Thyrotropin-Releasing Hormone Stimulates Phosphorylation of the Epidermal Growth Factor Receptor in GH3 Pituitary Cells
Ying-Hong Wang,
Shall F. Jue and
Richard A. Maurer
Department of Cell and Developmental Biology Oregon Health
Sciences University Portland, Oregon 97201
 |
ABSTRACT
|
---|
TRH has been found to stimulate tyrosine
phosphorylation of the epidermal growth factor (EGF) receptor. A
specific EGF receptor kinase inhibitor, tyrphostin AG1478,
substantially reduced TRH-stimulated tyrosine phosphorylation of the
EGF receptor. TRH-induced EGF receptor phosphorylation was found to
lead to the recruitment of the adapter proteins Grb2 and Shc. TRH
treatment also led to phosphorylation of the related receptor tyrosine
kinase, HER2. HER2 activation likely contributes to downstream
signaling events and enhances EGF receptor action. TRH-induced tyrosine
phosphorylation of the EGF receptor was reduced by incubation with a
protein kinase C (PKC) kinase inhibitor, GF109203X. EGF receptor
phosphorylation was required for full TRH-induced activation of
mitogen-activated protein kinase (MAPK) and stimulation of specific
transcriptional responses.
 |
INTRODUCTION
|
---|
The TRH receptor is a seven-transmembrane
Gq-coupled receptor (1). Activation of the
TRH receptor stimulates the activity of phospholipase Cß (2), leading
to the production of diacylglycerol and inositol 1,4,5-trisphosphate
(3). Increased intracellular levels of diacylglycerol and inositol
1,4,5 trisphosphate result in activation of protein kinase C (PKC) and
mobilization of intracellular calcium (4). TRH-induced signaling events
include activation of the mitogen-activated protein kinase (MAPK) in
both a PKC-dependent and PKC-independent manner (5, 6). While the
mechanisms that mediate TRH effects on MAPK activation have not been
completely determined, it has been observed that TRH induces rapid
tyrosine phosphorylation of Shc leading to activation of Ras (5). This
finding suggests that TRH-induced activation of MAPK may involve
activation of a tyrosine kinase, which in turn leads to activation of
Ras and the MAPK cascade. Thus the signaling pathways that allow the
Gq-coupled TRH receptor to activate MAPK may
overlap with the well studied pathways through which receptor tyrosine
kinases activate Ras and the MAPK cascade (7).
Interestingly, there is evidence that TRH and epidermal growth factor
(EGF) have overlapping activities in GH3 cells
(8, 9). Both EGF and TRH can stimulate PRL synthesis and inhibit GH
synthesis in GH3 cells (9). Long-term incubation
with either TRH or EGF can induce similar morphological changes in
GH3 cells (8, 9). The similar effects of TRH and
EGF in GH3 cells support the possibility that the
two signaling pathways may converge at some point.
In the present study we have further examined the possibility that TRH
may activate a tyrosine kinase in GH3 cells. We
report that TRH induces tyrosine phosphorylation of the EGF receptor.
Activation of the EGF receptor by TRH is accompanied by tyrosine
phosphorylation of the related receptor tyrosine kinase, HER2, as well
as the adapter protein, Shc. Blocking TRH-induced EGF receptor
activation was found to reduce activation of the MAPK cascade and
specific transcriptional events, demonstrating the necessary role of
the EGF receptor in mediating downstream effects of TRH.
 |
RESULTS
|
---|
TRH-Induced MAPK Activation Requires Ras
As a starting point for further analysis of the signaling pathways
mediating TRH effects on MAPK activation, we chose to examine the role
of Ras in mediating this response. Previous studies have shown that
both TRH and EGF can lead to a 2-fold increase in the percentage of Ras
that is bound with GTP (5). While these data strongly support a
possible role for Ras in mediating TRH-induced activation of MAPK, they
do not determine whether Ras is required for this response. To address
this question, GH3 cells were transiently
transfected with an expression vector for a dominant-negative, mutant
form of Ras (10) and epitope-tagged ERK2. The cells were treated with
TRH or EGF, and MAPK activity was determined by an immunocomplex kinase
assay. Transfection of the N17Ras mutant substantially reduced both
EGF- and TRH-induced MAPK activation (Fig. 1
). These findings offer evidence that
Ras is required for full TRH-induced MAPK activation.

View larger version (44K):
[in this window]
[in a new window]
|
Figure 1. TRH-Induced ERK Activation Is Ras Dependent
GH3 cells were transfected with 0.5 µg of an expression
vector for ERK2 tagged with a Myc epitope in the absence or presence of
1.5 µg of an expression vector for a dominant negative Ras (RasN17).
The cells were untreated (Control) or treated with 100 nM
TRH for 2.5 min or 10 nM EGF for 5 min. Myc-tagged ERK2 was
immunoprecipitated with the agarose-conjugated anti-Myc antibody and
assayed for ERK2 activity using
glutathione-S-transferase-Elk1 as a substrate. The
expression level of Myc-ERK2 was analyzed by immunoblotting Myc
immunoprecipitates with the anti-ERK2 antibody. This experiment has
been repeated twice with similar findings.
|
|
TRH Stimulates Tyrosine Phosphorylation of the EGF Receptor
and Recruitment of Adapter Proteins
A number of studies have shown that G protein-coupled receptors
can activate Ras and the MAPK cascade through activation of tyrosine
kinases (11, 12, 13, 14). As an initial step to explore possible regulation of
tyrosine kinase activity by TRH, we compared the effects of TRH and EGF
on phosphotyrosine-containing proteins in GH3
cell lysates. As expected, EGF treatment strongly increased the
phosphotyrosine content of several proteins (Fig. 2A
). The effects of TRH treatment were
more subtle, but TRH appeared to also stimulate an increase in the
phosphotyrosine content of several proteins. In particular, increased
phosphotyrosine content was detected for proteins of approximately 185,
170, and 66 kDa. These findings are consistent with an effect of TRH to
either activate a tyrosine kinase or inhibit a phosphotyrosine
phosphatase.

View larger version (35K):
[in this window]
[in a new window]
|
Figure 2. TRH Increases Phosphotyrosine Content of Several
Proteins in GH3 Cells
A, GH3 cells were serum-starved for 48 h and treated
with or without 100 nM TRH for 2.5 min. Cell lysates (100
µg protein) were resolved by denaturing PAGE and transferred to a
membrane. Phosphotyrosine-containing proteins were visualized by
immunoblotting with an antiphosphotyrosine antibody. B, GH3
cells were treated with TRH at varying times, and cell lysates were
prepared and immunoprecipitated with the antirat EGF receptor antibody
(provided by Dr. S. H. Earp). The immunoprecipitates were resolved
by denaturing PAGE, transferred to a membrane, and immunoblotted with
an antiphosphotyrosine antibody (P-Tyr Blot) and then reprobed with
anti-EGF receptor antibody (EGFR blot). These experiments have been
repeated at least three times with similar findings.
|
|
The TRH-inducible 170-kDa band comigrated with an EGF-inducible band.
As this is the appropriate size for the EGF receptor, this finding
suggests that TRH may stimulate phosphorylation of the EGF receptor. To
directly test this possibility, GH3 cells were
treated with TRH for varying times after which EGF receptor
phosphorylation was evaluated by immunoprecipitation of the receptor
followed by immunoblotting with antiphosphotyrosine antibody (Fig. 2B
).
TRH treatment was found to increase the phosphotyrosine content of the
EGF receptor at the earliest time point examined (2.5 min), and the
phosphotyrosine content was elevated for at least 1 h. Although
EGF was much more active than TRH in stimulating EGF receptor
phosphorylation, TRH stimulated an easily detectable increase in EGF
receptor phosphotyrosine content. As expected, EGF, but not TRH, was
able to stimulate EGF receptor phosphorylation in the Rat-1 cell line,
which does not contain TRH receptors (data not shown).
To investigate the nature of the TRH-induced tyrosine phosphorylation
of the EGF receptor, we tested whether a potent and specific inhibitor
of the EGF receptor, tyrophostin AG1478 (15, 16), had an effect on
TRH-induced EGF receptor tyrosine phosphorylation. AG1478 treatment
substantially reduced TRH-induced tyrosine phosphorylation of the EGF
receptor (Fig. 3A
). As AG1478 interacts
with the ATP binding site on the receptor (15, 16), this finding
suggests that TRH stimulates receptor autophosphorylation. To further
investigate whether TRH-induced tyrosine phosphorylation of the EGF
receptor reflects activation of the receptors intrinsic kinase
activity, we performed a kinase assay (17, 18) on EGF receptor
immunoprecipitates. TRH stimulated EGF receptor kinase activity, and
the TRH-induced increase in kinase activity was blocked by AG1478
treatment (Fig. 3B
). Similar results were obtained when EGF receptor
autophosphorylation was examined (data not shown).

View larger version (46K):
[in this window]
[in a new window]
|
Figure 3. TRH Stimulates EGF Receptor Intrinsic Kinase
Activity
A, GH3 cells were pretreated with 250 nM AG1478
or with the solvent used for AG1478 [dimethylsulfoxide (DMSO)] for 20
min and then treated with 100 nM TRH for varying times.
Cell lysates were immunoprecipitated with the antirat EGF receptor
antibody. The immunoprecipitates were resolved by denaturing PAGE,
transferred to a membrane, and immunoblotted with an
antiphosphotyrosine antibody (P-Tyr Blot) and then reprobed with
anti-EGF receptor antibody (EGFR blot). B, Analysis of TRH effects on
EGF receptor kinase activity. GH3 cells were treated with
100 nM TRH for 2.5 min. The EGF receptor was
immunoprecipitated and then assayed for kinase activity using myelin
basic protein as a substrate. These experiments have been repeated at
least twice with similar findings.
|
|
Recruitment and phosphorylation of adapter proteins are key events that
are essential for mediating the ability of the EGF receptor to activate
the Ras/MAPK pathway (19, 20, 21). Treatment of GH3
cells with TRH or EGF resulted in the apparent interaction of the
tyrosine-phosphorylated EGF receptor with a
Grb2-glutathione-S-transferase fusion protein (Fig. 4A
). As observed for TRH-induced
phosphorylation of the EGF receptor, TRH was much less effective than
EGF treatment in stimulating the interaction of Grb2 and the EGF
receptor. We also examined phosphorylation of Shc (Fig. 4B
). After TRH
treatment, tyrosine-phosphorylated proteins of 185, 66, and 52 kDa were
immunoprecipitated with antisera to Shc (Fig. 4B
). The
52- and 66-kDa proteins represent isoforms of Shc,
consistent with previous studies demonstrating TRH-induced tyrosine
phosphorylation of Shc (5). The 185-kDa phosphoprotein that was
coimmunoprecipitated likely represents the HER2 receptor tyrosine
kinase (see below). The tyrosine phosphorylation of the 185-, 66-, and
52-kDa proteins was greatly reduced by treatment with AG1478.

View larger version (45K):
[in this window]
[in a new window]
|
Figure 4. TRH Treatment Leads to Recruitment and
Phosphorylation of Adapter Proteins
A, TRH induces association between the EGF receptor and Grb2.
GH3 cells were treated with 100 nM TRH for 2.5
min or 10 nM EGF for 10 min. Cell lysates were incubated
with a resin containing a Grb2-glutathione-S-transferase
fusion protein. After washing, the resin was resolved by denaturing gel
electrophoresis, transferred to a membrane, and incubated with an
antiphosphotyrosine antibody (P-Tyr Blot). A phosphotyrosine-containing
protein with the appropriate size for the EGF receptor was detected as
shown. B, TRH-induced association with Shc. GH3 cells were
treated as above. Cell lysates were immunoprecipitated with 4 µg of
anti-Shc antibody and immunoblots probed with the antiphosphotyrosine
antibody (P-Tyr blot) and reprobed with the anti-Shc antibody (Shc
blot). These experiments have been repeated at least twice with similar
findings.
|
|
TRH Treatment Leads to Tyrosine Phosphorylation of HER2
In general, EGF receptor signaling is rapidly down-regulated due
to receptor-mediated endocytosis (22). In contrast, TRH-induced
activation of the EGF receptor persists for approximately 1 h,
implying that some event modulates the time course of activation. HER2,
also known as ErbB2 or neu, is a member of the ErbB family of receptor
tyrosine kinases and is most closely related to the EGF receptor
(ErbB1) (23). HER2 can form heterodimers with the EGF receptor and
enhance EGF-induced tyrosine phosphorylation of the EGF receptor and
potentiate and prolong EGF-induced signal transduction (24, 25, 26, 27, 28). To
explore the possibility that HER2 may play a role in TRH-induced EGF
receptor activation, we examined TRH effects on HER2 phosphorylation.
Treatment with TRH resulted in an increase in the phosphotyrosine
content of HER2, which persisted for at least 30 min (Fig. 5A
). To determine whether TRH-induced
tyrosine phosphorylation of HER2 was mediated by the EGF receptor, we
treated GH3 cells with the specific EGF receptor inhibitor, AG1478. At
a concentration of 250 nM, AG1478 strongly inhibited
TRH-induced tyrosine phosphorylation of HER2 (Fig. 5B
). As AG1478
inhibits HER2 only at much higher concentrations (>100
µM) (16), these data suggest that TRH-induced HER2
activation depends on activation of the EGF receptor. These findings
provide evidence that TRH induces HER2 phosphorylation in
GH3 cells, and the activation of HER2 may
contribute to prolonged phosphorylation of the EGF receptor.

View larger version (47K):
[in this window]
[in a new window]
|
Figure 5. TRH Treatment Stimulates HER2 Phosphophorylation
A, GH3 cells were treated with TRH for different times.
Cell lysates were immunoprecipitated with an anti-HER2 antibody, and
the proteins were separated by denaturing gel electrophoresis and
transferred to a membrane. Phosphotyrosine-containing proteins were
detected by immunoblotting with the antiphosphotyrosine antibody (P-Tyr
blot) and reprobed with an anti-HER2 antibody. B, TRH-induced HER2
activation is reduced by AG1478. Cells were pretreated with 250
nM AG1478 or the solvent used for AG1478 (DMSO) for 20 min
and then treated with 100 nM TRH or 10 nM EGF.
Cell lysates (1 mg) were immunoprecipitated with 4 µg of the
anti-HER2 antibody. Proteins were separated by denaturing gel
electrophoresis and transferred to a membrane.
Phosphotyrosine-containing proteins were detected by immunoblotting
with the antiphosphotyrosine antibody (P-Tyr blot) and reprobed with an
anti-HER2 antibody. These experiments have been repeated at least twice
with similar findings.
|
|
TRH-Stimulated Tyrosine Phosphorylation of the EGF Receptor
Requires PKC
Activation of phospholipase Cß by the TRH receptor leads to
increased intracellular concentration of diacylglycerol and subsequent
activation of PKC. To determine whether PKC lies in the pathway between
the TRH receptor and the EGF receptor, we pretreated cells with
GF109203X, a specific PKC inhibitor (29). Treatment of
GH3 cells with the PKC inhibitor reduced the
subsequent TRH-induced tyrosine phosphorylation of the EGF receptor
(Fig. 6
). Thus, PKC activation appears to
be necessary for TRH-induced EGF receptor transactivation.

View larger version (39K):
[in this window]
[in a new window]
|
Figure 6. TRH-Induced Phosphorylation of the EGF Receptor Is
PKC Dependent
GH3 cells were pretreated for 20 min with 1
µM GF109203X or the solvent used for the drug (DMSO). The
cells were then stimulated with 100 nM TRH for 2.5 min.
Tyrosine phosphorylation of the EGF receptor was analyzed by
immunoprecipitating with anti-EGF receptor antibody and then
immunoblotting with the antiphosphotyrosine antibody (P-Tyr blot) and
reprobing with EGF receptor antibody (EGFR blot). This experiment has
been repeated twice with similar findings.
|
|
Previous studies have shown that activation of Src family nonreceptor
tyrosine kinases by Gi-coupled receptors can
account for tyrosine phosphorylation of both the EGF receptor and Shc
(14). To study the mechanism of TRH-induced tyrosine phosphorylation of
the EGF receptor, we pretreated the cells with PP1, an inhibitor, which
is specific for the Src family tyrosine kinases (30). Although PP1
reduced the phosphotyrosine content of several proteins, it had little
effect on tyrosine phosphorylation of the EGF receptor (data not
shown). These results suggest that tyrosine phosphorylation of the EGF
receptor by TRH is not mediated by PP1-sensitive tyrosine kinases.
EGF Receptor Activity Is Required for TRH-Induced Activation of
MAPK and Specific Transcription
To test the role that EGF receptor phosphorylation plays in
mediating TRH effects on MAPK activation, we examined the effects of
AG1478 on MAPK activation. As determined by immunoblotting with an
anti-phospho-ERK antibody, TRH-induced MAPK activation was reduced by
pretreatment with AG1478 (Fig. 7A
). To
further examine the role that the EGF receptor plays, we used an
expression vector for a kinase-defective mutant of the EGF receptor
(HERK721A) (31). The HERK721A expression vector was transfected into
GH3 cells with an expression vector for
Myc-tagged ERK2, and ERK2 activity was then determined by an
immunocomplex assay (Fig. 7B
). TRH-induced ERK2 activation was
substantially reduced by the HERK721A expression vector.

View larger version (33K):
[in this window]
[in a new window]
|
Figure 7. EGF Receptor Activity Is Required for TRH-Induced
Activation of MAPK
A, The EGF receptor inhibitor, AG1478, reduces TRH-induced ERK
activation. GH3 cells were pretreated with 250
nM AG1478 for 20 min and treated with TRH for 2.5 min. ERK
activation was analyzed by immunoblotting with antiphospho-ERK
antibody. The expression level of ERKs was examined by reprobing the
membrane with anti-ERK1 antibody. B, An expression vector for a
kinase-defective EGF receptor mutant reduces TRH-induced ERK
activation. GH3 cells were transfected with an expression
vector for Myc-tagged ERK2 in the absence or presence of a vector for a
kinase-defective mutant of the EGF receptor (HERK721A). Cells were
treated with TRH or EGF. Cell lysates were immunoprecipitated with
antibody to the Myc epitope, and the immunocomplexes were assayed for
kinase activity. The expression level of Myc-ERK2 was analyzed by
immunoblotting the Myc immunoprecipitates with anti-ERK2 antibody.
These experiments have been repeated at least twice with similar
findings.
|
|
To determine the downstream importance of TRH-induced tyrosine
phosphorylation of the EGF receptor, we examined the effects of the
HERK721A vector on specific transcriptional responses to TRH. Previous
studies have shown that activation of the MAPK pathway is essential for
TRH effects on the transcriptional activity of a GAL-Elk1 fusion
protein as well as full induction of the PRL promoter (6). Transfection
of an expression vector for the kinase-defective EGF receptor
substantially reduced TRH-induced activation of Gal4-Elk1 activity
(Fig. 8A
) and more modestly reduced
induction of the PRL promoter (Fig. 8B
). As expected, neither TRH nor
the expression vector for the mutant EGF receptor affected the activity
of thymidine kinase reporter gene (Fig. 8C
). These data indicate that
the EGF receptors kinase activity is required for the full induction
of transcriptional responses by TRH. The partial inhibition on the PRL
promoter also suggests that TRH-induced EGF receptor phosphorylation is
not the only pathway that contributes to TRH-induced activation of this
transcriptional response.

View larger version (15K):
[in this window]
[in a new window]
|
Figure 8. EGF Receptor Kinase Activity Is Required for
TRH-Induced Transcriptional Events
GH3 cells were transfected with an empty expression vector
(control) or an expression vector for a kinase-defective mutant of the
EGF receptor (HERK721A). The cells also received an expression vector
for GAL4-Elk1 and a reporter gene containing 5 GAL4 binding sites (A)
or reporter genes containing the rat PRL promoter (B) or the herpes
simplex virus thymidine kinase promoter (C) linked to luciferase. All
cells also received a vector directing expression of
Renilla luciferase as an internal standard. Forty-eight
hours after transfection, the cells were treated with 100
nM TRH or 10 nM EGF and then collected for
luciferase assays 6 h later. All values are means ±
SEM for three separate transfections, which have been
corrected for transfection efficiency. This experiment has been
repeated three times with similar findings.
|
|
 |
DISCUSSION
|
---|
These studies provide evidence that TRH-induced phosphorylation
and activation of the EGF receptor play a role in activation of the
MAPK cascade leading to specific transcriptional events. An increasing
body of work has shown that tyrosine kinases may play a role as
downstream components of some G protein-coupled receptor pathways
(32, 33, 34, 35). In the present study, we demonstrate that TRH can stimulate
tyrosine phosphorylation of the EGF receptor in a time-dependent
manner. The use of specific inhibitors has provided evidence that
TRH-induced activation of the EGF receptor is required for several
downstream events including phosphorylation of adapter proteins,
activation of the MAPK pathway, and stimulation of Elk1 transcriptional
activation and full activation of PRL gene transcription.
Interestingly, very recent studies have provided evidence that GnRH
effects in cells of the gonadotrope lineage also involve the ability of
a Gq-coupled receptor to stimulate EGF receptor
phosphorylation leading to MAPK activation (36).
The ability of TRH to activate the EGF receptor appears to involve
autophosphorylation of the receptor. TRH-induced tyrosine
phosphorylation of the EGF receptor and the downstream activation of
MAPKs are inhibited by tyrphostin AG1478, which specifically inhibits
the EGF receptors kinase activity by competing for ATP binding.
Similar results were obtained when a kinase-inactive mutant of the EGF
receptor was used to block receptor signaling. AG1478 was also found to
reduce TRH-induced tyrosine phosphorylation of HER2 and Shc. These
findings provide evidence that TRH-induced activation of the EGF
receptor and downstream signaling events share many similarities with
EGF-induced activation of its receptor (19, 20, 21). Consistent with the
view that the TRH signaling pathway involves the EGF receptor
positioned upstream of the Ras-dependent MAPK pathway, expression of a
dominant-negative Ras mutant reduced TRH-induced MAPK activation.
TRH-induced phosphorylation of the EGF receptor is accompanied by
phosphorylation of the related receptor tyrosine kinase, HER2. HER2 is
a preferred partner of all members of the ErbB family (37), and HER2
can enhance signaling by the EGF receptor in several ways. HER2 can
increase the duration of EGF-induced tyrosine phosphorylation of the
EGF receptor by slowing the relatively fast endocytosis rate of the EGF
receptor (38). HER2 is very efficient in coupling to the MAPK pathway
(38), and HER2 can potentiate EGF-induced MAPK activation by recruiting
different SH2-containing substrates to the heterodimer complexes (27, 39). As the EGF receptor inhibitor, AG1478, reduces both TRH-induced
HER2 phosphorylation and MAPK activation, it is possible that HER2
contributes to the ability of TRH to regulate MAPK activity.
The molecular events that lead to TRH-induced phosphorylation of the
EGF receptor are unclear, although they appear to involve PKC. A
requirement for PKC activity has been shown by the ability of the PKC
inhibitor, GF109203X, to attenuate TRH-induced tyrosine phosphorylation
of the EGF receptor. However, the manner in which PKC contributes to
activation of the EGF receptor is unclear. It has been shown that PKC
can directly phosphorylate the EGF receptor on threonine 654 (40, 41).
However, this does not lead to EGF receptor activation but rather
inhibits tyrosine kinase activity of the receptor and reduces
high-affinity EGF binding (41, 42, 43). It may be that specific PKC
isozymes have different effects on the EGF receptor that account for
this apparent discrepancy. It is also possible that PKC may activate
the EGF receptor through inactivating a protein tyrosine phosphatase.
It has been shown that radiation, oxidants, and alkylating agents can
alter the phosphorylation of receptor tyrosine kinases through effects
on protein phosphatases (44). Recent studies have provided evidence
that the ability of G protein-coupled receptors to stimulate EGF
receptor phosphorylation may involve cleavage of heparin-binding
EGF-like growth factor (45). Cleavage of the heparin-binding EGF-like
growth factor by a metalloproteinase at a juxtamembrane site releases
an active form of the molecule (46). PKC (47) may play a role in
stimulating cleavage and activation of heparin-binding EGF-like growth
factor. We have found that 1,10-phenanthrolene, a metal chelator and
general inhibitor of metalloproteinases, reduces the ability of TRH to
activate MAPK (S. F. Jue, unpublished observations), suggesting
that cleavage of heparin-binding EGF-like growth factor may play a role
in mediating TRH-induced phosphorylation of the EGF receptor.
Although TRH and EGF share many signaling events in common, there
appears to be a substantial quantitative difference in the activation
of specific signaling steps. For instance, EGF has a much greater
effect than TRH on phosphorylation of the EGF receptor, HER2, Shc, and
Grb2. Nonetheless, TRH effects on EGF receptor phosphorylation are
clearly important as the dominant negative kinase-defective EGF
receptor, HERK721A, substantially reduced TRH-induced MAPK
activation as well as transcriptional responses. What accounts
for this apparent discrepancy between weak signal activation and an
important functional role? One aspect may involve an additive or
synergistic effect of multiple TRH-induced signaling events. For
instance, TRH treatment results in Ca2+ influx
through voltage-gated Ca2+ channels (48). Our
previous studies have provided evidence that Ca2+
influx contributes to MAPK activation in GH3
cells (6). It is possible that TRH initiates multiple signaling
pathways including EGF receptor phosphorylation and
Ca2+ influx that converge on MAPK activation.
The present findings provide new insights into the overlapping
biological activities of EGF and TRH in GH3
cells. Both EGF and TRH stimulate PRL synthesis (8, 9) and PRL gene
transcription (49, 50). TRH and EGF effects on expression of the PRL
gene are mediated via multiple, common DNA elements (51). Previous
studies have shown that both EGF and TRH can induce tyrosine
phosphorylation of Shc and activate the MAPK pathway (5). The present
study reveals a new level of signal convergence, which occurs at the
level of the EGF receptor. As both TRH and EGF activate the EGF
receptor and several common downstream signaling steps, it is not
surprising that TRH and EGF share some biological actions.
 |
MATERIALS AND METHODS
|
---|
Materials
Cell culture media and supplies were purchased from Life Technologies, Inc. (Gaithersburg, MD). Radioisotopes and
enhanced chemiluminescent reagents were purchased from DuPont New
England Nuclear (Boston, MA). Anti-ERK polyclonal antibodies,
anti-phospho-ERK monoclonal antibody, antiphosphotyrosine monoclonal
antibody, antihuman EGF receptor polyclonal antibody, anti-neu
polyclonal antibody, agarose-conjugated anti-Myc antibody,
agarose-conjugated glutathione-S-transferase-Grb2, and
horseradish peroxidase-coupled secondary antibodies were obtained from
Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Anti-Shc
polyclonal antibody was purchased from Transduction Laboratories, Inc. (Lexington, KY). Antirat EGF receptor antibodies were
kindly provided by Dr Shelton Earp (University of North Carolina at
Chapel Hill) or purchased from Calbiochem (La Jolla, CA).
The PKC inhibitor, GF109203X, and Src-like tyrosine kinase inhibitor,
PP1, were purchased from Calbiochem. AG1478 was from
Research Biochemicals International (Natick, MA); EGF was
obtained from Roche Molecular Biochemicals (Indianapolis,
IN). TRH was purchased from Peninsula Laboratories, Inc.
(Belmont, CA).
Cell Culture and Transfections
GH3 cells were cultured in DMEM
supplemented with 15% horse serum and 2.5% FBS, 100 U/ml penicillin,
and 100 µg/ml streptomycin. For transient transfection assays, 2.5 or
5 x 105 cells per well were subcultured in
six-well plates 1 day before the transfection. DNA was transfected into
these cells using lipofectamine (Life Technologies, Inc.)
according to a protocol provided by the manufacturer. In each
experiment, the total amount of transfected DNA per well was maintained
as a constant (usually 2 µg) by addition of empty expression vector
(either pCDNA3a or pRK5). After 5 h of treatment with the
lipofectamine/DNA mixture, an equal volume of serum-containing culture
medium was added. After an additional 18 h, the cells were
transferred to serum-free medium for a further 24 h before lysis
and analysis.
For reporter gene assays, two different reporters that can be assayed
independently were used in each experiment. Firefly luciferase
constructs (52) were used to assess transcriptional activation. A
reporter gene containing five Gal4 binding sites upstream of a minimal
promoter (53) was used to examine transcriptional activation of a
GAL4-Elk1 fusion protein (54). Firefly luciferase reporter genes
containing the proximal 255 bp of the 5'-flanking region of the PRL
gene (6) or a minimal herpes simplex virus thymidine kinase promoter
(55) have been reported previously. To correct for transfection
efficiency, the cells were also transfected with pRL, which expresses
Renilla luciferase. Renilla luciferase requires a
different substrate than the firefly luciferase and can be assayed
independently. The activities of firefly and Renilla
luciferase were determined using a protocol and reagents from
Promega Corp. (Madison, WI). Total firefly luciferase
light units were normalized to total Renilla luciferase activity.
Immunoprecipitation and Immunoblotting
Cells were lysed for 10 min on ice in lysis buffer [150
mM NaCl, 20 mM Tris, pH 7.5, 1% Triton X-100,
1 mM EDTA, 50 mM ß-glycerolphosphate, 10
mM NaF and 10% (vol/vol) glycerol] with freshly added 1
mM Na3VO4,and
1x Complete proteinase inhibitor (Roche Molecular Biochemicals). The cell lysates were centrifuged for 10 min at
12,000 x g. For immunoprecipitation experiments, 1 mg
of cell lysate was immunoprecipitated by incubation with the
appropriate antibody overnight at 4 C. Twenty microliters of
protein A/G agarose beads were then added for an additional 45 min.
Immune complexes were washed three times with the lysis buffer and
resuspended in SDS sample buffer [62.5 mM
Tris-HCl, pH 6.8, 2% (wt/vol) sodium dodecyl sulfate, 10% (vol/vol)
glycerol, 5% (vol/vol) ß-mercaptoethanol, and 0.05% (wt/vol)
bromophenol blue]. For immunoblotting, samples were resolved by
denaturing-polyacrylamide gel electrophoresis, transferred to a
polyvinylidene difluoride membrane, and incubated with the selected
antibody. Immunoblots were developed with enhanced chemiluminescent
reagents (DuPont New England Nuclear). For reprobing of immunoblots,
the membrane was stripped in 62.5 mM Tris, pH
6.8, 2% SDS, and 100 mM ß-mercaptoethanol at
50 C for 30 min.
Immunocomplex Kinase Assay
Immunoprecipitates were prepared as described above. For the ERK
kinase assay, immunoprecipitates were washed once in the ERK kinase
assay buffer (20 mM Tris pH 7.5, 10 mM
MgCl2, 0.1% Triton X-100, and 2 mM
EGTA). The kinase assays were carried out for 25 min at 30 C in 20 µl
of ERK kinase assay buffer supplemented with 1 µCi of
[
-32P] ATP and 5 µg
glutathione-S-transferase fusion with the carboxy-terminal
region of Elk1 (6). For the EGF receptor kinase assay, the
immunoprecipitates were washed once with EGF receptor kinase buffer (20
mM HEPES, pH 7.3, 10 mM
MnCl2, 1 mM dithiothreitol,
0.2 mM
Na3VO4). The kinase assays
were carried out for 10 min at 30 C in 20 µl of the EGFR kinase assay
buffer in the presence of 5 µCi of [
-32P]
ATP. For measuring the EGF receptors kinase activity by using an
exogenous substrate, 5 µg of myelin basic protein for each sample
were added to the reaction mixture. The kinase reactions were stopped
by adding SDS sample buffer, and the reactions were resolved on a
denaturing polyacrylamide gel. Phosphorylated proteins were detected by
autoradiography and quantitated using a PhosphorImager (Molecular Dynamics, Inc., Sunnyvale, CA).
 |
ACKNOWLEDGMENTS
|
---|
We thank Dr. Shelton H. Earp for the antirat EGF receptor
polyclonal antibody, Dr. Mihail Iordanov for the kinase-defective EGF
receptor construct, Dr. Geoffrey Cooper for the dominant-negative Ras
construct, and Dr. Christopher Marshall for the myc-ERK2
construct. We also thank Bobbi Maurer for assistance and aid in
preparing this manuscript.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Richard A. Maurer, Department of Cell and Developmental Biology, L215 Oregon Health Sciences University, 3181 Southwest Sam Jackson Park Road, Portland, Oregon 97201.
This research was supported by NIH Grant DK-40339 to R.A.M.
Received for publication March 24, 2000.
Revision received May 12, 2000.
Accepted for publication May 23, 2000.
 |
REFERENCES
|
---|
-
Gershengorn MC, Osman R 1996 Molecular and cellular
biology of thyrotropin-releasing hormone receptors. Physiol Rev 76:175191[Abstract/Free Full Text]
-
Aragay AM, Simon MI 1992 The G
q
and G
11 proteins couple the
thyrotropin-releasing hormone receptor to phospholipase C in
GH3 rat pituitary cells. J Biol Chem 267:2498324988[Abstract/Free Full Text]
-
Gershengorn MC 1985 Thyrotropin-releasing hormone action:
mechanism of calcium-mediated stimulation of prolactin secretion.
Recent Prog Horm Res 41:607653[Medline]
-
Gershengorn MC, Thaw C 1985 Thyrotropin-releasing hormone
(TRH) stimulates biphasic elevation of cytoplasmic free calcium in GH3
cells. Further evidence that TRH mobilizes cellular and extracellular
Ca2+. Endocrinology 116:591596[Abstract]
-
Ohmichi M, Sawada T, Kanda Y, Koike K, Hirota K, Miyake A,
Saltiel AR 1994 Thyrotropin-releasing hormone stimulates MAP kinase
activity in GH3 cells by divergent pathways. Evidence of a role for
early tyrosine phosphorylation. J Biol Chem 269:37833788[Abstract/Free Full Text]
-
Wang Y-H, Maurer RA 1999 A role for the mitogen-activated
protein kinase in mediating the ability of thyrotropin-releasing
hormone to stimulate the prolactin promoter. Mol Endocrinol 13:10941104[Abstract/Free Full Text]
-
Schlessinger J, Ullrich A 1992 Growth factor signaling by
receptor tyrosine kinases. Neuron 9:383391[Medline]
-
Hapgood J, Libermann TA, Lax I, Yarden Y, Schreiber AB, Naor
Z, Schlessinger J 1983 Monoclonal antibodies against epidermal growth
factor receptor induce prolactin synthesis in cultured rat pituitary
cells. Proc Natl Acad Sci USA 80:64516455[Abstract]
-
Johnson LK, Baxter JD, Vlodavsky I, Gospodarowicz D 1980 Epidermal growth factor and expression of specific genes: effects on
cultured rat pituitary cells are dissociable from the mitogenic
response. Proc Natl Acad Sci USA 77:394398[Abstract]
-
Feig LA, Cooper GM 1988 Inhibition of NIH 3T3 cell
proliferation by a mutant ras protein with preferential
affinity for GDP. Mol Cell Biol 8:32353243[Medline]
-
van Biesen T, Hawes BE, Luttrell DK, Krueger KM, Touhara K,
Porfiri E, Sakaue M, Luttrell LM, Lefkowitz RJ 1995 Receptor-tyrosine-kinase- and Gß
-mediated MAP kinase
activation by a common signaling pathway. Nature 376:781784[CrossRef][Medline]
-
Luttrell LM, Hawes BE, van Biesen T, Luttrell DK, Lansing TJ,
Lefkowitz RJ 1996 Role of c-Src tyrosine kinase in G protein-coupled
receptor- and Gß
subunit-mediated activation of
mitogen-activated protein kinases. J Biol Chem 271:1944319450[Abstract/Free Full Text]
-
Della Rocca GJ, van Biesen T, Daaka Y, Luttrelll DK, Luttrell
LM, Lefkowitz RJ 1997 Ras-dependent mitogen-activated protein kinase
activation by G protein-coupled receptors. Convergence of G1 and
Gq-mediated pathways on calcium/calmodulin Pyk2,
and Src kinase. J Biol Chem 272:1912519132[Abstract/Free Full Text]
-
Luttrell LM, Della Rocca GJ, van Biesen T, Luttrell DK,
Lefkowitz RJ 1997 Gß
subunits mediate Src-dependent
phosphorylation of the epidermal growth factor receptor. A scaffold for
G protein-coupled receptor-mediated Ras activation. J Biol Chem 272:46374644[Abstract/Free Full Text]
-
Fry DW, Kraker AJ, McMichael A, Ambroso LA, Nelson JM, Leopold
WR, Connors RW, Bridges AJ 1994 A specific inhibitor of the epidermal
growth factor receptor tyrosine kinase. Science 265:10931095[Medline]
-
Levitzki A, Gazit A 1995 Tyrosine kinase inhibiton: an
approach to drug development. Science 267:17821788[Medline]
-
Wang Q, Smith JB, Harrison ML, Gaehlan RL 1991 Identification
of tyrosine 67 in bovine brain myelin basic protein as a specific
phosphorylation site for thymus p561ck. Biochem Biophys Res Commun 178:13931399[Medline]
-
Moro L, Venturino M, Bozzo C, Silengo L, Altruda F, Beguinot
L, Tarone G, Defilippi P 1998 Integrins induce activation of EGF
receptor: role in MAP kinase induction and adhesion-dependent cell
survival. EMBO J 17:66226632[Abstract/Free Full Text]
-
Rozakis-Adcock M, McGlade J, Mbamalu G, Pelicci G, Daly R, Li
W, Batzer A, Thomas S, Brugge J, Pelicci PG, Schlessinger J, Pawson T 1992 Association of the Shc and Grb2/Sem5 SH2-containing proteins is
implicated in activation of the Ras pathway by tyrosine kinases. Nature 360:689692[CrossRef][Medline]
-
Buday L, Downward J 1993 Epidermal growth factor regulated
p21ras through the formation of complex of
receptor, Grb2 adapter protein, and Sos nucleotide exchange factor.
Cell 73:611620[Medline]
-
Pelicci G, Lanfrancone L, Grignani F, McGlad J, Cavallo F,
Forni G, Nicoletti I, Grignani F, Pawson T, Pelicci PG 1992 A novel
transforming protein (SHC) with an SH3 domain is implicated in
mitogenic signal transduction. Cell 70:93104[Medline]
-
Baulida J, Kraus MH, Alimandi M, Di Fiore PP, carpenter G 1996 All ErbB receptors other than the epidermal growth factor receptor are
endocytosis impaired. J Biol Chem 271:52515257[Abstract/Free Full Text]
-
Tzahar E, Yarden Y 1998 The ErbB-2/HER2 oncogenic receptor of
adenocarcinomas: from orphanhood to multiple stromal ligands. Biochim
Biophys Acta 1377:M25M37
-
Wada T, Qian X, Greene MI 1990 Intermolecular association of
the p185neu protein and EGF receptor modulates
EGF receptor function. Cell 61:13391347[Medline]
-
Pinkas-Kramarski R, Soussan L, Waterman H, Kevkowitz G, Alroy
I, Klapper L, Lavi S, Seger R, Ratzkin BJ, Sela M, Yarden Y 196
Diversification of Neu differentiation factor and epidermal growth
factor signaling by combinatorial receptor interactions. EMBO J 15:24512467
-
Kokai Y, Myers JN, Wada T, Brown VI, LeVea CM, Davis JG,
Dobashi K, Greene MI 1989 Synergistic interaction of p185c-neu and the
EGF receptor leads to transformation of rodent fibroblasts. Cell 58:287292[Medline]
-
Graus-Porta D, Beerli RR, Hynes NE 1995 Single-chain
antibody-mediated intracellular retention of ErbB-2 impairs Neu
differentiation factor and epidermal growth factor signaling. Mol Cell
Biol 15:11821191[Abstract]
-
Karunagaran D, Tazahar E, Beerli RR, Chen X, Graus-Porta D,
Ratzkin BJ, Seger R, Hynes NE, Yarden Y 1996 ErbB-2 is a common
auxiliary subunit of NDF and EGF receptors: implications for breast
cancer. EMBO J 15:254264[Abstract]
-
Toullec D, Pianetti P, Coste H, Bellevergue P, Grand-Perret T,
Ajakane M, Baudet V, Boissin P, Boursier E, Loriolle F, Duhamel L,
Charon D, Kirilovsky J 1991 The bisindolylmaleimide GF 109203X is a
potent and selective inhibitor of protein kinase C. J Biol Chem 266:1577115781[Abstract/Free Full Text]
-
Hanke JH, Gardner JP, Dow RL, Changelian PS, Brissette WH,
Weringer EJ, Pollok BA, Connelly PA 1996 Discovery of a novel, potent,
and Src family-selective tyrosine kinase inhibitor. Study of Lck- and
FynT-dependent T cell activation. J Biol Chem 271:695701[Abstract/Free Full Text]
-
Redemann N, Holzmann B, von Rüden T, Wagner EF,
Schlessinger J, Ullrich A 1992 Anti-oncogenic activity of
signalling-defective epidermal growth factor receptor mutants. Mol Cell
Biol 12:491498[Abstract]
-
Wan Y, Kurosaki T, Huang X-Y 1996 Tyrosine kinases in
activation of the MAP kinase cascade by G-protein-coupled
receptors. Nature 380:541544[CrossRef][Medline]
-
Lev S, Moreno H, Martinez R, Canoll P, Peles E, Musacchio JM,
Plowman GD, Rudy B, Schlessinger J 1995 Protein tyrosine kinase PYK2
involved in Ca2+-induced regulation of ion
channel and MAP kinase functions. Nature 376:737745[CrossRef][Medline]
-
Dikic I, Tokiwa G, Lev S, Courtneidge SA, Schlessinger J 1996 A role for Pyk2 and Src in linking G-protein-coupled receptors with MAP
kinase activation. Nature 383:547550[CrossRef][Medline]
-
Daub H, Weiss FU, Wallasch C, Ullrich A 1996 Role of
transactivation of the EGF receptor in signalling by G-protein-coupled
receptors. Nature 379:557570[CrossRef][Medline]
-
Grosse R, Roelle S, Herrlich A, Höhn J, Gudermann T 2000 Epidermal growth factor receptor tyrosine kinase mediates Ras
activation by gonadotropin-releasing hormone. J Biol Chem 275:1225112260[Abstract/Free Full Text]
-
Graus-Porta D, Beerli RR, Daly JM, Hynes NE 1997 ErB-2, the
preferred heterodimerization partner of all ErbB receptors, is a
mediator of lateral signaling. EMBO J 16:16471655[Abstract/Free Full Text]
-
Lenferink AEG, Pinkas-Kramarski R, van de Poll MLM, van Vugt
MJH, Waterman H, Sela M, van Zoelen EJJ, Yarden Y 1998 Differential
endocytic routing of homo- and hetero-dimeric ErbB tyrosine kinases
confers signaling superiority to receptor heterodimers. EMBO J 17:33853397[Abstract/Free Full Text]
-
Ben-Levy R, Paterson HF, Marshall CJ, Yarden Y 1994 A single
autophosphorylation site confers oncogenicity to the Neu/ErbB-2
receptor and enables coupling to the MAP kinase pathway. EMBO J 13:33023311[Abstract]
-
Davis RJ, Czech MP 1985 Platelet-derived growth factor mimics
phorbol diester action on epidermal growth factor receptor
phosphorylation at threonine-654. Proc Natl Acad Sci USA 82:40804084[Abstract]
-
Hunter T, Ling NC, Cooper JA 1984 Protein kinase C
phosphorylation of the EGF receptor at a threonine residue close to the
cytoplasmic face of the plasma membrane. Nature 311:480483[Medline]
-
Livneh E, Dull TJ, Berent E, Prywes R, Ullrich A, Schlessinger
J 1988 Release of a phorbol ester-induced mitogenic block by mutation
at Thr-654 of the epidermal growth factor receptor. Mol Cell Biol 8:23022308[Medline]
-
Friedman BA, Frackelton Jr AR, Ross AH, Connors JM, Fujiki H,
Sugimura T, Rosner MR 1984 Tumor promoters block tyrosine-specific
phosphorylation of the epidermal growth factor receptor. Proc Natl Acad
Sci USA 81:30343038[Abstract]
-
Knebel A, Rahmsdorf J, Ullirch A, Herrlich P 1996 Dephosphorylation of receptor tyrosine kinases as target of regulation
by radiation, oxidants or alkylating agents. EMBO J 18:53145325
-
Prenzel N, Zwick E, Daub H, Leserer M, Abraham R, Wallasch C,
Ullrich A 1999 EGF receptor transactivation by G-protein-coupled
receptors requires metalloproteinase cleavage of proHB-EGF. Nature 402:884888[CrossRef][Medline]
-
Suzuki M, Raab G, Moses MA, Fernandez CA, Klagsbrun M 1997 Matrix metalloproteinase-3 releases active heparin-binding EGF-like
growth factor by cleavage at a specific juxtamembrane site. J Biol
Chem 272:3173031737[Abstract/Free Full Text]
-
Izumi Y, Hirata M, Hasuwa H, Iwamoto R, Umata T, Miyado K,
Tamai Y, Kurisaki T, Sehara-Fujisawa A, Ohno S, Mekada E 1998 A
metalloprotease-disintegrin, MDC9/meltrin-
/ADAM9 and PKC
are
involved in TPA-induced ectodomain shedding of membrane-anchored
heparin-binding EGF-like growth factor. EMBO J 17:72607272[Abstract/Free Full Text]
-
Mantegazza M, Fasolato C, Hescheler J, Pietrobon D 1995 Stimulation of single L-type calcium channels in rat pituitary
GH3 cells by thyrotropin-releasing hormone. EMBO
J 14:10751083[Abstract]
-
Murdoch GH, Potter E, Nicolaisen AK, Evans RM, Rosenfeld MG 1982 Epidermal growth factor rapidly stimulates prolactin gene
transcription. Nature 300:192194[Medline]
-
Murdoch GH, Franco R, Evans RM, Rosenfeld MG 1983 Polypeptide hormone regulation of gene expression.
thyrotropin-releasing hormone rapidly stimulates both transcription of
the prolactin gene and the phosphorylation of a specific nuclear
protein. J Biol Chem 258:1532915335[Abstract/Free Full Text]
-
Berwaer M, Peers B, Nalda AM, Monget P, Davis JRE, Belayew
A, Martial JA 1993 Thyrotropin-releasing hormone and epidermal
growth factor induce human prolactin expression via identical
multiple cis elements. Mol Cell Endocrinol 92:17[Medline]
-
de Wet JR, Wood KV, DeLuca M, Helinski DR, Subramani S 1987 Firefly luciferase gene: structure and expression in mammalian cells.
Mol Cell Biol 7:725737[Medline]
-
Sun P, Enslen H, Myung PS, Maurer RA 1994 Differential
activation of CREB by Ca2+/calmodulin-dependent
protein kinases type II and type IV involves phosphorylation of a site
that negatively regulates activity. Genes Dev 8:25272539[Abstract]
-
Roberson MS, Misra-Press A, Laurance ME, Stork PJS, Maurer RA 1995 A role for mitogen-activated protein kinase in mediating
activation of the glycoprotein hormone
-subunit
gonadotropin-releasing hormone. Mol Cell Biol 15:35313539[Abstract]
-
Day RN, Maurer RA 1989 The distal enhancer region of the rat
prolactin gene contains elements conferring response to multiple
hormones. Mol Endocrinol 3:39[Abstract]