From the Howard Hughes Medical Institute and the Departments of Medicine and Biochemistry, Duke University Medical Center, Durham, North Carolina 27710, the ** NCI, National Institutes of Health, Rockville, Maryland 20857, and the § Department of Medicine (Nephrology), Medical University of South Carolina and the Ralph H. Johnson Veterans Affairs Medical Center, Charleston, South Carolina 29425
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ABSTRACT |
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Many receptors that couple to heterotrimeric
guanine nucleotide-binding (G) proteins mediate rapid activation of the
mitogen-activated protein kinases, Erk1 and Erk2. The
Gi-coupled serotonin (5-hydroxytryptamine (5-HT))
5-HT1A receptor, heterologously expressed in Chinese
hamster ovary or human embryonic kidney 293 cells, mediated rapid
activation of Erk1/2 via a mechanism dependent upon both Ras activation
and clathrin-mediated endocytosis. This activation was attenuated by
chelation of intracellular Ca2+ and
Ca2+/calmodulin (CAM) inhibitors or the CAM sequestrant
protein calspermin. The CAM-dependent step in the Erk1/2
activation cascade is downstream of Ras activation, because inhibitors
of CAM antagonize Erk1/2 activation induced by constitutively activated
mutants of Ras and c-Src but not by constitutively activated mutants of
Raf and MEK (mitogen and extracellular
signal-regulated kinase). Inhibitors of the classical CAM
effectors myosin light chain kinase, CAM-dependent protein
kinases II and IV, PP2B, and CAM-sensitive phosphodiesterase had no
effect upon 5-HT1A receptor-mediated Erk1/2 activation. Because clathrin-mediated endocytosis was required for
5-HT1A receptor-mediated Erk1/2 activation, we postulated a
role for CAM in receptor endocytosis. Inhibition of receptor
endocytosis by use of sequestration-defective mutants of
Receptors coupled to heterotrimeric guanine nucleotide-binding (G)
proteins, the largest known family of cell surface receptors, mediate
cellular responses to many extracellular stimuli, such as
neurotransmitters, peptide hormones, odorants, and photons (1). In
addition to regulating the generation of soluble second messengers,
many G protein-coupled receptors mediate proliferative or
differentiative signals in various cultured cell lines and tissues via
mitogen-activated protein
(MAP)1 kinases (2, 3). Erk1/2
MAP kinases are serine/threonine kinases involved in the activation of
nuclear transcription factors. Erk1/2 activity is regulated by
threonine/tyrosine phosphorylation, which is controlled by a highly
conserved phosphorylation cascade. Phosphorylation of Erk1/2 is
catalyzed by the MAP/Erk kinases 1 and 2 (MEK1 and MEK2) that are themselves phosphorylated and activated
by MEK kinases such as raf-1 oncogene family proteins. Activation of Raf-1 occurs as a consequence of membrane translocation, which can be mediated by the GTP-bound form of the small G protein, Ras.
Like the epidermal growth factor (EGF) receptor and other receptor
tyrosine kinases, many G protein-coupled receptors regulate Ras
function via tyrosine phosphorylation. Several Gi-coupled receptors stimulate pertussis toxin-sensitive,
Ras-dependent Erk activation through tyrosine
phosphorylation of adapter proteins, such as Shc and Gab1, and membrane
recruitment of the Ras guanine nucleotide exchange factor, mSos (2, 4).
Shc tyrosine phosphorylation and Erk activation by
Gi-coupled receptors are sensitive to inhibitors of
src family nonreceptor tyrosine kinases (4, 5), suggesting a
role for src kinases in mitogenic signaling by G
protein-coupled receptors. In some systems, Ca2+/CAM has
also been implicated in Gi- and Gq/11-coupled
receptor-mediated Erk activation (6-8).
Recent data have also suggested that clathrin-mediated endocytosis
plays a crucial, if poorly understood, role in Erk activation via EGF,
insulin-like growth factor-1 receptors (9, 10), lysophosphatidic acid
(LPA), and 5-HT mediates mitogenic effects in many cell types (13, 14). In CHO-K1
cells, the 5-HT1A receptor rapidly activates Erk via a
pathway that involves pertussis toxin-sensitive G protein Materials--
1,2-Bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic
acid (BAPTA), phorbol 12-myristate 13-acetate (PMA), and LPA were from Sigma. Fluphenazine, W-7, EGF, cyclosporine A, FK506, A23187, and
ionomycin were from Calbiochem. Basic fibroblast growth factor (bFGF)
was from Upstate Biotechnology (Lake Placid, NY). ML-7, ML-9, HA1077,
KN-62, KN-92, vinpocetine, and 8-methoxymethyl isobutylmethylxanthine were from Biomol (Plymouth Meeting, PA).
DNA Constructs--
The cDNAs for FLAG-tagged
Cell Culture and Transfection--
HEK-293 cells were maintained
in minimum essential medium with Earle's salts (Life Technologies,
Inc.) supplemented with 10% fetal bovine serum (FBS, Life
Technologies, Inc.) and 50 µg/ml gentamicin (Life Technologies, Inc.)
at 37 °C in a humidified 5% CO2 atmosphere. CHO-1A-27
cells (18) were maintained in Ham's F-12 nutrient mixture (Life
Technologies, Inc.) supplemented with 10% FBS, 50 µg/ml gentamicin,
and 400 µg/ml G418. Transfections were performed on 80-90%
confluent monolayers in 100-mm dishes, using calcium phosphate
coprecipitation for HEK-293 cells (23) and LipofectAMINE for CHO cells
(9 µl/µg of DNA, Life Technologies, Inc.) (15). Empty pcDNA3
vector was added to transfections to keep the total mass of DNA added
per dish constant within experiments. Prior to stimulation, cells were
incubated overnight in serum-free medium containing 0.1% bovine serum albumin.
Erk1/2 Phosphorylation and Kinase Assay--
Stimulations were
carried out at 37 °C in serum-starving medium. Monolayers were then
lysed directly using 200 µl/well Laemmli sample buffer.
Phosphorylation of Erk1/2 was detected by immunoblotting using rabbit
phosphospecific Erk IgG (New England Biolabs) as described previously,
except that the bands were visualized with Vistra ECF reagent
(Amersham Pharmacia Biotech) (13, 15). Membranes were stripped
and reprobed with rabbit anti-Erk2 IgG (Santa Cruz Biotechnology) to
quantitate total Erk2.
Alternatively, monolayers were lysed in 500 µl/well RIPA buffer (150 mM NaCl, 50 mM Tris-Cl, pH 8.0, 10 mM EDTA, 1% v/v Nonidet P-40, 0.5% w/v sodium
deoxycholate, 1 mM NaF, 1 mM sodium
pyrophosphate, 100 µM NaVO4, 1 mM
phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, 10 µg/ml
leupeptin), and then Erk2 was immunoprecipitated with rabbit anti-Erk2
IgG plus 50 µl of a 50% slurry of protein G plus/protein A-agarose
(Calbiochem). Immune complexes were washed twice in ice-cold RIPA
buffer and twice in kinase buffer (50 mM HEPES, pH 7.0, 10 mM MgCl2, 1 mM dithiothreitol).
Phosphorylation assays were performed as described previously using
myelin basic protein as the substrate (6, 13, 15). Both assays of ERK
activity yielded virtually identical results.
cAMP Production--
Cells were metabolically labeled with 3 µCi of [3H]adenine/ml for 6 h in medium containing
3% FBS, washed once in phosphate-buffered saline, and incubated in
serum-free medium with 1 mM isobutylmethylxanthine for 30 min at 37 °C. Following a 10-min exposure to agonist, reactions were
terminated by the addition of 1 ml/well ice-cold stop solution (0.1 mM cAMP, 4 nCi of [14C]cAMP/ml, 2.5%
perchloric acid). Cell lysates were neutralized in KOH, and total
[3H]cAMP production was assayed by anion exchange
chromatography (24, 25).
Inhibitors of Ca2+/CAM Impair 5-HT1A
Receptor-mediated Erk Activation in CHO-1A-27 Cells--
To examine
the role of Ca2+/CAM in Gi-coupled
receptor-mediated Erk activation, we employed CHO cells stably
expressing the 5-HT1A receptor (CHO-1A-27 cells) (18). In
this system, we have previously shown that 5-HT stimulation results in
rapid, Ras-dependent, activation of Erk2, which is mediated
via G
To determine whether CAM activity is required for receptor-mediated Erk
activation, CHO-1A-27 cells were pretreated with the pharmacologically
distinct CAM antagonists, fluphenazine and W-7. As shown in Fig.
1B, both agents impaired 5-HT- and bFGF-stimulated Erk2
activation. To confirm the specificity of the fluphenazine and W-7
effects, the experiments were repeated in HEK-293 cells transiently
coexpressing the 5-HT1A receptor with the CAM-binding protein calspermin (26), which has been shown to function as an
intracellular CAM
sequestrant.2 As shown in
Fig. 1C, calspermin expression attenuated Erk1/2 phosphorylation induced by 5-HT1A or endogenous EGF
receptors. Neither isoproterenol-stimulated intracellular cAMP
production nor 5-HT- and LPA-induced inhibition of forskolin-stimulated
cAMP production was affected by W-7 or fluphenazine, indicating that the CAM antagonists had no effect on receptor-G protein coupling (data
not shown).
Ca2+/CAM Antagonists Impair 5-HT1A
Receptor-mediated Erk Activation at a Point in the Pathway Downstream
of Ras Activation--
In many systems, the pathways of
Gi-coupled receptor- and receptor tyrosine kinase-mediated
Erk activation converge upstream of Ras activation (27). The finding
that CAM antagonists affected both receptor tyrosine kinase- and
5-HT1A receptor-mediated Erk activation suggested that
Ca2+/CAM was involved in regulating a common component of
the pathway. To determine the point in the signaling cascade at which
Ca2+/CAM is required, we assayed the effects of
fluphenazine and W-7 on Erk activation via constitutively active
mutants of Src, Ras, Raf, and MEK. As shown in Fig.
2, the constitutively activated proteins
Y530F-Src, V12-Ras, Raf-CAAX, or DD-MEK were sufficient to
induce chronic Erk activation in transiently transfected CHO-1A-27 cells. Fluphenazine or W-7 significantly inhibited Y530F-Src- and
V12-Ras-induced Erk2 activation, without affecting Raf-CAAX or DD-MEK. Because the Ca2+/CAM inhibitor-induced blockade
can be "bypassed" by activated Raf and MEK but not by activated Src
and Ras, these data suggest that Ca2+/CAM is required at a
point in the signaling cascade at the level of, or downstream of Ras
activation, but upstream of Raf and MEK.
Clathrin-mediated Endocytosis Is Required for 5-HT1A
Receptor-mediated Erk Activation--
A requirement for
clathrin-mediated endocytosis has recently been demonstrated for
activation of the Erk cascade via several receptors, including the EGF
(9) and insulin-like growth factor 1 (10) receptor tyrosine kinases,
and the LPA (11) and
To determine whether receptor endocytosis was required for
5-HT1A receptor-mediated Erk activation, we examined the
effects of dominant negative K44A-dynamin1 or
V53D- Ca2+/CAM Antagonists Impair 5-HT1A
Receptor-mediated Erk Activation by Interfering with Receptor
Endocytosis--
Ca2+/CAM regulates multiple enzymes,
including CAM-dependent protein kinases, myosin light chain
kinase, protein phosphatase 2B, and CAM-sensitive phosphodiesterases.
In addition, CAM is known to bind to neuronal clathrin light chains
(29), to interact with the actin microfilament attachment protein,
fodrin, a member of the spectrin family of membrane proteins (30, 31),
and to regulate the activity of the G protein receptor kinases GRK5 and
-6 (32-34). CAM also plays an as yet undefined role in endocytic events (35-37) and is important for endocytosis in adrenal chromaffin cells (38).
To test whether known enzymatic effectors of CAM participate in
5-HT1A receptor-mediated Erk activation, pharmacologic
inhibitors of several CAM effectors were assayed for the ability to
antagonize 5-HT-stimulated Erk2 activation in CHO-1A-27 cells.
Preincubation for 30 min with the myosin light chain kinase inhibitors
ML-7 (20 µM), ML-9 (50 µM), and HA1077 (50 µM); the protein phosphatase PP2B inhibitors cyclosporine
A (1 µM) and FK506 (1 µM); the
CAM-dependent protein kinase II and IV inhibitors KN-62 (10 µM) and KN-92 (10 µM); and the
CAM-sensitive phosphodiesterase type I inhibitors vinpocetine (50 µM) and 8-methoxymethyl isobutylmethylxanthine (20 µM) had no effect upon 5-HT1A
receptor-stimulated Erk activation (data not shown).
Because 5-HT1A receptor endocytosis is an obligate step in
the Erk activation cascade, we tested the hypothesis that
Ca2+/CAM is involved in receptor endocytosis.
Agonist-induced sequestration of HA-tagged 5-HT1A and
FLAG-tagged
Several authors have postulated a role for Ca2+/CAM in the
activation of the Erk cascade. Both
The mechanism whereby endocytosis of G protein-coupled receptors
contributes to activation of the Erk cascade is unclear. The appearance
of both Raf and
The finding that 5-HT1A receptor-mediated activation of the
Erk cascade is dependent upon receptor endocytosis indicates a novel
dimension in 5-HT receptor signaling. Endocytosis of G protein-coupled receptors has previously been viewed as a consequence of receptor desensitization (40), i.e. uncoupling of receptor from G
protein because of G protein-coupled receptor kinase phosphorylation
and arrestin binding. Our data suggest that the processes that
terminate 5-HT1A receptor coupling to G proteins
simultaneously contribute to the transduction of
Ras-dependent signals.
-arrestin1 and dynamin attenuated 5-HT1A
receptor-stimulated Erk1/2 activation. Inhibition of CAM prevented
agonist-dependent endocytosis of epitope-tagged 5-HT1A receptors. We conclude that
CAM-dependent activation of Erk1/2 through the
5-HT1A receptor reflects its role in endocytosis of the
receptor, which is a required step in the activation of MEK and
subsequently Erk1/2.
INTRODUCTION
Top
Abstract
Introduction
References
2-adrenergic receptors (11, 12). Inhibition
of
2-adrenergic receptor sequestration with dominant
inhibitory mutants of
-arrestin1 or dynamin
impairs Erk activation with no effect on receptor-G protein
coupling (12).
subunits, phosphatidylinositol 3'-kinase and src kinase, the Shc and Grb2 adapter proteins, mSos, Ras, and Raf (13, 15). Moreover,
5-HT1A receptors have been colocalized with adenylyl cyclase and G proteins in clathrin-rich brain vesicles (16). In this
report, we show that 5-HT1A receptor-mediated Erk
activation is sensitive to inhibitors of Ca2+/CAM and
clathrin-mediated endocytosis but not to inhibitors of the known CAM
effectors myosin light chain kinase, CAM-dependent protein
kinases II and IV, PP2B, and CAM-sensitive phosphodiesterases. Rather,
the Ca2+/CAM dependence of Erk activation derives from a
requirement for CAM in 5-HT1A receptor endocytosis. These
data suggest a previously unappreciated role for CAM in the
sequestration of G protein-coupled receptors and provide a mechanism
for cooperativity between Ca2+/CAM and tyrosine
phosphorylation in mitogenic signal transduction.
EXPERIMENTAL PROCEDURES
2-adrenergic receptor and HA-tagged 5-HT1A
receptor were cloned in our laboratory (17-19). Constitutively activated mutants V12-Ras and Raf-CAAX were the gifts of C. Der, and DD-MEK was the gift of R. Erikson. Constitutively activated Y530F-Src, in which the regulatory carboxyl-terminal tyrosine residue
has been mutated, was prepared as described (20-22). cDNAs encoding the dominant-interfering mutants
V53D-
-arrestin1 and K44A-dynamin were the kind gift of
M. Caron. The cDNA encoding the CAM-sequestering protein calspermin
was the gift of A. Means. Calspermin cDNA was subcloned into the
pcDNA3 vector via a NcoI-XbaI digest of
pCaMPL-calsp followed by blunt-end treatment with the Klenow fragment
of DNA polymerase I. The insert was placed into EcoRV-digested, calf intestinal alkaline phosphatase-treated
pcDNA3. Correct orientation was verified by diagnostic restriction digests.
2-Adrenergic and 5-HT1A Receptor
Sequestration--
Sequestration of epitope-tagged
2-adrenergic receptor and 5-HT1A receptor
was determined as agonist-induced loss of cell surface
immunofluorescence using fluorescence-assisted cell sorting (FACS)
(17). Cells expressing tagged receptors were exposed to 10 µM isoproterenol (
2-adrenergic receptor)
or 10 µM 5-HT (5-HT1A receptor) for 30 min at
37 °C before staining of cell surface receptors. Sequestration was
defined as the fraction of total cell surface receptors removed from
the plasma membrane following agonist treatment.
RESULTS AND DISCUSSION
subunits derived from pertussis toxin-sensitive
G proteins (13). As shown in Fig.
1A, stimulation of CHO-1A-27
cells with 5-HT resulted in a 7-fold stimulation of Erk2 kinase
activity compared with unstimulated cells. Stimulation of endogenous
fibroblast growth factor receptors and acute protein kinase C
activation with PMA also induced Erk2 activation. Pretreatment with
BAPTA, a cell-permeable Ca2+ sequestrant, in
Ca2+-free medium inhibited 5-HT1A receptor- and
bFGF-stimulated Erk2 activation, with no effect on the protein kinase
C-mediated signal.
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Fig. 1.
Effect of Ca2+ sequestration and
CAM antagonism on 5-HT1A receptor-mediated Erk
activation. A, CHO-1A-27 cells were treated either with
vehicle (control) or with BAPTA (25 µM). Cells were then
stimulated for 5 min with 5-HT (10 µM), bFGF (10 ng/ml),
or PMA (1 µM) prior to determination of Erk activity.
Values shown represent means ± S.E. from four separate
experiments each performed in duplicate. B, CHO-1A-27 cells
were pretreated for 15 min with vehicle (control), W-7 (50 µM), or fluphenazine (Flu, 10 µM). Cells were then stimulated with 5-HT (10 µM), bFGF (10 ng/ml), or PMA (1 µM) for 5 min prior to determination of Erk activity. Values shown represent
means ± S.E. from eight separate experiments, each performed in
duplicate. C, HEK-293 cells were cotransfected with cDNA
encoding HA-tagged 5-HT1A receptor (1 µg/dish) plus
either calspermin cDNA (5 µg/dish) or empty vector
(control, 5 µg/dish). After 24 h of serum starvation,
cells were stimulated with 5-HT (10 µM), EGF (10 ng/ml),
or PMA (1 µM) prior to determination of Erk activity.
Values shown represent means ± S.E. from two separate
experiments, each performed in duplicate. Data for all three
panels are expressed as -fold Erk activity in which the
values in unstimulated cells were defined as 1.0. NS, not
stimulated.
, indicates p < 0.05; *, indicates
p < 0.01 versus agonist alone
(black bars). Reverse Bonferroni correction was used.
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Fig. 2.
Effect of CAM antagonists on stimulation of
Erk1/2 phosphorylation in HEK-293 cells by constitutively activated
mutants of Src, Ras, Raf, and MEK. HEK-293 cells were transiently
transfected with cDNA encoding Y530F-Src (5 µg/dish), V12-Ras (5 µg/dish), Raf-CAAX (5 µg/dish), MEK1-DD (5 µg/dish),
or empty vector (5 µg/dish). Erk1 activity in these cells was
determined after 24 h of serum starvation. Data are expressed as
-fold Erk activity in which the values in unstimulated cells were
defined as 1.0. Values shown represent means ± S.E. from five
separate experiments, each performed in duplicate. *, indicates
p < 0.01 versus agonist alone
(black bars). Reverse Bonferroni correction was
used.
2-adrenergic (12) G protein-coupled
receptors. Specific inhibitors of G protein-coupled receptor
endocytosis, including dominant negative mutants of
-arrestin1 and dynamin, inhibit
2-adrenergic receptor-mediated Erk activation.
2-Adrenergic receptor stimulation results in the
appearance of both receptor and Raf kinase in a clathrin-rich endocytic
vesicle compartment (12). These data suggest that an endocytic process may be required to achieve activation of MEK and Erk kinases following the formation of an activated Ras-Raf complex on the plasma membrane.
-arrestin1, a
-arrestin1 mutant that
disrupts homologous desensitization and internalization of adrenergic
receptors (28), on 5-HT-stimulated Erk1/2 phosphorylation in
transiently transfected HEK-293 cells. Fig.
3 shows that both V53D-
-arrestin1 and K44A-dynamin1 attenuated
5-HT-stimulated Erk1/2 activation by the 5-HT1A receptor.
EGF receptor-induced Erk1/2 activation was sensitive to expression of
K44A-dynamin1, in accordance with previously published
findings (9), but not to expression of
V53D-
-arrestin1.
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Fig. 3.
Effect of endocytosis inhibitors on 5-HT- and
EGF-stimulated Erk1/2 activation in HEK-293 cells. Cells were
transiently transfected with cDNA encoding the HA-tagged
5-HT1A receptor (1 µg/dish) and cDNA encoding
V53D- -arrestin1 (5 µg/dish), K44A-dynamin1
(5 µg/dish), or empty vector (control, 5 µg/dish).
Serum-starved cells were stimulated with 5-HT (10 µM) or
EGF (10 ng/ml) for 5 min before determination of Erk activity. Data are
expressed as -fold Erk activity in which the values in unstimulated
cells were defined as 1.0. Values shown represent means ± S.E.
from three separate experiments each performed in duplicate.
NS, not stimulated.
, indicates p < 0.05;
*, indicates p < 0.01 versus agonist alone
(black bars). Reverse Bonferroni correction was
used.
2-adrenergic receptors was determined in the
presence of fluphenazine, W-7, or hypertonic sucrose. As shown in Fig.
4, both antagonists of
Ca2+/CAM impaired the sequestration of 5-HT1A
receptors in transiently transfected HEK-293 cells. These results were
duplicated for the
2-adrenergic receptor (data not
shown). The effect of the CAM inhibitors was comparable with that of
preincubation in hypertonic medium and expression of
K44A-dynamin1 (data not shown), both known inhibitors of
clathrin-mediated endocytosis. Coexpression of calspermin with
epitope-tagged 5-HT1A receptors also resulted in similar
abrogation of agonist-dependent receptor internalization (data not shown).
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Fig. 4.
Effect of CAM antagonists and hypertonic
sucrose on agonist-dependent sequestration of the
5-HT1A receptor in HEK-293 cells. Cells were
transiently transfected with cDNA encoding the HA-tagged
5-HT1A receptor (1 µg/dish). After 3 h of serum
starvation, cells were pretreated with vehicle, hypertonic sucrose (450 mM), fluphenazine (Flu, 10 µM), or
W-7 (50 µM) for 15 min. Cells were then stimulated with
5-HT (10 µM) for 35 min prior to determination of
HA-5-HT1A receptor cell surface expression by
fluorescence-assisted cell sorting. Data are expressed as percent of
cell surface receptors expressed in comparison with unstimulated cells.
Sequestration was calculated as the percent difference in cell surface
fluorescence when compared with unstimulated cells. Values shown
represent means ± S.E. from three separate experiments each
performed in triplicate. , indicates p < 0.05; *,
indicates p < 0.01 versus 5-HT alone
(black bar). Reverse Bonferroni correction was
used.
-adrenergic receptor-mediated Erk activation in HEK-293 cells (6) and angiotensin receptor-mediated Erk activation in vascular smooth muscle cells (8) are sensitive to CAM
inhibitors. Simultaneous expression of activated mutants of CaM kinase
IV and CaM kinase kinase is sufficient to induce weak activation of Erk
in NG-108 cells (39). We find that antagonism of Ca2+/CAM
using either pharmacologic inhibitors or the calcium sequestrant protein calspermin disrupts both Erk1/2 activation and agonist-induced endocytosis of the 5-HT1A receptor. The effects of CAM
inhibition are not mimicked by pharmacologic inhibition of CAM
effectors, including CaM kinase IV. Rather, our data indicate that the
effects of CAM antagonists on receptor endocytosis are sufficient to
account for the observed inhibition of Erk activation. However, because CAM inhibitors were somewhat more effective in preventing endocytosis than in blocking Erk activation, it is possible that there is an
additional endocytosis-independent Erk activation pathway.
2-adrenergic receptors in a
clathrin-rich light vesicle fraction following agonist exposure
suggests that the endocytic process may be required to transduce
signals between an activated Ras-Raf complex on the plasma membrane and
the MEK and Erk kinases in the cytosol (12). Our finding that CAM
antagonists inhibit Erk activation mediated by constitutively active
mutants of Src and Ras, but not Raf and MEK, supports such a model by localizing the putative endocytosis-dependent step in the
signaling cascade downstream of Ras activation. CAM has been shown to
bind to clathrin light (29, 41) and heavy chains (42) and to regulate
vesicle recycling by influencing vesicle tubulation (43) and
recruitment of clathrin to ligand-receptor complexes (44). Thus, CAM
inhibitors could prevent Erk activation by 1) preventing recruitment of
receptor-ligand complexes to clathrin, and/or 2) preventing successful
activation of Raf by Ras, and/or 3) preventing tubulation and vesicle
recycling. Certainly, other mechanisms might also be possible.
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ACKNOWLEDGEMENTS |
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We thank D. Addison and M. Holben for excellent secretarial assistance. We thank A. R. Means for the gifts of calspermin cDNA and anti-calspermin antibody, and insightful discussion.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grants DK52448 (to J. R. R.), HL16037 (to R. J. L.), and DK02352 and DK55524 (to L. M. L.) and by the Medical Research Service of the Department of Veterans Affairs (to J. R. R. and M. N. G.).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.
Supported by National Institutes of Health Medical Scientist
Training Program Grant T32GM-07171.
¶ Recipient of a fellowship from the American Heart Association.
Recipient of a type II merit award from the Department of
Veterans Affairs.
To whom correspondence should be addressed: 829 Clinical
Sciences Bldg., Medical University of South Carolina, 171 Ashley Ave.,
Charleston, SC 29425. Fax: 843-792-8399; E-mail:
raymondj{at}musc.edu.
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ABBREVIATIONS |
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The abbreviations used are: MAP, mitogen-activated protein; MEK, mitogen and extracellular signal-regulated kinase; EGF, epidermal growth factor; CAM, calmodulin; LPA, lysophosphatidic acid; CHO, Chinese hamster ovary; BAPTA, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; PMA, phorbol 12-myristate 13-acetate; bFGF, basic fibroblast growth factor; HA, hemagglutinin; FBS, fetal bovine serum.
2 A. R. Means, personal communication.
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REFERENCES |
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