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INTRODUCTION |
The mitogen-activated protein
(MAP)1 kinases, Erk-1 and
Erk-2, are phosphorylated and activated by a protein kinase cascade that can be initiated by both tyrosine kinase growth factor receptors and G-protein coupled receptors (GPCRs) (1, 2). Many of the same signal
transduction proteins are used by both growth factor receptors and
GPCRs in the activation of the MAP kinase pathway. The commonality
between these two receptor types has recently been extended to include
a role for ligand-mediated receptor endocytosis in the mechanism of
MAP kinase activation (3, 4).
Endocytosis of growth factor receptors, via clathrin-coated pits, is
known to play an essential role in Erk-1/2 activation (5, 6). Recent
studies on the
2-adrenergic receptor have also
demonstrated that clathrin/dynamin-mediated receptor endocytosis may
also be essential in the activation of the MAP kinase pathway by GPCRs
(3, 4). These studies indicated that ligand-induced
2-adrenergic receptor endocytosis provided a link
between the activated Raf-1 complex and MEK, the downstream kinase in
the phosphorylation cascade (3, 4). Inhibition of endocytosis was
sufficient to prevent Raf-1 activation of MEK and thereby block Erk-1/2
phosphorylation mediated by the
2-adrenergic receptor (3).
Because many GPCRs undergo ligand-induced endocytosis via a
clathrin/dynamin-mediated process (7) and that GPCRs activate the MAP
kinase pathway via Raf-1 (2), it has been suggested that GPCRs
generally employ receptor-mediated endocytosis in the mechanism of
activation of the MAP kinase pathway (4). This hypothesis, however, has
not been extensively tested for receptor subtypes other than the
2-adrenergic receptor. Importantly, different receptor
subtypes activate the MAP kinase pathway via distinct mechanisms. These
mechanisms depend on the G-protein subtype to which the GPCR is coupled
(8-13). Whether the differential mechanisms by which GPCRs activate
the MAP kinase pathway contribute to the role played by receptor
endocytosis has not yet been examined.
To address the question of the possibility that receptor endocytosis is
universally essential in the activation of the MAP-kinase pathway by
GPCRs, we investigate here the Gq/11-coupled m3-muscarinic receptor expressed in CHO cells. This receptor type shares a number of
properties with the
2-adrenergic receptor in that the
receptor is rapidly phosphorylated following agonist stimulation (14) and that it undergoes endocytosis in a
clathrin/dynamin-dependent process (15, 16). Using
inhibitors of endocytosis and a mutant receptor deficient in the
ability to undergo ligand-mediated endocytosis, we investigate the role
receptor endocytosis plays in the activation of Erk-1/2 by the
m3-muscarinic receptor. Further, we discuss the possibility that the
upstream mechanism of Raf-1 activation may play a role in determining
the involvement of receptor endocytosis in GPCR-mediated Erk-1/2 activation.
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MATERIALS AND METHODS |
Cell Culture--
Chinese hamster ovary cells (CHO) were
maintained in
-minimal essential media (MEM) supplemented with fetal
calf serum (10% v/v), fungizone (2.5 µg/ml), penicillin (100 IU/ml),
streptomycin (100 µg/ml) at 37 °C in a humidified incubator
containing 5% CO2. CHO-m3 cells expressing recombinant
human m3-muscarinic receptors (1.55 ± 0.04 pmol/mg of protein),
and CHO cells expressing the m3-muscarinic receptor mutant
m3(A349AAA352) (4.72 ± 0.08 pmol/mg
protein) were kind gifts from Dr. Wolfgang Sadee and Dr. Jelveh Lameh
(School of Pharmacy, University of California, San Francisco, CA). The
generation of the m3(A349AAA352) has been
previously described (17).
In experiments where endogenous PKC was down-regulated by phorbol
12,13-dibutyrate, cells were exposed to 1 µM phorbol
ester overnight prior to experimentation. Furthermore, in experiments where pertussis toxin was used, cells were incubated with 100 ng/ml
pertussis toxin overnight before experimentation.
Inositol 1,4,5-Trisphosphate (Ins(1,4,5)P3)
Determination--
Cells grown to confluence in 24-well plates were
washed with 250 µl of Krebs-Henseleit buffer (KHB, 10 mM
HEPES, pH 7.4, 118 mM NaCl, 4.3 mM KCl, 1.17 mM MgSO4·7H2O, 1.3 mM
CaCl2·2H2O, 25 mM
NaHCO3, 11.7 mM glucose). Cells were stimulated
with 1 mM carbachol for the indicated times. Reactions were
terminated by the addition of an equal volume of 1 M
trichloroacetic acid, and Ins(1,4,5)P3 was determined by a
radioreceptor assay previously described (18).
Receptor Phosphorylation--
Cells grown to confluence in
6-well plates were labeled with [32P]orthophosphate (50 µCi/ml) for 1 h. Cells were stimulated with 1 mM
carbachol for the times indicated, and the reactions were terminated by
rapid aspiration of media and the addition of 1 ml of ice-cold RIPA
buffer (10 mM Tris, 500 mM NaCl, 10 mM EDTA, 1% v/v Nonidet P-40, 0.1% v/v SDS, 0.5% w/v
Na-deoxycholate). The mutant m3(A349AAA352)
receptor expression levels were ~3-fold greater than wild type receptors. Therefore, to ensure that equivalent numbers of receptors were immunoprecipitated, the proteins were adjusted so that ~1 pmol
of solubilized receptors were used for each immunoprecipitation. The
receptors were immunoprecipitated from precleared lysates using 1 µg
of anti-muscarinic m3 receptor antibody (14). Immunocomplexes were
collected on protein A-Sepharose beads, and the beads were washed three
times with ice-cold TE buffer (10 mM Tris, pH 7.4, 2.5 mM EDTA). Immunocomplexes were resuspended in 2×
SDS-polyacrylamide gel electrophoresis sample buffer and placed in a
boiling water bath for 2 min. Proteins were resolved on an 8%
SDS-polyacrylamide electrophoresis gel. The gels were stained with
0.2% Coomassie Blue to ensure that there was equal
immunoprecipitation. The gels were then dried, and phosphorylated bands
were visualized by autoradiography.
Receptor Density Determination--
Confluent CHO cells grown in
24-well plates were serum-starved for 1 h in KHB with or without
endocytotic inhibitors and then stimulated with 1 mM
carbachol for the appropriate times. Reactions were terminated by
aspiration, and the cells were washed three times with ice-cold KHB.
Cells were incubated with ~0.14 µCi of [3H]N-methylscopolamine (NMS) overnight at
4 °C. Cells were washed two times in ice-cold KHB and solubilized by
the addition of 1 ml of ice-cold RIPA buffer, and receptor number was
determined by liquid scintillation counting. Nonspecific binding was
determined by the inclusion of 10 µM atropine.
Erk-1/2 Assay--
CHO cells grown to confluence in 6-well
plates were serum-starved for 1 h in KHB and then stimulated with
the appropriate agents. Stimulations were terminated by aspiration, and
cells were incubated for 10 min in lysis buffer (20 mM
Tris, pH 7.6, 0.5% Nonidet P-40, 250 mM NaCl, 3 mM EDTA, 3 mM EGTA, 1 mM
phenylmethylsulfonyl fluoride, 1 mM
Na3VO4, 1 mM dithiothreitol, 5 µg/ml benzamidine) at 4 °C. Solubilized CHO cell lysates were
pre-cleared by centrifuging at 20,000×g for 5 min.
Endogenous MAP kinase was immunoprecipitated using 0.2 µg of
anti-Erk-1/2 antiserum (Santa Cruz Biotechnology). Protein
A-Sepharose-immobilized MAP kinase was washed two times in lysis buffer
and two times in assay buffer (20 mM HEPES, pH 7.2, 20 mM
-glycerophosphate, pH 7.2, 10 mM
MgCl2, 1 mM dithiothreitol, 50 µM
Na3VO4). Washed pellets were resuspended in
assay buffer containing 2 µCi [32P]ATP, 20 µM ATP, 200 µM EGFr (peptide encompassing
region 661-681 of 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. 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.
Assay of Raf-1 Kinase Activity--
Confluent CHO cells seeded
in 6-well plates were serum-starved for 1 h in KHB and stimulated
with 1 mM carbachol for the appropriate times. Reactions
were terminated by rapidly aspirating the medium, and the cells were
lysed in gold lysis buffer (GLB, composition: 20 mM Tris,
pH 8.0, 137 mM NaCl, 5 mM EDTA, 1 mM EGTA, 15% glycerol, 1% (v/v) Triton X-100, 1 mM phenylmethylsulfonyl fluoride, 50 µg/ml benzamidine, 1 mM NaVO4, 10 mM
-glycerophosphate). Raf-1 was immunoprecipitated with 0.5 µg of
anti-Raf-1 antiserum (kind gift from Dr. C. Pritchard, Dept. of
Biochemistry, University of Leicester, UK). Immunocomplexes were washed
two times with ice-cold GLB and two times with ice-cold Raf-1 assay
buffer (25 mM HEPES, pH 7.2, 10 mM
MgCl2, 1 mM dithiothreitol, 1 mM
MnCl2). Washed immunocomplexes were resuspended in Raf-1
assay buffer containing 20 µM ATP, 2 µCi
[32P]ATP, 1.8 µg of Raf-1 peptide substrate. Assays
were subsequently performed as for the Erk assay.
Statistical Analysis--
Student's t test or
two-way analysis of variance was applied where indicated.
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RESULTS |
Effect of Inhibition of Receptor Endocytosis on m3-Muscarinic
Receptor Erk-1/2 Activation--
Stimulation of CHO-m3 cells
expressing the recombinant human m3-muscarinic receptor with the
cholinergic agonist carbachol (60 min, 1 mM) resulted in a
56% loss of cell surface receptors as determined using the binding of
the hydrophilic ligand [3H]N-methylscopolamine
([3H]NMS) (Fig. 1). Total
receptor number determined using [3H]NMS binding to a
broken cell preparation from stimulated or control cells remained
unchanged during this time (data not shown), indicating that the loss
of receptor binding at the cell surface was because of receptor
endocytosis (or internalization) rather than receptor degradation.
m3-Muscarinic receptor endocytosis could be completely inhibited
following pretreatment with concanavalin A and reduced by 81% by
cytochalasin D (Fig. 1).

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Fig. 1.
Inhibition of agonist-dependent
m3-muscarinic receptor endocytosis by concanavalin A and cytochalasin
D. CHO-m3 cells grown in 24-well plates were treated for 1 h
with vehicle, concanavalin A (250 µg/ml, con A) or
cytochalasin D (1 µM, cyto D). Cells were then
stimulated with 1 mM carbachol (CCH) for 1 h. The reactions were stopped by washing in ice-cold KHB. Radioligand
binding using a saturating concentration of the hydrophilic antagonist
[3H]NMS was performed overnight. Nonspecific binding was
determined in the presence of 10 µM atropine. The data
presented represent the mean ± S.E. for three experiments carried
out in duplicate.
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Stimulation of m3-muscarinic receptors resulted in an ~25-fold
increase in Erk-1/2 activity within 5 min, which fell to ~8.5-fold within 20 min (Fig. 2). Inhibition of
m3-muscarinic receptor endocytosis using concanavalin A and
cytochalasin D had no significant effect on m3-muscarinic receptor
activation of Erk-1/2 (Fig. 2).

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Fig. 2.
m3-Muscarinic receptor stimulation of the
Erk-1/2 activity is unaffected by inhibitors of endocytosis.
CHO-m3 cells were pretreated for 1 h with either vehicle, 250 µg/ml concanavalin A (solid bars, concanavalin A;
gray bars, + concanavalin A) (A) or 1 µM cytochalasin D (solid bars, cytochalasin
D; gray bars, + cytochalasin D) (B). Cells were
then stimulated with 1 mM carbachol for 0, 5, and 20 min,
after which the cells were lysed and Erk-1/2 activity was determined.
The data presented represent the mean ± S.E. for three
experiments carried out in duplicate.
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Characterization of the m3(A349AAA352)
Receptor Mutant--
Previous studies have demonstrated that mutation
of a region in the third intracellular loop of the m3-muscarinic
receptor (S349ASS352
A349AAA352)
resulted in a receptor that was unable to undergo receptor endocytosis (17). This was confirmed in the present study where expression of the
m3(A349AAA352) mutant in CHO cells resulted in
a receptor that showed no significant endocytosis following 60 min of
agonist treatment (Fig. 3A).
In contrast, wild type receptors were internalized by 51.1 ± 5.1% following 60-min stimulation with carbachol (Fig. 3A).
The m3(A349AAA352) receptor did, however,
couple to the phospholipase C (PLC) pathway as evident by a peak and
plateau profile of Ins(1,4,5)P3 production (Fig.
3B). Comparison of the peak Ins(1,4,5)P3
response revealed a numerical but not significant reduction in
Ins(1,4,5)P3 for the m3(A349AAA352)
receptor compared with wild type receptor (p = 0.073, n = 6, unpaired Student's t test).
Furthermore, the m3(A349AAA352) receptor also
underwent rapid agonist-mediated phosphorylation (Fig. 3C),
which was maximal after ~30 s and was maintained for at least 30 min
(data not shown). Densitometric analysis revealed that phosphorylation
of the m3(A349AAA352) receptor was ~3-fold
greater than the wild type receptor. Note that in the data presented in
Fig. 3C, the receptor number was normalized at the point of
immunoprecipitation so that an equal number of wild type and
m3(A349AAA352) mutant receptors were
immunoprecipitated (see "Materials and Methods").

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Fig. 3.
The m3-muscarinic receptor mutant
m3(A349-AAA352) is unable
to undergo agonist-mediated endocytosis but is still coupled to
phospholipase C and receptor-specific kinases. A, receptor
internalization of wild type m3-muscarinic receptors (  ) and
m3(A349-AAA352) mutant receptors (- - - -)
was determined by incubation of cells grown on 24-well plates with 1 mM carbachol for the indicated times. The reactions were
stopped by washing cells in ice-cold KHB buffer, and cell surface
receptor number was determined by radioligand binding using
[3H]NMS. The data presented represent the mean ± S.E. for three experiments carried out in duplicate. B,
inositol (1,4,5)-trisphosphate was determined in cells expressing wild
type m3-muscarinic (  ) receptor and
m3(A349-AAA352) (  ) receptor using a
radioreceptor assay. Cells were stimulated with carbachol (1 mM) for the times indicated. The data presented represent
the mean ± S.E. for three experiments carried out in duplicate.
C, CHO cells expressing wild type or
m3(A349-AAA352) receptors were labeled with
[32P]orthophosphate and stimulated with carbachol (1 mM) for 30 and 60 s. Receptors were then solubilized
and adjusted so that the number of receptors used in the
immunoprecipitated were equivalent (~1
pmol/receptor/immunoprecipitation). The gels were stained with 0.2%
Coomassie Blue before drying to ensure that immunoprecipitation was
equivalent between lanes. The results are representative of at least
three experiments.
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Activation of Erk-1/2 Activity by the Wild Type and
m3(A349AAA352) Receptor--
Despite the fact
that the m3(A349AAA352) receptor did not
undergo endocytosis, the m3(A349AAA352)
receptor stimulated Erk-1/2 activation with a time course that was
similar to the wild type receptor (Fig.
4A). The
m3(A349AAA352) receptor did, however, show a
slight lag evident at 1 min (p < 0.05) and a
significantly more robust response at 20 min (p < 0.01) when compared with the wild type receptor. The
agonist-concentration dependence between the wild type and
m3(A349AAA352) receptor was not significantly
different (p = 0.82, two-way analysis of variance)
(Fig. 4B).

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Fig. 4.
Activation of Erk-1/2 by the wild type
m3-muscarinic receptor and the mutant
m3(A349-AAA352).
A, time course for wild type m3-muscarinic (  )
receptor and mutant m3(A349-AAA352)
(- - - -) receptor-mediated activation of Erk-1/2. Cells were
stimulated for the times indicated with carbachol (1 mM).
B, dose-dependence of Erk-1/2 activation by m3-muscarinic
(  ) and m3(A349-AAA352) (- - - -)
receptors stimulated with carbachol (CCH) for 5 min.
EC50 values for wild type m3-muscarinic receptors and
m3(A349-AAA352) receptors were 9.5 and 22.7 µM, respectively, and were not statistically different
from each other (p = 0.82, two-way analysis of
variance). The data presented represent the mean ± S.E. for three
experiments carried out in duplicate.
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The wild type and m3(A349AAA352) receptor
activation of Erk-1/2 could be completely blocked by the MEK-1/2
inhibitor PD98053 (Calbiochem, data not shown). Furthermore, both the
wild type and m3(A349AAA352) receptor Erk-1/2
responses were mediated, at least in part, by PKC. Down-regulation of
PKC using phorbol ester pretreatment reduced the wild type and
m3(A349AAA352) receptor responses by 87 and
77%, respectively (Fig. 5). In control
experiments, PKC down-regulation completely inhibited the Erk-1/2
response to phorbol 12,13-dibutyrate (Fig. 5). Inhibition of PKC using
the inhibitor Ro-318220 reduced wild type and
m3(A349AAA352) receptor responses by 86 and
56%, respectively (Fig. 5). Once again in control experiments,
Ro-318220 completely inhibited the Erk-1/2 response to phorbol
12,13-dibutyrate (data not shown).

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Fig. 5.
Characterization of Erk-1/2 activation by
wild type m3-muscarinic and
m3(A349-AAA352)
receptors. Panels A-C represent wild type muscarinic
receptors, and panels D-F represent
m3(A349-AAA352) receptors. A and
D, cells were either treated overnight (~12 h) with
phorbol 12,13-dibutyrate (1 µM, PDBu) or with vehicle.
Cells were then stimulated with carbachol (1 mM,
CCH), phorbol 12,13-dibutyrate (1 µM, PDBu),
or vehicle for 5 min. Cells were then lysed and Erk-1/2 activity
determined. B and E, cells plated on 6-well
dishes were stimulated for 5 min with carbachol (1 mM,
CCH) or vehicle. In the appropriate experiments, the PKC
inhibitor Ro-318220 (10 µM, Ro) was added 10 min before the stimulatory agents. The reaction was then stopped by
addition of lysis buffer. Erk-1/2 was immunoprecipitated from the
lysate and used in an in vitro kinase assay to determine the
activity of Erk-1/2. C-F, cells were either treated
overnight with pertussis toxin (100 ng/ml, PTX) or vehicle.
Cells were stimulated for 5 min with carbachol (1 mM,
CCH) and then lysed, and Erk-1/2 activity was determined.
Basal levels of Erk-1/2 activity in control cells was 145.5 ± 23.5 fmol/mg/min and for pertussis toxin-treated cells 101.6 ± 4.1 fmol/mg/min. The data presented represent the mean ± S.E. for
three experiments carried out in duplicate.
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Previous studies have demonstrated that Gq/11-coupled
muscarinic receptors expressed in CHO cells activated Erk-1/2 via a pertussis toxin-insensitive G-protein (11, 13). This was confirmed in
the present study where both m3(A349AAA352) and
wild type receptor-stimulated Erk-1/2 activity was unaffected by
pertussis toxin pretreatment (Fig. 5).
The ability of the muscarinic receptor to activate Raf-1, the upstream
kinase in the MAP kinase cascade, was also investigated. m3-Muscarinic
receptor activation stimulated an ~5-fold increase in Raf-1 kinase
activity (basal = 125 fmol of phosphate incorporated/mg/min, n = 2) that peaked at 1-5 min followed by a plateau
phase (~3-fold over basal) evident at 10 min and extending for at
least 20 min.
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DISCUSSION |
The present study demonstrates that the Gq/11-coupled
m3-muscarinic receptor expressed in CHO cells activates the MAP-kinase pathway (Erk-1/2) via a mechanism that is independent of receptor endocytosis. These data conflict with recent reports, based on studies
on the
2-adrenergic receptor, that have suggested that receptor endocytosis is essential for GPCR activation of the MAP-kinase pathway (3, 4).
Our conclusion is supported by the findings that inhibition of
m3-muscarinic receptor endocytosis by cytochalasin D and concanavalin A
did not affect the ability of the receptor to activate Erk-1/2. Furthermore, a receptor mutant (m3(A349AAA352)
that did not undergo endocytosis in response to agonist exposure was
still able to stimulate Erk-1/2 activity. Importantly, with the
exception of receptor endocytosis, the
m3(A349AAA352) mutant behaved with many of the
characteristics associated with the wild type m3-muscarinic receptor in
that it was able to couple to the PLC-pathway and undergo
agonist-sensitive phosphorylation.
The model for
2-adrenergic receptor-mediated activation
of the MAP-kinase pathway recently proposed by Lefkowitz and colleagues is thought to represent a general scheme for GPCR stimulation of
Erk-1/2 (3, 4).
2-Adrenergic receptor stimulation
results in receptor phosphorylation by receptor-specific kinases
(e.g. GRK-2/3) and protein kinase A (19). Although
previously linked with receptor desensitization,
2-adrenergic receptor phosphorylation has recently been
shown to play a role in endocytosis and activation of the MAP-kinase
pathway. Phosphorylation at GRK-2/3 sites allows for the recruitment of
-arrestin which acts as an adaptor protein linking the receptor to
clathrin and hence the clathrin/dynamin endocytic pathway (20, 21).
Protein kinase A phosphorylation acts as a molecular switch allowing
for the
2-adrenergic receptor to act via
Gi-proteins to stimulate the MAP-kinase pathway (22) in a
manner similar to more conventional Gi-coupled receptors (for example m2-muscarinic). Dominant negative mutants of
-arrestin and dynamin, as well as chemical inhibitors of endocytosis, prevented both
2-adrenergic receptor endocytosis and Erk-1/2
activation (3). The point at which receptor endocytosis links with the MAP-kinase pathway is at the activation of MEK-1/2 by Raf-1 (3). It has
been suggested that the activated Raf-1 complex once associated with
the plasma membrane requires internalization to enable phosphorylation of MEK-1/2 (3, 4).
Because receptor endocytosis is a common phenomenon among GPCRs (7) and
that GPCR activation of the MAP-kinase pathway occurs through Raf-1
(2), it has been suggested that the role for receptor endocytosis in
Erk-1/2 activation described for the
2-adrenergic
receptor may generally be applicable to all GPCRs (4). Despite this,
however, the data presented here clearly demonstrate that activation of
the MAP-kinase pathway by m3-muscarinic receptors does not require
receptor endocytosis.
Muscarinic receptors coupled via Gq/11 to the PLC pathway
have previously been shown to undergo endocytosis via a
clathrin/dynamin pathway (15, 16, 23). Consistent with these findings
are data presented here that demonstrate the ability of concanavalin A
and cytochalasin D, reagents known to disrupt clathrin-mediated endocytosis (24), to inhibit m3-muscarinic receptor internalization. Furthermore, we have previously reported that the m3-muscarinic receptor is rapidly phosphorylated following agonist stimulation (14),
possibly by CK1
(25). The role phosphorylation plays in
m3-muscarinic receptor endocytosis is unclear. It is interesting to
note, however, that in the present study we show that the process of
receptor phosphorylation is not sufficient to ensure endocytosis since, despite the m3(A349AAA352) receptor
mutant undergoing agonist-mediated phosphorylation, this receptor is
unable to endocytose. In fact, the
m3(A349AAA352) receptor was phosphorylated to a
level ~3-fold greater than the wild type receptor. The apparent
hyper-phosphorylation of the m3(A349AAA352)
receptor may be explained by findings from previous studies that
suggest a role for receptor endocytosis in dephosphorylation of GPCRs
(26). If endocytosis is important for muscarinic receptor dephosphorylation, then a mutant receptor unable to undergo endocytosis may "accumulate" in the phosphorylated form. Whether this is the reason for the hyper-phosphorylation of the
m3(A349AAA352) receptor is presently under investigation.
The m3-muscarinic receptor expressed in CHO-m3 cells, therefore, shares
some characteristics in common with the
2-adrenergic receptor that is known to be important in
2-adrenergic
receptor-mediated MAP-kinase activation. Namely, the receptor undergoes
rapid phosphorylation following agonist stimulation and is internalized
via a clathrin/dynamin-dependent pathway. However, despite
these similarities, the receptors do not share a common role for
receptor endocytosis in MAP-kinase activation.
The m3-muscarinic and
2-adrenergic receptors do show
divergent mechanisms of activation of the MAP-kinase pathway at the level of the upstream activation of Raf-1. In some cell types, Gq/11-coupled receptors including muscarinic receptors,
have been shown to activate Erk-1/2 in a pertussis toxin-insensitive
manner, via PKC, in a process that is independent of Ras but dependent on Raf-1 (11, 12, 13). The data presented here demonstrates that the
m3-muscarinic receptor-stimulated Erk-1/2 activity is reduced by PKC
inhibition and is unaffected by pertussis toxin, suggesting that a
similar mechanism is in operation for the m3-muscarinic receptor
expressed in CHO-m3 cells. Furthermore, the ability of the
m3-muscarinic receptors to activate Raf-1 in this study indicates that
the process of Erk-1/2 activation by m3-muscarinic receptors in CHO-m3
cells is mediated via Raf-1 activation of MEK. This is supported by the
fact that the MEK inhibitor PD98053 blocked m3-muscarinic
receptor-mediated Erk-1/2 activation. In contrast with the
Gq/11-coupled muscarinic receptor subtypes, the mechanism of Raf-1 activation by
2-adrenergic receptors involves

-subunits derived from pertussis toxin-sensitive G-proteins
activating cytosolic tyrosine kinases that are able to phosphorylate
adaptor proteins involved in the recruitment of Ras exchange factors
and the activation of Ras (21).
The common thread, therefore, in the activation of Erk-1/2 by GPCR
subtypes, and indeed growth factor receptors, is that they all operate
through the activation of Raf-1 (2). The difference lies in the
mechanisms employed in the upstream activation of Raf-1. The argument
tested in this report is as follows. If receptor endocytosis is
essential for Raf-1 activation of MEK, which appears to be the case for
some growth factor receptors (5, 6) and the
2-adrenergic
receptor (3), then it might be predicted that this would be true for
GPCRs generally because they undergo ligand-dependent
endocytosis and all stimulate Erk-1/2 via Raf-1. Our data, however, do
not support this hypothesis and demonstrate that receptor endocytosis
is not universally essential in the mechanism of GPCR activation of the
MAP kinase pathway. The reason for the differences in the role played
by endocytosis between the various GPCR subtypes may lie at the
differential upstream mechanisms employed in the activation of Raf-1,
e.g. whether via PKC or 
-subunits.