From the Department of Cell Physiology and Pharmacology, University of Leicester, P. O. Box 138, Medical Sciences Building, University Road, Leicester, LE1 9HN, United Kingdom
Received for publication, September 27, 2000, and in revised form, November 9, 2000
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We investigated the role played by
agonist-mediated phosphorylation of the Gq/11-coupled
M3-muscarinic receptor in the mechanism of activation of
the mitogen-activated protein kinase pathway, ERK-1/2, in
transfected Chinese hamster ovary cells. A mutant of the
M3-muscarinic receptor, where residues
Lys370-Ser425 of the third intracellular loop
had been deleted, showed a reduced ability to activate the ERK-1/2
pathway. This reduction was evident despite the fact that the receptor
was able to couple efficiently to the phospholipase C second messenger
pathway. Importantly, the ERK-1/2 responses to both the wild-type
M3-muscarinic receptor and
It is now clear that mitogenic signals mediated by the
mitogen-activated protein
(MAP)1 kinases, ERK-1 and
ERK-2, can be initiated by both receptor-tyrosine kinases (RTKs) and by
the heptahelical G-protein-coupled receptors (GPCRs). The activation of
the ERK-1/2 pathway by GPCRs is mediated by any one of a number of
mechanisms (1) probably reflecting the diversity of receptors within
this large gene family. These mechanisms appear quite distinct; for
example, ERK-1/2 activation has been shown to proceed via a tyrosine
kinase-dependent mechanism for some receptors and a
tyrosine kinase-independent manner for others (2, 3). Despite this
diversity, common features do exist, the most prominent of which is
that GPCRs activate ERK-1/2 by acting initially through "classical"
heterotrimeric G-protein signaling pathways (4). For example,
stimulation of ERK-1/2 by Gi-coupled receptors, such as
M2-muscarinic, and Similarly, Gq/11-coupled receptors that stimulate
phospholipase C and the subsequent hydrolysis of phosphatidylinositol
4,5-bisphosphate to produce the second messengers inositol
1,4,5-trisphosphate (Ins(1,4,5)P3) and diacylglycerol,
activate the ERK-1/2 pathway via Gq/11-heterotrimeric
G-proteins. In this case there is evidence for the involvement of both
Studies have also indicated that the Ca2+ mobilization arm
of the phospholipase C signaling pathway is important in the activation of ERK-1/2 by Gq/11-coupled receptors. Bradykinin, LPA
(25), and These studies indicate that the mechanism for Gq/11-coupled
receptor-mediated ERK-1/2 activation is dependent on the coupling of
the receptor to Gq/11-heterotrimeric G-proteins and
subsequent phospholipase C signaling through Ca2+
mobilization and PKC activation. A further component in the activation of the ERK-1/2 pathway by GPCRs has recently been suggested from studies on the In the present paper, we investigate the role played by receptor
phosphorylation in the activation of the ERK-1/2 pathway by the
Gq/11-coupled M3-muscarinic receptor. This
receptor is rapidly phosphorylated on serine following agonist
occupation (33). However, in contrast to the
Cell Culture--
CHO cell lines were grown in medium consisting
of Generation of the Dominant Negative Mutant of CK1 Generation of the Third Intracellular Loop Peptide (3i-Loop
Peptide)--
The sequence encoding amino acids
Ser345-Leu463 from the third intracellular
loop of the M3-muscarinic receptor was cloned into BamHI and EcoRI sites in pcDNA-3 (Invitrogen)
as described previously (35).
Generation of the M3-Muscarinic Receptor
Deletion Mutant
Transient Transfections of CHO Cells--
Cells were plated onto
6-well dishes 24 h before transfection. Cells (15-20% confluent)
were transfected with either 3 µg of F-CK1 Quantification of M3-Muscarinic Receptor
Expression--
M3-Muscarinic receptor expression on
intact plated-down cells was determined using a saturating
concentration of the hydrophilic muscarinic antagonist
[3H]N-methyl scopolamine
([3H]NMS, ~0.5 nM) as described previously
(35). Nonspecific binding was determined in the presence of 20 µM atropine and was < 3% of the total binding.
Mass Ins(1,4,5)P3 Determination--
Cells grown in
24-well dishes were washed with Krebs/HEPES buffer (HEPES (10 mM), NaCl (118 mM), KHPO3 (1.17 mM), KCl (4.3 mM), MgSO4·7 (1.17 mM), CaCl2 (1.3 mM),
NaHCO3 (25.0 mM), glucose (11.7 mM), pH 7.4) and challenged with agonist for the
appropriate times. Incubations were terminated by rapid aspiration,
addition of ice-cold 0.5 M trichloroacetic acid, and
transfer to an ice-bath. After 15 min, the supernatant was removed and
neutralized by addition of EDTA and freon/tri-n-octylamine
as described previously (36). Extracts were brought to pH 7 by addition
of NaHCO3 and stored at 4 °C until analysis.
Ins(1,4,5)P3 mass measurements were performed using a
radio-receptor assay described previously (37).
Erk-1/2 Assay--
CHO cells grown to confluence in 6-well
plates were serum-starved for 1 h in Krebs/HEPES buffer and then
stimulated with the appropriate agents. Stimulation was terminated by
aspiration, and cells were incubated for 10 min in lysis buffer (Tris
(20 mM), Nonidet P-40 (0.5%), NaCl (250 mM),
EDTA (3 mM), EGTA (3 mM), phenylmethylsulfonyl
fluoride (1 mM), Na3VO4 (1 mM), dithiothreitol (1 mM), benzamidine (5 µg/ml), pH 7.6) at 4 °C. Solubilized CHO cell lysates were
pre-cleared by centrifuging at 14,000 rpm for 5 min. Endogenous MAP
kinase was immunoprecipitated using 0.2 µg of anti-Erk-1/2 antiserum
(Santa Cruz). Protein A-Sepharose immobilized MAP kinase was washed
twice in lysis buffer and twice in assay buffer (HEPES (20 mM), Determination of Intracellular Ca2+ Concentrations
([Ca2+]i)--
Confluent monolayers of cells
in 175 cm2 flasks were harvested and resuspended in 2.5 ml
of Krebs/HEPES buffer. A 0.5-ml aliquot of this was removed for
determination of cellular autofluorescence. Fura-2-acetoxymethyl ester
(Fura-2-AM, 5 µM) was added to the remaining 2 ml, which
was then left for ~40 min at room temperature with gentle mixing.
Supernatant containing extracellular Fura-2-AM was removed following
gentle centrifugation of the 0.5-ml aliquots. Cells were resuspended in
a cuvette containing 3 ml of Krebs/HEPES buffer at 37 °C. Using a
Perkin-Elmer LS-5B spectrofluorimeter with a cuvette water jacket to
maintain the temperature at 37 °C, emission at 509 nm was recorded
following excitation at both 340 and 380 nm. The excitation ratio was
recorded every 1 s and converted to
[Ca2+]i as previously reported (38) using
0.1% Triton X-100 in the presence of a saturating [Ca2+]
to determine Rmax and the addition of EGTA to
determine Rmin. Cells were challenged with
10-50 µl of agonist. Initial experiments were conducted in the
presence of 1.3 mM extracellular [Ca2+] (as
represented in Fig. 4B). In experiments to determine the potency of intracellular Ca2+ mobilization by the full
agonist methacholine (represented in Fig. 4C), the
experiments were conducted in Ca2+-free medium where the
Krebs/HEPES buffer had been supplemented with EGTA to reduce
extracellular [Ca2+] to ~100 nM (determined
using Fura-2). This was to ensure that the ability of the agonist to
mobilize intracellular Ca2+ stores was being measured,
because any changes in intracellular Ca2+ concentrations
under these conditions would have been the result of release of
Ca2+ from intracellular stores with no contribution being
made form an influx of extracellular Ca2+.
ERK-1/2 Activation by a Phosphorylation-deficient Mutant of the
M3-Muscarinic Receptor--
Previous studies
from our laboratory and others (21-24) have shown that
M3-muscarinic receptors activate the ERK-1/2 pathway in a
PTX-insensitive, PKC-dependent manner. The time course for ERK-1/2 activation peaks at 5 min then falls to a plateau, which is
maintained for at least 20 min (21). To test whether receptor phosphorylation plays a role in the regulation of the ERK-1/2 pathway,
a mutant M3-muscarinic receptor was used where residues Lys370-Ser425 of the third intracellular loop
of the human M3-muscarinic receptor had been deleted. This
mutant receptor, termed
Concentration-response analysis of CHO cells expressing the wild-type
M3-muscarinic receptor (CHO-m3 cells) showed a
receptor-mediated ERK-1/2 activation with a half-maximal response
(EC50) to the agonist carbachol of 45 ± 1.3 nM (n = 3, ± S.E., Fig.
1.) This is very similar to the
EC50 value that we obtained previously using another
distinct M3-muscarinic receptor-transfected CHO cell line
(21). In contrast to the wild-type receptor, the mutant receptor showed
a rightward shift in the ERK-1/2 concentration-response curve to
carbachol (Fig. 1). The EC50 values for the two clonal cell
lines expressing the
To test for the possibility of clonal variation between the wild-type
CHO-m3 cells and mutant receptor cell lines, concentration-response curves for serum-induced ERK-1/2 activation were carried out. The
concentration-response curves for serum-activated ERK-1/2 in the mutant
receptor CHO cell lines were not significantly different from that of
the CHO-m3 cells (Fig. 2). This indicated
that there was no clonal difference in the ERK-1/2 pathway stimulated
by serum.
PKC Dependence of Muscarinic ERK-1/2 Responses--
We have
previously shown that the wild-type M3-muscarinic
receptor-mediated ERK-1/2 response is dependent on PKC because
inhibition of PKC using Ro-318220 or down-regulation of PKC reduced the
muscarinic-ERK-1/2 response by >90% (21). The
Coupling of the
We have reported previously that despite the fact that the
The ability of the Effect of the CK1
Control experiments were designed to test the ability of F-CK1
It is interesting to note that in experiments where phorbol esters were
used to stimulate ERK-1/2 activity in CHO-m3 cells the F-CK1
Analysis of the ERK-1/2 concentration-response curves to carbachol
demonstrated that in addition to reducing the maximal response the
3i-loop peptide and F-CK1 Despite intensive research, the mechanisms employed by GPCRs in
the activation of the ERK-1/2 pathway are generally poorly understood.
One reason for this is that GPCRs are able to employ a number of
diverse mechanisms in the activation of ERK-1/2 depending on the
receptor type and the cellular environment (4). For example,
M1-muscarinic receptor ERK-1/2 responses have been shown to
operate in both a Ras-dependent (6) and Ras-independent (3)
fashion using a mechanism, which in some cell types, employs tyrosine
phosphorylation (2) and in others acts in a tyrosine kinase-independent
manner (3). To add a further level of complexity, it has now become
clear that a number of Gq/11-coupled receptors can
simultaneously employ at least two independent mechanisms to activate
the ERK-1/2 pathway (20, 24, 39). Despite this diversity there is one
overriding common feature in the mechanisms employed by
Gq/11-coupled receptors, namely, the involvement of the
Gq/11-heterotrimeric G-proteins and the subsequent
activation of the phospholipase C signaling pathway. Both the
Ins(1,4,5)P3/Ca2+ mobilization and
diacylglycerol/PKC arms of the phospholipase C signaling pathway have
been implicated to play a role and in many instances appear to provide
the primary signal that links receptor activation to the initiation of
the ERK-1/2 pathway.
We have shown previously that Gq/11-coupled
M3-muscarinic receptors expressed in CHO cells stimulate
the ERK-1/2 pathway in a PKC-dependent manner (21). This
was confirmed in the present study and is consistent with previous
reports from other laboratories (22-24) and would suggest that
activation of PKC by the M3-muscarinic receptor is
sufficient to stimulate ERK-1/2. This conclusion could be applied to a
large number of Gq/11-coupled receptors that show PKC-dependent activation of ERK-1/2, such as prostaglandin
F2 Thus, one model for ERK-1/2 activation by Gq/11-coupled
receptors, including the M3-muscarinic receptor, would be
that receptor-mediated PKC activation is sufficient to provide the
signal that elicits the ERK-1/2 response.
Our data, however, using the Lys370-Ser425 receptor mutant were
dependent on the activity of protein kinase C. Our results, therefore,
indicate the existence of two mechanistic components to the ERK-1/2
response, which appear to act in concert. First, the activation of
protein kinase C through the diacylglycerol arm of the phospholipase C
signaling pathway and a second component, absent in the
Lys370-Ser425 receptor mutant, that is
independent of the phospholipase C signaling pathway. The reduced
ability of the
Lys370-Ser425 receptor
mutant to activate the ERK-1/2 pathway correlated with an ~80%
decrease in the ability of the receptor to undergo agonist-mediated phosphorylation. Furthermore, we have previously shown that
M3-muscarinic receptor phosphorylation can be inhibited by
a dominant negative mutant of casein kinase 1
and by expression of a
peptide corresponding to the third intracellular loop of the
M3-muscarinic receptor. Expression of these inhibitors of
receptor phosphorylation reduced the wild-type
M3-muscarinic receptor ERK-1/2 response. We conclude that phosphorylation of the M3-muscarinic receptor on sites
in the third intracellular loop by casein kinase 1
contributes to the mechanism of receptor activation of ERK-1/2 by working in concert
with the diacylglycerol/PKC arm of the phospholipase C signaling pathway.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2A-adrenergic receptors, is pertussis
toxin-sensitive indicating a role of Gi-proteins (5-7). It
is proposed that liberation of
-subunits from
Gi-proteins is responsible for the initiation of tyrosine
phosphorylation (3, 8), possibly by the activation of Src or Src-like
tyrosine kinases (9, 10, 11), that ultimately results in
Ras-dependent ERK-1/2 activation (3, 6, 7, 12).
-subunits (6, 13, 14) and G
q/11-subunits (3, 10,
12, 14, 15). Furthermore, the activation of ERK-1/2 by these receptors
appears to be dependent on PKC because inhibition of PKC either
abolishes (3, 15-17) or significantly diminishes (18-20) the ERK-1/2
response to Gq/11-coupled receptors. This is particularly
apparent for the M3-muscarinic receptor where the ERK-1/2
response is blocked by >85% by either PKC inhibition or PKC
down-regulation (21-24).
1B-adrenergic (10) receptor-stimulated ERK-1/2
responses were shown to be dependent on changes in intracellular
Ca2+. Receptor-mediated Ca2+ mobilization is
proposed to activate the Ca2+/PKC-sensitive tyrosine
protein kinase, Pyk2 (26), which is thought to act upstream of Ras in
the Erk-1/2 pathway (10, 25). In the case of receptors such as the
angiotensin AT1 (27), bradykinin (28), CCKA
(18), chemokine CXCR-1/2 (19), and purinergic P2Y2
receptors (20, 29), the activation of ERK-1/2 is proposed to be via
transactivation of RTKs, a process that is dependent on
Ca2+ mobilization and subsequent activation of Pyk2 or
related kinases.
2-adrenergic receptor where receptor
phosphorylation has been shown to play a central role. The
2-adrenergic receptor is phosphorylated by both PKA and
the G-protein coupled receptor kinases (GRKs) (30). PKA phosphorylation
of the receptor on sites on the third intracellular loop has been
proposed to act as a "molecular switch" coupling the receptor to
Gi-proteins and subsequently the activation of the ERK-1/2
pathway via the generation of
-subunits (31). The
2-adrenergic receptor can also be phosphorylated in an
agonist-dependent manner by the GRKs, particularly GRK-2.
This has classically been considered to result in the recruitment of
-arrestin and receptor desensitization (30). However, recent studies
have shown that
-arrestin can act as an adaptor protein recruiting
activated c-Src to the plasma membrane in a process that is essential
in the activation of the ERK-1/2 pathway by the
2-adrenergic receptor (32).
2-adrenergic receptor, which is phosphorylated by the
GRKs, M3-muscarinic receptors are phosphorylated in an
agonist-dependent manner on sites in the third
intracellular loop by casein kinase 1
(CK1
) (34, 35). Deletion of
a region of the third intracellular loop of the human
M3-muscarinic receptor (Lys370-Ser425) reduced receptor
phosphorylation by ~80% (35). Furthermore, expression of a dominant
negative mutant of CK1
or a peptide corresponding to the third
intracellular loop of the receptor, reduced receptor phosphorylation
(35). Using these reagents in the present study, we investigate the
role played by agonist-mediated receptor phosphorylation in the
activation of the ERK-1/2 pathway.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
MEM supplemented with 10% fetal calf serum, 100 IU/ml
penicillin, 100 µg/ml streptomycin, and 2.5 µg/ml fungizone. Cells
were grown in a 5% CO2, 95% air, humidified incubator at
37 °C. The
Lys370-Ser425 receptor mutant
clone 2 was maintained in blasticidine (5 µg/ml).
(F-CK1
K46R)--
The dominant negative mutant of CK1
(F-CK1
K46R) was generated by point mutagenesis of the lysine residue
at position 46, which represents the invariant lysine at the ATP
binding site of CK1
. The lysine residue was mutated to an arginine
as described previously (35).
Lys370--
Ser425
Two
stably transfected CHO cell lines expressing the
M3-muscarinic receptor deletion mutant
Lys370-Ser425 were used in the present
study. Clone 1 was generated by digestion of the
M3-muscarinic receptor coding sequence contained in
pcDNA-3 (Invitrogen) with HindIII and then religating
the plasmid. This removed the coding sequence for amino acids
Lys370-Ser425 inclusive, but maintained the
reading frame of the remaining cDNA. This construct was transfected
into CHO cells, and clones were selected using medium supplemented with
G-418 (200 µg/ml). The second clone used (clone 2) originated from
another transfection where the cDNA encoding the
Lys370-Ser425 receptor mutant was subcloned
into pcDNA-6 (Invitrogen). Clones from this transfection were
selected using medium supplemented with blasticidine (5 µg/ml).
K46R or 3i-loop peptide
per well using 8 µl of Fugene 6 transfection reagent (Roche Molecular
Biochemicals). Cells were used 48 h after transfection. Using a
green fluorescent protein construct, we estimated that the transfection
efficiency was ~70%.
-glycerophosphate (20 mM),
MgCl2 (10 mM), dithiothreitol (1 mM), Na3VO4 (50 µM),
pH 7.2). Washed pellets were resuspended in assay buffer containing 2 µCi of [32P]ATP, 20 µM ATP, 200 µM EGFr (peptide encompassing region 661-681 of the EGF
receptor), and reactions were left to proceed for 20 min at 37 °C.
Reactions were terminated by the addition of 25% trichloroacetic acid
and spotted onto P81 phosphocellulose paper squares (Whatman). Squares
were washed four times with 0.05% orthophosphoric acid and once with
acetone, and radioactivity associated with the EGFr was determined by
liquid scintillation counting.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Lys370-Ser425, had
previously been demonstrated to show an ~80% decrease in its ability
to undergo agonist-mediated phosphorylation (35). Two stably
transfected CHO cell lines were prepared expressing the
Lys370-Ser425 receptor at levels comparable
with the wild-type controls (Bmax values in
fmols of receptor/mg protein: wild type = 908 ± 124,
Lys370-Ser425 mutant clone 1 = 782 ± 67, mutant clone 2 = 1209 ± 10).
Lys370-Ser425 mutant
were 660 ± 100 nM and 300 ± 100 nM
(n = 3, ± S.E.) for clones 1 and 2, respectively.
These EC50 values were significantly different from the
wild-type receptor values (p < 0.05, Student's t test). In addition to a reduction in the potency of
carbachol, there was also a reduction in the maximal ERK-1/2 response
with clone 1 showing a 23 ± 7% reduction and clone 2 a
61 ± 5% reduction (n = 3, ± S.E.) in the
maximal carbachol response compared with wild-type receptor controls
(Fig. 1). The time course for activation of ERK-1/2 was not, however,
significantly different between the control and mutant receptors (data
not shown).
View larger version (16K):
[in a new window]
Fig. 1.
Activation of ERK-1/2 by wild-type
M3-muscarinic receptors and the deletion mutant
Lys370-Ser425. Stably
transfected CHO cells expressing either the wild-type human
M3-muscarinic receptor or the deletion mutant
Lys370-Ser425 were stimulated for 5 min in
the presence of varying concentrations of carbachol (CCH).
The reaction was terminated by addition of lysis buffer, and ERK-1/2
activity was determined. Shown are the concentration-response curves
for wild-type receptor and two separate clones: clone 1 (A),
clone 2 (B), expressing the
Lys370-Ser425 receptor mutant. The data
presented represent the mean ± S.E. for three experiments.
View larger version (16K):
[in a new window]
Fig. 2.
Serum-mediated ERK-1/2 responses. Stably
transfected CHO cells expressing either the wild-type human
M3-muscarinic receptor or the deletion mutant
Lys370-Ser425 were stimulated for 20 min in
the presence of varying concentrations of fetal calf serum. The
reaction was terminated by addition of lysis buffer, and ERK-1/2
activity was determined. Shown are the concentration-response curves
for wild-type receptor and two separate clones: clone 1 (A),
clone 2 (B), expressing the
Lys370-Ser425 receptor mutant. The data
represent the mean ± S.E. of at least three experiments.
Lys370-Ser425 receptor mutant response also
appeared to be sensitive to PKC inhibition in a manner similar to the
wild-type receptor. The phorbol 12,13-dibutyrate ERK-1/2 responses in
the CHO-m3 cells and cells expressing
Lys370-Ser425 receptor mutant (clone 2)
were completely inhibited by the PKC inhibitor Ro-318220 (Fig.
3). The ERK-1/2 responses to carbachol in
the CHO-m3 cells and the cells expressing the
Lys370-Ser425 receptor mutant were
inhibited (~90%) by Ro-318220 (Fig. 3).
View larger version (14K):
[in a new window]
Fig. 3.
PKC-dependence of ERK-1/2 responses.
Cells were pretreated with either vehicle or the PKC-inhibitor
Ro-318220 (10 µM, Ro) for 10 min prior to
stimulation with 1 mM carbachol (CCH) or 1 µM phorbol 12,13-dibutyrate (PDBu) or
nonstimulated (Control). Stimulation was for 5 min after
which reactions were terminated by addition of lysis buffer, and
ERK-1/2 activity was determined. The data represent the mean ± S.E. of three experiments.
Lys370-Ser425 Receptor
Mutant to the Phospholipase C Signaling Pathway--
We have
previously reported that the
Lys370-Ser425
receptor mutant showed agonist and antagonist binding characteristics
that were not significantly different from the wild-type receptors
(35). We have also reported that the
Lys370-Ser425 receptor mutant is coupled to
the phospholipase C pathway in a manner analogous to the wild-type
receptor. For example, the time course of Ins(1,4,5)P3
generation of both the wild-type M3-muscarinic receptor and
the
Lys370-Ser425 receptor mutant peaks
within 5-10 s of agonist stimulation and reaches a plateau phase after
60 s, which is maintained for at least 5 min (35). Significantly,
we have shown previously that the
Lys370-Ser425 receptor mutant appears to
give a more robust Ins(1,4,5)P3 response than the wild-type
receptor, suggesting that the receptor may be more efficiently coupled
to phospholipase C (35). In the present study, this characteristic is
evident by an ~3.0-fold greater production of
Ins(1,4,5)P3 at maximal agonist concentration (Fig.
4A).
View larger version (14K):
[in a new window]
Fig. 4.
Coupling of the wild-type
M3-muscarinic receptor and
Lys370-Ser425 receptor
mutant to the phospholipase C pathway. A,
Ins(1,4,5)P3 generation was determined in cells that had
been stimulated with carbachol (1 mM) for varying times.
The data represent the mean ± S.E. of three experiments carried
out in duplicate. B, time-course of the changes in free
intracellular Ca2+ concentrations
([Ca2+]i) determined in cell suspensions
loaded with the Ca2+ indicator Fura2-AM and stimulated with
the full agonist methacholine (1 mM). C,
concentration-response curve of the peak Ca2+ response
following stimulation with methacholine. The data represent the
mean ± S.E. of at least three experiments.
Lys370-Ser425 receptor mutant was able to
drive a larger Ins(1,4,5)P3 response, the potency of the
full agonist carbachol to mediate an Ins(1,4,5)P3 response
was not significantly different between the mutant and wild-type
receptors, which had EC50 values of 9.71 ± 1.9 µM and 7.14 ± 3.2 µM
(n = 3, ± S.E.), respectively (35).
Lys370-Ser425 receptor
mutant (clone 1) to mobilize intracellular Ca2+ was also
tested. The time course for receptor-mediated increases in
intracellular Ca2+ for both mutant and wild-type receptors
were similar (Fig. 4B). Interestingly, in contrast to the
Ins(1,4,5)P3 response, there was no significant difference
in the magnitude of the Ca2+ mobilization response between
the wild-type and
Lys370-Ser425 receptor
mutant. Similarly, the concentration-response curves for peak
Ca2+ mobilization were not significantly different with
EC50 values of 166 ± 70 nM and 258 ± 50 nM (n = 3, ± S.E.) for the wild-type M3-muscarinic receptor and
Lys370-Ser425 receptor mutant, respectively
(Fig. 4C). (Note; in these Ca2+ mobilization
experiments the full agonist methacholine was used. Both methacholine
and carbachol are full agonists at the M3-muscarinic receptor and produce almost identical responses.)
Dominant Negative Mutant (F-CK1
-K46R) and
the 3i-Loop Peptide on M3-Muscarinic
Receptor-mediated ERK-1/2 Activation--
Our previous studies had
shown that CK1
was able to phosphorylate the
M3-muscarinic receptor in an agonist-dependent
manner (34). Furthermore, we demonstrated that transient expression of
a dominant negative mutant of CK1
(F-CK1
-K46R) was able to reduce
receptor phosphorylation by ~40% (35). In these earlier studies, we
also showed that expression of a peptide corresponding to the third
intracellular loop of the M3-muscarinic receptor (Ser345-Leu463), named the 3i-loop peptide,
resulted in inhibition of receptor phosphorylation by >70% (35). To
test the role that receptor phosphorylation might play in ERK-1/2
activation we transiently transfected F-CK1
-K46R and the 3i-loop
peptide into CHO-m3 cells stably expressing the
M3-muscarinic receptor. Expression of F-CK1
-K46R and the
3i-loop peptide resulted in the reduction of the carbachol-mediated ERK-1/2 response by 53.9 ± 7.7% and 49.4 ± 3.2%,
respectively (Fig. 5A).
View larger version (22K):
[in a new window]
Fig. 5.
Effect of the CK1
dominant negative mutant (F-CK1
K46R) and
the 3i-loop peptide on the M3-muscarinic ERK-1/2
response. CHO-m3 cells stably expressing recombinant
M3-muscarinic receptors (A) or native CHO-K1
cells (B), were transiently transfected with the CK1
dominant negative mutant, F-CK1
K46R (K46R) or the 3i-loop peptide
(3i-loop) corresponding to
Ser345-Leu463 of the third intracellular loop
of the M3-muscarinic receptor or were sham transfected
(Control). 48 h after transfection, cells were
stimulated with 1 mM carbachol (CCH, A) or 1 µM phorbol 12,13-dibutyrate (PDBu, B), for 5 min. Reactions were terminated using lysis buffer, and ERK-1/2 activity
was determined. The data represent the mean ± S.E. of three
experiments.
-K46R
or the 3i-loop peptide to inhibit nonreceptor-mediated ERK-1/2
activation. Hence, the effect of transient transfection of
F-CK1
-K46R or the 3i-loop peptide on the phorbol 12,13-dibutyrate ERK-1/2 response in native CHO-K1 cells was tested. It was found that
neither F-CK1
-K46R or the 3i-loop peptide had any significant effect
on the phorbol ester-mediated ERK-1/2 response in these cells (Fig.
5B).
-K46R
construct inhibited the phorbol ester response by 31% (data not
shown). The fact that the F-CK1
-K46R construct had very little
effect on the phorbol ester response in CHO-K1 cells, but a significant
effect in CHO-m3 cells would suggest that in CHO-m3 cells the
M3-muscarinic receptor itself might contribute to the
phorbol ester ERK-1/2 response. This may be because of the fact that
phorbol esters are able to mediate phosphorylation of the
agonist-unoccupied M3-muscarinic receptor (33). This and
other possibilities are presently under investigation.
-K46R significantly (p < 0.05, Student's t test) reduced the potency of carbachol by
15.7-fold and 1.8-fold, respectively (Fig.
6).
View larger version (15K):
[in a new window]
Fig. 6.
ERK-1/2 concentration-response curves in
CHO-m3 cells transiently transfected with the CK1
dominant negative mutant (F-CK1
K46R) and
the 3i-loop peptide. CHO-m3 cells stably expressing recombinant
M3-muscarinic receptors were transiently transfected with
the CK1
dominant negative mutant, F-CK1
K46R (K46R)
(A), or the 3i-loop peptide (3i-loop peptide, B)
corresponding to Ser345-Leu463 of the third
intracellular loop of the M3-muscarinic receptor. 48 h
after transfection cells were stimulated with varying concentrations of
carbachol (CCH) for 5 min. Reactions were terminated using lysis
buffer, and ERK-1/2 activity was determined. The data represent the
mean ± S.E. of three experiments.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(15), P2Y2-purinergic (20, 29), CCK (18),
M1-muscarinic,
1-adrenergic (3), and
bradykinin (17) receptors. Furthermore, the ability of phorbol esters
to increase ERK-1/2 activity (40) provides evidence that simply
stimulating PKC is sufficient to drive the activation of ERK-1/2.
Lys370-Ser425
M3-muscarinic receptor mutant would suggest that this
simple model is not correct. Deletion of
Lys370-Ser425 in the third intracellular loop
of the human M3-muscarinic receptor resulted in a reduction
in the ability of the receptor to stimulate ERK-1/2 activity. This
reduction was evident despite the fact that the receptor was
efficiently coupled to the phospholipase C signaling pathway. In fact
this study, consistent with our previous report (35), demonstrates that
the
Lys370-Ser425 receptor mutant is more
efficiently coupled to the phospholipase C pathway than the wild-type
receptor. This suggests that simply activating the diacylglycerol/PKC
arm of the phospholipase C signaling pathway was not in itself
sufficient to drive a full Gq/11-coupled receptor ERK-1/2
response. It is interesting to note that the ERK-1/2 response mediated
by the
Lys370-Ser425 receptor mutant was
still sensitive to PKC inhibition. Thus, the
Lys370-Ser425 receptor mutant ERK-1/2
response still has an absolute requirement for the activation of PKC
but appears to be unable to employ an additional mechanism that is
independent of Gq/11-activated phospholipase C signaling.
This additional mechanism (Fig. 7,
Mechanism 2) appears to act in concert with PKC to elicit a
full ERK-1/2 response.
View larger version (21K):
[in a new window]
Fig. 7.
Scheme of the mechanisms involved in the
activation of the ERK-1/2 pathway by M3-muscarinic
receptors. Our data have identified two mechanisms involved in the
activation of the ERK-1/2 pathway by M3-muscarinic
receptors expressed in CHO cells. Mechanism 1 is
PKC-dependent and is essential in the activation of
ERK-1/2. Inhibition of Mechanism 1 (e.g. inhibition of PKC
with Ro-318220) prevents activation of ERK-1/2 despite the fact that
Mechanism 2 is still intact. Mechanism 2, therefore, will not elicit an
ERK-1/2 response alone. However, Mechanism 2 does operate in concert
with Mechanism 1 to give a full ERK-1/2 response. Hence a receptor that
is only able to activate Mechanism 1 (i.e. the
Lys370-Ser425 receptor mutant or the
wild-type M3-muscarinic receptor expressed together with
the 3i-loop peptide or F-CK1
K46R) will give a less than maximal
ERK-1/2 response. CK1
, casein kinase 1
;
DAG, diacylglycerol; InsP3, inositol
1,4,5-trisphosphate; PIP2, phosphatidylinositol
4,5-bisphosphate; PLC, phospholipase C.
These data, therefore, support a model that identifies two mechanisms in the activation of ERK-1/2 (Fig. 7). Mechanism 1 is PKC-dependent and is absolutely required for ERK-1/2 activation but when operating alone is only able to mediate a partial ERK-1/2 response. Mechanism 2 is PKC-independent and although is unable to elicit an ERK-1/2 response when operating alone, it is able to act in concert with Mechanism 1 to give a full ERK-1/2 response.
The most prominent PKC-independent mechanism assigned to
Gq/11-coupled receptor activation of ERK-1/2 is via the
activity of the Ca2+-sensitive tyrosine kinase Pyk2 or
related kinases (25). Ins(1,4,5)P3-dependent increases in intracellular Ca2+ has been demonstrated to
stimulate Pyk2 activity resulting in "transactivation" of RTKs and
subsequent activation of the ERK-1/2 pathway (18-20, 27-29). We can,
however, eliminate the involvement of this process in the explanation
of the results obtained with the
Lys370-Ser425 receptor mutant for two
reasons. First, the muscarinic receptor ERK-1/2 response in CHO cells
is independent of changes in intracellular Ca2+ (22)
suggesting that Pyk2 is not involved in the M3-muscarinic receptor response in these cells. Second, GPCR transactivation of RTKs
via Pyk2 is a process that involves Ins(1,4,5)P3-mediated increases in intracellular Ca2+ (26). Because the
Lys370-Ser425 receptor couples efficiently
to the phospholipase C pathway, stimulating Ca2+
mobilization in an identical manner to the wild-type receptor, the
involvement of a Ca2+-sensitive mechanism would not explain
the lack of responsiveness of this receptor mutant.
Hence, the data presented here identifies a novel component of the M3-muscarinic receptor ERK-1/2 response that is independent of activation of the Gq/11/phospholipase C pathway and dispels the notion that Gq/11-coupled receptors mediate ERK-1/2 activation by solely stimulating PKC or activating tyrosine phosphorylation via Ins(1,4,5)P3-dependent increases in intracellular Ca2+.
We next tested the possibility that the novel component of the
M3-muscarinic receptor ERK-1/2 response involved
agonist-mediated phosphorylation of the receptor. Our earlier studies
had shown that the M3-muscarinic receptor is rapidly
phosphorylated on serine in an agonist-dependent manner
(33). Extensive studies by our group have identified CK1 as a
cellular kinase able to phosphorylate the M3-muscarinic
receptor (also the M1-muscarinic receptor and rhodopsin) in
an agonist-dependent manner (34, 35, 41, 42). These
studies established for the first time a mechanism for
agonist-dependent phosphorylation of GPCRs that was
distinct from that of the GRKs. During these studies we suggested that
sites within the third intracellular loop of the
M3-muscarinic receptor were important for the
phosphorylation of the receptor. To test this we generated the
Lys370-Ser425 receptor mutant, which lacked
eight potential serine phospho-acceptor sites and the putative CK1
binding site (His374-Val391) (35). Consistent
with our hypothesis, the
Lys370-Ser425
receptor mutant was reduced in its ability to undergo
agonist-dependent phosphorylation by ~80% (35).
The reduced ability of the Lys370-Ser425
receptor mutant to undergo agonist-mediated phosphorylation correlates
with the reduction in the receptor ERK-1/2 response and suggests that
there is a link between receptor phosphorylation and activation of the
ERK-1/2 pathway. It is of course possible that deletion of residues
Lys370-Ser425 removes a domain involved in the
ERK-1/2 response but which is not connected with receptor
phosphorylation. This, in itself is an intriguing possibility and one
that is being actively tested in our laboratory at the moment. However,
our data to date is consistent with the hypothesis that phosphorylation
of the M3-muscarinic receptor is involved in the
PKC-dependent activation of the ERK-1/2 pathway.
We further investigated the role of receptor phosphorylation in the
M3-muscarinic receptor-mediated ERK-1/2 response by
inhibiting phosphorylation of the wild-type receptor. We have
previously demonstrated that inhibition of CK1-mediated
M3-muscarinic receptor phosphorylation could be achieved
using either a dominant negative mutant of CK1
, F-CK1
-K46R, or
expression of a region of the third intracellular loop of the
M3-muscarinic receptor (3i-loop peptide) that acted as a
pseudo-substrate for CK1
(35). In the present study, expression of
these constructs resulted in rightward shift in the
concentration-response curve for carbachol-mediated ERK-1/2 activation
and a reduction in the maximal ERK-1/2 response. The effect of these
inhibitors of receptor phosphorylation appeared to be specific for the
M3-muscarinic-mediated ERK-1/2 response because expression
of these constructs in CHO-K1 cells did not greatly affect the phorbol
ester-mediated ERK-1/2 response. Furthermore, previously we have shown
that F-CK1
-K46R did not prevent the receptor from coupling to the
phospholipase C pathway but in fact increased the ability of the
receptor to activate phospholipase C (35). Thus, the depressed ERK-1/2
response observed in the presence of inhibitors of receptor
phosphorylation is receptor specific and produces a response in the
wild-type receptor that is very similar to that observed for the
phosphorylation-deficient
Lys370-Ser425
receptor mutant. These data suggest, therefore, that agonist-mediated phosphorylation of the M3-muscarinic receptor contributes
to the mechanism of ERK-1/2 activation.
This conclusion is supported by recent reports linking phosphorylation
of the 2-adrenergic receptor to the regulation of ERK-1/2 activity. PKA-mediated phosphorylation of the
2-adrenergic receptor has been demonstrated to act as a
"molecular switch" resulting in the coupling of the receptor to the
ERK-1/2 pathway via Gi-protein
-subunits (31).
Furthermore, agonist-mediated GRK-2 phosphorylation has been shown to
recruit a
-arrestin·c-Src complex to the
2-adrenergic receptor (32). Preventing the ability of
-arrestin to interact with c-Src inhibits
2-adrenergic receptor-mediated ERK-1/2 activation,
suggesting that recruitment of c-Src to the phosphorylated
2-adrenergic receptor via
-arrestin is essential in
the mechanism of activation of ERK-1/2 (32). Hence, the data we present
here indicates that the M3-muscarinic receptor, in common
with the
2-adrenergic receptor, employs agonist-mediated receptor phosphorylation in the mechanism of activation of the ERK-1/2 pathway.
In conclusion, we propose that agonist-mediated receptor
phosphorylation via CK1 initiates a process that acts in concert with PKC to mediate a full M3-muscarinic receptor ERK-1/2
response (Fig. 7). The exact nature of the mechanism initiated by
receptor phosphorylation is presently unclear but appears not to
involve Gq/11 heterotrimeric G-proteins nor the activation
of the phospholipase C second messenger signaling cascade. We are
presently pursuing the possibility that phosphorylation of sites in the
third intracellular loop of the M3-muscarinic receptor
recruits an adaptor protein that is important in the activation of the
ERK-1/2 pathway in a manner analogous to
-arrestin·c-Src and the
2-adrenergic receptor.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Prof. Nahorski whom, together with
Drs. A. B. Tobin and G. B. Willars, initiated the work on the
Lys370-Ser425 receptor mutant.
![]() |
FOOTNOTES |
---|
* This work was supported by Wellcome Trust Grant No. 047600/Z/96.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.: 0116-2522935;
Fax: 0116-2523996; E-mail: TBA@le.ac.uk.
Published, JBC Papers in Press, November 16, 2000, DOI 10.1074/jbc.M008827200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
MAP, mitogen-activated protein;
[Ca2+]i, intracellular calcium concentration;
CK1, casein kinase 1
;
ERK, extracellular-regulated protein kinases;
GPCR, G-protein-coupled
receptor;
GRK, G-protein-coupled receptor kinase;
Ins(1, 4,5)P3, inositol (1,4,5)-trisphosphate;
PKC, protein
kinase C;
PKA, cAMP-dependent protein kinase;
RTK, receptor-tyrosine kinases;
CHO, Chinese hamster ovary cells.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Malarkey, J., Belham, C. M., Paul, A., Graham, A., Scott, P. H., and Plevin, R. (1995) Biochem. J. 309, 361-375[Medline] [Order article via Infotrieve] |
2. | Wan, Y., Kurosaki, T., and Huang, X-Y. (1996) Nature 380, 541-544[CrossRef][Medline] [Order article via Infotrieve] |
3. |
Hawes, B. E.,
van Biesen, T.,
Koch, W. J.,
Luttrell, L. M.,
and Lefkowitz, R. J.
(1995)
J. Biol. Chem.
270,
17148-17153 |
4. |
Gutkind, J. S.
(1998)
J. Biol. Chem.
273,
1839-1842 |
5. |
Winitz, S.,
Russell, M.,
Qian, N-X.,
Gardner, A.,
Dwyers, L.,
and Johnson, G. L.
(1993)
J. Biol. Chem.
268,
19196-19199 |
6. | Crespo, P., Xu, N., Simmonds, W. F., and Gutkind, J. S. (1994) Nature 369, 418-420[CrossRef][Medline] [Order article via Infotrieve] |
7. |
Koch, W. J.,
Hawes, B. E.,
Allen, L. F.,
and Lefkowitz, R. J.
(1994)
Proc. Natl. Acad. Sci. U. S. A.
91,
12706-12710 |
8. |
Lopez-Ilasaca, M.,
Crespo, P.,
Pellici, P. G.,
Gutkind, J. S.,
and Wetzker, R.
(1997)
Science
275,
394-397 |
9. |
Luttrell, L. M.,
Hawes, B. E.,
van Biesen, T.,
Luttrell, D. K.,
Lansing, T. J.,
and Lefkowitz, R. J.
(1996)
J. Biol. Chem.
271,
19443-19450 |
10. |
Della Rocca, G. J.,
van Biesen, T.,
Daaka, Y.,
Luttrell, D. K.,
Luttrell, L. M.,
and Lefkowitz, R. J.
(1997)
J. Biol. Chem.
272,
19125-19132 |
11. |
Della Rocca, G. J.,
Maudsley, S.,
Daaka, Y.,
Lefkowitz, R. J.,
and Luttrell, L. M.
(1999)
J. Biol. Chem.
274,
13978-13984 |
12. |
Faure, M.,
Voyno-Yasenetskaya, T. A.,
and Bourne, H. R.
(1994)
J. Biol. Chem.
269,
7851-7854 |
13. |
Palomero, T.,
Barros, F.,
Del Camino, D.,
Viloria, C. G.,
and De La Pena, P.
(1998)
Mol. Pharmacol.
53,
613-622 |
14. |
Launay, J-M.,
Birraux, G.,
Bondoux, D.,
Callebert, J.,
Choi, D-S.,
Loric, S.,
and Maroteaux, L.
(1996)
J. Biol. Chem.
271,
3141-3147 |
15. |
Watanabe, T.,
Waga, I.,
Honda, Z-i.,
Kurokawa, K.,
and Shimizu, T.
(1995)
J. Biol. Chem.
270,
8984-8990 |
16. |
Zou, Y.,
Komuro, I.,
Aikawa, R.,
Kudoh, S.,
Shiojima, I.,
Hiroi, Y.,
Mizuno, T.,
and Yazaki, Y.
(1996)
J. Biol. Chem.
271,
33592-33597 |
17. | Velarde, V., Ullian, M. E., Morinelli, T. A., Mayfield, R. K., and Jaffa, A. A. (1999) Am. J. Physiol. 277, C253-C261[Medline] [Order article via Infotrieve] |
18. |
Tapia, J. A.,
Ferris, H. A.,
Jensen, R. T.,
and Garcia, L. J.
(1999)
J. Biol. Chem.
274,
31261-31271 |
19. |
Venkatakrishnam, G.,
Salgia, R.,
and Groopman, J. E.
(2000)
J. Biol. Chem.
275,
6868-6875 |
20. |
Soltoff, S. P.,
Avraham, H.,
Avraham, S.,
and Cantley, L. C.
(1998)
J. Biol. Chem.
273,
2653-2660 |
21. |
Budd, D. C.,
Rae, A.,
and Tobin, A. B.
(1999)
J. Biol. Chem.
274,
12355-12360 |
22. | Wylie, P. G., Challiss, R. A. J., and Blank, J. L. (1999) Biochem. J. 338, 619-628[CrossRef][Medline] [Order article via Infotrieve] |
23. | Kim, J-Y., Yang, M-S., Oh, C-D., Kim, K-T., Kang, S-S., and Chun, J-S. (1999) Biochem. J. 337, 275-280[CrossRef][Medline] [Order article via Infotrieve] |
24. | Slack, B. E. (2000) Biochem. J. 348, 381-387[CrossRef][Medline] [Order article via Infotrieve] |
25. | Dikic, I., Tokiwa, G., Lev, S., Courtneidge, S. A., and Schlessinger, J. (1996) Nature 383, 547-550[CrossRef][Medline] [Order article via Infotrieve] |
26. | Lev, S., Moreno, H., Martinez, R., Canoll, P., Peles, E., Musacchio, J. M., Plowman, G. D., Rudy, B., and Schlessinger, J. (1995) Nature 276, 737-745 |
27. |
Eguchi, S.,
Numaguchi, K.,
Iwasaki, H.,
Matsumoto, T.,
Yamakawa, T.,
Utsunomiya, H.,
Motley, E. D.,
Kawakatsu, H.,
Owada, K. M.,
Hirata, Y.,
Marumo, F.,
and Inagami, T.
(1998)
J. Biol. Chem.
273,
8890-8896 |
28. |
Zwick, E.,
Daub, H.,
Aoki, N.,
Yamaguchi-Aoki, Y.,
Tinhofer, I.,
Maly, K.,
and Ullrich, A.
(1997)
J. Biol. Chem.
272,
24767-24770 |
29. |
Soltoff, S. P.
(1998)
J. Biol. Chem.
273,
23110-23117 |
30. | Pitcher, J. A., Freedman, N. J., and Lefkowitz, R. J. (1998) Annu. Rev. Biochem. 67, 653-692[CrossRef][Medline] [Order article via Infotrieve] |
31. | Daaka, Y., Luttrell, L. M., and Lefkowitz, R. J. (1997) Nature 390, 88-91[CrossRef][Medline] [Order article via Infotrieve] |
32. |
Luttrell, L. M.,
Ferguson, S. S. G.,
Daaka, Y.,
Miller, W. E.,
Maudsley, S.,
Della Rocca, G. J.,
Lin, F-T,
Kawakatsu, H.,
Owada, K.,
Luttrell, D. K.,
Caron, M. G.,
and Lefkowitz, R. J.
(1999)
Science
283,
655-660 |
33. |
Tobin, A. B.,
and Nahorski, S. R.
(1993)
J. Biol. Chem.
268,
9817-9823 |
34. |
Tobin, A. B.,
Totty, N. F.,
Sterlin, A. E.,
and Nahorski, S. R.
(1997)
J. Biol. Chem.
272,
20844-20849 |
35. |
Budd, D. C.,
McDonald, J. E.,
and Tobin, A. B.
(2000)
J. Biol. Chem.
275,
19667-19675 |
36. | Tobin, A. B., Lambert, D. G., and Nahorski, S. R. (1992) Mol. Pharmacol. 42, 1042-1048[Abstract] |
37. | Challiss, R. A. J., Batty, I. H., and Nahorski, S. R. (1988) Biochem. Biophys. Res. Commun. 157, 684-691[Medline] [Order article via Infotrieve] |
38. | Grynkiewicz, G., Poenie, M., and Tsien, R. Y. (1985) J. Biol. Chem. 260, 3440-3450[Abstract] |
39. |
Xiong, L.,
Lee, J. W.,
Graves, L. M.,
and Earp, H. S.
(1998)
EMBO J.
17,
2574-2583 |
40. |
Cobb, M. H.,
and Goldsmith, E. J.
(1995)
J. Biol. Chem.
270,
14843-14846 |
41. |
Tobin, A. B.,
Keys, B.,
and Nahorski, S. R.
(1996)
J. Biol. Chem.
271,
3907-3916 |
42. | Waugh, M. G., Challiss, R. A. J., Berstein, G., Nahorski, S. R., and Tobin, A. B. (1999) J. Biochem. 338, 175-183[CrossRef] |