 |
INTRODUCTION |
The signaling mechanism of the angiotensin II (Ang
II)1 type 1 (AT1)
receptor has traditionally been portrayed to be dependent on
heterotrimeric G proteins, including G
q and
G
i proteins and their downstream targets, primarily
phospholipase C (1). This results in inositol triphosphate generation,
which in turn causes an increase in intracellular calcium
concentrations and diacylglycerol formation, leading to activation of
protein kinase C. However, recent investigations revealed that tyrosine
phosphorylation is also intimately involved in AT1 receptor signaling
(2-6). Ang II-induced ERK1/2 activation, for example, requires
tyrosine kinase activation, including Src family tyrosine kinases (7,
8) and epidermal growth factor receptor (EGFR) (9, 10). It is unclear,
however, how AT1 receptors, which lack intrinsic tyrosine kinase
activities, are able to stimulate tyrosine kinases.
We have recently shown that an AT1 receptor second intracellular loop
mutant, lacking heterotrimeric G protein coupling, is able to activate
Src tyrosine kinase (11). This suggests that heterotrimeric G
protein-independent mechanisms are able to activate Src. Furthermore,
increasing lines of evidence suggest that the carboxyl terminus
(C-tail) of the AT1 receptor plays an important role in the AT1
receptor signaling (11, 12). For example, ligand binding to the AT1
receptor induces physical association of the C-tail of the AT1 receptor
with Jak2, thereby causing phosphorylation and translocation of STAT to
the nucleus (13). Other signaling molecules, including phospholipase
C
and SHP-2, also have been shown to interact with the C-tail of the
AT1 receptor (14, 15). These results suggest that direct interaction
between the heterotrimeric G protein-coupled receptor and intracellular
signaling molecules may play an important role in mediating activation
of downstream-signaling mechanisms.
Accumulating data suggests that EGFR is involved in signal transduction
of many G protein-coupled receptors, including the AT1 receptor (9, 10)
(for review, see Ref. 16). Ang II induces tyrosine phosphorylation of
EGFR and its association with Shc and Grb2, leading to subsequent
activation of the Ras-Raf-MEK-ERK1/2 pathway (9). Although several
signaling mechanisms are involved in Ang II-induced activation of EGFR
(9, 17-20), whether or not direct interaction between the AT1 receptor
and intracellular signaling molecules is required for EGFR activation
and, if so, the amino acid sequence of the AT1 receptor mediating Ang
II-induced EGFR activation has not been identified.
To elucidate the molecular mechanism of Ang II-induced EGFR activation,
we investigated the structural requirements of the AT1 receptor and the
associating signaling mechanism leading to transactivation of EGFR. Our
results indicate that tyrosine 319 at the conserved YIPP motif in the
carboxyl terminus of the AT1 receptor plays an essential role in
mediating Ang II-induced transactivation of EGFR and cell proliferation.
 |
EXPERIMENTAL PROCEDURES |
Materials--
Ang II was purchased from Peninsula. Anti-FLAG M2
affinity gel was from Sigma. Horseradish peroxidase-conjugated
anti-phosphotyrosine monoclonal antibody (RC20H) and anti-EGF receptor
monoclonal antibody were from Transduction Laboratories. Anti-v-Src
monoclonal antibody was from Calbiochem. Anti-EGF receptor sheep
polyclonal antibody was from Upstate Biotechnology. Rabbit anti-ERK1/2
polyclonal antibody was from Zymed Laboratories Inc.,
and rabbit anti-active ERK1/2 polyclonal antibody was from Promega.
Horseradish peroxidase-conjugated anti-rabbit IgG and anti-mouse IgG
antibodies were from Cell Signaling Technology. Anti-AT1 receptor
antibody was from Santa Cruz Biotechnology. Dowex AG1-X8 formate resin
was from Bio-Rad.
3-[4-Iodotyrosyl-125I]Ang II and
myo-[3H]inositol were from Amersham Biosciences. Enolase
was from Roche Molecular Biochemicals. AG1478 was from Biomol.
Plasmids--
The full-length rat AT1a wild type receptor
(AT1-WT) cDNA subcloned into pcDM8 was obtained from Dr. J. Harrison. The following AT1a receptor mutants were generated by using
PCR and QuikChange (Stratagene): AT1-Y319F, tyrosine 319 was replaced
with phenylalanine; AT1-Y319E, tyrosine 319 was replaced with glutamic
acid; AT1-(1-338), the carboxyl terminus of the AT1 receptor
(339-359) was truncated; AT1-(1-311), the carboxyl terminus of the
AT1 receptor (312-359) was truncated. Mammalian expression plasmid
encoding the AT1 receptor carboxyl terminus peptide was generated by
subcloning cDNA encoding AT1-(292-359) into a mammalian expression
vector pHM6 (Roche Molecular Biochemicals), which has an HA tag in the
amino terminus. Site-directed mutagenesis was performed to generate
HA-AT1-(292-359)-Y319F using QuikChange. The DNA sequence of all
constructs was confirmed by DNA sequence analyses. Expression plasmids
for EGFR and JAK2 were provided by Dr. A. Yoshimura (Kyushu University,
Fukuoka, Japan). Plasmid for Myc-SHP-2 was provided by Dr. S. Sano
(Mitsubishi Kasei Institute of Life Science, Tokyo, Japan). Plasmid
encoding Myc-dominant negative SHP-2 (SHP-2-(1-225)) was generated by
PCR, and the PCR product was subjected to TA cloning using pCR3.1
vector (Invitrogen). Plasmid encoding dominant negative Src
(K296R/Y528F) and its control vector (pUSE) were purchased from
Upstate Biotechnology.
Cell Cultures, Transfection, and Receptor Binding
Assays--
COS-7 cells were maintained in Dulbecco's modified
Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, 100 units/ml penicillin G, and 100 µg/ml streptomycin at 37 °C in a
humidified 5% CO2 atmosphere. Transfections were performed
on 70% confluent monolayers in 60-mm dishes for immunoprecipitation
and Src kinase assays or in 35-mm dishes for ERK1/2 assays. For
transient transfection, 2.5 ml of Opti-MEM I (Invitrogen) containing 4 µg of DNA, 8 µl of LipofectAMINE Plus reagent, and 12 µl of
LipofectAMINE was used for a 60-mm dish. One ml of Opti-MEM I
containing 2 µg of DNA, 6 µl of LipofectAMINE Plus reagent, and 4 µl of LipofectAMINE was used for a 35-mm dish. Empty
pcDNA 3.0 plasmid was added as needed to keep the total amount of
DNA constant per transfection. Cells were incubated in serum-free
Opti-MEM at 37 °C for 3 h. COS-7 cells were then incubated with
10% fetal bovine serum in DMEM and incubated overnight. Transfected
cells were serum-starved in serum-free DMEM for 24-36 h before
stimulation. Assays were performed 48 h after transfection.
Saturation binding curves were determined using a modification of the
whole cell receptor binding assay (16) as described previously (11).
The Bmax and the dissociation constant
(Kd)
3-[4-iodotyrosyl-125I]AngII binding was
determined by using Prism 3.0 (GraphPad). Protein assay was performed
on each sample using the Bio-Rad protein assay kit.
Cardiac fibroblast cultures were prepared as described previously (17).
In brief, hearts were removed from 1-day-old Crl:(WI)BR-Wistar rats
(Charles River Laboratories) and subjected to digestion with collagenase type IV (Sigma), 0.1% trypsin (Invitrogen), and 15 µg/ml
DNase I (Sigma). Cell suspensions were applied on a discontinuous Percoll gradient (1.060/1.086 g/ml) and subjected to centrifugation at
3000 rpm for 30 min (18). The layer containing primarily non-cardiac
myocytes was removed and subjected to the preplating procedure for
1 h. The supernatant was discarded, and the attached cells were
cultured in the media containing DMEM/F-12 supplemented with 10% fetal
bovine serum. Cells were passed twice to enrich for cardiac
fibroblasts. Cells were cultured in serum-free conditions for 48 h
before experiments.
Immunoprecipitation and Immunoblotting--
Cell stimulation was
carried out at 37 °C in serum-free medium. After stimulation, COS-7
cells were scraped and lysed in CHAPS buffer (150 mM NaCl,
40 mM HCl-Tris, pH 7.5, 1% Triton X-100, 0.1% CHAPS, 10%
glycerol, 2 mM EDTA, 0.1 mM NaVO4,
1 mM NaF, 0.5 mM
4-(2-aminoethyl)benzenesulfonyl fluoride, 0.5 µg/ml aprotinin, 0.5 µg/ml leupeptin) for immunoprecipitation or hypoosmotic lysis buffer
(25 mM NaCl, 25 mM Tris, pH 7.5, 0.5 mM EGTA, 10 mM sodium pyrophosphate, 1 mM Na3VO4, 10 mM NaF,
0.5 mM 4-(2-aminoethyl)benzenesulfonyl fluoride, 0.5 µg/ml aprotinin, 0.5 µg/ml leupeptin) for ERK1/2 kinase assays.
Cell lysates were incubated on ice for 10 min and subjected to
centrifugation for 30 min. Protein concentrations of the supernatants
were adjusted to be 1 mg/ml with the lysis buffer. For
immunoprecipitation of EGFR-FLAG, the cell lysates (500 µg) were
incubated with 40 µl of anti-FLAG M2 affinity gel at 4 °C for
2 h. For immunoprecipitation of endogenous EGFR, the cell lysates
were incubated with 4 µg of anti-EGFR monoclonal antibody for 1 h followed by protein G-agarose (30 µl of slurry) for 45 min. The
immune complexes were washed 3 times with lysis buffer and denatured in
Laemmli sample buffer. After SDS-PAGE, samples were transferred onto
polyvinylidene fluoride microporous membranes (Millipore). Immunoblots
were performed as described previously. Phosphorylated levels of the
EGFR, the AT1 receptor, or ERK1/2 were analyzed by immunoblotting with
anti-phosphospecific antibody and scanning densitometry, and the
results were expressed as fold increase compared with the control.
Anti-phospho-Tyr-319 AT1 Receptor--
Polyclonal
anti-phospho-specific antibody was generated by injecting
Ac-QLLK(pY)IPPKAKS(Ahx)C-amide (pY is phosphotyrosine; Ahx, is a
six-carbon spacer) into rabbits as an immunogen. Generated antibody was then subjected to affinity purification
(BIOSOURCE International).
Phosphoinositide Production--
Measurement of
inositol phosphates (IPx) was based upon the method of Berridge
et al. (19) as described previously (11). Cells were
incubated with myo-[3H]inositol (10 µCi/ml) in DMEM for
24 h at 37 °C. Labeling was terminated by aspirating the
medium, rinsing cells with oxygenated reaction buffer (142 mM NaCl, 30 mM Hepes buffer, pH 7.4, 5.6 mM KCl, 3.6 mM NaHCO3, 2.2 mM CaCl2, 1.0 mM MgCl2,
and 1 mg/ml D-glucose), and harvesting cells with
phosphate-buffered saline, 0.02%EDTA. Cells were centrifuged twice
(300 × g, 5 min) in reaction buffer, and the pellet
was resuspended in an equal volume of reaction buffer containing 60 mM LiCl. Stimulation of IPx production was initiated by
mixing 0.25 ml of cell suspension with 0.25 ml of 0-100 nM
Ang II in reaction buffer (without LiCl). The mixture was incubated for
30 min at 37 °C, then 0.5 ml of ice-cold 20% trichloroacetic acid
was added. Precipitates were pelleted (4100 × g, 20 min), and the trichloroacetic acid-soluble fraction was transferred to
new tubes, washed with water-saturated diethyl ether, and neutralized
with NaHCO3. IPx were isolated by adsorption to 0.5 ml of
Dowex AG1-X8 formate resin slurry and rinsed 5 times with 3 ml of
unlabeled 5 mM myoinositol followed by elution with 1 ml of
1.2 M ammonium formate, 0.1 M formic acid. The
elutes were counted by liquid scintillation counter in 5 ml of ScintiVerse.
Src Kinase Assay--
The tyrosine kinase activity of Src was
determined by the immune complex kinase assay using enolase as a
substrate as described previously (7, 11). Cell lysates were prepared
in a lysis buffer (150 mM NaCl, 15 mM HEPES, pH
7.0, 1% deoxycholic acid, 1% IGEPAL, 0.1% SDS, 0.1 mM
NaVO4, 1 mM NaF, 0.5 mM
4-(2-aminoethyl)benzenesulfonyl fluoride, 0.5 µg/ml aprotinin, 0.5 µg/ml leupeptin). The cell lysates containing equal amount of protein
(750 µg) were incubated with anti-v-Src monoclonal antibody at
4 °C for 1 h. Protein G-Sepharose was then added. The
immunoprecipitates were washed twice with lysis buffer without SDS or
deoxycholic acid and then washed once with kinase buffer (50 mM HEPES, pH 7.6, 0.1 mM EDTA, 10 mM MnCl2, 0.015% Brig 35). Pellets were
incubated for 15 min at 37 °C in the kinase buffer with 1 µCi of
[
-32P]ATP and 0.25 µg of enolase as a substrate. The
reaction was terminated by the addition of Laemmli sample buffer on
ice. Reaction mixtures were boiled and subjected to 12% SDS-PAGE
followed by autoradiography. Results were analyzed by densitometry.
Adenovirus Vectors--
Adenovirus-mediated transduction was
performed as described previously (20). Cells grown in 60-mm dishes
were transduced with an adenovirus vector harboring dominant-negative
Ras (Ad5.N17Ras) (courtesy of Dr. M. Schneider, Baylor College of
Medicine, Houston, TX) at a multiplicity of infection of 100. For a
control study, Ad5/
E1sp1B (courtesy of Dr. B. French, University of
Virginia, Charlottesville, VA) was used. Adenovirus vectors harboring
the wild type AT1 receptor or AT1-Y319F were generated by using the AdEasy system (21). All experiments were performed 48 h after transduction.
Cell Proliferation Experiments--
Cells were plated at a
density of 0.3 × 106/well in six-well plates. Twelve
hours after plating, cells were serum-starved for 12 h and then
stimulated with Ang II (10
7 M) in the
presence or absence of AG1478 (250 nM) for 36 h. Ang II or AG1478 was added every 12 h. After stimulation, cells were washed twice with phosphate-buffered saline. The cell layer was scraped
with 1 ml of standard sodium citrate containing 0.25% SDS and vortexed
extensively. Total DNA content was determined by the Hoechst dye method
as described previously (17).
Statistics--
Data are given as the mean ± S.E.
Statistical analyses were performed using the analysis of variance. The
post-test comparison was performed by the method of Tukey. Significance
was accepted at p < 0.05 level.
 |
RESULTS |
Ang II Activates EGFR in COS-7 Cells Expressing the Wild Type AT1
Receptor--
Ang II stimulation of COS-7 cells without transfection
of the AT1 receptor did not activate either endogenous or transfected EGFRs (not shown). By contrast, in COS-7 cells transfected with the AT1
receptor, Ang II, caused time-dependent increases in
tyrosine phosphorylation of either endogenous (not shown) or
transfected EGFRs (Fig. 1A).
Tyrosine phosphorylation of EGFR by Ang II was observed within 3 min,
reached a peak around 5 min, and lasted for more than 60 min (Fig.
1B (n = 7) and 2.8 ± 0.1-fold at 60 min (n = 3)). This suggests that stimulation of the AT1
receptor causes transactivation of EGFR in COS-7 cells.

View larger version (27K):
[in this window]
[in a new window]
|
Fig. 1.
Ang II transactivates EGFR. COS-7 cells
co-transfected with AT1R and EGFR-FLAG were treated with Ang II
(10 7 M) for the indicated time. Cell
lysates were subjected to immunoprecipitation (IP) with
antibody against FLAG and immunoblotted with antibodies against
phosphotyrosine (PY) or EGFR. A, shown is the
representative of the experiments. B, fold increase of
phosphorylation level of EGFR is shown. n = 7.
|
|
Tyrosine 319 in the Carboxyl Terminus of the AT1 Receptor Plays an
Important Role in Ang II-induced Activation of EGFR--
Because it
has been suggested that the C-tail of the AT1 receptor plays an
important role in mediating cell-signaling mechanisms of the AT1
receptor (11), we examined the role of the AT1 receptor C-tail in Ang
II-induced transactivation of EGFR. We co-transfected EGFR-FLAG and
carboxyl-terminal-truncated AT1 receptors into COS-7 cells, and EGFR
was immunoprecipitated with anti-FLAG antibody. Although Ang II caused
significant increases in tyrosine phosphorylation of EGFR in cells
transfected with AT1-(1-338), it failed to do so in cells transfected
with AT1-(1-311) (Fig. 2A).
These results suggest that the amino acid sequence located between
amino acid 312 and 337 of the AT1 receptor is required for activation
of EGFR by Ang II stimulation.

View larger version (38K):
[in this window]
[in a new window]
|
Fig. 2.
Tyrosine 319 at the carboxyl terminus of the
AT1 receptor is required for Ang II-induced EGFR activation.
A, COS-7 cells were co-transfected with EGFR-FLAG and either
AT1-(1-338) or AT1-(1-311). Forty-eight hours after transfection,
cells were treated with Ang II (10 7 M) for 5 min. Cell lysates were immunoprecipitated (IP) with antibody
against FLAG and immunoblotted with antibody against phosphotyrosine
(PY) or EGFR. The left panel is the
representative of the experiments. In lane 5, EGFR-FLAG was
not transfected. In the right panel, values are the fold
increase relative to the unstimulated control. n = 3. B, comparison of the amino acid sequence of the carboxyl
terminus AT1 receptor containing the conserved YIPP motif in various
species. C, COS-7 cells were co-transfected with EGFR-FLAG
and wild type AT1 receptor (WT) or AT1-Y319F
(Y319F). Forty-eight hours after transfection, cells were
treated with Ang II (10 7 M) for 5 min. Cell
lysates were subjected to immunoprecipitation with antibody against
FLAG and immunoblotted with antibody against phosphotyrosine
(PY) or EGFR. The left panel is representative of
the experiments. For the control (lane 5), EGFR-FLAG was not
transfected. In the right panel, values are the fold
increase relative to the unstimulated control obtained from six
experiments.
|
|
Between amino acids 312 and 337 of the AT1 receptor, the
YIPP-(319-322) motif is evolutionarily conserved in AT1 receptors cloned from many species (Fig. 2B). It has been shown that
several signaling molecules directly or indirectly associate with the YIPP motif in the AT1 receptor (13, 14). To test the role of this
conserved motif in Ang II-induced EGFR activation, we made a mutant
where a tyrosine residue at position 319 in this motif was mutated to
phenylalanine (AT1-Y319F). Interestingly, Ang II-induced activation of
EGFR was abolished in COS-7 cells transfected with AT1-Y319F (Fig.
2C).
AT1 Receptor Y319E Mutant Increases Basal EGFR Activities in COS-7
Cells--
To test if phosphorylation of tyrosine 319 is involved in
activation of EGFR, we mutated tyrosine 319 to glutamate (Y319E) to
mimic the status of phosphorylation. Expression of AT1-Y319E in COS-7
cells significantly increased phosphorylation of EGFR in basal
conditions, and Ang II stimulation failed to increase phosphorylation
of EGFR (Fig. 3A). These
results suggest that phosphorylation of tyrosine 319 may be involved in
Ang II-induced activation of EGFR.

View larger version (36K):
[in this window]
[in a new window]
|
Fig. 3.
AT1-Y319E increases the base-line activity of
EGFR. A, COS-7 cells were co-transfected with EGFR-FLAG
and either wild type AT1 receptor (WT) or AT1-Y319E.
Forty-eight hours after transfection, cells were treated with Ang II
(10 7 M) for 5 min. Cell lysates were
immunoprecipitated (IP) with antibody against FLAG and
immunoblotted with antibody against phosphotyrosine (PY) or
EGFR. The left panel shows a representative immunoblot. In
lane 5, EGFR-FLAG was not transfected. In the right
panel, values are the fold increase relative to the unstimulated
control obtained from four independent experiments. B and
C, COS-7 cells were transfected with either WT or AT1-Y319F.
Forty-eight hours after transfection, cells were treated with Ang II
(10 7 M) for the indicated durations. In
B, AT1 receptors were immunoprecipitated with anti-phospho
Tyr-319 AT1 receptor-specific antibody and immunoblotted with the same
antibody. A representative immunoblot is shown in the upper
panel. In lane 7, neither WT nor AT1-Y319F was
transfected. In the lower panel, values are the fold
increase relative to the unstimulated control obtained from three
independent experiments. In C, the duplicated samples were
subjected to immunoprecipitation using anti-(total) AT1 receptor
(AT1R) antibody and immunoblotted with the same antibody.
n = 3.
|
|
Tyrosine 319 Is Phosphorylated in Response to Ang II
Stimulation--
To test if tyrosine 319 of the AT1 receptor is
phosphorylated in vivo in response to Ang II stimulation, we
generated phosphotyrosine 319-specific anti-AT1 receptor antibody
(anti-phosphotyrosine 319 antibody). Either wild type AT1 receptor
(AT1-WT) or AT1-Y319F was transfected in COS-7 cells. The AT1 receptor
phosphorylated at tyrosine 319 was immunoprecipitated by the
anti-phosphotyrosine 319 antibody and immunoblotted with the same
antibody. Although AT1-WT was not detected by anti-phosphotyrosine 319 antibody in unstimulated cells, it was detected after the cells were
stimulated with Ang II for 3 min (Fig. 3B). Phosphorylation
of tyrosine 319 was transient and returned to the basal level at 5 min.
No apparent signals of phosphotyrosine 319 were detected in samples
obtained from cells expressing AT1-Y319F (Fig. 3B),
suggesting that the signal found in AT1-WT was most likely from
phosphorylated Tyr-319. Duplicate samples were subjected to
immunoprecipitation using anti-(total)-AT1 receptor antibody and
immunoblotted with the same antibody. The result confirmed that similar
amounts of the AT1 receptor were solubilized in each sample (Fig.
3C). These results indicate that tyrosine 319 in AT1-WT is
transiently phosphorylated in response to Ang II stimulation.
Ang II-induced Activation of EGFR in AT1-WT Is Inhibited in the
Presence of AT1 Carboxyl Terminus Minigene--
To confirm that
tyrosine 319 of the AT1 receptor is important for Ang II-induced
transactivation of EGFR, we examined the effect of overexpression of a
mini-gene-containing AT1 receptor carboxyl terminus peptide (HA-AT1-C)
upon Ang II-induced EGFR activation in COS-7 cells. Expression of
HA-AT1-C inhibited Ang II-induced activation of EGFR in COS-7 cells
(Fig. 4A). Overexpression of
HA-AT1-C alone did not inhibit activation of EGFR by EGF treatment (Fig. 4B), suggesting that the effect of HA-AT1-C is
stimulus-specific. Furthermore, a mini-gene-containing HA-AT1-C, whose
tyrosine 319 is mutated to phenylalanine (HA-AT1-C-Y319F), failed to
inhibit Ang II-induced EGFR activation in COS-7 cells (Fig.
4A). These results further support the notion that tyrosine
319 in the AT1 receptor plays an essential role in mediating EGFR
activation by the AT1 receptor.

View larger version (31K):
[in this window]
[in a new window]
|
Fig. 4.
AT1 receptor carboxyl terminus peptide blocks
Ang II-induced EGFR activation. COS-7 cells were co-transfected
with EGFR-FLAG, wild type AT1 receptor (AT1-WT), and either the
carboxyl terminus (292-359) of the wild type AT1a receptor
(C-tail WT) or the carboxyl terminus of AT1a-Y319F (C
tail Y319F). Forty-eight hours after transfection, cells were
treated with Ang II (10 7 M) in A
or EGF (10 ng/ml) in B for 5 min. Cell lysates were prepared
and immunoprecipitated (IP) with antibodies against FLAG and
immunoblotted with antibodies against phosphotyrosine (PY)
or EGFR. In the control (lane 5) cells, EGFR-FLAG was not
transfected. n = 3.
|
|
Ang II Stimulates Interaction between AT1 Receptors and EGFR, Which
Was Inhibited in the Presence of Dominant Negative SHP-2--
We next
examined if the AT1 receptor and EGFR physically associate with each
other. We co-transfected AT1 receptors and EGFR into COS-7 cells and
stimulated the cells with Ang II. EGFR immunoprecipitates were
immunoblotted with anti-AT1 receptor antibody. Interestingly, AT1
receptors were co-immunoprecipitated with EGFR in samples stimulated
with Ang II for 3 min (2.7 ± 0.2-fold versus 0 min, n = 4, Fig.
5A). This interaction between
the AT1 receptor and EGFR was transient, since it was not observed at 5 min. Equal amounts of EGFRs were immunoprecipitated in each sample, and
the immunoprecipitated EGFR was tyrosine-phosphorylated in response to
Ang II stimulation, consistent with the results shown in Fig. 1 (Fig.
5A). Because it has been shown that SHP-2 is associated with
AT1 receptor at the YIPP motif (14, 15), we examined if disrupting
interaction between the AT1 receptor and SHP-2 affects interaction
between the AT1 receptor and EGFR and abolishes Ang II-induced
activation of EGFR. Ang II-dependent interaction between the AT1 receptor and EGFR and activation of EGFR were both inhibited in
the presence of dominant negative SHP-2 (22) (Fig. 5A).
These results suggest that SHP-2 plays an essential role in mediating Ang II-induced activation of EGFR.

View larger version (47K):
[in this window]
[in a new window]
|
Fig. 5.
Ang II stimulates the interaction between AT1
receptors and EGFR, which was inhibited in the presence of dominant
negative SHP-2. A, COS-7 cells were co-transfected with
EGFR-FLAG, wild type AT1 receptor (AT1-WT), and either
dominant negative SHP-2 (DN-SHP2) or control plasmid
(pcDNA). In the control (lane 7) cells, no
plasmid was transfected. Forty-eight hours after transfection, cells
were treated with Ang II (10 7 M) for the
indicated durations. Cell lysates were subjected to immunoprecipitation
(IP) with antibodies against FLAG and immunoblotted with
antibodies against phosphotyrosine (PY), EGFR, or AT1
receptor (AT1R). n = 4. B, COS-7
cells were co-transfected with EGFR-FLAG and AT1-Y319F. In the control
cells (lane 4), no plasmid was transfected. Cell lysates
were subjected to immunoprecipitation and immunoblotting as described
in A. n = 4.
|
|
Because tyrosine 319 plays an essential role in mediating Ang
II-induced activation of EGFR, we attempted to determine if EGFR can
interact with AT1-Y319F. EGFR and AT-Y319F were co-transfected into
COS-7 cells, and cells were stimulated with Ang II. EGFR was
immunoprecipitated and then immunoblotted with anti-AT1 receptor antibody. We did not detect the AT1 receptor in the EGFR
immunoprecipitates (Fig. 5B). As expected, EGFR was not
phosphorylated in response to Ang II stimulation in these experiments.
These results suggest that tyrosine 319 plays an essential role in
mediating Ang II-dependent interaction between the AT1
receptor and EGFR.
Mutation of Tyrosine 319 Does Not Affect Other Signaling
Mechanisms--
We examined if other signaling mechanisms are also
affected in the AT1-Y319F mutant. Ang II caused significant levels of
IPx accumulation in cells transfected with AT1-Y319F, which was not statistically different from those in cells transfected with AT1-WT (Fig. 6A). This suggests that
AT1-Y319F maintains coupling with the G
q-phospholipase C
pathway. This also suggests that activation of IPx alone is not
sufficient for the AT1 receptor to mediate EGFR activation.

View larger version (28K):
[in this window]
[in a new window]
|
Fig. 6.
Ang II causes IPx accumulation and activation
of Src and JAK2 in cells expressing AT1-Y319F. A and
B, COS-7 cells were transfected with either wild type AT1a
receptor (WT) or AT1-Y319F (Y319F). A,
cells were labeled with myo-[3H]inositol and incubated
with Ang II (10 7 M) for 30 min. The amount of
IPx, including inositol monophosphate, inositol bisphosphates, and
inositol triphosphates, was determined and normalized to that of
WT-expressing cells without Ang II stimulation. n = 3. B, cell lysates were immunoprecipitated (IP) with
anti v-Src antibody. Immune complex Src assays were performed.
Phosphorylation of enolase by the immune complex is shown. In
lane 4, cells were transfected with empty vector. In
lane 8, anti-v-Src antibody was not included in the
immunoprecipitation as a negative control (NC).
n = 5. C, COS-7 cells were transfected with
FLAG-JAK2 and either WT or Y319F. Cell lysates were immunoprecipitated
with antibody against FLAG and immunoblotted with antibody against
phosphotyrosine (PY). For the control (lane 1),
FLAG- JAK2 was not transfected. n = 3.
|
|
We attempted to determine if activation of other tyrosine kinases is
also affected in AT1-Y319F. Ang II caused significant increases in Src
activities in cells transfected with either AT1-WT or AT1-Y319F
(2.9 ± 0.1-fold in WT, 2.7 ± 0.2-fold in Y319F at 5 min)
(Fig. 6B). Ang II also caused similar levels of increases in
tyrosine phosphorylation of transfected JAK2-FLAG in cells expressing
either AT1-WT or AT1-Y319F (3.5 ± 0.1-fold in WT, 3.4 ± 0.2-fold in Y319F at 5 min) (Fig. 6C). These results suggest that Ang II-induced activation of some tyrosine kinases, including Src
and JAK2, is preserved in cells expressing AT1-Y319F.
EGFR Plays an Important Role in Ang II-induced ERKs Activation by
AT1-WT, Whereas Src Mediates Ang II-induced ERKs Activation by
AT1-Y319F--
Ang II activated ERKs (3.5 ± 0.1-fold,
p < 0.05 versus control) in COS-7 cells
transfected with AT1-WT (Fig.
7A). AG1478, a specific
inhibitor for EGFR, abolished Ang II-induced ERK activation in COS-7
cells transfected with AT1-WT (Fig. 7A), suggesting that EGFR plays an essential role in ERK activation by AT1-WT in COS-7 cells. Surprisingly, however, Ang II was still able to activate ERKs in
AT1-Y319F (3.6 ± 0.2-fold, p < 0.05 versus control), where Ang II-induced EGFR activation is
abolished. Because AG1478 did not affect Ang II-induced ERKs activation
in cells transfected with AT1-Y319F (3.4 ± 0.1-fold,
p < 0.05 versus control) (Fig. 7A), activation of ERKs by AT1-Y319F was not due to residual
activation of EGFR. These results suggest that although the AT1
receptor normally activates ERK through EGFR, the AT1 receptor mutant, which does not activate EGFR, still activates ERKs through an (back-up)
EGFR-independent mechanism.

View larger version (36K):
[in this window]
[in a new window]
|
Fig. 7.
ERKs are activated by a
Src-Ras-dependent mechanism in cells expressing Y319F.
Activation of ERKs (p42 and p44) was evaluated by immunoblot analyses
using anti-phospho-specific ERK antibody. To show equal loading,
membranes were stripped and re-probed with antibody against total ERKs.
A, COS-7 cells were transfected with either WT or Y319F.
Forty-eight hours after transfection, cells were pretreated with either
vehicle or AG1478 (250 nM) for 30 min. Cells were then
treated with or without Ang II (10 7 M) for 5 min. n = 4. B, COS-7 cells were transfected
with either WT or Y319F together with either dominant-negative Src
(DN-Src) or empty vector pUSE (Upstate Biotechnology) and
then stimulated with or without Ang II (10 7
M) for 5 min. n = 4. C, COS-7
cells were transfected with WT or Y319F. Cells were then transduced
with either control adenovirus or adenovirus harboring dominant
negative Ras (DN-Ras). Forty-eight hours after transduction,
cells were stimulated with or without Ang II (10 7
M) for 5 min. n = 3.
|
|
Besides EGFR, tyrosine kinase Src also plays an important role in
mediating ERK activation in some cell types. To identify the (back-up)
mechanism by which AT1-Y319F activates ERKs, we examined the role of
Src in Ang II-induced ERK activation. Expression of dominant negative
Src did not affect Ang II-induced ERK activation (3.6 ± 0.2-fold,
p < 0.05 versus control) in COS-7 cells
expressing AT1-WT. By contrast, dominant negative Src abolished Ang
II-induced ERK activation in COS-7 cells expressing AT1-Y319F (1.0 ± 0.1-fold, not significant versus control) (Fig.
7B). This suggests that Src compensates the loss of EGFR
activation for Ang II-induced ERK activation by AT1-Y319F.
It has been shown that both EGFR- and Src-dependent
activation of ERKs is mediated by Ras. Ang II-induced activation of
ERKs in COS-7 cells expressing either AT1-WT or AT1-Y319F was abolished in the presence of dominant negative Ras (Fig. 7C),
suggesting that the mechanisms of ERK activation by AT1-WT and
AT1-Y319F are likely to converge at the level of or upstream of Ras.
Overexpression of the AT1-Y319F Mutant Abolishes Ang II-induced
Cell Proliferation in Cardiac Fibroblasts--
Our results presented
thus far indicated that AT1-Y319F selectively lacks Ang II-induced
transactivation of EGFR, whereas it maintains activation of some, if
not all, signaling molecules, including IPx, Src, JAK2, and ERKs. To
test if AT1-Y319F mutant has cellular functions different from AT1-WT,
we expressed either AT1-WT or AT1-Y319F in primary cultured cardiac
fibroblasts by adenovirus-mediated gene delivery. An adenovirus
harboring an irrelevant sequence was used as a control vector. Cell
cultures with comparable levels of expression of either AT1-WT or
AT1-Y319F were used for these experiments. Overexpression of AT1-WT in
cardiac fibroblasts increased Ang II-induced EGFR activation. By
contrast, overexpression of AT1-Y319F failed to enhance Ang II-induced
activation of EGFR, suggesting that Tyr-319 plays an essential role in
mediating Ang II-induced EGFR activation in cardiac fibroblasts (Fig.
8A). Under these conditions,
overexpression of the AT1-WT in cardiac fibroblasts significantly
enhanced Ang II-induced cell proliferation, which was determined by the
total DNA content, compared with control virus transduced cells (Fig.
8B). By contrast, overexpression of AT1-Y319F abolished
small increases in cell proliferation found in control virus-infected
cardiac fibroblasts (Fig. 8B). To determine the role of EGFR
activation in Ang II-induced cardiac fibroblast proliferation, we
treated the cells with AG1478. Treatment with AG1478 completely
abolished the Ang II-induced cell proliferation in both control
virus-transduced and AT1-WT-transduced cardiac fibroblasts (Fig.
8B). These results suggest that the cell-signaling mechanism
mediated by Tyr-319 in the AT1 receptor, including activation of EGFR,
is required for Ang II-induced cell proliferation in cardiac
fibroblasts.

View larger version (31K):
[in this window]
[in a new window]
|
Fig. 8.
The role of tyrosine 319 in the AT1 receptor
in Ang II-induced cell proliferation in cardiac fibroblasts.
A, neonatal rat cardiac fibroblasts were transduced with
either adenovirus harboring wild type AT1 receptor (WT) or
AT1-Y319F (Y319F). Forty-eight hours after transduction,
cells were treated with Ang II (10 7 M) for 5 min. Cell lysates were subjected to immunoprecipitation (IP)
with or without antibody against EGFR and immunoblotted with antibodies
against phosphotyrosine (PY) or EGFR. For the control (C),
antibody against EGFR was not added to the immunoprecipitation.
B, Ang II-induced cell proliferation was enhanced in cells
overexpressing WT, whereas it was abolished in AT1-Y319F. Neonatal rat
cardiac fibroblasts were transduced with either control adenovirus or
adenovirus harboring WT or Y319F. Forty-eight hours after transduction,
cells were stimulated with Ang II (10 7 M) in
the presence or absence of AG1478 (250 nM) for an
additional 36 h. Cell numbers were estimated by total DNA content
and normalized to those in control virus-transduced cells without Ang
II treatment. NC, negative control; NS, not
significant (n = 6).
|
|
 |
DISCUSSION |
Tyrosine 319 Is Specifically Required for Ang II-induced EGF
Receptor Activation--
The YIPP motif found in the AT1 receptor is
conserved in all members of the AT1 receptor family so far cloned,
suggesting that this motif is involved in important cellular functions
of the AT1 receptor. This motif is also found in the platelet-derived growth factor
and
receptors and is involved in
ligand-dependent activation of phospholipase C
(23).
Although it has been shown that several signaling molecules associate
with the motif in the AT1 receptor (13-15, 24, 25), the specific
requirement of tyrosine 319 for activation of downstream protein
kinases has not been clearly demonstrated in vivo. Because
Ang II-induced activation of Src and JAK2 was not affected in
AT1-Y319F, the requirement of tyrosine 319 in the AT1 receptor seems
specific for activation of EGFR among the activation of major tyrosine
kinases. The structural requirements of the AT1 receptor in Ang
II-induced EGFR have not been previously determined.
It has been shown that the AT1 receptor is tyrosine-phosphorylated by
ligand binding (14, 26). However, the tyrosine residue phosphorylated
by ligand binding has not been identified in vivo. Because
signaling molecules containing the SH2 domain interact with the YIPP
motif of the AT1 receptor, it has been speculated that tyrosine 319 is
phosphorylated (13-15). By using anti-phosphotyrosine 319-specific AT1
receptor antibody, we demonstrated that tyrosine 319 is phosphorylated
in response to Ang II.
Although it has been speculated that ligand-dependent
phosphorylation of the AT1 receptor may modulate the activities of
downstream signaling molecules besides internalization of the receptor,
this has not been clearly demonstrated. In fact, it has been recently shown that phosphorylation of the AT1 receptor by G protein-coupled receptor kinase does not play an essential role in Ang II-induced cell
signaling (27). In our study, because expression of AT1-Y319E increased
basal levels of EGFR phosphorylation and because Ang II failed to show
additive effects on EGFR activation by Y319E, phosphorylation of
tyrosine 319 seems to mediate Ang II-induced activation of the EGFR. At
present we do not know which tyrosine kinase is responsible for
phosphorylation of tyrosine 319.
The Mechanism of EGFR Activation by the AT1
Receptor--
Ca2+-dependent mechanisms (9),
other tyrosine kinases, such as Src and Pyk2 (28-30), metalloproteases
(for review, see Ref. 31), and reactive oxygen species (32) have been
proposed as mechanisms of Ang II-induced activation of EGFR in various
cell types. The requirement of tyrosine 319 in the AT1 receptor for EGFR activation found in the present investigation may not contradict these previous observations but may represent another requirement for
EGFR activation by AT1 receptors. Because production of IPx and
activation of other kinases such as Src and JAK2 by Ang II are
preserved in AT1-Y319F, activation of these molecules alone is not
sufficient for Ang II-induced EGFR activation.
Growing lines of evidence suggest that heterotrimeric G protein-coupled
receptors directly interact with intracellular signaling molecules (33,
34). The mini-gene containing AT1-C, but not AT1-C-Y319F, effectively
blocked Ang II-induced activation of EGFR. Thus, protein-protein
interaction at the AT1-C containing tyrosine 319 may mediate Ang
II-induced EGFR activation. It has been shown in vitro that
the SH2 domain of SHP2 interacts with the YIPP motif, where Tyr-319 is
located, once tyrosine is phosphorylated (13, 15). Our results suggest
that the AT1 receptor and EGFR transiently interact with each other in
a ligand binding-dependent manner, and the timing of their
interaction coincides with that of Tyr-319 phosphorylation. Therefore,
we speculate that SHP2 binds to Tyr-319 when Tyr-319 is phosphorylated,
thereby acting as a scaffold protein. Because dominant negative SHP-2
(22) was able to inhibit both AT1 receptor-EGFR interaction and Ang II-induced transactivation of EGFR, it is likely that SHP-2 mediates AT1 receptor-EGFR interaction. It has been recently shown that EGFR
associates with
-adrenergic receptor in a ligand
binding-dependent manner, possibly through a scaffold
protein,
-arrestin (35). Thus, both SHP-2 and
-arrestin work as
scaffold proteins to induce interaction between G protein-coupled
receptors and EGFR for transactivation of EGFR. This hypothesis is
consistent with a recent observation that assembling signaling
molecules, including the AT1 receptor and EGFR, by scaffolding proteins
such as caveolin is required for Ang II-induced transactivation of EGFR
(36). It should be noted that phosphorylation of EGFR persists even
after interaction between the AT1R and EGFR is lost. We speculate that
once phosphorylation of EGFR is initiated, the activity of EGFR may be
maintained through autophosphorylation until other mechanisms of
inactivation are activated.
Hypothesis about the Back-up Mechanism--
Although Ang II
binding to the AT1-WT activates ERKs through an
EGFR-dependent mechanism, binding to AT1-Y319F still
activates ERKs through an Src-dependent mechanism despite
the fact that AT1-Y319F failed to activate EGFR. The cellular mechanism
by which the Src-dependent mechanism compensates for the
loss of EGFR activation to activate ERKs only in cells expressing
AT1-Y319F is unclear at present. One possible explanation would be that
EGFR may sequestrate molecules leading to Ras activation, such as Grb2,
when tyrosine 319 of the AT1 receptor is available. It should be noted
that it has been previously shown that tyrosine kinases are able to mediate Ang II-induced ERK activation only when a protein kinase C-dependent mechanism of ERK activation is blocked (37).
Thus, tyrosine kinases may in general work as a back-up mechanism for the AT1 receptor to maintain the activity of ERKs.
The Role of Tyrosine 319 in Cell Proliferation in Cardiac
Fibroblasts--
Our results show that tyrosine 319 of the AT1
receptor plays an essential role in mediating Ang II-induced cell
proliferation in cardiac fibroblasts. Although we have shown in this
work that tyrosine 319 of the AT1 receptor plays a key role in Ang
II-induced EGFR activation in cardiac fibroblasts, this does not
exclude the possibility that activation of other molecules may also
depend upon tyrosine 319. However, considering the fact that the
EGFR-specific inhibitor AG1478 completely abolished Ang II-induced cell
proliferation, it is likely that the effect of the mutation at tyrosine
319 is primarily mediated through its effect upon Ang II-induced EGFR activation. The signaling mechanisms of Ang II-induced cellular responses have been primarily studied by using either specific chemical
inhibitors or inhibitor molecules such as dominant negatives (10). Our
results suggest that the AT1 receptor mutant, which has a selective
defect in the signaling mechanism, can be used to elucidate the
cellular function of the signaling mechanisms activated by the AT1 receptors.