 8). However, the roles of intermediate
signaling molecules such as Pyk2 and the EGF-R in this pathway have not
been clearly defined. Our results show that GnRH causes rapid, marked,
and transient phosphorylation of ERK1/2 through transactivation of the
EGF-R in GT1-7 cells. The signaling response to GnRH also involves the
PKC-dependent phosphorylation of two nonreceptor tyrosine
kinases, Src and Pyk2. To date, there has been no indication that this
mechanism, with consequent transactivation of the EGF-R, is operative
during GPCR-induced MAPK signaling in neuronal cells.
The requirement for growth factor receptor transactivation in
GnRH-induced ERK1/2 phosphorylation in other cell types is
controversial. Whereas observations in immortalized pituitary
gonadotrophs (
T3-1 cells) and COS-7 cells expressing GnRH receptors
have suggested that GnRH-induced ERK1/2 activation involved
transactivation of the EGF-R, more recent studies found no role of
EGF-R transactivation in the phosphorylation of MAPK in
T3-1 cells
(9) and HeLa cells expressing GnRH-R (23). Our data show that EGF-Rs
are abundantly expressed in GT1-7 cells and, when stimulated, undergo marked autophosphorylation, leading to activation of ERK1/2. More significantly, transactivation of the EGF-R kinase is essential for
GnRH-induced ERK1/2 activation in GT1-7 neurons, which is completely
prevented by the selective EGF-R kinase inhibitor AG1478. Furthermore,
GnRH causes selective phosphorylation of the EGF-R at
Tyr1173, a target site for Src action (35) and at
Tyr1068, a binding site for the Grb2/Src homology 2 domain
(36) (Fig. 6).
Whereas involvement of the EGF-R in GPCR-mediated ERK1/2 activation is
well documented (14, 17, 18, 22), the molecular mechanisms responsible
for this cascade are not clearly defined. Depending on the cell type,
EGF-R transactivation has been reported to be mediated by
Gi protein 
-subunits (40, 41), Ca2+ (26,
30), PKC (10, 37, 42), and heparin-binding EGF (HB-EGF) released by
matrix metalloproteinases (43, 44). However, the latter does not appear
to be a universal mechanism for transactivation of the EGF-R by GPCRs
(17, 45). Recently, the metabotropic glutamate receptor-5 has been
shown to directly interact with the EGF-R, bypassing phospholipase
C
, PKC, and Ca2+ (46). In the present study, the use of
pharmacological inhibitors and constitutively active and dominant
negative mutants of relevant signaling molecules has defined PKC, Src,
and Pyk2 as critical factors in GnRH-mediated EGF-R transactivation and
the consequent increase in ERK1/2 phosphorylation.
Tyrosine kinases implicated in cell signaling include Src
family kinases, RTKs such as the EGF-R, the FAK family, Pyk2, and JAK
kinases. Pyk2 belongs to the FAK family and is activated by tyrosine
phosphorylation in response to several GPCRs (39) as well as by stress
stimuli (47) and membrane depolarization (36). Pyk2 has also been
implicated in the regulation of ion channels (48), cell adhesion and
motility, neurite outgrowth (38, 39), and the induction of long term
potentiation in CA1 hippocampal cells (49). Under various experimental
conditions, Pyk2 has been shown to participate in the activation of all
three major MAPKs: ERK1/2 (26, 48, 50-52), p38 MAPK (28), and Jun
N-terminal kinase (27). However, no evidence about the role of Pyk2 in GnRH-induced ERK1/2 activation has been available. Our data have established that GnRH causes marked stimulation of Pyk2 phosphorylation and that overexpression of a kinase-inactive Pyk2 mutant impairs ERK1/2
activation by GnRH and PMA (Figs. 9 and 10). We have also demonstrated
that Pyk2 acts upstream of the EGF-R, since EGF failed to activate Pyk2
and dominant negative Pyk2 had no effect on EGF-induced responses.
Moreover, GnRH-induced phosphorylation of Pyk2 was not affected by
AG1478 (Fig. 11).
The present data also show that GnRH enhances the association of Pyk2
with c-Src (Fig. 10), an interaction that results from binding of the
autophosphorylated Tyr402 of Pyk2 to the Src homology 2 domains of c-Src (50-52). Expression of wild type Pyk2 induces
phosphorylation of Shc and increases its association with Grb2 (52), a
finding consistent with GnRH-induced phosphorylation of the EGF-R at
Tyr1068 (Fig. 6). On the other hand, a mutant form of Pyk2
that cannot complex with c-Src behaves as a dominant negative inhibitor
of GPCR-stimulated ERK1/2 activation (50). Our studies using both the
selective Src inhibitor, PP2, and the C-terminal Src kinase, Csk, have
demonstrated the essentiality of Src in the activation of ERK1/2 by
GnRH and PMA (Fig. 8). In contrast, the lack of effect of Src
inhibition on EGF-induced ERK1/2 activation indicates that Src acts
upstream of EGF-R in GT1-7 cells. Since Src was essential for
GnRH-induced phosphorylation of the EGF-R, these findings indicate that
activation of Src/Pyk2 has a critical role in transducing signals from
the GnRH-R to EGF-R transactivation in GnRH neuronal cells. These
results are consistent with recent studies in fibroblasts from knockout
mice showing that Src kinases are critical for activation of Pyk2 and
that Src and Pyk2 are indispensable for EGF-R activation by GPCRs (31).
In contrast, ERK1/2 activation by GnRH in
T3-1 pituitary
gonadotrophs was independent of Pyk2 as well as EGF-R transactivation.
Instead, it was primarily mediated through the direct activation of
Raf-1 by PKC and to a lesser extent by Ras activation that was
dependent on dynamin and Src (9). Similarly, whereas ERK1/2 activation
by endothelin-1 in rat mesangial cells involved Pyk2, it was
independent of EGF-R activation (28). It is clear that the matrix of
signaling molecules utilized during GPCR stimulation is highly variable
among different cell types, in which several specific patterns of
interactions and phosphorylations are now becoming evident.
Earlier studies on the role of PKC isoforms in GnRH action have
demonstrated activation of PKC
and -
(53) and PKC
2, -
, -
, and -
in
T3-1 cells (54) and activation of PKC
and -
in rat pituitary cells (55). However, no information was available about the specific PKC isoform(s) involved in GnRH-induced ERK activation in GT1-7 cells. We found that GT1-7 cells contain PKC isoforms
,
,
, and
and that both GnRH and PMA increased
the translocation of PKC
and -
to the cell membranes and enhanced their association with Src (Figs. 3 and 8). Moreover, GnRH-stimulated phosphorylation of Pyk2 and ERK1/2 in GT1-7 cells is primarily dependent on PKC, since both pharmacological PKC inhibition and PKC
depletion by PMA abolished ERK1/2 activation by GnRH and PMA but not by
EGF (Figs. 2, 7, and 8). Overexpression of dominant negative PKC
also attenuated ERK1/2 activation by GnRH (Fig. 4), but not by EGF
(data not shown). These data suggest that whereas PKC is an important
mediator of GnRH-induced signals, its stimulatory action is upstream of
the EGF-R in GT1-7 cells. GPCR-mediated activation of Pyk2 and ERK1/2
is reported to be dependent on both Ca2+ (26, 30, 41, 50)
and PKC (19, 37, 39, 51). PKC is also known to cause activation of Src
(24) and the EGF-R (10, 24, 44, 45) in several cell types. In fact,
PKC
and -
undergo direct physical and functional interactions
with the EGF-R and Pyk2, respectively (56, 57). Thus, PKC can stimulate signaling cascades by targeting a variety of intermediary proteins.
In many cells, the pathways of GPCR- and RTK-mediated
ERK1/2 activation converge primarily at the level of the EGF-R (20, 22,
41, 58). Following agonist-induced tyrosine phosphorylation of the
EGF-R, the signaling pathways involved in ERK1/2 activation by GPCRs
and EGF-Rs appear to be identical (14, 20). This also applies to the
action of GnRH in GT1-7 cells. In conclusion, our results show that
GnRH causes rapid phosphorylation of ERK1/2 through transactivation of
the EGF-R. Such cross-regulation between the GnRH and EGF receptors
occurs through the rapid activation of PKC, Src, and Pyk2 by GnRH. Our
data support the view that agonist stimulation of neuronal GnRH
receptors induces the assembly of a multiprotein signaling complex that
includes PKC
/
, Src/Pyk2, and the EGF-R. A summary of the manner
in which GnRH causes phosphorylation of ERK1/2 in GT1-7 cells is shown
in Fig. 12.

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Fig. 12.
Schematic representation of the signaling
pathways involved in GnRH action in GT1-7 hypothalamic neurons.
GnRH-R stimulation leads to activation of
Gq/phospholipase C and translocation of PKC / to the
membrane. Agonist stimulation increases the association of PKC with Src
and that of Src with Pyk2. The activated Src-Pyk2 complex
associates with and activates EGF-R, leading to recruitment of adaptor
proteins, such as Shc-Grb2-SOS, and subsequent activation of the ERK1/2
cascade.
|
|
 |
FOOTNOTES |
*
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.
¶
To whom correspondence should be addressed: ERRB, NICHD, Bldg.
49, Rm. 6A36, National Institutes of Health, Bethesda, MD 20892-4510. Tel.: 301-496-2136; Fax: 301-480-8010; E-mail:
catt@helix.nih.gov.
Published, JBC Papers in Press, November 21, 2002, DOI 10.1074/jbc.M208783200
 |
ABBREVIATIONS |
The abbreviations used are:
GnRH, gonadotropin-releasing hormone;
Csk, C-terminal Src kinase;
EGF, epidermal growth factor;
EGF-R, epidermal growth factor receptor;
ERK1/2, extracellularly regulated MAPKs 1 and 2;
ERK1/2-P, phosphorylated ERK1/2;
GPCR, G protein-coupled receptor;
PMA, phorbol
12-myristate 13-acetate;
Pyk2, proline-rich tyrosine kinase;
PKM, dominant-negative Pyk2 mutant;
RTK, receptor tyrosine kinase;
PKC, protein kinase C;
FAK, focal adhesion kinase;
PBS, phosphate-buffered
saline;
bFGF, basic fibroblast growth factor;
HB-EGF, heparin-binding
EGF;
PP2, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine;
BAPTA, 1,2-bis (2-aminophenoxy)ethane-N,N,N',N'-tetraacetic
acid.
 |
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