From the Howard Hughes Medical Institute and the Departments of Medicine and Biochemistry, Box 3821, Duke University Medical Center, Durham, North Carolina 27710 and the § Geriatrics Research, Education and Clinical Center, Durham Veterans Affairs Medical Center, Durham, North Carolina 27705
Received for publication, February 9, 2001, and in revised form, April 3, 2001
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
"Transactivation" of epidermal growth factor
receptors (EGFRs) in response to activation of many G protein-coupled
receptors (GPCRs) involves autocrine/paracrine shedding of
heparin-binding EGF (HB-EGF). HB-EGF shedding involves
proteolytic cleavage of a membrane-anchored precursor by incompletely
characterized matrix metalloproteases. In COS-7 cells,
G protein-coupled receptors
(GPCRs)1 can employ multiple
distinct pathways to activate the ERK/MAPK cascade. At
least one of these pathways, demonstrated for both Gi- and
Gq-coupled receptors, involves transactivation of
classical receptor tyrosine kinases (RTKs) such as the epidermal growth
factor receptor (EGFR) (1-3). A characteristic feature of
transactivation-dependent ERK activation is that stimulation of
the GPCR leads to activation of the intrinsic tyrosine kinase activity
of the RTK. Whereas the early steps leading from GPCR activation to
tyrosine phosphorylation of the RTK have remained largely unknown, once
transactivation of the RTK has occurred, RTK- and GPCR-mediated ERK
activation are indistinguishable. Thus, tyrosine phosphorylation of the
EGFR leads to the rapid tyrosine phosphorylation of adapter proteins
such as SHC and Gab1 and the recruitment Grb2-Sos1 complexes to the
activated RTK. The Ras guanine nucleotide exchange factor, Sos1,
catalyzes the exchange of GDP for GTP on the low molecular weight G
protein, Ras. Ras activation, in turn, initiates the phosphorylation
cascade consisting of Raf, MEK, and ERK.
Recently, Prenzel et al.(4) have demonstrated that
GPCR-stimulated tyrosine phosphorylation of the EGFR involves release
of a soluble EGFR ligand, heparin-binding EGF (HB-EGF). HB-EGF is a
single transmembrane-spanning protein that is proteolytically cleaved
at a juxtamembrane site leading to the "shedding" of a soluble EGFR
ligand that activates the EGFR in an autocrine/paracrine manner (5).
GPCR-mediated HB-EGF release and EGFR transactivation are inhibited by
CRM197 (4), a non-toxic diphtheria toxin mutant, which
selectively binds and inactivates HB-EGF (6). HB-EGF shedding is also
sensitive to the non-selective metalloprotease inhibitor, batimistat,
indicating that one or more, as yet unknown, matrix metalloproteases
function as novel effectors of GPCR signaling (4).
Little is presently known about how GPCRs control metalloprotease
activity to induce regulated HB-EGF shedding. In this study, we have
developed a mixed culture system to study the mechanism by which
Materials--
LipofectAMINE and all tissue culture reagents
were from Life Technologies. CRM197 was purchased from Sigma and
pertussis toxin from List Biologicals. PP2 and tyrphostin AG1478 were
from Calbiochem. Monoclonal HA.11 anti-hemagglutinin (HA) affinity
beads were from Covance. The anti-phospho-ERK1/2 antibody was from Cell
Signaling, and the anti-ERK1/2 antibody was from Upstate Biotechnology.
Peroxidase-conjugated donkey anti-rabbit secondary antibody was from
Amersham Pharmacia Biotech, and SuperSignal chemiluminescence reagent
(Pierce) was used to develop blots.
Plasmids--
The plasmid encoding GFP-ERK2 was the gift
of K. A. DeFea and N. W. Bunnett (University of California at San
Francisco) and that encoding HA-ERK2 was the gift of J. Pouyssegur
(University of Nice). The plasmid encoding K97A MEK1 came from E. G.
Krebs (University of Washington) and the plasmid encoding c-Src kinase (CSK) came from H. Hanafusa (Rockefeller University). The
plasmid encoding GST- Cell Culture and Transfections--
COS-7 cells were grown
in Dulbecco's modified Eagle's medium supplemented with 10% fetal
bovine serum and 100 µg/ml gentamicin in a humified 95% air, 5%
CO2 incubator at 37 °C. Transient transfection of COS-7
cells was carried out at 70-80% confluency on the day of
transfection. Cells in 10-cm plates were transfected with a total of
5-10 µg of DNA using a 1:6 ratio (w/v) of LipofectAMINE per dish
following the manufacturer's instructions. One day following transfections, the cells were trypsinized and transferred to six-well dishes as described. Transfected cells were serum-starved overnight in
Dulbecco's modified Eagle's medium supplemented with 10 mM HEPES and 0.1% bovine serum albumin prior to stimulation.
Immunoprecipitation and Immunoblotting--
Serum-starved
transfected cells in six-well dishes were pretreated with the
appropriate concentrations of inhibitors as indicated in the figure
legends. Additionally, all cells were pretreated with the
As shown in Fig. 1B, stimulation of co-cultures with the
selective
To determine the role of endogenous EGFRs in
In HB-EGF expressing CHO-K1 cells, shedding of HB-EGF in response to
phorbol esters is sensitive to the MEK inhibitor, PD98059 (8).
Similarly, in neutrophils,
N-formylmethionyl-leucyl-phenylalanine (fMLP)-stimulated
L-selectin shedding is blocked by inhibitors of ERK or p38
MAP kinase signaling (9). These data suggest that MAP kinase activation
may be required to initiate metalloprotease-dependent shedding. In contrast, EGFR activation in response to Gi-
and Gq/11-coupled receptor activation in fibroblasts is
insensitive to PKC inhibitors (4). To test whether in our system, ERK
activity is required for G
To determine the role of heterotrimeric G protein subunits in
Src family nonreceptor tyrosine kinases are also known to play a role
in GPCR-mediated ERK activation via transactivated EGFR (2, 3, 10).
Uncertainty remains, however, as to whether these kinases are involved
in regulating the release of HB-EGF or in the downstream response of
the transactivated EGFR. As shown in Fig.
5A, treatment of co-cultures
with the Src kinase inhibitor PP2 inhibits
In HeLa cells, the ability of EGFR to mediate activation of the ERK
cascade has previously been shown to involve clathrin-mediated endocytosis (12). Unlike many GPCRs, the With the system developed in these experiments, the
transactivation pathway to ERK activation can be isolated from other
pathways of ERK activation and intermediate steps localized as either
upstream or downstream of HB-EGF shedding and the EGFR. Fig.
7 depicts a model of
2A-adrenergic receptors (ARs) stimulate ERK
phosphorylation via two distinct pathways, a transactivation pathway
that involves the release of HB-EGF and the EGFR and an alternate
pathway that is independent of both HB-EGF and the EGFR. We have
developed a mixed culture system to study the mechanism of
GPCR-mediated HB-EGF shedding in COS-7 cells. In this system,
2AAR expressing "donor" cells are co-cultured with
"acceptor" cells lacking the
2AAR. Each population
expresses a uniquely epitope-tagged ERK2 protein, allowing the
selective measurement of ERK activation in the donor and acceptor
cells. Stimulation with the
2AR selective agonist
UK14304 rapidly increases ERK2 phosphorylation in both the donor and
the acceptor cells. The acceptor cell response is sensitive to
inhibitors of both the EGFR and HB-EGF, indicating that it results from
the release of HB-EGF from the
2AAR-expressing donor
cells. Experiments with various chemical inhibitors and dominant
inhibitory mutants demonstrate that EGFR-dependent
activation of the ERK cascade after
2AAR stimulation requires G
subunits upstream and
dynamin-dependent endocytosis downstream of HB-EGF shedding
and EGFR activation, whereas Src kinase activity is required both for
the release of HB-EGF and for HB-EGF-mediated ERK2 phosphorylation.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2A-adrenergic receptors (ARs) control HB-EGF shedding in
COS-7 cells. In our model system, the release of HB-EGF from donor
cells expressing
2AARs is detected by measuring the
response in acceptor cells lacking the GPCR. This permits signaling
events upstream of HB-EGF shedding to be physically dissociated from those involved in the downstream response to endogenously generated paracrine signals. By selectively introducing dominant negative mutants
of putative intermediate proteins into either the donor or acceptor
cell pool, we have been able to determine their involvement in either
the release of, or response to, HB-EGF. We find that
2AAR-mediated shedding of HB-EGF requires both the
release of G
subunits and Src kinase activity, while the response
to HB-EGF is dependent upon the EGFR, Src kinases, and
clathrin-mediated endocytosis.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
ARKCT was constructed in our laboratory by
excising a BamHI/NotI fragment from pRK5/
ARKCT
and ligating it into pEBG. The effects pEBG/
ARKCT and pRK5/
ARKCT
were indistinguishable, and these constructs were used interchangeably.
All other constructs were prepared in our laboratory.
-adrenergic receptor antagonist, propranolol (1 µM), to block the activation of endogenous
receptors in COS-7 cells. Cells were exposed to agonist at 37 °C for the times indicated in
the figure legends, washed once with ice-cold phosphate-buffered saline, lysed in 300 µl of glycerol lysis buffer (5 mM
HEPES, 250 mM NaCl, 10% (v/v) glycerol, 0.5% Nonidet
P-40, 2 mM EDTA, 100 µM NaV04, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, 10 µg/ml aprotinin), and clarified by centrifugation. Before
immunoprecipitation, 50 µl of the cell lysate was transferred to a
separate tube and an equal volume of 2× Laemmli sample buffer was
added to provide samples for the determination of GFP-ERK2
phosphorylation and plasmid expression. HA-ERK2 was immunoprecipitated
using 20 µl of a 50% slurry of anti-HA affinity beads that had been
mixed with an equal volume of CL4B-Sepharose and rotated overnight at 4 °C. Immune complexes were washed twice with cold glycerol lysis buffer and denatured in 2× Laemmli sample buffer. Immunoprecipitated proteins were resolved by protein electrophoresis on 4-20%
SDS-polyacrylamide gels (Invitrogen) and transferred to polyvinylidene
difluoride membranes (PerkinElmer Biosystems). Phospho-ERK1/2 was
detected using a 1:3000 dilution of a rabbit polyclonal phospho-ERK1/2 specific antibody (New England Biolabs), and total ERK1/2 was detected
using a 1:2000 dilution of an ERK1/2 antibody (Upstate Biotechnology).
Blots were probed with a 1:5000 dilution of a donkey anti-rabbit
horseradish peroxidase-conjugated secondary antibody (Amersham
Pharmacia Biotech). Proteins were visualized using Supersignal
chemiluminescence reagent (Pierce), and the autoradiographs were
quantitated using a Fluor-S MultiImager (Bio-Rad).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2AARs Employ At Least Two Distinct Mechanisms to
Activate the ERK Cascade--
Fig.
1A schematically depicts the
mixed culture system employed to dissociate
transactivation-dependent ERK activation from EGFR-independent ERK activation after
2AAR stimulation.
Two populations of COS-7 cells were prepared by transient transfection.
Donor cells expressed the
2AAR, a receptor that is not
endogenously expressed in COS-7 cells, along with HA epitope-tagged
ERK2 (HA-ERK2), to allow ERK phosphorylation in the receptor-expressing
population to be independently assayed. Acceptor cells in which the
2AAR was not introduced were transfected with green
fluorescent protein-tagged ERK2 (GFP-ERK2) to uniquely identify the ERK
pool in those cells. Following transfection, the transfected cell
populations were mixed and plated at high density using a ratio of
donor/acceptor cells of 1:1.5. Following agonist stimulation, cell
lysates were prepared, and anti-HA immunoprecipitation and subsequent
immunoblotting were used to isolate the HA-ERK2 expressed in the donor
cells. GFP-ERK2 phosphorylation in the acceptor cell pool was
determined by immunoblotting of the whole cell lysates, since GFP-ERK2
could be resolved from the endogenous ERK pool based upon its slower electrophoretic mobility. This system permitted the simultaneous monitoring of ERK activation in the two separate populations of cells
from the same dish.
View larger version (24K):
[in a new window]
Fig. 1.
Co-culture system for measuring ERK
phosphorylation simultaneously in two populations of cells, donor cells
that express the 2AAR and
co-cultured acceptor cells that do not. A, schematic
representation of the co-culture system. Donor COS-7 cells in 10-cm
dishes were transfected with 50 ng of
2AAR/pRK5 along
with 1 µg of HA-ERK2/pCDNA3, whereas acceptor COS-7 cells were
transfected with 1 µg of ERK2/pEGFP, which encodes a GFP-tagged form
of ERK2 as described under "Experimental Procedures." One day after
transfection, cells were trypsinized, and the donor and acceptor cells
were cultured together in 6-well dishes at high density. Cells were
serum-starved overnight before stimulation with 1 µM
UK14304. Cells were then harvested and analyzed as described under
"Experimental Procedures." Lysates and immunoprecipitations were
analyzed with both phospho-ERK1/2 and total ERK1/2 antibodies to ensure
that ERK expression was not affected by the presence of coexpressed
plasmids. B, time course of
2AAR-stimulated
ERK phosphorylation in donor and acceptor cells. Co-cultured donor and
acceptor cells were stimulated for the indicated times with 1 µM UK14304 at 37 °C
and then harvested as indicated under "Experimental Procedures."
The experiment shown is representative of four independent
experiments.
2AR agonist UK14304 leads to a
time-dependent increase in phospho-ERK2 immunoreactivity
both in donor cells and in acceptor cells. The time course of ERK2
phosphorylation was similar in both populations, with maximal
activation occurring within 5 min of agonist application. The acceptor
cell response was more transient, however, with ERK2 phosphorylation
returning to near basal levels within 30 min, whereas ERK2
phosphorylation was maintained in the donor cells for at least 2 h. Control experiments (not shown) demonstrated that UK14304 failed to
induce ERK2 phosphorylation in COS-7 cells in the absence of
transfected
2AAR and that responses in the acceptor
cells were only seen when the cells were cultured at high density.
These data are consistent with a paracrine response in the acceptor
cells that is mediated in response to the release of a soluble factor
from the donor cells.
2AAR-mediated ERK2 activation, we examined the extent to
which ERK activation in both donor and acceptor cells was sensitive to
inhibitors of HB-EGF shedding and of EGFR signaling. Fig.
2 shows the results of experiments in
which co-cultures were pretreated with either tyrphostin AG1478, a
specific inhibitor of the EGFR tyrosine kinase (7), or with CRM197, a
non-toxic diphtheria toxin analog that selectively binds to the
EGF-like domain of HB-EGF (6). In the
2AAR-expressing
donor cells, maximally efficacious concentrations of CRM197 and
tyrphostin AG1478 each inhibited only about 30% of UK14304-stimulated
ERK activation (black bars). In contrast, the acceptor cell
response in the same co-culture was almost completely blocked by either
inhibitor (white bars). Whereas the
2AAR-stimulated ERK response in the donor cells was only
partially sensitive to tyrphostin AG1478, comparable EGF-stimulated ERK
activated was essentially completely inhibited by tyrphostin
AG1478 (data not shown). These data suggest that
2AARs
can employ at least two mechanisms to stimulate ERK2 phosphorylation in
COS-7 cells, a direct pathway that is independent of HB-EGF shedding
and the EGFR, and a transactivation-dependent pathway that
requires both HB-EGF shedding and EGFR activation. In cells expressing
the
2AAR, the EGFR-independent pathway predominates.
Pretreating cells with CRM197 and tyrphostin AG1478 in combination did
not result in any further reduction in the donor cell response,
indicating that HB-EGF shedding can account for all of the
transactivation-dependent ERK activation in both donor and
acceptor cells (data not shown).
View larger version (33K):
[in a new window]
Fig. 2.
2AAR-stimulated ERK
activation in donor and acceptor cells is differentially sensitive to
inhibitors of HB-EGF shedding and EGFR tyrosine kinase activity.
A, cells that were transiently transfected as
described were pretreated with either 250 nM tyrphostin
AG1478 (AG) or 10 µg/ml CRM197 for 20 min prior to a 5 min
stimulation with 1 µM UK14304 (UK) at
37 °C. A representative experiment demonstrating the effects of
AG1478 and CRM197 on ERK2 activation in cells that express
2AAR (HA IP (Donor Cells)) or in
co-cultured cells that do not express the
2AAR
(LYSATE (Acceptor Cells)) (A).
B, results shown are the mean ± S.E. of seven
independent experiments that were normalized to the percent maximal
response found in cells that were not pretreated with any
inhibitor.
2AAR-stimulated HB-EGF
shedding, we transiently transfected the dominant inhibitory MEK1
inhibitor, K97A MEK1, into the donor cells and measured ERK activation
in both the donor and the acceptor cells. As shown in Fig.
3, expression of K97A MEK1 in donor cells
markedly attenuated ERK2 activation in the donor cell pool but had no
effect on ERK2 activation in the acceptor cells. These data support the
hypothesis that the EGFR-independent and HB-EGF-dependent
pathways of ERK2 activation employed by
2AAR are
mechanistically independent. Tyrphostin AG1478 and CRM197 treatment
blocks HB-EGF-mediated ERK activation in acceptor cells while not
affecting the transactivation-independent component of the donor cell
response (Fig. 2). Conversely, expression of K97A MEK1 in donor cells
blocks the donor, but not the acceptor, cell response, indicating that
ERK2 activation in the donor cells is not required for HB-EGF shedding
(Fig. 3).
View larger version (11K):
[in a new window]
Fig. 3.
Effect of dominant negative K97AMEK1 on
2AAR-stimulated ERK activation in donor
and acceptor cells. Cells were transfected as described in the
legend to Fig. 1 with the addition of 5 µg of the MEK1 dominant
inhibitory construct K97A MEK1 transfected into the donor cells.
Following co-culture, donor and acceptor cells were stimulated with 1 µM UK14304 (UK) for 5 min at 37 °C prior to
harvesting. Results shown are the mean ± S.E. of five independent
experiments and were normalized to the percent maximal response of
cells that were not transfected with K97A MEK1.
Subunits, Src Kinases, and Clathrin-mediated Endocytosis
Are Involved in
2AAR-mediated EGFR
Transactivation--
In the co-culture system,
2AAR-mediated ERK activation in acceptor cells reflects
the release of HB-EGF from the donor cell pool. For ERK activation to
occur in acceptor cells, signaling events leading to the release of
HB-EGF must occur in donor cells, whereas events involved in the
response to HB-EGF must occur in the acceptor cells. Thus, by
introducing dominant inhibitory mutants of putative intermediate
proteins selectively into either donor or acceptor cells, intermediates
upstream and/or downstream of HB-EGF shedding and EGFR activation can
be unambiguously identified.
2AAR-mediated EGFR transactivation, we assayed the
effects of pertussis toxin and the G
subunit sequestrant
polypeptide
ARKCT on the ability of acceptor cells to respond
to
2AAR stimulation. As shown in Fig.
4, both pertussis toxin pretreatment and
expression of the
ARKCT peptide in donor cells inhibited ERK
activation in both the donor cell and acceptor cell populations.
Expression of
ARKCT in acceptor cells had no effect upon
2AAR-mediated ERK2 phosphorylation in either the donor
or acceptor cells (not shown). These data indicate that consistent with
prior reports (3), G
subunits derived from pertussis
toxin-sensitive Gi/o proteins are necessary for
2AAR-mediated transactivation of the EGFR.
View larger version (10K):
[in a new window]
Fig. 4.
Effect of pertussis toxin and
ARKCT expression on
2AAR-stimulated ERK activation in donor
and acceptor cells. For the pertussis toxin experiments, donor
cells were transfected with plasmids encoding the
2AAR
and HA-ERK2 as described. The co-cultured donor and acceptor cells were
treated for 16 h with 100 ng/ml pertussis toxin prior to agonist
stimulation. To test the inhibitory effects of
ARKCT on
2AAR-stimulated ERK activation in the donor and the
acceptor cells, donor cells were transfected with plasmids encoding the
2AAR, HA-ERK2, and 5-10 µg of a plasmid encoding
ARKCT. For both the pertussis toxin and
ARKCT experiments, cells
were exposed to 1 µM UK14304 (UK) for 5 min at
37 °C prior to harvesting the cells. Results shown are the mean ± S.E. of seven experiments with pertussis toxin and nine experiments
with
ARKCT. The result in each experiment was normalized to the
response in cells that were not exposed to any inhibitor.
2AAR-mediated
ERK2 phosphorylation in both donor and acceptor cells. To distinguish
between the potential roles of Src kinases upstream or downstream of
HB-EGF shedding, two different expressible Src inhibitors were
selectively transfected into either the donor or the acceptor cells. As
shown in Fig. 5B, the dominant inhibitory mutant K298M
c-Src, when expressed in donor cells, significantly inhibited
2AAR-stimulated ERK activation in both the donor and in
the acceptor cells. When K298M c-Src is expressed in acceptor cells,
2AAR-stimulated ERK activation in the acceptor cells is
attenuated, whereas ERK activation in the donor cells is unaffected. As
shown in Fig. 5C, comparable results were obtained when
donor and acceptor cells were transfected with the physiological c-Src
inhibitor, CSK, which inhibits c-Src activity by phosphorylating the
regulatory carboxyl-terminal tyrosine of Src family kinases (11). These
data are consistent with a role for Src kinase activity in both the
direct and EGFR transactivation-dependent pathways.
Moreover, in the transactivation pathway, c-Src is required both for
2AAR-mediated HB-EGF shedding and for
HB-EGF-dependent ERK activation via the transactivated
EGFR, because inhibiting Src kinase activity in either the donor cell
or the acceptor cell blocks the acceptor cell response.
View larger version (17K):
[in a new window]
Fig. 5.
Effect of c-Src inhibitors on
2AAR-stimulated ERK activation in donor
and acceptor cells. A, cells transfected as described
were pretreated with 5 µM PP2 for 20 min prior to
stimulation with 1 µM UK14304 (UK) for 5 min
at 37 °C. Results shown are the mean ± S.E. of five
independent experiments that were normalized to the percent maximal
response found in cells that were not pretreated with PP2.
B, cells were transfected with 3-5 µg of the dominant
inhibitory mutant K298M c-Src transfected in either donor cells or in
acceptor cells. Cells were stimulated with 1 µM UK14304
for 5 min at 37 °C prior to harvesting. Results shown are the
mean ± S.E. of four independent experiments and were normalized
to the percent maximal response of cells that were not transfected with
K298M c-Src. C, cells were transfected with 5 µg of
plasmid DNA encoding the c-Src inhibitory kinase (CSK) in
either the donor cells or in the acceptor cells. Cells were stimulated
with 1 µM UK14304 for 5 min at 37 °C prior to
harvesting. Results shown are the mean ± S.E. of seven
independent experiments and were normalized to the percent maximal
response of cells that were not transfected with CSK.
2AAR does not
undergo agonist-induced internalization (13). However, in
2A-expressing COS-7 cells, UK14304 stimulation causes
both EGFR internalization and ERK2 activation that is sensitive to
inhibitors of clathrin-mediated endocytosis (14). Thus, we have
previously proposed that inhibitors of clathrin-mediated endocytosis
inhibit ERK activation not at the level of the GPCR itself but rather
downstream of the EGFR (14). To test this hypothesis definitively, a
dominant inhibitory form of dynamin I (K44A dynamin I) was expressed in
either donor or acceptor cells, and the effect on ERK2 phosphorylation
in the acceptor cells was measured. As shown in Fig.
6, K44A dynamin I expression in donor
cells did not affect ERK activation in acceptor cells, whereas
transfecting K44A dynamin I into the acceptor cells attenuated
the
2AAR-stimulated ERK activation. These data not only
dissociate the early steps of
2AAR activation from the
requirement for clathrin-mediated endocytosis but also demonstrate that
in the transactivation pathway dynamin-dependent endocytosis is
required downstream of the EGFR.
View larger version (15K):
[in a new window]
Fig. 6.
Effect of K44A dynamin I expression in donor
or acceptor cells on
2AAR-stimulated ERK activation in
acceptor cells. Cells were transfected with 5 µg of plasmid DNA
encoding either wild-type or K44A dynamin I (K44A DynI), an
inhibitor of clathrin-mediated endocytosis, in donor or in acceptor
cells. Cells were stimulated with 1 µM UK14304 for 5 min
at 37 °C prior to harvesting. Results shown are the mean ± S.E. of seven independent experiments and were normalized to the
percent maximal response in the acceptor cells when either the donor or
the acceptor cells were transfected with wild-type dynamin I.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2AAR-stimulated ERK activation that is consistent with
our data. In response to
2AAR stimulation, ERK
activation proceeds simultaneously via both a direct pathway and a
transactivation-dependent pathway. The initial steps of
both pathways are indistinguishable and involve both G
subunits
released from pertussis toxin-sensitive G proteins and Src kinase
activity. Following c-Src activation, the direct and the
transactivation pathways diverge. The direct pathway leads to MEK
activation and ERK phosphorylation that is independent of EGFR kinase
activity. In the transactivation pathway, Src kinase activity is
required for the induction of HB-EGF shedding, which is mediated
by one or more as yet unidentified matrix metalloproteases. Consistent
with previous reports (2, 3, 14), both c-Src activity and
dynamin-dependent endocytosis are required downstream of
the transactivated EGFR. Thus, Src kinases are apparently involved both
upstream of HB-EGF shedding and downstream of the EGFR in the pathway
of transactivation-dependent ERK activation.
View larger version (19K):
[in a new window]
Fig. 7.
Model of
2AAR-mediated ERK activation that
proceeds via both a direct pathway and via a
transactivation-dependent pathway. In the donor cells,
both the direct pathway and the transactivation-dependent
pathway contribute to
2AAR-mediated ERK activation,
whereas in the acceptor cells,
2AAR-stimulated ERK
activation proceeds only via HB-EGF-dependent EGFR
transactivation. Both pathways require G
subunits released from
pertussis toxin-sensitive G proteins and Src kinase activity. The
pathways, however, diverge downstream of c-Src. The response of
acceptor cells to HB-EGF is dependent upon both Src kinase activity and
dynamin function.
To date, the mechanisms underlying G activation of Src kinases in
response to
2AAR stimulation remain unknown. In the case of the
2-adrenergic receptor (15), the CXCR1 receptor
(16), and the NK-1 receptor (17), the binding of Src family
kinases to
-arrestin results in recruitment of an activated
tyrosine kinase to the GPCR following agonist stimulation. Other
mechanisms of GPCR-mediated Src activation must exist, however. For
example the
3-adrenergic receptor, which does not
interact with
-arrestins, recruits Src kinase via a direct
interaction between the kinase and intracellular domains of the
receptor (18). Like the
3-adrenergic receptor,
2AARs do not undergo clathrin-mediated endocytosis (14,
18), and the mechanism for
2AAR-stimulated c-Src
activation remains enigmatic.
Previously, HB-EGF shedding has been shown to be downstream of phorbol
12-myristate 13-acetate (PMA)-stimulated ERK activation (8, 9).
However, in our system, expression of K97A MEK1 in donor cells almost
completely abolished ERK activation in the donor cells without
affecting ERK activation in the acceptor cells. Although a role for ERK
activation in 2AAR-stimulated HB-EGF shedding cannot be
entirely ruled out, our data strongly suggest that the direct and
transactivation pathways diverge downstream of c-Src but upstream of
MEK activation and that ERK activation probably does not contribute to
2AAR-stimulated HB-EGF shedding in COS-7 cells.
An attractive hypothesis is that c-Src in the donor cells is a proximal
regulator of the unknown metalloprotease responsible for HB-EGF
shedding. Several metalloproteases of the ADAM family, which
have been shown to regulate shedding of other EGFR ligands, including
transforming growth factor , have proline-rich Src-homology (SH3)
binding domains in their cytoplasmic tails. Src family members are
known to bind many of their substrates via SH3 domain interactions. Recently, activation of a c-Src family kinase was shown to regulate the
ADAM-dependent shedding of the L1 adhesion molecule (19). In this case, L1 adhesion molecule shedding is regulated by two distinct pathways, a protein kinase C-dependent
pathway that is ERK-dependent and a pervanadate pathway
that is dependent on c-Src activation but independent of ERK
activation. L1 shedding, then, may be analogous to HB-EGF shedding in
which there are at least two pathways, an ERK-dependent
pathway downstream of PKC activation and an ERK-independent pathway
that involves c-Src activation. In the case of the
2AAR,
it is not ERK activation but rather activation of c-Src that is the
major pathway contributing to HB-EGF shedding. Potentially, then, the
metalloprotease responsible for GPCR-stimulated HB-EGF shedding may
contain proline-rich SH3 binding domains.
Previous reports have implicated Src kinase activity in the cellular response to transactivated EGFRs. Activation of c-Src downstream of the EGFR may be involved in regulating catalytic activity of the EGFR and/or regulating dynamin-dependent endocytosis of the EGFR or a downstream intermediate. c-Src has been shown to phosphorylate two residues on the EGFR that increase the catalytic activity of the EGFR and regulate EGF-stimulated mitogenesis (20). GPCR-induced tyrosine phosphorylation of the adapter proteins Shc and Gab1 (2, 3) requires the activity of both the EGFR and Src kinases. c-Src may also regulate internalization of the EGFR by phosphorylating two components of the clathrin-dependent endocytosis system, clathrin and dynamin. Mutations of two tyrosines in dynamin that are phosphorylated by c-Src diminish EGFR internalization and ERK activation (21). Similarly, blocking EGF-stimulated c-Src-dependent phosphorylation of clathrin also inhibits EGFR internalization (22).
This model of transactivation-dependent ERK activation
clearly dissociates endocytosis of the GPCR from the requirement for dynamin-dependent endocytosis in
transactivation-dependent ERK activation. Our data
demonstrate that all of the early steps in transactivation-mediated ERK
activation up to and including activation of the metalloprotease are
independent of clathrin-mediated endocytosis. In the acceptor cells,
dynamin-dependent endocytosis is required either at or
downstream of EGFR phosphorylation. Previous studies have demonstrated
that, in the case of the 2-adrenergic receptor, all of
the steps leading up to and including Raf activation were intact in
cells expressing a dominant inhibitory form of dynamin (23, 24),
suggesting that the Raf-MEK interface is the step regulated by
dynamin-dependent endocytosis.
While helping to clarify the mechanisms whereby GPCRs mediate
activation of the ERK cascade, our data do not provide insight into the
functional significance of the direct and
transactivation-dependent pathways. Why should such seeming
redundancy exist? One possibility is that the consequences of ERK
activation are determined to a significant degree by the mechanism by
which they are activated. Activation of the ERK cascade via different
pathways may provide a mechanism for regulating either the time course
or spatial distribution of ERK activity, resulting in distinctly
different consequences for the cell. Some evidence for this is provided
by the finding that wild-type PAR-2 receptors, which cause
-arrestin-dependent activation of a cytosolic pool of
ERK1/2, do not mediate mitogenic responses in KNRK cells. In contrast,
mutant PAR-2 receptors that cannot bind
-arrestin, but still
activate ERK1/2 through a calcium-dependent pathway,
stimulate nuclear translocation of the ERK and provoke a proliferative
response (25). Further experimentation with the system presented here
should provide insight into the relevance of GPCR-mediated ERK
activation in a host of physiological and pathophysiological conditions.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Donna Addison, Mary Holben, and Julie Turnbough for excellent secretarial assistance and Francine Roudabush for valuable technical assistance.
![]() |
FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grants HL16037 (to R. J. L.) and DK55524 (to L. M. L).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.
These authors contributed equally to this work.
¶ Investigator of the Howard Hughes Medical Inst. To whom correspondence should be addressed. Tel.: 919-684-2974; Fax: 919-684-8875; E-mail: Lefko001@receptor-biol.duke.edu.
Published, JBC Papers in Press, April 4, 2001, DOI 10.1074/jbc.M101303200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: GPCR, G protein-coupled receptor; EGFR, epidermal growth factor receptor; HA, hemagglutinin; MAP, mitogen-activated protein; CSK, c-Src kinase; GFP, green fluorescent protein; HB-EGF, heparin-binding EGF; ERK, extracellular signal-regulated kinase; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; RTK, receptor tyrosine kinase.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Daub, H., Weiss, F. U., Wallasch, C., and Ullrich, A. (1996) Nature 379, 557-560[CrossRef][Medline] [Order article via Infotrieve] |
2. |
Daub, H.,
Wallasch, C.,
Lankenau, A.,
Herrlich, A.,
and Ullrich, A.
(1997)
EMBO J.
16,
7032-7044 |
3. |
Luttrell, L. M.,
Della Rocca, G. J.,
van Biesen, T.,
Luttrell, D. K.,
and Lefkowitz, R. J.
(1997)
J. Biol. Chem.
272,
4637-4644 |
4. | Prenzel, N., Zwick, E., Daub, H., Leserer, M., Abraham, R., Wallasch, C., and Ullrich, A. (1999) Nature 402, 884-888[CrossRef][Medline] [Order article via Infotrieve] |
5. | Raab, G., and Klagsbrun, M. (1997) Biochim. Biophys. Acta 1333, F179-99[CrossRef][Medline] [Order article via Infotrieve] |
6. |
Mitamura, T.,
Higashiyama, S.,
Taniguchi, N.,
Klagsbrun, M.,
and Mekada, E.
(1995)
J. Biol. Chem.
270,
1015-1019 |
7. | Levitzki, A., and Gazit, A. (1995) Science 267, 1782-1788[Medline] [Order article via Infotrieve] |
8. |
Gechtman, Z.,
Alonso, J. L.,
Raab, G.,
Ingber, D. E.,
and Klagsbrun, M.
(1999)
J. Biol. Chem.
274,
28828-28835 |
9. |
Fan, H.,
and Derynck, R.
(1999)
EMBO J.
18,
6962-6972 |
10. |
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 |
11. | Matsuda, M., Mayer, B. J., Fukui, Y., and Hanafusa, H. (1990) Science 248, 1537-1539[Medline] [Order article via Infotrieve] |
12. |
Vieira, A. V.,
Lamaze, C.,
and Schmid, S. L.
(1996)
Science
274,
2086-2089 |
13. |
Daunt, D. A.,
Hurt, C.,
Hein, L.,
Kallio, J.,
Feng, F.,
and Kobilka, B. K.
(1997)
Mol. Pharmacol.
51,
711-720 |
14. |
Pierce, K. L.,
Maudsley, S.,
Daaka, Y.,
Luttrell, L. M.,
and Lefkowitz, R. J.
(2000)
Proc. Natl. Acad. Sci. U. S. A.
97,
1489-1494 |
15. |
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,
655-661 |
16. | Barlic, J., Andrews, J., Kelvin, A., Bosinger, S., DeVries, M., Xu, L., Dobransky, T., Feldman, R., Ferguson, S., and Kelvin, D. (2000) Nat. Immunol. 1, 227-233[CrossRef][Medline] [Order article via Infotrieve] |
17. |
DeFea, K. A.,
Vaughn, Z. D.,
O'Bryan, E. M.,
Nishijima, D.,
Dery, O.,
and Bunnett, N. W.
(2000)
Proc. Natl. Acad. Sci. U. S. A.
97,
11086-11091 |
18. |
Cao, W.,
Luttrell, L. M.,
Medvedev, A. V.,
Pierce, K. L.,
Daniel, K. W.,
Dixon, T. M.,
Lefkowitz, R. J.,
and Collins, S.
(2000)
J. Biol. Chem.
275,
38131-38134 |
19. |
Gutwein, P.,
Oleszewski, M.,
Mechtersheimer, S.,
Agmon-Levin, N.,
Krauss, K.,
and Altevogt, P.
(2000)
J. Biol. Chem.
275,
15490-15497 |
20. |
Biscardi, J. S.,
Maa, M. C.,
Tice, D. A.,
Cox, M. E.,
Leu, T. H.,
and Parsons, S. J.
(1999)
J. Biol. Chem.
274,
8335-8343 |
21. |
Ahn, S.,
Maudsley, S.,
Luttrell, L. M.,
Lefkowitz, R. J.,
and Daaka, Y.
(1999)
J. Biol. Chem.
274,
1185-1188 |
22. | Wilde, A., Beattie, E. C., Lem, L., Riethof, D. A., Liu, S. H., Mobley, W. C., Soriano, P., and Brodsky, F. M. (1999) Cell 96, 677-687[Medline] [Order article via Infotrieve] |
23. |
Kranenburg, O.,
Verlaan, I.,
and Moolenaar, W. H.
(1999)
J. Biol. Chem.
274,
35301-35304 |
24. |
Daaka, Y.,
Luttrell, L. M.,
Ahn, S.,
Della Rocca, G. J.,
Ferguson, S. S.,
Caron, M. G.,
and Lefkowitz, R. J.
(1998)
J. Biol. Chem.
273,
685-688 |
25. |
DeFea, K. A.,
Zalevsky, J.,
Thoma, M. S.,
Dery, O.,
Mullins, R. D.,
and Bunnett, N. W.
(2000)
J. Cell Biol.
148,
1267-1281 |