Activation of MAPK by TRH Requires Clathrin-Dependent Endocytosis and PKC but Not Receptor Interaction with ß-Arrestin or Receptor Endocytosis
Jeffrey Smith,
Run Yu and
Patricia M. Hinkle
Department of Pharmacology and Physiology and the Cancer Center,
University of Rochester School of Medicine and Dentistry, Rochester,
New York 14642
Address all correspondence and requests for reprints to: Dr. Patricia M. Hinkle, Department of Pharmacology and Physiology, Box 711, University of Rochester Medical Center, 601 Elmwood Avenue, Rochester, NY 14642. E-mail: Patricia_Hinkle{at}urmc.rochester.edu
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ABSTRACT
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To determine whether the interaction of the TRH receptor with
ß-arrestin is necessary for TRH activation of MAPK, cells expressing
either intact or truncated, internalization-defective TRH receptors
were transfected with a ß-arrestin-green fluorescent protein
conjugate. In cells expressing the wild-type pituitary TRH
receptor, TRH caused translocation of the ß-arrestin-green
fluorescent protein conjugate from the cytosol to the plasma membrane
within 30 sec. After 5 min, the ß-arrestin-green fluorescent protein
conjugate was visible in vesicles, where it colocalized with
rhodamine-labeled TRH. In hypertonic sucrose, the ß-arrestin-green
fluorescent protein conjugate translocated to the plasma membrane after
TRH addition but did not internalize. In cells expressing the truncated
TRH receptor, TRH did not cause translocation of the ß-arrestin-green
fluorescent protein conjugate. TRH activated MAPK strongly in cells
expressing intact or truncated TRH receptors, indicating that the
receptor does not need to bind ß-arrestin or internalize. MAPK
activation by TRH, epidermal growth factor, and phorbol ester was
strongly inhibited by hypertonic sucrose and concanavalin A, which
block movement of proteins into coated pits and coated pit assembly.
Hypertonic sucrose did not affect MAPK activation in cells
overexpressing MAPK kinase 1. Dominant negative dynamin, which
blocks conversion of coated pits to vesicles, also reduced receptor
internalization and TRH activation of MAPK. TRH activation of MAPK
required PKC but was insensitive to pertussis toxin and did not require
ras, epidermal growth factor receptor kinase, or PI3K. These results
show that the TRH receptor itself does not need to bind ß-arrestin or
undergo sequestration to activate MAPK but that the endocytic pathway
must be intact.
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INTRODUCTION
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TRH ACTS THROUGH a G protein-coupled
receptor (GPCR) to stimulate the release and synthesis of thyrotropin
and PRL. The pituitary TRH receptor is coupled to Gq/11, and TRH causes
an increase in PLC activity and subsequent mobilization of
intracellular calcium and activation of PKC. TRH also stimulates MAPK,
both in pituitary cells expressing endogenous receptors and in a number
of cell lines expressing transfected receptors (1, 2, 3, 4).
MAPK activation is required for TRH stimulation of PRL transcription
(4).
Activation of many GPCRs leads to an increase in MAPK activity, but the
molecular mechanisms involved are not fully understood (5, 6). ß-Arrestin binds to agonist-activated GPCRs after their
phosphorylation by receptor kinases and links the activated GPCRs to
clathrin, thereby targeting them to the endocytic pathway
(7, 8, 9). It has been proposed that it is actually the
receptor-ß-arrestin complex that signals to MAPK for the well studied
ß2-adrenergic receptor (10). Formation of the
ß-arrestin-receptor complex appears to be essential for MAPK
activation. Specifically, it has been suggested that the
receptor-ß-arrestin complex acts as a scaffold binding src, a
nonreceptor tyrosine kinase, and that src transduces the signal from
the GPCR to ras, activating the MAPK cascade (10, 11).
Components of the MAPK cascade, including raf, MAPK kinase (MEK), and
MAPK, have been identified in isolated endocytic vesicles. Because some
GPCRs coupled to all families of G proteins interact with ß-arrestin
and subsequently undergo endocytosis, the involvement of ß-arrestin
in signaling to MAPK may be of widespread importance (12).
Supporting the model are the findings that both dominant negative
dynamin and dominant negative ß-arrestin suppress activation of MAPK
by the M1 muscarinic receptor, which normally couples via Gq/11 to
phospholipid turnover (13), and that dominant negative
dynamin partially inhibits MAPK activation by the Gq-coupled GnRH
receptor (14). In addition, dominant negative dynamin
blocks activation of MAPK by the serotonin 1A and
-opioid
receptors (15, 16). The wild-type proteinase-activated
receptor 2 also appears to activate MAPK through an internalized
receptor-ß-arrestin-raf complex (17). The requirement
for receptor endocytosis is not universal, however, since several
receptors have been shown to activate MAPK without internalization
(18, 19, 20, 21, 22).
Once TRH binds, the receptor binds ß-arrestin and internalizes
extensively through a clathrin-dependent pathway (23, 24, 25, 26, 27, 28).
In this study, we have asked whether ß-arrestin-receptor interaction
and subsequent receptor endocytosis are required for activation of MAPK
by TRH. We show that ß-arrestin binding and endocytosis of the TRH
receptor are not required per se for TRH to activate MAPK,
but that a functional endocytic pathway and TRH activation of PKC are
necessary.
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RESULTS
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Interaction of TRH Receptors with ß-Arrestin-Green Fluorescent
Protein Conjugate (ß-arr-GFP)
To determine whether an interaction between the activated TRH
receptor and ß-arrestin is necessary for the activation of MAPK, we
compared the ability of intact and internalization-defective TRH
receptors to bind to ß-arrestin and increase MAPK activation. HEK293
cells stably expressing wild-type or truncated TRH receptors were
transiently transfected with ß- arr-GFP and then exposed to 1
µM TRH. Cells stably transfected with intact TRH
receptors (293TRHR cells) or truncated, internalization-defective TRH
receptors terminating at residue 334 (293TRHR-C335Stop cells) bound
4.16 ± 0.13 and 3.69 ± 0.23 pmol
[3H]MeTRH/mg protein, respectively, at 10
nM radioligand. Cells expressing these receptors respond to
TRH with an increase in IP3 production and a robust calcium signal
(24, 29). [3H]MeTRH bound to
wild-type receptor was rapidly and extensively internalized after
binding, whereas radioligand bound to truncated receptor was not
internalized (Fig. 1
).

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Figure 1. Internalization of [3H]MeTRH
Cells were preincubated at 37 C with: no drug (Control), 0.45
M sucrose for 20 min (Sucrose) or 250 µg/ml concanavalin
A for 1 h (Con A); 5 nM [3H]MeTRH was
then added in the continued presence of drug and internalized
[3H]MeTRH was measured at intervals. Experiments were
performed on duplicate dishes, and all errors were within symbol
size.
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We used ß-arr-GFP to monitor the interaction between the receptor and
ß-arrestin in live cells (Fig. 2
).
Before stimulation, ß-arr-GFP was evenly distributed in the cytosol.
In 293TRHR cells, ß-arr-GFP rapidly translocated from the cytosol to
the plasma membrane after exposure to TRH. Translocation was readily
apparent within 15 sec. ß-arr-GFP began to appear in cytoplasmic
vesicles after 5 min, and it all appeared to be in intracellular
vesicles after 20 min of continuous TRH treatment (Fig. 2
, left
panels). TRH was able to translocate ß-arr-GFP at concentrations
as low as 1 nM (data not shown). When 293TRHR
cells were incubated in HBSS containing 0.45 M
sucrose, which inhibits clathrin mobilization, ß-arr-GFP still
translocated to the plasma membrane after TRH addition but remained
there. In 293TRHR-C335Stop cells, which do not internalize TRH, there
was no change in the localization of ß-arrestin after TRH
stimulation. These results show that the wild-type TRH receptor
interacts strongly with ß-arrestin but the truncated receptor does
not.

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Figure 2. Translocation of ß-arr-GFP by TRH in Cells
Expressing Intact or Internalization-Defective Receptors
Cells expressing TRH receptors were transiently transfected with
ß-arr-GFP. After 2448 h, cells were placed in HBSS, and the
localization of ß-arr-GFP was followed microscopically. Shown are
series of images of the same cells before (0), 1, or 20 min after the
addition of 1 µM TRH at 37 C. TRHR indicates 293TRHR
cells; Truncated TRHR, 293TRHR-C335Stop cells; TRHR+Sucrose, 293TRHR
cells in HBSS supplemented with 0.45 M sucrose for 20 min.
Similar results have been obtained in multiple experiments, and the
results were not affected by overnight serum starvation. TRH had no
effect on localization of ß-arr-GFP in HEK293 cells that had not been
transfected with the TRH receptor.
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Internalization of TRH Receptors with ß-arr-GFP
We next asked whether the TRH-receptor complex and ß-arr-GFP are
localized in the same vesicles (Fig. 3
).
Rhodamine-labeled TRH (Rhod-TRH), which is a weak agonist for the TRH
receptor, was used at a low concentration to minimize nonspecific
binding. After 25 min, most of the Rhod-TRH was visible in endocytic
vesicles in the 293TRHR cells (top panels). Although much of
the ß-arr-GFP remained in the cytosol, some ß-arr-GFP appeared in
vesicles, and these vesicles colocalized almost perfectly with the
Rhod-TRH vesicles. In the 293TRHR-C335Stop cells, Rhod-TRH did not
internalize, and none of the ß-arr-GFP was observed moving to the
plasma membrane or in endocytic vesicles (bottom
panels).

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Figure 3. Colocalization of Rhod-TRH and ß-arr-GFP
Cells in HBSS were incubated with Rhod-TRH at 37 C for 2025 min and
washed. Rhod-TRH (left panels) and ß-arr-GFP
(middle panels) fluorescence images were recorded with
appropriate filters. The images were overlaid and orange
or yellow color show where the two molecules were
colocalized (right panels). There was no significant
bleedthrough between these two settings. TRHR indicates 293TRHR cells
transfected with ß-arr-GFP; Truncated TRHR, 293TRHR-C335Stop cells
transfected with ß- arr-GFP.
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Activation of MAPK by Intact and Truncated TRH Receptors
MAPK activity was followed by immunoblotting with antibodies
against the phosphorylated, active forms of MAPK (ERK1/2). TRH
stimulated MAPK phosphorylation in 293TRHR cells in a dose- and
time-dependent manner (Fig. 4
).
Activation was evident with as little as 1 nM TRH and
apparently maximal at 1 µM TRH. MAPK activation by 1
µM TRH peaked between 2 and 5 min and subsided after 10
min. In subsequent experiments, we used either 1 µM or 10
nM TRH for 5 min at 37 C to activate MAPK. At 5 min, the
receptor-ß-arrestin complex was fully formed and largely in
intracellular vesicles, although some surface receptor/ß-arrestin
complex remained (data not shown). The MAPK response to TRH in 293TRHR
cells was comparable to the response to maximally effective
concentrations of the phorbol ester 12-O-tetradecanoylphorbol
13-acetate (TPA), which activates PKC, or epidermal growth factor
(EGF), which activates the EGF receptor tyrosine kinase (Fig. 5a
). TRH activated MAPK strongly in cells
expressing the truncated receptor, indicating that an interaction with
ß-arrestin is not required (Fig. 5a
).

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Figure 4. Activation of MAPK in 293TRHR Cells
Replicate dishes of 293TRHR cells were serum-starved overnight and then
treated with vehicle or TRH at 37 C in HBSS and lysed, and proteins
were resolved by SDS-PAGE. Activated MAPK was identified by
immunoblotting using phospho-MAPK specific antibody, which labeled 44-
and 42-kDa bands, ERK1 and ERK2, respectively. Total MAPK was shown to
be the same in all samples on parallel blots that were probed with
antibody against total MAPK. a, Cells were incubated
with 0 to 10 µM TRH for 5 min. b, cells
were incubated with 1 µM TRH for 040 min.
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Figure 5. Activation of MAPK via an Internalization-Defective
TRH Receptor
Cells were serum-starved overnight and then treated as follows.
a, Cells were placed at 37 C in either HBSS (left
lanes) or HBSS supplemented with 0.45 M sucrose
(right lanes); after 20 min, cells were treated for 5
min with no additions, 1 µM TRH, 1 µM TPA,
or 10 nM EGF. b, 293TRHR cells were
incubated for 1 h without (Control) or with 250 µg/ml
concanavalin A (Con A) before treatment with no additions, 1
µM TRH, 1 µM TPA, or 10 nM EGF
for 5 min. c, 293TRHR cells were transfected without
(TRHR-MEK) or with 1 µg/ml of a plasmid encoding MEK1
(TRHR+MEK) and placed in HBSS. Left lanes, 0.45
M sucrose was added for 0, 10, 30, or 60 min. Right
lanes, 50 µM PD98059 was added for 0, 10, 30, or
60 min. Films of the gels grouped with a bracket were
exposed identically; a lighter exposure of the gel containing TRHR+MEK
is also shown, noted by the asterisk. d, Cells were
serum starved overnight in the absence (Control) or presence of 1
µM TPA to deplete PKC (PKC Depl). The cells were then
placed in HBSS and treated for 5 min with or without 1 µM
TRH. TRHR indicates 293TRHR cells; truncated TRHR, 293TRHR-C335Stop
cells.
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Role of the Endocytic Pathway in MAPK Activation
We next measured MAPK activation in the presence of hypertonic
sucrose, which permits the interaction between the TRH receptor and
ß-arrestin (Fig. 2
) but prevents clathrin mobilization and
endocytosis (23, 30). Hypertonic sucrose delayed
internalization of the TRH receptor, measured by the conversion of
receptor-bound [3H]MeTRH to an
acid/salt-resistant form (Fig. 1
) and fluorescence microscopy using
Rhod-TRH (23). Sucrose does not impair the ability of TRH
to stimulate phosphoinositide turnover or a calcium response
(24). In 293TRHR cells, hypertonic sucrose treatment
increased basal MAPK activity slightly by itself but severely inhibited
activation of MAPK by TRH, EGF, or TPA (Fig. 5a
). Sucrose also
abrogated activation of MAPK by diverse signals in cells expressing the
truncated receptor. We also used concanavalin A, a lectin, to block the
endocytic pathway. Concanavalin A slowed [3H]MeTRH
internalization (Fig. 1
), although it was not as effective as sucrose,
and it inhibited MAPK responses to TRH, EGF, and TPA (Fig. 5b
).
Dominant negative forms of dynamin (K44A) or ß- arrestin
(319418) were also tested for their effects on TRH-induced receptor
internalization and MAPK activation (Fig. 6
). To assess the effectiveness of the
dominant negative proteins, we cotransfected HEK293 cells stably
expressing an epitope-tagged TRH receptor with plasmids encoding GFP
and either K44A dynamin or 319418 ß-arrestin. The hemagglutinin
(HA) epitope was at the amino terminus of the receptor where it would
not be expected to interfere with ß- arrestin interactions. We
then exposed cells to vehicle or TRH for 5 min, and fixed and localized
receptors immunocytochemically with antibody against the HA epitope.
Receptor localization was measured in GFP-positive cells, since these
had been successfully transfected. In cells transfected with the
control plasmid, receptor was predominantly on the plasma membrane of
82% of untreated cells but only 35% of cells treated with TRH for 5
min (Fig. 6a
). In cells transfected with either K44A dynamin or
319418 ß-arrestin, TRH did not change the fraction of cells with
predominantly cell surface receptor, indicating that the dominant
negative proteins had blocked ligand-driven receptor internalization.
When internalization was assessed by conversion of receptor-bound
[3H]MeTRH to an acid/salt resistant form (Fig. 6b
), dominant negative dynamin and ß-arrestin could still be seen to
inhibit internalization (Fig. 6b
), but their effects were less
pronounced. Much of the receptor-bound
[3H]MeTRH was acid/salt resistant in cells
expressing K44A dynamin and 319418 ß-arrestin even though by
microscopy the receptor appeared to be on the plasma membrane. This
suggests that the hormone-receptor complex forms a stable,
acid/salt-resistant complex before it moves into an endosome, and that
dominant negative dynamin and ß-arrestin block at a late step after
the receptor has become acid stable.

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Figure 6. Effect of Dominant Negative Dynamin and
ß-Arrestin on TRH Internalization and MAPK Activation
A, HEK293 cells stably expressing an epitope-tagged TRH receptor were
transiently transfected with 0.6 µg GFP and with 3 µg of either
pcDNA3, K44A dynamin, or 319418 ß-arrestin and grown for 24 h.
Cells were serum-starved overnight and then treated with vehicle or 1
µM TRH for 5 min, fixed, and stained with monoclonal
antibody against the HA epitope. Slides were coded and then evaluated
by an observer unaware of treatment group. Cells positive for GFP
fluorescence ( half of the cells) were divided into those with
receptor clearly localized on the plasma membrane and those with
receptor localized partially or entirely intracellularly. B, HEK293
cells were transiently transfected with 1 µg TRH receptor and 3 µg
pcDNA3, K44A dynamin, or 319418 ß-arrestin. Dishes were incubated
with 5 nM [3H]MeTRH for 1 h at 0 C in
HBSS and washed. The fraction of specifically bound
[3H]MeTRH internalized was determined before and after a
subsequent 5-min incubation at 37 C based on resistance to an acid/salt
wash. C, HEK293 cells were transfected with 1 µg TRH receptor and 3
µg pcDNA3 (lanes 1 and 2), or with 1 µg TRH receptor and 3 µg
K44A dynamin (lanes 3 and 4) or 3 µg 319418 ß-arrestin (lanes 5
and 6). The next day, cells were changed to medium lacking serum
16 h before stimulation. Dishes were rinsed with HBSS and treated
with or without 10 nM TRH for 5 min and assayed for
activated MAPK (upper blot) or total MAPK
(lower blot).
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Dominant negative dynamin and dominant negative ß-arrestin both
blunted the MAPK response to 10 nM TRH (Fig. 6c
). Neither
dominant negative protein decreased total MAPK, which is shown in the
lower blot. Based on densitometry, 10
nM TRH stimulated an average 2.4-fold increase in
phosphorylation of ERK1/2 in these experiments (n = 4). K44A
dynamin and 319418 ß-arrestin both reduced basal levels of
activated MAPK. K44A dynamin reduced the TRH-stimulated increase in
phosphorylated MAPK to an average of 39 ± 14% of control and
319418 ß-arrestin to 82 ± 19% of control.
Pathways leading from EGF, TPA, and TRH to MAPK activation are believed
to converge at raf-1, where PKC acts directly. Because hypertonic
sucrose blocks MAPK activation by all three agents, it presumably
inhibits at raf-1 or a site distal to it. To pinpoint the site, we
determined whether hypertonic sucrose prevented MEK from activating
MAPK. In control 293TRHR cells, basal MAPK activity was low and was
slightly increased by hypertonic sucrose (Fig. 5c
). When MEK1 was
overexpressed, basal MAPK activity was very high, as expected (Fig. 5c
), and was not inhibited by addition of hypertonic sucrose over 60
min. In contrast, the MEK inhibitor PD98059 inhibited MAPK rapidly and
significantly in cells overexpressing MEK (Fig. 5c
). These results
indicate that hypertonic sucrose does not prevent MEK from activating
MAPK and imply that sucrose instead inhibits raf-1 activation of
MEK.
Importance of PKC, PI3K, and EGF Receptor Kinase
Because ß-arrestin interaction and receptor endocytosis were not
required for MAPK activation by TRH, we examined the requirement for
PKC, which has been shown to be responsible for 5060% of MAPK
activation in pituitary and COS cells (2, 3, 4). Overnight
treatment with a high concentration of a phorbol ester, which
down-regulates some isoforms of PKC, prevented activation of MAPK by 10
nM TRH in cells expressing intact or truncated TRH
receptors (Fig. 5d
). Down-regulation of PKC had no effect on the
TRH-induced translocation of ß-arr-GFP (data not shown).
Staurosporine and GF109203X (bisindolylmaleimide I), two PKC
inhibitors, blocked MAPK activation by 10 nM TRH and
partially inhibited MAPK activation by 1 µM TRH in
293TRHR cells (data not shown).
PD98059, a selective MEK inhibitor, effectively blocked MAPK activation
by TRH, TPA, and EGF (Fig. 7a
). To assess
the requirement for ras in TRH activation of MAPK, we expressed a
dominant negative form of ras, N17 ras. N17 ras markedly reduced EGF
activation of MAPK but had relatively little effect on the TRH response
(Fig. 7b
). Wortmannin and LY294002, both inhibitors of PI3K, block MAPK
activation by some GPCRs (31). Wortmannin has been
reported to inhibit TRH activation of MAPK in pituitary GH3 cells
(1). These inhibitors had no effect on TRH activation of
MAPK but significantly reduced stimulation by serum (Fig. 7c
). The
specific tyrphostin AG1478, an inhibitor of the tyrosine kinase
activity of the EGF receptor (32), has been reported to
prevent MAPK activation by some receptors coupled to Gq
(33). AG1478 inhibited EGF but not TRH stimulation of MAPK
activity (Fig. 7d
). Likewise, the general tyrosine kinase inhibitor,
genistein, inhibited EGF but not TRH activation. TRH activation of MAPK
was completely insensitive to pertussis toxin (Fig. 7e
).

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Figure 7. Involvement of ras, PI3K, and the EGF Receptor in
TRH Activation of MAPK in 293TRHR Cells
a, Cells were incubated with vehicle, 1 µM TRH, 1
µM TPA, or 10 nM EGF for 5 min; in the
right lanes, 50 µM PD98059 was added 30
min before other drugs. b, Cells were mock transfected or transfected
with N17ras and then incubated in serum-free medium overnight, placed
in HBSS at 37 C, and treated with vehicle, 0.5 nM EGF (EGF
0.5), 1 nM EGF (EGF 1), or 10 nM TRH (TRH) for
5 min. Similar results were obtained in experiments where 10
nM EGF was used. c and d, Cells were serum starved
overnight and then placed in HBSS and incubated for 10 min with no
drug, 100 nM wortmannin, 25 µM LY294002, or
100 nM AG1478, as shown. Cells were then treated for 5 min
with vehicle, 10 nM TRH, 10 nM EGF, or 30%
FCS. In the lower blot, cells were incubated with 100
µM genistein for 30 min and then exposed to 10
nM EGF or 1 µM TRH for 5 min.
e, Cells were incubated with 50 ng/ml pertussis toxin
overnight, placed in HBSS at 37 C, and treated with vehicle or 1
µM TRH for 5 min.
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DISCUSSION
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GPCRs have been classified according to their ability to bind
various arrestins, a characteristic dictated by the cytoplasmic
carboxy-terminal region of the receptor (34). The
pituitary TRH receptor falls into class B, the group of receptors able
to bind all three arrestins, ß-arrestin1, ß-arrestin2, and visual
arrestin. Groarke and co-workers (25) showed that
ß-arrestin cointernalizes with TRH receptors in HEK293 cells, and we
found that it cointernalizes with rhodamine-labeled TRH (Fig. 3
). Taken
together, these results indicate that it is a complex containing at
least hormone, TRH receptor, and ß-arrestin that internalizes in
HEK293 cells. We have found that TRH also causes translocation of
ß-arr-GFP to the membrane and then to vesicles in GH3 cells, which
express endogenous TRH receptors.1 A large
number of GPCRs have been shown to interact with ß-arrestin; in some
cases (ß2-adrenergic, dopamine D1A, and
endothelin ETA receptors), the ß-arrestin does
not internalize with the receptor, whereas in others (neurotensin and
angiotensin AT1A receptors) it does (34, 35). In contrast, ß-arrestin remains on the plasma membrane
and does not move into endocytic vesicles with rhodamine-labeled
TRH when the TRH receptor is expressed in Fq/11 fibroblasts from mice
lacking G
q and G
11 (36). The difference between
Fq/11 and pituitary and HEK293 cells probably results from either the
low level of receptor expression in the Fq/11 cells or differences in
the pools of endogenous ß-arrestin.
TRH is capable of activating MAPK just as effectively through a
truncated receptor, which does not bind ß-arrestin or internalize, as
through the full-length receptor, proving that receptor-ß-arrestin
interaction is not necessary for the activation of MAPK by TRH. Similar
results have been reported for several other GPCRs
(18, 19, 20, 21, 22). Interaction of activated receptor with
ß-arrestin is also not sufficient for MAPK activation by TRH, because
the wild-type TRH receptor could not activate MAPK after PKC was
down-regulated even though ß-arrestin translocation was intact. We
cannot formally eliminate the possibility that the wild-type TRH
receptor activates MAPK through a complex containing ß-arrestin and
the truncated TRH receptor activates MAPK by a different,
arrestin-independent mechanism. In fact, this appears to be the case
for a mutated form of the proteinase-activated receptor 2
(17). However, the finding that intact and truncated TRH
receptors both require active PKC suggests that they activate MAPK by
the same pathway. Our results on MAPK activation by the TRH receptor
differ from the results reported for the ß2-adrenergic receptor,
wild-type proteinase-activated receptor 2, and M1 muscarinic receptor,
which must interact with ß-arrestin to activate src and subsequently
MAPK (13, 17, 37). Based on currently available data, it
appears that the role of receptor-ß-arrestin interaction in MAPK is
receptor- and perhaps cell type-specific.
Hypertonic sucrose prevents the recruitment of clathrin and interferes
with normal coated pit formation and endocytosis (30).
Because hypertonic sucrose and concanavalin A, another inhibitor of
endocytosis, inhibited MAPK activation by internalization-defective as
well as intact TRH receptors, these inhibitors did not block MAPK
activation by inhibiting TRH receptor internalization per
se. Instead, our results suggest that even though endocytosis of
the TRH receptor itself is not essential for TRH activation of MAPK,
early steps in the endocytic pathway may be required. Hypertonic
sucrose and concanavalin A would both be expected to prevent the
assembly of proteins in coated pits, and both treatments caused a
global inhibition of MAPK activation, reducing the responses to
activators of a GPCR, a receptor tyrosine kinase and PKC. Dominant
negative dynamin would be expected to block conversion of coated pits
to coated vesicles, and it too inhibited TRH activation of MAPK.
Because sucrose, concanavalin A, and K44A dynamin inhibit the endocytic
pathway by very different mechanisms, it seems likely that they
inhibited MAPK activation as a result of their effects on
clathrin-dependent endocytosis and not because of other actions. The
results are consistent with the idea that proteins in the MAPK cascade
need to assemble at coated pits or endocytic vesicles for effective
MAPK activation (19, 38), although the TRH receptor itself
does not need to bind ß-arrestin or undergo internalization. Such a
model is a variant on schemes that envision assembly of GPCR,
ß-arrestin, src, and proteins in the MAPK cascade in endosomes.
Since the pathways leading to MAPK activation by EGF and phorbol esters
converge on raf-1, and TRH activation of MAPK requires PKC, which acts
at the level of raf-1, hypertonic sucrose appears to inhibit at raf-1
or a site distal to it. Daaka and co-workers (37) showed
that raf-1 activation is not prevented by inhibiting clathrin-dependent
endocytosis, and we showed that MAPK activation caused by
overexpression of MEK is not prevented by hypertonic sucrose. These
findings suggest that hypertonic sucrose blocks the MAPK cascade at the
level of raf activation of MEK. This conclusion appears to conflict
with results of Kranenburg and co-workers (19), who
reported that dominant negative dynamin abrogates activation of MAPK by
multiple signals but does not prevent activation of ras, raf, or MEK.
Instead, they find that dominant negative dynamin blocks the transfer
of phosphorylated, active MEK from the plasma membrane to the cytosol
in endocytic vesicles. Our results are compatible with this model if
overexpression of MEK eliminates the need for clathrin-dependent
translocation of activated MEK. Some GPCRs signal to MAPK by causing
activation of the EGF receptor, which must undergo endocytosis for MAPK
stimulation (33, 39), and others via src
(40, 41, 42). However, TRH does not signal by these pathways
because MAPK responses are completely insensitive to the tyrphostin
AG1478 and to the broad specificity tyrosine kinase inhibitor
genistein. It remains unclear why endocytosis is essential for MAPK
activation in some systems but not in others (13, 16, 18, 19, 21, 22, 37).
TRH acts via Gq/11 to activate PLC and increase the concentration
of diacylglycerol, which activates PKC, and IP3, which releases
calcium. The fact that prolonged phorbol ester pretreatment nearly
eliminated the MAPK response to TRH in HEK 293 cells indicates that PKC
isoforms subject to down-regulation are critical for the MAPK response
in this cell type. PKC has been reported to account for about
two-thirds of MAPK activation by TRH in pituitary cells
(2) and COS-7 cells (3). In COS-7 cells, the
PKC-independent activation of MAPK by TRH appears to be insensitive to
pertussis toxin, independent of ras activation, but dependent on G
protein ß
-subunits (3). We have found that the
truncated TRH receptor can activate MAPK in COS-7 cells (data not
shown), indicating that an interaction with ß-arrestin is again not
necessary.
In summary, we have shown that TRH activates MAPK in a manner that does
not require interaction between the receptor and ß-arrestin but does
require PKC. Early steps in the endocytic pathway appear to be
important for MAPK activation by multiple signal pathways. Our findings
emphasize the diversity in signal transduction pathways used by members
of the GPCR superfamily to activate the MAPK cascade.
 |
MATERIALS AND METHODS
|
---|
Materials
HBSS and LipofectAmine were purchased from Life Technologies, Inc. (Gaithersburg, MD). Plasmids encoding a
conjugate pß-arr-GFP protein (12) and K44A
dynamin were gifts from Dr. Marc Caron (Duke University, Durham, NC).
Other plasmids were generously provided by the following: N17ras from
Dr. Ian Macara (University of Virginia, Charlottesville, VA), intact
and truncated TRH receptors from Marvin C. Gershengorn (Weill Medical
College of Cornell University, New York, NY), and 318419 ß-arrestin
from Dr. Jeffrey L. Benovic (Thomas Jefferson University, Philadelphia,
PA). A plasmid encoding an HA-tagged TRH receptor was constructed by
PCR, adding two HA sequences separated by a Gly after the initial Met,
and inserted in pcDNA3.2 Plasmid encoding
MEK1 was purchased from Stratagene (La Jolla, CA) and
plasmid encoding GFP was from CLONTECH Laboratories, Inc.
(Palo Alto, CA). Rhod-TRH was synthesized as previously described
(23). Antibodies against phospho-MAPK and total MAPK were
from Cell Signaling Technology (Beverly, MA). TRH, GF1095203X, and
PD98059 were from Calbiochem (San Diego, CA); cell
attachment matrix was from Upstate Biotechnology, Inc.
(Lake Placid, NY); and pertussis toxin, genistein, EGF, concanavalin A,
staurosporine, and TPA were from Sigma (St. Louis, MO).
LY294002 and wortmannin were purchased from BioMol (Plymouth Meeting,
PA). Horseradish peroxidase- labeled antibody to rabbit IgG and
[3H]MeTRH (64 Ci/mmol) were obtained from
Dupont/NEN Life Science Products (Boston, MA); and
enhanced chemiluminescence reagent was from Amersham Pharmacia Biotech (Arlington Heights, IL).
Cell Culture and Transfection
Properties of HEK293 cells stably expressing the wild-type mouse
TRH receptor (293TRHR cells) and HEK293 cells stably expressing an
internalization-defective, truncated mouse TRH receptor (Cys 335
converted to a stop codon) (293TRHR-C335Stop cells) have been described
previously (36). HEK293 cell lines were grown in DMEM
supplemented with 5% FBS and transfected with LipofectAmine according
to the manufacturers instructions. After transient transfection,
between 20 and 60% of cells expressed detectable GFP or ß-arr-GFP.
We also carried out several experiments to estimate the efficiency of
cotransfection. When cells were cotransfected with plasmids encoding
epitope-tagged receptors and GFP at ratios of
3:1, 95% of the
GFP-positive cells also stained for the receptor by
immunocytochemistry.
Microscopy
293TRHR and 293TRHR-C335Stop cells were transfected with
pß-arr-GFP (1 µg/6 cm dish). Cells were then split onto
ECL-coated coverslips. On the second or third day after transfection,
cells were stimulated at 37 C for up to 20 min with 1 µM
TRH in normal HBSS buffered with 15 mM HEPES to pH 7.4
(HBSS) or buffered HBSS supplemented with 0.45 M sucrose
and localization of ß-arr-GFP was followed microscopically. In some
experiments, cells were coincubated with Rhod-TRH (1:100,
400
nM, in HBSS) at 37 C for 25 min. Cells expressing an
HA-tagged TRH receptor were fixed with paraformaldehyde and incubated
with a 1:1,000 dilution of monoclonal antibody to the HA epitope
(Covance Laboratories, Inc., Princeton, NJ) followed by a
1:500 dilution of goat rhodamine-labeled antimouse IgG (American
Qualex, LaMiranda, CA), as previously described (23).
GFP fluorescence was detected with fluorescein filters and Rhod-TRH and
secondary antibody with rhodamine filters, using a 40x objective on a
Nikon inverted microscope (Nikon, Melville, NY) with a
Micromax camera (Princeton Instruments, Trenton, NJ).
Bleedthrough was negligible under the conditions used. Images were
analyzed using Metamorph software (Universal Imaging, Media, PA).
Activation of MAPK
293TRHR and 293TRHR-C335Stop cells plated on coated 35- or 60-mm
dishes were serum-starved for 24 h before stimulation. In other
experiments HEK293 cells were transiently transfected with receptor
and/or dominant negative constructs, grown in complete medium for
1 d and then serum-starved overnight before use. Cells were then
washed with HBSS and incubated with TRH or other drugs in HBSS at 37 C,
usually for 5 min, placed on ice, and immediately lysed with SDS-PAGE
sample buffer (150 µl/6 cm dish). Equal amounts of sample (10 to 20
µl) were loaded into each lane of 10% SDS-PAGE gels. After
electrophoresis, proteins were transferred to nitrocellulose membranes,
and the membranes were blocked and probed with phospho-MAPK-
specific polyclonal antibody (1:1,000) and subsequently with goat
antirabbit IgG horseradish peroxidase (1:5,000) and visualized with
enhanced chemiluminescence reagents. Samples of the cell extracts were
run on separate gels and blotted with antibody against total MAPK to
ensure that the amounts of MAPK protein were equal within each
treatment group. All experiments have been replicated two to ten times
with comparable results.
TRH Binding
To measure receptor density, cells plated on replicate 35-mm
dishes were incubated for 1 h in HBSS containing 10 nM
[3H]MeTRH, and specific binding was determined
as previously described (26). Protein was determined by
the Lowry method using BSA as standard. Radioligand internalization was
determined by measuring the fraction of specifically bound
[3H]MeTRH resistant to washing with ice-cold
0.2 N acetic acid/0.5 M NaCl (26).
Nonspecific binding, measured in the presence of a 1,000-fold excess of
unlabeled hormone, has been subtracted from all points.
 |
ACKNOWLEDGMENTS
|
---|
We are grateful to Caroline Perkowski and John Puskas for
excellent technical assistance.
 |
FOOTNOTES
|
---|
This work was supported in part by NIH Grants DK-19974 and Cancer
Center Core Research Grant CA-11098. R.Y. was supported by a Wilmot
Fellowship.
Abbreviations: ß-arr-GFP, ß-arrestin-green fluorescent
protein conjugate; EGF, epidermal growth factor; GFP, green fluorescent
protein; GPCR, G protein-coupled receptor; MEK, MAPK kinase; Rhod-TRH,
rhodamine-labeled TRH; TPA, 12-O-tetradecanoylphorbol
13-acetate.
1 Yu, R., and P. M. Hinkle, unpublished
results. 
2 Zhu, C.-C., and P. M. Hinkle, manuscript in
preparation. 
Received for publication January 28, 2000.
Accepted for publication May 29, 2001.
 |
REFERENCES
|
---|
-
Kanasaki H, Fukunaga K, Takahashi K, Miyazaki K, Miyamoto
E 1999 Mitogen-activated protein kinase activation by stimulation with
thyrotropin-releasing hormone in rat pituitary GH3 cells. Biol Reprod 61:319325[Abstract/Free Full Text]
-
Ohmichi M, Sawada T, Kanda Y, et al. 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]
-
Palomero T, Barros F, del Camino D, Viloria CG, de la Pena P 1998 A G protein ß
dimer-mediated pathway contributes to
mitogen-activated protein kinase activation by thyrotropin-releasing
hormone receptors in transfected COS-7 cells. Mol Pharmacol 53:613622[Abstract/Free Full Text]
-
Wang YH, 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]
-
Luttrell LM, van Biesen T, Hawes BE, et al. 1997 G-protein-coupled receptors and their regulation: activation of the MAP
kinase signaling pathway by G-protein- coupled receptors. Adv
Second Messenger Phosphoprotein Res 31:263277[Medline]
-
Gutkind JS 1998 Cell growth control by G protein- coupled
receptors: from signal transduction to signal integration. Oncogene 17:13311342[CrossRef][Medline]
-
Ferguson SS, Barak LS, Zhang J, Caron MG 1996 G-protein-coupled receptor regulation: role of G-protein-coupled
receptor kinases and arrestins. Can J Physiol Pharmacol 74:10951110[CrossRef][Medline]
-
Ferguson SS, Downey WE, Colapietro AM, Barak LS, Menard L,
Caron MG 1996 Role of ß-arrestin in mediating agonist-promoted G
protein-coupled receptor internalization. Science 271:363366[Abstract]
-
Goodman Jr OB, Krupnick JG, Santini F, et al. 1996 ß-Arrestin acts as a clathrin adaptor in endocytosis of the
ß2-adrenergic receptor. Nature 383:447450[CrossRef][Medline]
-
Luttrell LM, Ferguson SS, Daaka Y, et al. 1999 ß-Arrestin-dependent formation of ß2 adrenergic receptor-Src
protein kinase complexes. Science 283:655661[Abstract/Free Full Text]
-
Maudsley S, Pierce KL, Zamah AM, et al. 2000 The
ß(2)-adrenergic receptor mediates extracellular signal-regulated
kinase activation via assembly of a multi- receptor complex with
the epidermal growth factor receptor. J Biol Chem 275:95729580[Abstract/Free Full Text]
-
Barak LS, Ferguson SS, Zhang J, Caron MG 1997 A
ß-arrestin/green fluorescent protein biosensor for detecting G
protein-coupled receptor activation. J Biol Chem 272:2749727500[Abstract/Free Full Text]
-
Vogler O, Nolte B, Voss M, Schmidt M, Jakobs KH, van Koppen CJ 1999 Regulation of muscarinic acetylcholine receptor sequestration and
function by ß-arrestin. J Biol Chem 274:1233312338[Abstract/Free Full Text]
-
Benard O, Naor Z, Seger R 2000 Role of dynamin, Src and Ras in
the PKC-mediated activation of ERK by gonadotropin-releasing hormone.
J Biol Chem 16:16[CrossRef]
-
Della Rocca GJ, Mukhin YV, Garnovskaya MN, et al. 1999 Serotonin 5-HT1A receptor-mediated Erk activation requires
calcium/calmodulin-dependent receptor endocytosis. J Biol Chem 274:47494753[Abstract/Free Full Text]
-
Ignatova EG, Belcheva MM, Bohn LM, Neuman MC, Coscia CJ 1999 Requirement of receptor internalization for opioid stimulation of
mitogen-activated protein kinase: biochemical and immunofluorescence
confocal microscopic evidence. J Neurosci 19:5663[Abstract/Free Full Text]
-
DeFea KA, Zalevsky J, Thoma MS, Dery O, Mullins RD, Bunnett NW 2000 ß-Arrestin-dependent endocytosis of proteinase-activated
receptor 2 is required for intracellular targeting of activated ERK1/2.
J Cell Biol 148:12671281[Abstract/Free Full Text]
-
DeGraff JL, Gagnon AW, Benovic JL, Orsini MJ 1999 Role of
arrestins in endocytosis and signaling of
2-adrenergic receptor
subtypes. J Biol Chem 274:1125311259[Abstract/Free Full Text]
-
Kranenburg O, Verlaan I, Moolenaar WH 1999 Dynamin is required
for the activation of mitogen-activated protein (MAP) kinase by MAP
kinase kinase. J Biol Chem 274:3530135304[Abstract/Free Full Text]
-
Li JG, Luo LY, Krupnick JG, Benovic JL, Liu-Chen LY 1999 U50,488H-induced internalization of the human
opioid receptor
involves a ß-arrestin- and dynamin- dependent mechanism.
Receptor internalization is not required for mitogen-activated protein
kinase activation. J Biol Chem 274:1208712094[Abstract/Free Full Text]
-
Yang W, Wang D, Richmond A 1999 Role of clathrin-mediated
endocytosis in CXCR2 sequestration, resensitization, and signal
transduction. J Biol Chem 274:1132811333[Abstract/Free Full Text]
-
Whistler JL, von Zastrow M 1999 Dissociation of functional
roles of dynamin in receptor-mediated endocytosis and mitogenic signal
transduction. J Biol Chem 274:2457524578[Abstract/Free Full Text]
-
Ashworth R, Yu R, Nelson EJ, Dermer S, Gershengorn MC, Hinkle
PM 1995 Visualization of the thyrotropin-releasing hormone receptor and
its ligand during endocytosis and recycling. Proc Natl Acad Sci USA 92:512516[Abstract]
-
Yu R, Hinkle PM 1998 Signal transduction, desensitization, and
recovery of responses to thyrotropin-releasing hormone after inhibition
of receptor internalization. Mol Endocrinol 12:737749[Abstract/Free Full Text]
-
Groarke DA, Wilson S, Krasel C, Milligan G 1999 Visualization
of agonist-induced association and trafficking of green fluorescent
protein-tagged forms of both ß-arrestin-1 and the
thyrotropin-releasing hormone receptor-1. J Biol Chem 274:2326323269[Abstract/Free Full Text]
-
Hinkle PM, Kinsella PA 1982 Rapid temperature-dependent
transformation of the thyrotropin-releasing hormone-receptor complex in
rat pituitary tumor cells. J Biol Chem 257:54625470[Free Full Text]
-
Drmota T, Gould GW, Milligan G 1998 Real time visualization of
agonist-mediated redistribution and internalization of a green
fluorescent protein-tagged form of the thyrotropin-releasing hormone
receptor. J Biol Chem 273:2400024008[Abstract/Free Full Text]
-
Groarke DA, Drmota T, Bahia DS, Evans NA, Wilson S, Milligan G 2001 Analysis of the C-terminal tail of the rat thyrotropin-releasing
hormone receptor-1 in interactions and cointernalization with
ß-arrestin 1-green fluorescent protein. Mol Pharmacol 59:375385[Abstract/Free Full Text]
-
Yu R, Hinkle PM 1997 Desensitization of thyrotropin-releasing
hormone receptor-mediated responses involves multiple steps. J Biol
Chem 272:2830128307[Abstract/Free Full Text]
-
Hansen SH, Sandvig K, van Deurs B 1993 Clathrin and HA2
adaptors: effects of potassium depletion, hypertonic medium, and
cytosol acidification. J Cell Biol 121:6172[Abstract]
-
Graness A, Adomeit A, Heinze R, Wetzker R, Liebmann C 1998 A
novel mitogenic signaling pathway of bradykinin in the human colon
carcinoma cell line SW-480 involves sequential activation of a Gq/11
protein, phosphatidylinositol 3-kinase ß, and protein kinase C
.
J Biol Chem 273:3201632022[Abstract/Free Full Text]
-
Levitzki A, Gazit A 1995 Tyrosine kinase inhibition: an
approach to drug development. Science 267:17821788[Medline]
-
Daub H, Wallasch C, Lankenau A, Herrlich A, Ullrich A 1997 Signal characteristics of G protein-transactivated EGF receptor. EMBO J 16:70327044[Abstract/Free Full Text]
-
Oakley RH, Laporte SA, Holt JA, Caron MG, Barak LS 2000 Differential affinities of visual arrestin, ß arrestin 1, and ß
arrestin 2 for G protein-coupled receptors delineate two major classes
of receptors. J Biol Chem 275:1720117210[Abstract/Free Full Text]
-
Zhang J, Barak LS, Anborgh PH, Laporte SA, Caron MG, Ferguson
SSG 1999 Cellular trafficking of G protein-coupled
receptor/ß-arrestin endocytic complexes. J Biol Chem 274:1099911006[Abstract/Free Full Text]
-
Yu R, Hinkle PM 1999 Signal transduction and hormone-dependent
internalization of the thyrotropin-releasing hormone receptor in cells
lacking Gq and G11. J Biol Chem 274:1574515750[Abstract/Free Full Text]
-
Daaka Y, Luttrell LM, Ahn S, et al. 1998 Essential role for G
protein-coupled receptor endocytosis in the activation of
mitogen-activated protein kinase. J Biol Chem 273:685688[Abstract/Free Full Text]
-
Pierce KL, Maudsley S, Daaka Y, Luttrell LM, Lefkowitz RJ 2000 Role of endocytosis in the activation of the extracellular
signal-regulated kinase cascade by sequestering and nonsequestering G
protein-coupled receptors. Proc Natl Acad Sci USA 97:14891494[Abstract/Free Full Text]
-
Daub H, Weiss FU, Wallasch C, Ullrich A 1996 Role of
transactivation of the EGF receptor in signalling by G-protein-coupled
receptors. Nature 379:557560[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]
-
Chen YH, Pouyssegur J, Courtneidge SA, Van
Obberghen-Schilling E 1994 Activation of Src family kinase activity by
the G protein-coupled thrombin receptor in growth-responsive
fibroblasts. J Biol Chem 269:2737227377[Abstract/Free Full Text]
-
Simonson MS, Herman WH 1993 Protein kinase C and
protein tyrosine kinase activity contribute to mitogenic signaling by
endothelin-1. Cross-talk between G protein-coupled receptors and
pp60c-src. J Biol Chem 268:93479357[Abstract/Free Full Text]