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and R2ß CRH Receptor Subtypes and Stimulation of Gq-Proteins
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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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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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 50–70% 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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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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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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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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). 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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).
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 5–10 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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, 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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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) (2–12 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 (2–12 h).
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cells were pretreated with the PLC inhibitor U73122 (20 min,
10 µM) before stimulation with UCN (100 nM).
U73122 treatment led to a 60–80% 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 70–90% (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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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 Gq 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 80–100% more
potent than CRH, both at submaximal (10 nM) and maximal
(100 nM) concentrations.
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). Consistent with
our previous findings, the response to UCN was 60–80% 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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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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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 42–50 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 Gs, Gq, Gi, G12, 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
Gs, Gi, Gq, and Go (23). The data presented in this study
indicate that the UCN stimulation of MAPK via R1 and R2ß CRH
receptor subtypes requires Gq-, but not Gs-, Gi-, or Go-,
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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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.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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-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 (50–100 µg of protein) were
incubated with 125I-oCRH or
125I-UCN (0.2–2 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 95–100 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 (0.1–1000
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 (1–1000 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 100–200 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 90–95% 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 manufacturer’s 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 30–50%. 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 2–5 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).
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FOOTNOTES |
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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.
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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receptor. Mol Endocrinol 12:1077–1085-32P]GTP azidoanilide. Methods Enzymol 195:286–299[Medline]
activates
RAF-1 by direct phosphorylation. Nature 364:249–252[CrossRef][Medline]
subunits. Nature 369:418–420[CrossRef][Medline]
subunit
activates Ras, Raf and MAP kinase in HEK293 cells. FEBS Lett 368:183–187[CrossRef][Medline]
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 I. V. Ivanova, R. Schubert, D. B. Duridanova, T. B. Bolton, L. T. Lubomirov, and H. S. Gagov Heart/Cardiac Muscle: Cocaine- and amphetamine-regulated transcript (CART) peptide as an in vivo regulator of cardiac function in Rana ridibunda frog Exp Physiol, November 1, 2007; 92(6): 1037 - 1046. [Abstract] [Full Text] [PDF]
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 Dan Jin, Ping He, Xingji You, Xiaoyan Zhu, Ling Dai, Qian He, Chunmin Liu, Ning Hui, Jinyan Sha, and Xin Ni Expression of Corticotropin-Releasing Hormone Receptor Type 1 and Type 2 in Human Pregnant Myometrium Reproductive Sciences, September 1, 2007; 14(6): 568 - 577. [Abstract] [PDF]
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 S. V. Wu, P.-q. Yuan, L. Wang, Y. L. Peng, C.-Y. Chen, and Y. Tache Identification and Characterization of Multiple Corticotropin-Releasing Factor Type 2 Receptor Isoforms in the Rat Esophagus Endocrinology, April 1, 2007; 148(4): 1675 - 1687. [Abstract] [Full Text] [PDF]
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A. Punn, M. A. Levine, and D. K. Grammatopoulos Identification of Signaling Molecules Mediating Corticotropin-Releasing Hormone-R1{alpha}-Mitogen-Activated Protein Kinase (MAPK) Interactions: The Critical Role of Phosphatidylinositol 3-Kinase in Regulating ERK1/2 But Not p38 MAPK Activation Mol. Endocrinol., December 1, 2006; 20(12): 3179 - 3195. [Abstract] [Full Text] [PDF]
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 M. Breuiller-Fouche and G. Germain Gene and protein expression in the myometrium in pregnancy and labor. Reproduction, May 1, 2006; 131(5): 837 - 850. [Abstract] [Full Text] [PDF]
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 E. W. Hillhouse and D. K. Grammatopoulos The Molecular Mechanisms Underlying the Regulation of the Biological Activity of Corticotropin-Releasing Hormone Receptors: Implications for Physiology and Pathophysiology Endocr. Rev., May 1, 2006; 27(3): 260 - 286. [Abstract] [Full Text] [PDF]
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 P. Florio, M. Torricelli, L. Galleri, G. De Falco, E. Leucci, G. Calonaci, E. Picciolini, G. Ambrosini, E. A. Linton, and F. Petraglia High Fetal Urocortin Levels at Term and Preterm Labor J. Clin. Endocrinol. Metab., September 1, 2005; 90(9): 5361 - 5365. [Abstract] [Full Text] [PDF]
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S. R. Sooranna, N. Engineer, J. A. Z. Loudon, V. Terzidou, P. R. Bennett, and M. R. Johnson The Mitogen-Activated Protein Kinase Dependent Expression of Prostaglandin H Synthase-2 and Interleukin-8 Messenger Ribonucleic Acid by Myometrial Cells: The Differential Effect of Stretch and Interleukin-1{beta} J. Clin. Endocrinol. Metab., June 1, 2005; 90(6): 3517 - 3527. [Abstract] [Full Text] [PDF]
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T. Teli, D. Markovic, M. A. Levine, E. W. Hillhouse, and D. K. Grammatopoulos Regulation of Corticotropin-Releasing Hormone Receptor Type 1{alpha} Signaling: Structural Determinants for G Protein-Coupled Receptor Kinase-Mediated Phosphorylation and Agonist-Mediated Desensitization Mol. Endocrinol., February 1, 2005; 19(2): 474 - 490. [Abstract] [Full Text] [PDF]
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A. Chen, M. Perrin, B. Brar, C. Li, P. Jamieson, M. DiGruccio, K. Lewis, and W. Vale Mouse Corticotropin-Releasing Factor Receptor Type 2{alpha} Gene: Isolation, Distribution, Pharmacological Characterization and Regulation by Stress and Glucocorticoids Mol. Endocrinol., February 1, 2005; 19(2): 441 - 458. [Abstract] [Full Text] [PDF]
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J. Chen and H. S. Randeva Genomic Organization of Mouse Orexin Receptors: Characterization of Two Novel Tissue-Specific Splice Variants Mol. Endocrinol., November 1, 2004; 18(11): 2790 - 2804. [Abstract] [Full Text] [PDF]
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 P. J. Peeters, H. W. Gohlmann, I. Van den Wyngaert, S. M. Swagemakers, L. Bijnens, S. U. Kass, and T. Steckler Transcriptional Response to Corticotropin-Releasing Factor in AtT-20 Cells Mol. Pharmacol., November 1, 2004; 66(5): 1083 - 1092. [Abstract] [Full Text] [PDF]
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D. Wietfeld, N. Heinrich, J. Furkert, K. Fechner, M. Beyermann, M. Bienert, and H. Berger Regulation of the Coupling to Different G Proteins of Rat Corticotropin-releasing Factor Receptor Type 1 in Human Embryonic Kidney 293 Cells J. Biol. Chem., September 10, 2004; 279(37): 38386 - 38394. [Abstract] [Full Text] [PDF]
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 M. S. Soloff, Y.-J. Jeng, M. Ilies, S. L. Soloff, M. G. Izban, T. G. Wood, N. I. Panova, G. V.N. Velagaleti, and G. D. Anderson Immortalization and characterization of human myometrial cells from term-pregnant patients using a telomerase expression vector Mol. Hum. Reprod., September 1, 2004; 10(9): 685 - 695. [Abstract] [Full Text] [PDF]
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K. L. Parham, S. Zervou, E. Karteris, R. D. Catalano, R. W. Old, and E. W. Hillhouse Promoter Analysis of Human Corticotropin-Releasing Factor (CRF) Type 1 Receptor and Regulation by CRF and Urocortin Endocrinology, August 1, 2004; 145(8): 3971 - 3983. [Abstract] [Full Text] [PDF]
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B. K. Brar, A. Chen, M. H. Perrin, and W. Vale Specificity and Regulation of Extracellularly Regulated Kinase1/2 Phosphorylation through Corticotropin-Releasing Factor (CRF) Receptors 1 and 2{beta} by the CRF/Urocortin Family of Peptides Endocrinology, April 1, 2004; 145(4): 1718 - 1729. [Abstract] [Full Text] [PDF]
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N. Papadopoulou, J. Chen, H. S. Randeva, M. A. Levine, E. W. Hillhouse, and D. K. Grammatopoulos Protein Kinase A-Induced Negative Regulation of the Corticotropin-Releasing Hormone R1{alpha} Receptor-Extracellularly Regulated Kinase Signal Transduction Pathway: The Critical Role of Ser301 for Signaling Switch and Selectivity Mol. Endocrinol., March 1, 2004; 18(3): 624 - 639. [Abstract] [Full Text] [PDF]
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E. Karteris, E. W. Hillhouse, and D. Grammatopoulos Urocortin II Is Expressed in Human Pregnant Myometrial Cells and Regulates Myosin Light Chain Phosphorylation: Potential Role of the Type-2 Corticotropin-Releasing Hormone Receptor in the Control of Myometrial Contractility Endocrinology, February 1, 2004; 145(2): 890 - 900. [Abstract] [Full Text] [PDF]
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N. Bayatti, J. Zschocke, and C. Behl Brain Region-Specific Neuroprotective Action and Signaling of Corticotropin-Releasing Hormone in Primary Neurons Endocrinology, September 1, 2003; 144(9): 4051 - 4060. [Abstract] [Full Text] [PDF]
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C. Li, P. Chen, J. Vaughan, A. Blount, A. Chen, P. M. Jamieson, J. Rivier, M. S. Smith, and W. Vale Urocortin III Is Expressed in Pancreatic {beta}-Cells and Stimulates Insulin and Glucagon Secretion Endocrinology, July 1, 2003; 144(7): 3216 - 3224. [Abstract] [Full Text] [PDF]
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M. H. Perrin, M. R. DiGruccio, S. C. Koerber, J. E. Rivier, K. S. Kunitake, D. L. Bain, W. H. Fischer, and W. W. Vale A Soluble Form of the First Extracellular Domain of Mouse Type 2beta Corticotropin-releasing Factor Receptor Reveals Differential Ligand Specificity J. Biol. Chem., April 25, 2003; 278(18): 15595 - 15600. [Abstract] [Full Text] [PDF]
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P. Florio, L. Cobellis, J. Woodman, F. M. Severi, E. A. Linton, and F. Petraglia Levels of Maternal Plasma Corticotropin-Releasing Factor and Urocortin During Labor Reproductive Sciences, July 1, 2002; 9(4): 233 - 237. [Abstract] [PDF]
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S. Zervou, E. Karteris, E.W. Hillhouse, and R.W. Old Steroids mediate the expression of cytoplasmic and membrane-linked components in human myometrial cells Mol. Hum. Reprod., July 1, 2002; 8(7): 597 - 605. [Abstract] [Full Text] [PDF]
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E. Dermitzaki, C. Tsatsanis, A. Gravanis, and A. N. Margioris Corticotropin-releasing Hormone Induces Fas Ligand Production and Apoptosis in PC12 Cells via Activation of p38 Mitogen-activated Protein Kinase J. Biol. Chem., March 29, 2002; 277(14): 12280 - 12287. [Abstract] [Full Text] [PDF]
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 E. Aggelidou, E. W. Hillhouse, and D. K. Grammatopoulos Up-regulation of nitric oxide synthase and modulation of the guanylate cyclase activity by corticotropin-releasing hormone but not urocortin II or urocortin III in cultured human pregnant myometrial cells PNAS, February 14, 2002; (2002) 52296399. [Abstract] [Full Text] [PDF]
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 W. A. Pedersen, R. Wan, P. Zhang, and M. P. Mattson Urocortin, But Not Urocortin II, Protects Cultured Hippocampal Neurons from Oxidative and Excitotoxic Cell Death via Corticotropin-Releasing Hormone Receptor Type I J. Neurosci., January 15, 2002; 22(2): 404 - 412. [Abstract] [Full Text] [PDF]
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 A. SLOMINSKI, J. WORTSMAN, A. PISARCHIK, B. ZBYTEK, E. A. LINTON, J. E. MAZURKIEWICZ, and E. T. WEI Cutaneous expression of corticotropin-releasing hormone (CRH), urocortin, and CRH receptors FASEB J, August 1, 2001; 15(10): 1678 - 1693. [Abstract] [Full Text] [PDF]
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E. Aggelidou, E. W. Hillhouse, and D. K. Grammatopoulos Up-regulation of nitric oxide synthase and modulation of the guanylate cyclase activity by corticotropin-releasing hormone but not urocortin II or urocortin III in cultured human pregnant myometrial cells PNAS, March 5, 2002; 99(5): 3300 - 3305. [Abstract] [Full Text] [PDF]
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