Urocortin, but Not Corticotropin-Releasing Hormone (CRH), Activates the Mitogen-Activated Protein Kinase Signal Transduction Pathway in Human Pregnant Myometrium: An Effect Mediated via R1
and R2ß CRH Receptor Subtypes and Stimulation of Gq-Proteins
Dimitris K. Grammatopoulos,
Harpal S. Randeva,
Michael A. Levine,
Efrosini S. Katsanou and
Edward W. Hillhouse
Sir Quinton Hazell Molecular Medicine Research Centre (D.K.G.,
H.S.R., E.S.K., E.W.H.) Department of Biological Sciences
University of Warwick Coventry, CV4 7AL, United Kingdom
The Johns Hopkins University School of Medicine Division of
Pediatric Endocrinology and the Ilyssa Centre for Cellular and
Molecular Endocrinology Department of Pediatrics Baltimore,
Maryland 21287
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ABSTRACT
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CRH and CRH-related peptides such as urocortin
mediate their actions in the human myometrium via activation of two
distinct classes of CRH receptors, R1 and R2. These heptahelical
receptors are able to stimulate a number of different intracellular
signals; one key mediator of G protein-activated intracellular
signaling is the cascade of p42/p44, mitogen-activated protein kinase
(MAPK). We therefore hypothesized that activation of MAPK might mediate
CRH and or/urocortin actions in the myometrium.
In cultured human pregnant myometrial cells, urocortin but not CRH was
able to induce MAPK phosphorylation and activation, suggesting that in
the human myometrium these two peptides have distinct actions and
biological roles. To identify the particular receptor subtypes
mediating this phenomenon, all known CRH receptors present in the human
myometrial cells were stably expressed individually in HEK293 and CHO
cells, and their ability to activate MAPK was tested. The R1
and
R2ß, but not the R1ß, R1c, or R1d, receptor subtypes were able to
mediate urocortin-induced MAPK activation. The signaling components
were further investigated; activation of Gs, Go, or Gi proteins did not
appear to be involved, but activation of Gq with subsequent production
of inositol triphosphates (IP3) and protein
kinase C (PKC) activation correlated with MAPK phosphorylation. Studies
on Gq protein activation using
[
-32P]-GTP-
-azidoanilide and
IP3 production in cells expressing the R1
or
R2ß CRH receptors demonstrated that urocortin was 10 times more
potent than CRH. Moreover, urocortin (UCN) generated peak responses
that were 5070% greater than CRH in activating the Gq protein and
stimulating IP3 production.
In conclusion, UCN acting thought multiple receptor subtypes can
stimulate myometrial MAPK via induction of the Gq/phospholipase
C/IP3/PKC pathway, whereas CRH-induced
activation of this pathway appears to be insufficient to achieve MAPK
activation.
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INTRODUCTION
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In mammals, CRH, the hypothalamic peptide that regulates the
stress response via activation of the pituitary adrenal axis (1), is
also involved in the neuroendocrine control of immune response and can
regulate cardiovascular function, cognitive function, ingestive
behavior, reproductive function, and pregnancy and labor (2, 3, 4, 5, 6). The
multiple actions of CRH in various tissues are mediated by specific CRH
receptors that are distributed throughout the brain and peripheral
tissues. Distinct CRH actions require interactions of ligands to two
classes of CRH receptors, termed R1 and R2 (7, 8) that are encoded by
unique genes. Alternative processing of nascent transcripts from each
of these genes generates several protein variants (9, 10, 11). CRH belongs
to a family of peptides that include the recently discovered mammalian
peptide urocortin (UCN) (12), the frog peptide sauvagine, and the fish
peptide urotensin I. The existence of these structurally related
peptides enables a diversity of CRH receptor actions in specific target
tissues. Interestingly, UCN binds and activates the CRH-R2 family of
receptors with higher binding affinity and potency than CRH, suggesting
that UCN may be the preferred or native ligand for these receptors.
During human pregnancy, CRH derived from the placenta and intrauterine
tissues (13, 14, 15) is secreted into the maternal circulation. Our studies
on the human myometrium have led us to hypothesize that during
pregnancy circulating CRH has a "protective" role, by preventing
uterine contractions (16). Human myometrium expresses an extensive
variety of CRH receptor subtypes, both types R1 and R2 (17), and CRH
effects are mediated primarily via activation of Gs proteins and
stimulation of the adenylate cyclase-cAMP second messenger system (18).
Additional evidence suggests that UCN is also expressed in placental
and other intrauterine tissues (19, 20) but is not secreted into the
maternal circulation, raising the possibility that UCN acts in a local
and paracrine manner via CRH receptors and may also modulate myometrial
contractility (21).
We recently showed that native CRH receptors in human myometrium
(22) and stably expressed CRH-R1
in HEK293 cells (23) can activate
multiple G proteins, namely Gs
, Gi
, Go
, Gq
, and Gz
,
suggesting that CRH may regulate diverse signaling pathways. The
CRH-R1
and the CRH-R2
receptor subtypes have been shown to
activate the p42/p44 mitogen-activated protein kinase (MAPK) signaling
cascade after stimulation with sauvagine (24). MAPK belongs to a family
of serine-threonine kinases comprised of p44 and p42 MAPKs, which are
activated via phosphorylation of both tyrosine and threonine residues
by diverse stimuli and are expressed ubiquitously in eukaryotic cells
where they play an important role in the regulation of processes such
as cell proliferation, differentiation, and apoptosis (25, 26). In
addition, MAPK has been proposed to be involved in the regulation of
myometrial contractility by uterotonins (27, 28), as
oxytocin-stimulated uterine contractions can be partially
inhibited by MAPK inhibitors and MAPK appears to be involved in the
activation of phospholipase A2, which stimulates the release of
arachidonic acid from cellular lipids with subsequent stimulation of
prostaglandin production.
In view of these findings we sought to investigate the role of CRH and
UCN in the activation of MAPK in human myometrial cells. In addition,
to characterize in detail this interaction and identify the particular
receptor subtypes mediating this phenomenon, all known CRH receptor
subtypes present in the human myometrial cells (29) were stably
expressed individually in HEK293 or CHO cells and investigated for
their ability to activate the MAPK signaling pathway.
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RESULTS
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MAPK Activation in Cultured Human Pregnant Myometrial Cells
Cultured human myometrial cells that had been obtained from lower
segment myometrial biopsies at term before the onset of labor were
incubated with various peptides (oxytocin,10 nM; CRH,100
nM; UCN, 100 nM; or sauvagine, 100
nM) for 10 min, and cell lysates were assayed for MAPK
activation. Treatment with UCN and sauvagine led to an increase in
phospho-MAPK, with p42 appearing to be the predominant form (Fig. 1a
). Similar results were obtained with
oxytocin, which was used as a tissue-specific positive control based on
previous studies that demonstrated oxytocin-stimulated increase in MAPK
in human pregnant myometrium (30). Sauvagine was used as a
receptor-specific positive control since it has been previously shown
to activate the p42/p44 MAPK signaling cascade in HEK293 cells stably
expressing CRH-R1 and -R2 receptor subtypes (24). Despite increased
levels of phospho-MAPK, the total amount of immunoreactive p42/p44 MAPK
was unchanged. CRH was unable to activate MAPK in these cells, however,
even at concentrations up to 1 µM.

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Figure 1. Effect of (A) CRH, CRH-Like Peptides (upper
panel), Oxytocin (lower panel), and (B) PKA or
PKC Activators (Forskolin and PMA, Respectively) on MAPK Activation in
Human Pregnant Myometrial Cells
Cells were stimulated with either CRH-agonists (100 nM) for
10 min, oxytocin (110 nM), forskolin (500
µM), and PMA (200 ng/ml) for 20 min. After cell lysis and
centrifugation, supernatants were subjected to SDS-PAGE and
immunoblotted with antibody for phospho-p44/42 to determine the
phosphorylated/activated p44/42 MAPK as described in Materials
and Methods. Alternatively, the same samples were immunoblotted
with antibody for p44/42 MAPK to determine total MAPK as a control.
Identical results were obtained from five independent myometrial cell
preparations.
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As MAPK can be activated by various signaling cascades (31), we used
forskolin, which directly activates the adelylyl cyclase, and phorbol
myristate acetate (PMA), an activator of PKC, to obtain information
about the intracellular mechanisms involved in MAPK activation in these
cultured cells. Although forskolin (500 µM) failed to
stimulate MAPK, PMA (200 nM) caused a marked increase in
MAPK p42/p44 activity (Fig. 1b
).
The characteristics of UCN-stimulated MAPK activation in the cultured
myometrial cells were further investigated. The effect of UCN was found
to be time- and concentration-dependent. The UCN effect was maximal
after 510 min of treatment and returned to basal levels after 60 min
of treatment (Fig. 2a
). The UCN-dependent
increase in MAP activation was significant only at concentrations
greater than 10 nM and was maximal at concentrations of 100
nM (Fig. 2b
). Higher concentrations did not produce any
further stimulation (data not shown), and there was no increase in the
total amount of p42/p44 MAPK immunoreactivity. In addition, the CRH
receptor antagonist astressin (1 µM) was able to
significantly inhibit the stimulatory action of UCN (Fig. 2c
),
indicating that the UCN effect was mediated via activation of CRH
receptors.

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Figure 2. Characteristics of UCN-Induced MAPK Activation in
Myometrial Cells
Time course (A) and dose response (B) of UCN-induced MAPK activation in
human pregnant myometrial cells. C, Effect of the CRH receptor
antagonist astressin on UCN-induced MAPK activation in human pregnant
myometrial cells. Cells were stimulated with either UCN (100
nM) for 5, 10, or 60 min or varying concentrations of UCN
(100 pM to 100 nM) for 10 min or
UCN (100 nM) in the presence or absence of astressin (1
µM) for 10 min. After cell lysis and centrifugation,
supernatants were subjected to SDS-PAGE and immunoblotted with antibody
for phospho-p44/42 to determine the phosphorylated/activated p44/42
MAPK as described in Materials and Methods.
Alternatively, the same samples were immunoblotted with antibody for
p44/42 MAPK to determine total MAPK as a control (A, lower
panel). The intensity of the bands corresponding to p42 and p44
MAPK were quantitated with an imaging densitometer (B, lower
panel). Identical results were obtained from five independent
myometrial cell preparations. *, P < 0.05 compared
with basal.
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Since cultured human myometrial cells express at least five CRH-R
subtypes (i.e. R1
, R1ß, R1c, R1d, and R2ß) (29), each
CRH-R was stably expressed individually in HEK293 or CHO cells, and the
activation of MAPK by CRH or UCN was further investigated.
MAPK Activation in Mammalian Cells Expressing Individual CRH
Receptors
In HEK293 or CHO cells, untransfected or stably expressing
different CRH-R subtypes, MAPK p42/p44 activity was stimulated by PMA
(200 nM) but not by forskolin (500 µM) or
choleragen (12 h, 500 ng/ml) (data not shown), confirming the existence
of functional MAPK signaling cascades with similar characteristics to
those in the cultured myometrial cells.
Studies in HEK293 or CHO cells expressing the CRH-R1
receptor
subtype (293-R1
or CHO-R1
) showed that UCN but not CRH was able
to increase phospho-MAPK p42/p44 immunoreactivity, with p42 appearing
to be the predominant form (Fig. 3a
). The
UCN effect was found to be time- and concentration-dependent with
characteristics similar to those found in the human myometrial cells
(data not shown). Although CRH at concentrations up to 1
µM failed to stimulate MAPK,
125I-tyro-CRH bound to the
R1
receptor subtype with binding affinity comparable to
125I-tyro-UCN, as assessed
by Scatchard analysis (1.25 ± 0.4 vs. 1.05 ± 0.3
nM, respectively) (Fig. 3b
). Subsequent studies
in HEK293 cells expressing various CRH-R receptor subtypes revealed
that UCN was able to activate MAPK in 293-R2ß cells, but not in cells
expressing the CRH-R1ß, -R1c, or -R1d receptor subtypes, even at
pharmacological concentrations of urocortin (10
µM). By contrast, CRH failed to activate MAPK
in all receptor subtypes tested (Table 1
). Furthermore, although UCN has been
shown to have greater binding affinity [dissociation constant
(Kd) for the R2ß than the R1
receptor
subtype (0.6 ± 0.2 vs. 1.9 ± 0.6 for the R2ß
and R1
, respectively], the sensitivity and potency of MAPK
activation was found to be similar via both receptors (sensitivity: 10
nM of UCN; potency: 3.1 ± 0.4 times above
basal at a UCN concentration of 100 nM) (data not
shown). In addition, in 293-R1c cells UCN or CRH was found to have very
low binding affinity for UCN or CRH, and these ligands stimulated only
a small cAMP response, consistent with the finding that the 40-amino
acid deletion in the N-terminal domain of CRH-R1c impairs the binding
ability of this receptor. Both peptides could exert a significant cAMP
production only at concentrations greater than 1
µM, and maximum cAMP responses ranged between
4 ± 1.1- and 5 ± 1.5-fold above basal (at a peptide
concentration of 1 µM) (Fig. 4
). Neither CRH nor UCN elicited an
increase in IP3 production or MAPK activity.
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Table 1. Effect of CRH and UCN on MAPK and Adenylyl
Cyclase Activation from Various CRH Receptor Subtypes (Expressed in
Human Pregnant Myometrial Cells) Individually Expressed in HEK 293
Cells
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Figure 4. cAMP Release from HEK-293 Cells Stably Transfected
with CRH-R1c Receptor Subtype (50 µg Protein) in the Presence of
Different Concentrations of h/rCRH or Urocortin
Results are expressed as the mean ± SEM of four
estimations from three independent experiments. *,
P < 0.05 compared with basal.
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No MAPK activation was observed upon addition of UCN (up to 1
µM) (data not shown) to untransfected HEK293 cells.
Characterization of the Intracellular Components Involved in MAPK
Activation
Because activation of the CRH-R1
receptor by CRH or UCN leads
to interaction with at least four G proteins, Gs, Gi, Gq, and Go (23),
we sought to identify which G proteins were involved in
UCN/CRH-R1
/MAPK activation. The involvement of Gs
proteins was
excluded by the finding that activation of the Gs-adenylyl cyclase-cAMP
pathway (cholera toxin or forskolin) in 293-R1
cells did not induce
MAPK activation. To evaluate possible involvement of Gi or Go proteins,
we used pertussis toxin (PTX) (212 h, final concentration, 100 ng/ml)
to inactivate Gi/Go proteins via ADP ribosylation. Pertussis treatment
of 293-R1
cells had no effect on UCN-induced MAPK activation (Fig. 5a
); however, in all PTX incubation
periods, a reduction (P < 0.05) in basal MAPK activity
was found to be present. PTX inactivation of Gi and Go was confirmed by
examining the ability of UCN to induce 32P-GTP-AA
incorporation to these G protein
-chains. As expected, UCN-induced
incorporation of 32P-GTP-AA into the
PTX-sensitive G proteins, Gi and Go, was dramatically inhibited (Fig. 5b
) by PTX at all incubation periods used (212 h).

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Figure 5. Investigation of Gi/Go Protein Involvement on
UCN-MAPK Interaction
A, Effect of pertussis-toxin pretreatment on UCN-induced activation of
MAPK in 293-R1 cells. Cells were pretreated with or without PTX (100
ng/ml for 212 h) at 37 C at 5% CO2 and were then washed
and stimulated with UCN (100 nM) for 10 min. After cell
lysis and centrifugation, supernatants were subjected to SDS-PAGE and
immunoblotted with antibody for phospho-p44/42 to determine the
phosphorylated/activated p44/42 MAPK (upper panel) as
described in Materials and Methods. Alternatively, the
same samples were immunoblotted with antibody for p44/42 MAPK to
determine total MAPK as a control. The intensity of the bands
corresponding to p42 MAPK was quantitated with an imaging densitometer
(lower panel). Identical results were obtained from
three independent experiments. *, P < 0.05
compared with untreated basal; +, P <
0.05 compared with untreated UCN-stimulated. B, Effect of PTX
pretreatment (final concentration, 100 ng/ml) on UCN-induced
photolabeling (with GTP-AA) of G i and G o proteins from membranes
prepared from 293-R1 cells. Cells were cultured in six-well plates
in the presence or absence of PTX for 6 h at 37 C at 5%
CO2, followed by cell membrane preparation and addition of
UCN (100 nM) for 5 min at 30 C. Then GTP-AA was added,
followed by UV cross-linking and immunoprecipitation of the
G -subunits using specific antibodies. Proteins were resolved on
SDS-PAGE gels, followed by autoradiography and densitometry scanning
for quantitation of agonist-induced photolabeling of specific
G -subunits. Identical results were obtained from six independent
experiments. *, P < 0.05 compared with untreated
basal.
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These data suggested that the UCN effect might be mediated via Gq
proteins and implicated involvement of the phospholipase C
(PLC)-IP3-PKC signaling pathway. To confirm this,
293-R1
cells were pretreated with the PLC inhibitor U73122 (20 min,
10 µM) before stimulation with UCN (100 nM).
U73122 treatment led to a 6080% reduction in UCN-induced MAPK
activation (Fig. 6a
), confirming that
activation of PLC, likely through Gq-proteins, is required for UCN
action. The role of PKC in UCN activation of MAPK was investigated
after prolonged stimulation with PMA (12 h, 200 ng/ml) to deplete PKC
(32). In this experimental paradigm the UCN-induced increase in
phospho-MAPK p42/p44 immunoreactivity was reduced by 7090% (Fig. 6b
), confirming that UCN activates MAPK predominantly via a
PKC-mediated phenomenon. Similar results were obtained with myometrial
cells and 293-R2ß cells, suggesting a common signaling pathway of
myometrial R1
and R2ß receptor subtypes after stimulation by UCN
(data not shown).

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Figure 6. Effect of (A) PLC Inhibition Using U73122 or (B)
PKC Depletion on UCN-Induced Activation of MAPK in 293-R1 Cells
Cells were pretreated with or without PMA (200 ng/ml for 12 h) or
with or without U73122 (10 µM for 20 min). Cells were
then washed and stimulated with UCN (100 nM) for 10 min.
After cell lysis and centrifugation, supernatants were subjected to
SDS-PAGE and immunoblotted with antibody for phospho-p44/42 to
determine the phosphorylated/activated p44/42 MAPK (a, b, upper
panel) as described in Materials and Methods.
Alternatively, the same samples were immunoblotted with antibody for
p44/42 MAPK to determine total MAPK as a control (b, lower
panel). The intensity of the bands corresponding to p42 MAPK
was quantitated with an imaging densitometer (a, lower
panel). Identical results were obtained from three independent
experiments. *, P < 0.05 compared with basal;
+, P < 0.05 compared with untreated
UCN-stimulated.
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In previous studies we had shown that CRH treatment of 293-R1
stimulates Gq proteins and IP3 production (23),
and yet in the present study CRH failed to stimulate MAPK activity.
This apparent discrepancy was investigated in more detail in
experiments studying the concentration of CRH or UCN needed to activate
G protein signaling. Gq protein activation was investigated by using
the nonhydrolyzable GTP analog 32P-GTP-AA to
label receptor-activated G proteins, followed by immunoprecipitation
with specific G protein
-chain antibodies (33). As shown in Fig. 7
, CRH or UCN exhibited substantial
differences in their ability to radiolabel the 40-kDa G
q protein in
293-R1
cells. Both CRH and UCN activated Gq at a threshold
concentration of about 1 nM, with maximal
32P-GTP-AA incorporation obtained at a
concentration of 100 nM. However, UCN was 80100% more
potent than CRH, both at submaximal (10 nM) and maximal
(100 nM) concentrations.
In addition, activation of Gq protein by CRH or UCN correlates
with activation of phospholipase C and increased generation of
IP3 . We found that CRH or UCN treatment
stimulated a significant increase in inositol phosphate production,
with a threshold of 1 nM and a maximum response at 100
nM (Fig. 8a
). Consistent with
our previous findings, the response to UCN was 6080% more potent
than that to CRH (maximum 150 ± 21% of basal for CRH and
235 ± 34% of basal for UCN), which resulted in a 95 ± 17%
stimulation of basal PKC activity, whereas CRH had no effect on PKC
activity (Fig. 8a
, inset). By contrast, both peptides have
comparable potency, increasing cAMP with a threshold of 100
pM and maximum response at 100 (maximum 90
± 14-fold above basal for CRH and 104 ± 25-fold above basal for
UCN) (Fig. 8b
).

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Figure 8. IP3 Accumulation (A) and cAMP Release
(B) from HEK-293 Cells Stably Transfected with CRH-R1 Receptor
Subtype (50 µg Protein) in the Presence of Different Concentrations
of CRH or UCN
Inset, PKC activation by CRH or UCN in 293-R1 cells.
Cells were stimulated with CRH or UCN (100 nM for 10 min)
and after cell lysis and centrifugation the PKC activity was determined
using a nonradioactive, ELISA-based protein kinase C assay kit. Results
are representative of one receptor clone and are expressed as the
mean ± SEM of four estimations from three independent
experiments. *, P < 0.05 compared with basal;
+, P < 0.05 compared with
each other.
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DISCUSSION
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The purpose of this study was to determine whether CRH and
CRH-related peptides are able to activate MAPK in human pregnant
myometrium. In cultured human pregnant myocytes, UCN, but not CRH, was
a potent stimulator of MAPK activity. This effect appears to be
mediated via the R1
and R2ß CRH receptor subtypes as shown by
studies in which all CRH-R subtypes known to be present in the
cultured myocytes were individually transfected into HEK293 cells. This
is the first demonstration that the CRH-R1
receptor subtype, which
binds both CRH and UCN with equipotent affinity, can differentially
activate intracellular signaling cascades based on the peptide ligand,
leading to either selective activation (UCN-MAPK) or
nonselective activation (UCN/CRH-adenylate cyclase). This might have
wider implications for understanding the biological actions of the two
peptides and their receptors. Identical results were obtained in
experiments using CRH-R subtypes stably expressed in CHO cells,
confirming that this finding is not due to artifacts of the
heterologous expression system used. In addition, the CRH-R2ß
receptor subtype appeared to possess similar properties; this, however,
is less surprising considering that this receptor subtype
preferentially binds UCN over CRH.
Although at present the mechanism whereby the R1 receptor can exhibit
such selective activation in signaling cascades is unknown, it is
likely that CRH and UCN bind to distinct domains within the receptor
leading to conformational changes, which facilitate coupling to
particular G proteins and activation of intracellular signaling
molecules. Several studies have identified important sequences for
high-affinity agonist binding to CRH-R1 in the N terminus (amino acids
4250 and 76 to 84) and in the extracellular (EC) domains of the R1
receptor (34, 35). Also, regions important for high-affinity CRH
binding have been localized to the second and fourth EC domain, the
junctions of the third EC domain/fifth transmembrane domain (TMD), and
second EC domain/third TMD (36, 37). The same three regions have been
shown to influence the binding of UCN and sauvagine, but to different
degrees (38). Regions in the third EC domain, such as Asp254, appear to
be important for sauvagine but not CRH or urocortin binding (38),
demonstrating that different agonists differentially interact with some
of the same regions of the CRH-R1.
Previous studies have demonstrated that CRH and CRH-like ligands are
capable of modulating the MAPK system; in ovine anterior pituitary
cells, CRH can stimulate and in mouse pituitary tumor AtT20 cells
inhibit MAPK activity (39). In addition, in CHO cells stably expressing
the subtypes R1
or R2
, sauvagine has been shown to activate MAPK
(24). Most interestingly, in primary cardiac myocyte cultures, UCN
acting via activation of MAPK is able to protect against ischemic and
reperfusion injury (40). However, very little is known about the
intracellular pathways involved in these events initiated by CRH
receptor activation. Studies in other 7TMD receptors/MAPK interactions
have shown that in different cellular systems and under certain
conditions several G proteins, such as G
s, G
q, G
i, G
12, and
Gß
-subunits, can regulate MAPK activity (41). In earlier studies
we showed that CRH-R1
receptors that are stimulated by CRH or UCN
can couple to and activate at least four different G proteins, namely
G
s, G
i, G
q, and G
o (23). The data presented in this study
indicate that the UCN stimulation of MAPK via R1
and R2ß CRH
receptor subtypes requires G
q-, but not G
s-, G
i-, or G
o-,
proteins. Further support of this signaling pathway derives from our
experiments showing that the CRH receptor subtypes, R1ß and R1d,
which are unable to couple to Gq protein, are unable to activate the
MAPK signaling pathway. The mechanism by which PKC directly activates
the MAPK cascade remains unclear; it has been shown that activated PKC
can directly phosphorylate and activate Raf1 (42), by a mechanism
partially dependent on Ras, leading to stimulation of the MAPK cascade
(43). Interestingly, neither PKC depletion nor PLC inhibition totally
blocked MAPK activation, thus raising the possibility that additional
mechanisms, such as activation of ß
-subunits of G
proteins in a Ras-dependent, PKC-independent process (44), which has
been found to be active in HEK293 cells (45), might be involved in the
UCN/MAPK interaction.
Having established that the activated CRH-R1
receptor can interact
with MAPK via a Gq/PLC/PKC signal transduction pathway, we investigated
ligand-R1
receptor interactions to identify differences between CRH
and UCN binding that might explain the inability of CRH to stimulate
MAPK phosphorylation. Although CRH and UN were equipotent in binding to
the R1
and activating the Gs/AC/cAMP pathway, UCN was more potent in
activating the Gq proteins and IP3 production and
subsequently PKC, reinforcing the view that binding of UCN to the R1
receptor subtype causes a distinctive conformational change that
preferentially couples to Gq proteins and leads to activation of the
PLC/IP3/PKC/MAPK cascade (Fig. 9
). Although the stoichiometric details
of this signal generating pathway are not known, CRH-induced activation
of PLC appears to be insufficient to achieve activation of PKC or MAPK.
Similar findings have been previously reported in the human pregnant
myometrium at term (22). This raises the possibility that CRH and
urocortin, acting via the same receptor subtypes, may have distinct
functional roles during human pregnancy and labor. Thus, the
ligand-receptor complex, rather than the ligand alone, may play a
fundamental physiological role in determining signal specificity.

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Figure 9. Schematic Representation of the Proposed Mechanism
For MAPK Activation by CRH-Related Agonists in Human Pregnant
Myometrium
According to this model UCN can bind with equal or higher affinity with
CRH to the CRH-R1 and CRH-R2ß receptor subtypes, respectively,
initiating a series of diverse intracellular events. UCN acting via
both receptor subtypes can stimulate the Gq-protein/
PLC/IP3 pathway leading to MAPK activation by a
PKC-dependent mechanism. CRH acting via the same receptor subtypes is
able to stimulate the same intracellular pathway but with reduced
potency, which appears to be insufficient for the downstream
stimulation of MAPK. Both peptides can stimulate the adenylate cyclase
(AC) pathway via activation of Gs proteins (with equal potency when
bound to CRH-R1 or greater for UCN when bound to CRH-R2ß) leading
to elevation of cAMP, but this pathway appears not to be involved in
MAP kinase activation. The modulators of the intracellular components
used in this study are represented in italics.
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The UCN effect on myometrial MAPK activation was rapid and transient.
This is usually associated with cellular growth and proliferation,
whereas prolonged activation is involved in cell differentiation (46, 47). Activation of myometrial MAPK has also been implicated in the
regulation of myometrial contractility, particularly as a target of
several uterotonins such as oxytocin, prostaglandins, and endothelin-1
(27, 28, 48). Myometrial MAPK has also been shown to be activated by
oxytocin via a PTX-dependent but PKC-independent mechanism, which leads
to cyclooxygenase-2 expression and increased production of
prostaglandins (49). These data indicate that UCN may play an important
and distinct role in the regulation of myometrial contractions during
labor (21). The physiological circulating concentrations of UCN do not
change during pregnancy and are in the range of 2.63.6 pM
(50). However, local production of urocortin from intrauterine tissues
(19) during pregnancy raises the possibility that myometrial cells
might be exposed to much higher concentrations of urocortin.
Our data demonstrate that UCN and sauvagine can interact with specific
CRH receptor subtypes to acutely stimulate myometrial MAPK in part
through the Gq-coupled PKC pathway. In contrast, CRH can interact with
the same receptor subtypes but is unable to generate a similar effect,
raising the possibility that these two ligands can induce different
conformations in the same receptor with different signaling
consequences. In the human pregnant myometrium, activation of MAPK
appears to be involved in the development of myometrial contractility,
suggesting that UCN and CRH have distinct roles in the mechanism of
human labor. Further molecular characterization of the receptor-ligand
complex for CRH and related peptides will help to elucidate the
functional role of these peptides and provide the basis for their
proposed "dual" role (maintenance of myometrial relaxation and
stimulation of contractility) (16) during pregnancy and labor.
 |
MATERIALS AND METHODS
|
---|
Chemicals
Radioiodinated ovine-tyr-CRH, radioiodinated UCN, human UCN,
human/rat CRH, sauvagine, and oxytocin were obtained from
Peninsula Laboratories, Inc. (Merseyside, UK). The
mammalian expression vector pCI-neo was obtained from Promega Corp. UK Ltd (Chilworth Research Centre, Southampton, UK).
Dithiothreitol, GDP, forskolin, cholera and pertussis toxins,
2-[N-morpholino]ethane sulfonic acid, 1,4-dioxane,
triethylamine, and all other chemicals were purchased from
Sigma Chemical Company Ltd (Poole, Dorset, UK). Waters
Sep-Pak C18 columns were obtained from Millipore Corp. Ltd
(Watford, Herts, UK). The PhosphoPlus p44/42 MAPK antibody kit was
purchased from New England Biolabs, Inc. Ltd (Hitchin,
Hertfordshire, UK). Polyclonal G-protein rabbit antibodies,
[3H]myo-inositol and the cAMP assay kits were
obtained from NEN Life Science Products
(Hertfordshire, UK). PMA and U73122 were obtained from
Calbiochem (La Jolla, CA). Protein-A Sepharose beads
(CL-4B) were purchased from Pharmacia Biotech (Uppsala,
Sweden). [
-32P]-GTP, enhanced
chemiluminescence (ECL), the DNA sequencing kit, and
35S-
-ATP were obtained from Amersham International (Amersham Place, Little Chalfont, Buckinghamshire,
UK). 4-Azidoanilide-HCl, 1-(3-dimethylamino
propyl)-3-ethylenecarbodiimide hydrochloride (NDEC) was purchased from
Aldrich Chemical Co. (Dorset, UK). PCR and cloning
reagents were purchased from Life Technologies Ltd
(Renfrewshire, UK). The DNA 3'-end labeling kit was purchased from
Roche Molecular Biochemicals (Bell Lane, UK). Synthetic
oligonucleotide probes and enzymes were purchased from Life Technologies (Paisley, UK). All other chemicals were purchased
from BDH (Poole, UK).
Subjects and Culture of Myocytes
Pregnant myometrial biopsies (n = 7) were obtained from
women undergoing elective cesarean section at term before the onset of
labor for nonmaternal problems. The biopsy site was standardized to the
upper margin of the lower segment of the uterus in the midline. This
provides the closest approximation to the upper segment of the uterus.
These studies were approved by the local ethical committee, and
informed consent was obtained from all patients.
The tissue was immediately placed in 20 ml of ice-cold DMEM culture
medium containing 200 IU penicillin/ml, 200 mg streptomycin/ml.
Myocytes were prepared by enzymatic dispersion as previously described
(29). The cells were cultured at 37 C in a humidified atmosphere of
95% air and 5% CO2 until confluent.
Stable Transfection of HEK293 and CHO Cells
Human CRH-R1 or R2 receptor subtypes (23) were subcloned into
the expression vector pCI-neo (Promega Corp.), which
contains the human cytomegalovirus immediate early promoter. HEK293 or
CHO cells were transfected using Lipofectamine reagent (Life Technologies). The cells were grown in DMEM in the presence of
G418 (500 µg/ml) to select for transfected cells and those surviving
were subcultured.
Membrane Preparation and CRH RRA
Confluent HEK293 or CHO cells were washed with PBS and lysed
with 0.2% NaCl. The cells were homogenized in extraction buffer A (10
mM Tris-HCl, 1 mM EDTA, 1 mM
phenylmethylsulfonylfluoride, 10 mM
MgCl2, 0.1% BSA, and 0.1% bacitracin, pH 7.2).
The homogenate was centrifuged at 600 x g for 30 min
at 4 C to remove nuclei and unbroken cells. The supernatant was
collected and centrifuged at 40,000 x g for 60 min at
4 C. The pellet was rinsed twice, resuspended in binding buffer B (10
mM Tris-HCl, 1 mM EDTA, 10
mM MgCl2, 0.1% BSA, and
0.1% bacitracin, pH 7.2), and aliquoted (50 µg in 50 µl aliquots)
in microfuge tubes.
For receptor analysis, membranes (50100 µg of protein) were
incubated with 125I-oCRH or
125I-UCN (0.22 nM) and unlabeled
r/h CRH or UCN (1,000 molar excess) in 50 µl of binding buffer B. The
tubes were incubated at 22 C for 120 min. The reaction was terminated
by adding 1 ml/tube of ice-cold 20% polyethylene glycol (PEG). After
centrifugation at 10,000 x g for 15 min at 4 C, the
pellets were washed once with 20% PEG and radioactivity was quantified
in a
-counter (Packard Instruments, Meriden, CT) at 70%
efficiency. Nonspecific binding was 18 ± 5% of the total added
radioactivity.
The binding data were analyzed using the EBDA program (51) and LIGAND
(52) (EBDA/LIGAND, Elsevier-Biosoft, Cambridge, UK).
MAPK Activation and Western Blot Analysis
Cells (myometrial, HEK293, or CHO) were cultured in six-well
dishes for 2 days in DMEM containing 0.5% FCS. The confluent cells
were washed with DMEM containing 0.5% FCS and incubated in fresh
medium for a further 30 min before addition of agonists. At the end of
the incubation the medium was aspirated and the cells were washed twice
with PBS containing 1 mM NaF. Cells were lysed by the
addition of 100 µl SDS-PAGE sample buffer containing 62.5
mM Tris-HCl (pH 6.8), 2% (wt/vol) SDS, 10% glycerol, and
50 mM dithiothreitol. The solubilized material was then
removed from dishes and sonicated for 15 sec, heated to 95100 C for 5
min, and cooled on ice. Before electrophoresis the extracts were
centrifuged at 4,000 rpm for 5 min to remove insoluble material. After
electrophoresis through 10% PAGE gels, the resolved proteins were
transferred to polyvinylidene difluoride membrane at 100 mA for 90 min.
The membrane was then blocked with Tris-buffered saline containing 5%
nonfat dry milk and 0.1% Tween 20 at room temperature for 1 h and
subsequently incubated overnight at 4 C in primary antibody solution
(phospho-MAPK, 1:1000, total MAPK, 1:2000 in TBS containing 5% nonfat
dry milk, 0.1% Tween-20). Polyvinylidene difluoride membranes were
washed three times with TBS-Tween 20 (0.1%) and incubated with goat
antirabbit antibody conjugated with horseradish peroxidase (1:1000 in
blocking solution) for 1 h at room temperature. After three washes
with TBS-Tween 20 (0.1%), antibody binding was detected by enhanced
chemiluminescence (ECL).
Second Messenger Studies
293-R1 or 293-R2 cells were seeded in 96-well dishes and
cultured until 95% confluency. Before treatments, cells were washed
once with 200 µl DMEM containing 0.1% BSA, followed by preincubation
with DMEM containing 0.5 mM 3-isobutyl-1-methylxanthine for
30 min. Cells were then stimulated with hCRH or hUCN (0.11000
nM) for 15 min at 37 C; reactions were terminated by
addition of 0.1 M HCl. After an overnight freeze/thaw
cycle, the cAMP levels were measured in the supernatants using RIA. The
sensitivity of the assay was 0.025 pmol/liter and the precision was as
follows: intraassay CV, 2.9%; and interassay CV, 9.7%.
For the inositol phosphate stimulation assay, cells were seeded in
six-well dishes and subcultured in DMEM until 95% confluency. After
incubation with inositol-free DMEM containing
[3H]myo-inositol (10 µCi/well) for 24 h,
cells were washed with inositol-free DMEM once and preincubated with
inositol-free DMEM containing 0.1% BSA and 30 mM LiCl for
30 min at 37 C. Phosphoinositide turnover was stimulated with hCRH or
UCN (11000 nM) in the presence of 30 mM LiCl,
and the reactions were terminated by addition of
chloroform/methanol/hydrochloric acid (50:100:1) at specified time
intervals. After transferring to borosilicate glass tubes and
centrifugation, the upper phase was applied to Prefilled Poly-Prep
columns (AG 1-X8 resin 100200 mesh chloride from Bio-Rad Laboratories, Inc. York, UK), and
[3H]IPs were resolved and quantified as
previously described (53, 54). The radioactivity was measured by a
ß-counter.
Measurement of PKC Activity
293-R1
or CHO-R1
cells were seeded in six-well dishes and
cultured until 9095% confluency (density
107cells per well). Cells were incubated with CRH
or UCN (100 nM) in DMEM, for 10 min at 37 C. Cells were
then collected in ice-cold PBS and after centrifugation the pellet was
suspended in sample preparation buffer containing 50 mM
Tris-HCl, 10 mM benzamidine, 5 mM EDTA, 10
mM EGTA, 50 mM ß-mercaptoethanol, 1
mM phenylmethylsulfonylfluoride, pH 7.5, followed by
sonication on ice four to five times for 10 sec. Samples were
centrifuged at 100,000 x g for 60 min at +4 C, and the
PKC activity in each sample was measured using a nonradioactive,
protein kinase assay kit (Calbiochem, San Diego, CA)
according to the manufacturers instruction. This enzyme-linked
immunosorbent assay (ELISA)-based assay kit utilizes a immobilized
synthetic PKC pseudosubstrate and a biotinylated monoclonal antibody
that recognizes the phosphorylated form of the peptide.
Synthesis of GTP-AA and Photolabeling of G
Subunits
GTP-AA was synthesized following a method described previously
(23). Fractions containing GTP-AA were combined, evaporated to dryness,
and stored at -70 C for up to 1 month. The overall yield of GTP-AA
varied from 3050%. All procedures were performed in a darkened
room.
293-R1 or 293-R2 cell membranes (100 µg) were incubated with or
without varying concentrations of h/rCRH or UCN for 5 min at 30 C
before the addition of 5 µCi of
[
-P32]-GTP-AA in 120 µl of 50
mM HEPES buffer, pH 7.4, containing 30 mM KCl,
10 mM MgCl2, 1 mM
benzamidine, 5 µM GDP, and 0.1 mM EDTA, in a
darkened room. After incubation at 30 C, membranes were collected by
centrifugation and resuspended in 100 µl of the above buffer
containing 2 mM glutathione, placed on ice, and exposed to
UV light (254 nm) at a distance of 5 cm for 5 min.
G Protein Immunoprecipitation
GTP-AA-labeled 293-R1 or 293-R2 cell membranes were precipitated
by centrifugation, solubilized, immunoprecipitated, and electrophoresed
following a method described previously (23). The gels were then
stained with Coomassie Blue, dried using a slab gel dryer, and exposed
to Fuji Photo Film Co., Ltd (Tokyo, Japan) x-ray
film at -70 C for 25 days.
Statistical Analysis
Data are shown as the mean ± SEM of each
measurement. Comparison between group means was performed by ANOVA, and
P < 0.05 was considered significant. The relative
density of the bands was measured by optical density scanning using the
software Scion Image-Beta 3b for Windows (Scion Corp., Frederick,
MD).
 |
FOOTNOTES
|
---|
Address requests for reprints to: Dr. D. Grammatopoulos, Sir Quinton Hazell Molecular Medicine Research Centre, Department of Biological Sciences, The University of Warwick, Gibbet Hill Road, Coventry CV4 7AL, UK. E-mail: chdg{at}dna.bio.warwick.ac.uk
This work was supported by a Wellcome Trust Career Development Award to
D.G. E.H. is the Warwickshire Private Hospitals Charitable
Trust Chair of Medicine.
Received for publication June 19, 2000.
Revision received August 25, 2000.
Accepted for publication September 14, 2000.
 |
REFERENCES
|
---|
-
Vale W, Spiess J, Rivier C, Rivier J 1981 Characterization of a 41-residue ovine hypothalamic peptide that
stimulates secretion of corticotropin and ß-endorphin. Science 213:13941397[Medline]
-
Orth DN 1992 Corticotropin-releasing hormone in humans.
Endocr Rev 13:164191[Medline]
-
De Souza EB 1995 Corticotropin-releasing factor receptors:
physiology, pharmacology, biochemistry and role in central nervous
system and immune disorders. Psychoneuroendocrinol 20:789819[CrossRef][Medline]
-
Webster EL, Torpy DJ, Elenkov IJ, Chrousos GP 1998 Corticotropin-releasing hormone and inflammation. Ann NY Acad Sci 840:2132[Abstract/Free Full Text]
-
Chrousos GP, Torpy DJ, Gold PW 1998 Interactions between the
hypothalamic-pituitary-adrenal axis and the female reproductive system:
clinical implications. Ann Intern Med 129:229240[Abstract/Free Full Text]
-
McLean M, Bisits A, Davies J, Woods R, Lowry P, Smith R 1995 A placental clock controlling the length of human pregnancy. Nat Med 1:460463[Medline]
-
Chen R, Lewis KA, Perrin MH, Vale WW 1993 Expression cloning
of a human corticotropin-releasing-factor receptor. Proc Natl Acad Sci
USA 90:89678971[Abstract]
-
Liaw CW, Lovenberg TW, Barry G, Oltersdorf T, Grigoriadis
DE, De Souza EB 1995 Cloning and characterization of the human
corticotropin-releasing factor-2 receptor complementary
deoxyribonucleic acid. Endocrinology 137:7277[Abstract]
-
Ross PC, Kostas CM, Ramabhadren TV 1994 A variant the human
corticotropin-releasing factor (CRF) receptor: cloning, expression and
pharmacology. Biochem Biophys Res Commun 205:18361842[CrossRef][Medline]
-
Valdenaire O, Giller T, Breu V, Gottowik J, Kilpatrick G 1997 A new functional isoform of the human CRF2
receptor for corticotropin-releasing factor. Biochim et Biophys Acta 1352:129132[Medline]
-
Kostich WA, Chen A, Sperle K, Largent BL 1998 Molecular
identification and analysis of a novel human corticotropin-releasing
factor (CRF) receptor: the CRF2
receptor. Mol Endocrinol 12:10771085[Abstract/Free Full Text]
-
Vaughan J, Donaldson C, Bittencourt J, Perrin MH, Lewis K,
Sutton S, Chan R, Turnbull AV, Lovejoy D, Rivier C, Rivier J, Sawchenko
PE, Vale W 1995 Urocortin, a mamalian neuropeptide related
to fish urotensin I and to corticotropin-releasing factor. Nature 378:287292[CrossRef][Medline]
-
Petraglia F, Sawchenko PE, Rivier J, Vale W 1987 Evidence for
local stimulation of ACTH-secretion by corticotropin-releasing factor
in human placenta. Nature 328:717719[CrossRef][Medline]
-
Clifton VL, Telfer JF, Thompson AJ, Cameron IT, Teoh TG, Lye
SJ, Challis JR 1998 Corticotropin-releasing hormone and
proopiomelanocortin-derived peptides are present in human
myometrium. J Clin Endocrinol Metab 83:37163721[Abstract/Free Full Text]
-
Makrigiannakis A, Psychoyos A, Zoumakis E, Margioris AN,
Stournaras C, Gravanis A 1997 Endometrial corticotropin-releasing
hormone: expression, regulation, and potential physiological
implications. Ann NY Acad Sci 816:11628[Abstract]
-
Grammatopoulos D, Hillhouse EW 1999 Role of
corticotropin-releasing hormone in the onset of labour. Lancet 353:15461549[CrossRef]
-
Grammatopoulos DK, Dai Y, Chen J, Karteris E, Papadopoulou N,
Easton AJ, Hillhouse EW 1998 Human CRH receptor:
differences in subtype expression between pregnant and non-pregnant
myometria. J Clin Endocrinol Metab 83:25392544[Abstract/Free Full Text]
-
Grammatopoulos D, Stirrat GM, Williams SA, Hillhouse EW 1996 The biological activity of the corticotropin-releasing hormone
receptor-adenylate cyclase complex in human myometrium is reduced at
the end of pregnancy. J Clin Endocrinol Metab 81:745751[Abstract]
-
Petraglia F, Florio P, Gallo R, Simoncini T, Saviozzi M, Di
Blasio AM, Vaughan J, Vale W 1996 Human placenta and fetal
membranes express human urocortin mRNA and peptide. J Clin
Endocrinol Metab 81:38073810[Abstract]
-
Glynn BP, Wolton A, Rodriguez-Linares B, Phaneuf S, Linton EA 1998 Urocortin in pregnancy. Am J Obstet Gynecol 179: 533539
-
Petraglia F, Florio P, Benedetto C, Marozio L, Di Blasio AM,
Ticconi C, Piccione E, Luisi S, Genazzani AR, Vale W 1999 Urocortin stimulates placental adrenocorticotropin and prostaglandin
release and myometrial contractility in vitro. J Clin
Endocrinol Metab 84:14201423[Abstract/Free Full Text]
-
Randeva HS, Grammatopoulos DK, Hillhouse EW, Differential
activation of G-proteins by CRH and urocortin in human pregnant
myometrium. Program of the 81st Annual Meeting of The Endocrine
Society, San Diego, CA, 1999 (Abstract P3460)
-
Grammatopoulos DK, Dai Y, Randeva HS, Levine MA, Karteris E,
Easton AJ, Hillhouse EW 1999 A novel spliced variant of the
type 1 corticotropin-releasing hormone receptor with a deletion in the
seventh transmembrane domain present in the human pregnant term
myometrium and fetal membranes. Mol Endocrinol 13:2189202[Abstract/Free Full Text]
-
Rossant CJ, Pinnock RD, Hughes J, Hall MD, McNulty S 1999 Corticotropin-releasing factor type 1 and type 2alpha receptors
regulate phosphorylation of calcium/cyclic adenosine
3',5'-monophosphate response element-binding protein and activation of
p42/p44 mitogen-activated protein kinase. Endocrinology 140:15251536[Abstract/Free Full Text]
-
Cowley S, Paterson H, Kemp P, Marshall CJ 1994 Activation of
MAP kinase kinase is necessary and sufficient for PC12 differentiation
and for transformation of NIH 3T3 cells. Cell 77:841852[Medline]
-
Lindquist JM, Rehnmark S 1998 Ambient temperature regulation
of apoptosis in brown adipose tissue. Erk1/2 promotes
norepinephrine-dependent cell survival. J Biol Chem 273:3014730156[Abstract/Free Full Text]
-
Nohara A, Ohmichi M, Koike K, Masumoto N, Kobayashi M, Akahane
M, Igekami H, Hirota K, Miyake A, Murata Y 1996 The role of
mitogen-activated protein kinase in oxytocin-induced contraction of
uterine smooth muscle in pregnant rat. Biochem Biophys Res Commun 229:938944[CrossRef][Medline]
-
Ohmichi M, Koike K, Kimura A, Masuhara K, Ikegami H, Ikebuchi
Y, Kanzaki T, Touhara K, Sakaue M, Kobayashi Y, Akabane M, Miyake A,
Murata Y 1997 Role of mitogen-activated protein kinase
pathway in prostaglandin F2alpha-induced rat puerperal uterine
contraction. Endocrinology 138:31033111[Abstract/Free Full Text]
-
Grammatopoulos D, Hillhouse EW 1999 Basal and
interleukin-1ß prostaglandin production from cultured human
myometrial cells: differential regulation by corticotropin-releasing
hormone. J Clin Endocrinol Metab 84:22042211[Abstract/Free Full Text]
-
Ohmichi M, Koike K, Nohara A, Kanda Y, Sakamoto Y, Zhang ZX,
Hirota K, Miyake A 1995 Oxytocin stimulates mitogen-activated protein
kinase activity in cultured human puerperal uterine myometrial cells.
Endocrinology 136:20822087[Abstract]
-
Naor Z, Benard O, Seger R 2000 Activation of MAPK cascades by
G-protein-coupled receptors: the case of gonadotropin-releasing hormone
receptor. Trends Endocrinol Metabol 11:9199[CrossRef][Medline]
-
Han XB, Conn PM 1999 The role of protein kinases A and C
pathways in the regulation of mitogen-activated protein kinase
activation in response to gonadotropin-releasing hormone receptor
activation. Endocrinology 140:22412251[Abstract/Free Full Text]
-
Offermanns S, Schultz G, Rosenthal W 1991 Identification of
receptor-activated G-proteins with photoreactive GTP analog
[
-32P]GTP azidoanilide. Methods Enzymol 195:286299[Medline]
-
Wille S, Sydow S, Palchaudhuri MR, Spiess J, Dautzenberg FM 1999 Identification of amino acids in the N-terminal domain of
corticotropin-releasing factor receptor 1 that are important
determinants of high-affinity ligand binding. J Neurochem 72:38895[CrossRef][Medline]
-
Perrin MH, Sutton S, Bain DL, Berggren WT, Vale WW 1998 The
first extracellular domain of corticotropin releasing factor-R1
contains major binding determinants for urocortin and astressin.
Endocrinol 139:566570[Abstract/Free Full Text]
-
Sydow S, Flaccus A, Fischer A, Spiess J 1999 The role of the
fourth extracellular domain of the rat corticotropin-releasing factor
receptor type 1 in ligand binding. Eur J Biochem 259:5562[Abstract/Free Full Text]
-
Liaw CW, Grigoriadis DE, Lovenberg TW, De Souza EB, Maki RA 1997 Localization of ligand-binding domains of human
corticotropin-releasing factor receptor; a chimeric receptor approach.
Mol Endocrinol 11:980985[Abstract/Free Full Text]
-
Liaw CW, Grigoriadis DE, Lorang MT, De Souza EB, Maki RA 1997 Localization of agonist- and antagonist-binding domains of human
corticotropin-releasing factor receptors. Mol Endocrinol 11:204853[Abstract/Free Full Text]
-
Li H, Robinson PJ, Kawashima S, Funder JW, Liu JP 1998 Differential regulation of MAP kinase activity by
corticotropin-releasing hormone in normal and neoplastic corticotropes.
Int J Biochem Cell Biol 30:13891401[CrossRef][Medline]
-
Brar BK, Jonassen AK, Stephanou A, Santilli G, Railson J,
Knight RA, Yellon DM, Latchman DS 2000 Urocortin protects against
ischemic and reperfusion injury via a MAPK-dependent pathway. J
Biol Chem 275:850814[Abstract/Free Full Text]
-
Naor Z, Benard O, Seger R 2000 Activation of MAPK cascades by
G-protein-coupled receptors: the case of gonadotropin-releasing hormone
receptor. Trends Endocrinol Metabol 11:9199[CrossRef][Medline]
-
Kolch W, Heidecker G, Kochs G, Hummel R, Vahidi H, Mischak H,
Finkenzeller G, Marme D, Rapp UR 1993 Protein kinase C
activates
RAF-1 by direct phosphorylation. Nature 364:249252[CrossRef][Medline]
-
Marais R, Light Y, Mason C, Paterson H, Olson MF, Marshall CJ 1998 Requirement of Ras-GTP-Raf complexes for activation of Raf-1 by
protein kinase C. Science 280:109112[Abstract/Free Full Text]
-
Crespo P, Xu N, Simonds WF, Gutkind JS 1994 Ras-dependent
activation of MAP kinase pathway mediated by G-protein ß
subunits. Nature 369:418420[CrossRef][Medline]
-
Ito A, Satoy T, Kaziro Y, Itoh H 1995 G protein ß
subunit
activates Ras, Raf and MAP kinase in HEK293 cells. FEBS Lett 368:183187[CrossRef][Medline]
-
Traverse S, Gomez N, Paterson H, Marshall C, Cohen P 1992 Sustained activation of the mitogen-activated protein (MAP) kinase
cascade may be required for differentiation of PC12 cells. Comparison
of the effects of nerve growth factor and epidermal growth factor.
Biochem J 288:351355[Medline]
-
Dikic I, Schlessinger J, Lax I 1994 PC12 cells overexpressing
the insulin receptor undergo insulin-dependent neuronal
differentiation. Curr Biol 4:702708[Medline]
-
Kimura A, Ohmichi M, Takeda T, Kurachi H, Ikegami H, Koike K,
Masuhara K, Hayakawa J, Kanzaki T, Kobayashi M, Akabane M, Inoue M,
Miyake A, Murata Y 1999 Mitogen-activated protein kinase
cascade is involved in endothelin-1-induced rat puerperal uterine
contraction. Endocrinology 140:722731[Abstract/Free Full Text]
-
Molnar M, Rigo Jr J, Romero R, Hertelendy F 1999 Oxytocin
activates mitogen-activated protein kinase and up-regulates
cyclooxygenase-2 and prostaglandin production in human myometrial
cells. Am J Obstet Gynecol 181:4249[Medline]
-
Watanabe F, Oki Y, Ozawa M, Masuzawa M, Iwabuchi M, Yoshimi T,
Nishiguchi T, Iino K, Sasano H 1999 Urocortin in human
placenta and maternal plasma. Peptides 20:205209[CrossRef][Medline]
-
McPherson G 1983 A practical computer based approach to the
analysis of radioligand binding experiments. Prog Biomed 17:107114
-
Munson P, Rodgbard D 1980 LIGAND: a versatile computerised
approach for characterization of ligand binding systems. Anal Biochem 107:220239[Medline]
-
Bone EA, Fretten P, Palmer S, Kirk CJ, Michell RH 1984 Rapid
accumulation of inositol phosphates in isolated rat superior cervical
sympathetic ganglia exposed to V1-vasopressin and
muscarinic cholinergic stimuli. Biochem J 221:803811[Medline]
-
Europe-Finner GN, Newell PC 1987 Cyclic AMP stimulates
accumulation of inositol trisphosphate in dictyostelium. J Cell
Sci 87:221229[Abstract]