Circulation Research. 2000;86:729-736
(Circulation Research. 2000;86:729.)
© 2000 American Heart Association, Inc.
The Third Cytoplasmic Loop of the Angiotensin II Type 1 Receptor Exerts Differential Effects on Extracellular SignalRegulated Kinase (ERK1/ERK2) and Apoptosis via Ras- and Rap1-Dependent Pathways
Judith Haendeler,
Mari Ishida,
Laszlo Hunyady,
Bradford C. Berk
From the Center for Cardiovascular Research, University of Rochester,
Rochester, NY. Present address of M.I. is Department of Clinical Laboratory,
Hiroshima University School of Medicine, Japan; present address of L.H. is
Department of Physiology, Semmelweis University of Medicine, Budapest,
Hungary.
Correspondence to Bradford C. Berk, MD, PhD, Center for Cardiovascular Research, University of Rochester, 601 Elmwood Ave, Box 679, Rochester, NY 14642. E-mail bradford_berk{at}urmc.rochester.edu
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Abstract
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AbstractThe third cytoplasmic
loop of the angiotensin
(Ang) II type 1 receptor
(AT
1) is important for receptor coupling
to G
proteins and activation of downstream events. Therefore,
we determined
whether specific AT
1 sequences were required for
kinase
activation and inhibition of apoptosis by transfecting
wild-type
(AT1Rwt) and mutated AT
1 into 293 cells. Ang II
stimulated a
19.4-fold increase in extracellular signalregulated
kinase
(ERK1/ERK2) activity in 293 cells transfected with AT1Rwt.
However,
in 293 cells that expressed a receptor in which amino acids
221
and 222 were deleted (AT1R[Del221/222]), Ang IImediated
ERK1/ERK2
activation was inhibited by >85%. In contrast, c-Jun
NH
2-terminal
protein kinase (JNK) activation was similar in
AT1Rwt- and AT1R(Del221/222)-transfected
cells. Activation of ERK1/ERK2
by AT1Rwt was independent of
Ca
2+, whereas the low level of
ERK1/ERK2 activation by AT1R(Del221/222)
was completely
Ca
2+ dependent. Activation of ERK1/ERK2 in AT1Rwt
required
Ras, whereas AT1R(Del221/222) required Rap1. These
results demonstrate
the presence of 2 different pathways for
ERK1/ERK2 activation by Ang
II, which differ in their requirements
for Ca
2+ and small G
proteins (Ras versus Rap1). Furthermore,
Ang II prevented serum
deprivationinduced apoptosis in
cells transfected
with AT1Rwt but not AT1R(Del221/222). AKT
was only
phosphorylated by Ang II in AT1Rwt-transfected cells.
Overexpression
of constitutively active AKT significantly reduced serum
deprivationinduced
apoptosis in cells transfected
with AT1R(Del221/222). This study
shows for the first time a direct
link between kinase activation
and inhibition of apoptosis
dependent on amino acids 221 and
222 in the third cytoplasmic loop of
the AT
1.
Key Words: angiotensin II apoptosis AT1 kinases
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Introduction
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Angiotensin II (Ang II), an octapeptide pressor hormone,
activates
cellular events that may contribute to the
pathogenesis of cardiovascular
disease.
1
The physiological actions of Ang II in adults are
mediated
largely via the Ang II type 1 receptor
(AT
1).
2 AT
1 is
a G proteincoupled
receptor (GPCR). GPCRs share a common basic
structure of 7 transmembrane
helices connected by alternating
cytoplasmic and extracellular
loops. Structure-function
analysis of the AT
1 has revealed
important
functional domains for signal transduction in the third
cytoplasmic
loop.
3 AT
1 couples
signaling events via heterotrimeric G proteins,
which occurs primarily
through the G
q/11 group of G proteins
and the
third cytoplasmic loop. Recent studies revealed that
a synthetic
peptide representing the proximal part of the third
cytoplasmic
loop (residues 216 to 230) was able to activate
purified G proteins,
whereas a peptide composing the distal loop
(residues 229 to
240) had no effect.
4 In addition, the
third cytoplasmic loop
appears to be necessary for Ang IImediated
conformational
changes in the AT
1 that result in
receptor activation.
3
Activation of AT1 by Ang II stimulates protein
synthesis and cell hypertrophy.5 6 7 Signaling
events required for the growth-promoting effects of Ang II downstream
of the AT1 include calcium mobilization and
stimulation of kinases such as p38, c-Jun
NH2-terminal protein kinase (JNK), and
extracellular signalregulated kinase (ERK1/ERK2).7 8 9 10
Furthermore, Ang II signaling through the AT1
also activates phosphatidylinositol 3-kinase (PI3K), which
leads to activation of the serine/threonine kinase AKT, also known as
protein kinase B (AKT).11 Activation of PI3K and
AKT is associated with growth stimulation by Ang II.12
Recent studies have demonstrated the antiapoptotic and
promitogenic functions of ERK1/ERK2 and AKT in different
cells.13 14 In contrast, JNK is activated by
inflammatory cytokines, reactive oxygen species, and Ang
II.10 Activation of JNK is associated with
proapoptotic effects.15 Therefore, the present
study was performed to elucidate the signaling pathways that are
dependent on the third cytoplasmic loop of the
AT1 and the effect of mutations in the third loop
on activation of kinases and inhibition of apoptosis.
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Materials and Methods
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Cell Culture, cDNA Constructs, and Transfection
293 cells were maintained in DMEM containing 10%
heat-inactivated
FBS. The plasmids for the wild-type
AT
1 (AT1Rwt) and the deletion
mutants were
subcloned as described previously.
16 Dominant-negative
RasN17,
Rap1N17, and CDC42N17 were generated as described
previously.
17 18 Plasmid DNAs were transfected into 293
cells using lipofectamine/Plus
according to the manufacturers
protocol. The final amount
of transfected DNA for a 60-mm dish was
adjusted to 1.3 µg
by pcDNA3.1-LacZ.
[Sar,Ile]Ang II Binding to Intact Cells
To determine the expression level and structural integrity of
the mutant receptor, the number of Ang II binding sites was determined
as described previously.16
Immunoblotting
After incubation with the indicated stimuli, cells were lysed in
lysis buffer (in mmol/L, Tris-HCl 20 [pH 7.5], NaCl 150, KCl
2.5, Na3VO4 2, NaF 50, DTT
2, and benzamidine 2) for 20 minutes and scraped off the plates. After
removing the cell debris, equal amounts of protein were loaded on
SDS-PAGE and then transferred to polyvinylidene
difluoride membranes. Immunoblotting was
performed with monoclonal antibodies directed against
phospho-ERK1/ERK2, phospho-AKT, and phospho-JNK (New England Biolabs);
ERK1/ERK2 and AT1 (Santa Cruz Biotechnology); and
Ras and Rap1 (Transduction Laboratories). Antibodies were detected by
the enhanced chemiluminescence system (Amersham).
Preparation of Recombinant Proteins
The Raf1-RBD- or the RalGDS-RBD-construct was transformed
into Escherichia coli D5
. Protein production was
initiated by addition of 1 mmol/L isopropyl
ß-D-thiogalactopyranoside to the culture.
Fusion proteins were affinity purified on gluthathione Sepharose 4B to
obtain glutathione S-transferase (GST)Raf1-Ras-binding
domain (RBD) to isolate RasGTP or GST-RalGDS-RBD
to isolate Rap1GTP.
Ras and Rap1 Activation Assays
Cells were lysed in lysis buffer containing 10 mmol/L
MgCl2. GST-Raf1-RBD or GST-RalGDS-RBD was added
to the supernatant and incubated overnight at 4°C with slight
agitation. After washing the beads 3 times with lysis buffer, Laemmli
sample buffer was added and samples were resolved on a 12%
SDS-PAGE.
JNK Activation Assay
JNK activity was measured with a commercially available kit
based on phosphorylation of recombinant c-Jun (New
England Biolabs).
Detection of Cell Death
Cells were cotransfected with 0.9 µg of
AT1 plasmids and 0.4 µg of pcDNA3.1-LacZ using
lipofectamine/Plus (GIBCO-BRL). After a 24-hour incubation, to allow
protein expression, apoptosis was induced by serum
deprivation for 24 hours. The dishes were
centrifuged to pellet-detached cells. Transfected living and
apoptotic cells were identified by ß-galactosidase staining.
Cells were fixed in 2% formaldehyde/0.2%
glutaraldehyde, and ß-galactosidase activity was
determined by incubation with 40 µg/mL X-gal for 24 hours at 37°C.
The transfection efficiency with 1.3 µg of pcDNA3.1-LacZ was 90±4%,
and the efficiency for the cotransfection was 71±5%. For
morphological staining of nuclei, cells were stained with DAPI as
described previously.19 Cell apoptosis was also
measured by terminal
deoxynucleotidyltransferasemediated
dUTP nick end labeling (TUNEL) staining and DNA laddering as described
previously.20
Statistical Analysis
Data are expressed as mean±SEM from
3 independent
experiments. Statistical analysis was performed with ANOVA
followed by modified least significant difference test.
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Results
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Effect of Ang II on ERK1/ERK2 Activation in 293 Cells Transfected
With AT1Rwt or AT1R(Del221/222)
We investigated the effect of a deletion in the third cytoplasmic
loop
of the AT
1 on ERK1/ERK2 activation by Ang
II. A series of experiments
supports a critical role for a conserved
apolar residue in the
third cytoplasmic loop (Leu222) in
agonist-induced activation
of the AT
1 and
possibly many other GPCRs.
21 There is also a
conserved
motif in the third cytoplasmic loop (amino acids 221
and 222)
present in both AT
1 and
AT
2, and absent in other GPCRs,
suggesting
important Ang IIspecific effects.
22 Therefore,
we have
chosen to determine the effects on Ang II signal transduction
of
deleting amino acids 221 and 222 in the AT
1. We
determined
that the expression and binding capacity of AT1R(Del221/222)
and
AT1Rwt for Ang II were equivalent (93±12% for AT1R[Del221/222])
compared
with AT1Rwt (data not shown).
21 However, coupling
to G proteins
by AT1R(Del221/222) was altered, because Ang II failed to
stimulate
phospholipid hydrolysis.
21 In the present
experiments, AT1Rwt
and AT1R(221/222) were transfected into 293 cells,
which do
not express AT
1. Equivalent protein
expression was verified
by Western blot analysis (Figure 1A

). To prove that Ang IIinduced
ERK1/ERK2
activation was due to activation of the
AT
1, ERK1/ERK2 activation
was blocked by
preincubation with losartan, an AT
1
antagonist
(Figure 1B

).

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Figure 1. Effect of Ang II on ERK1/ERK2 activation. A,
Equal expression of AT1Rwt and AT1R(Del221/222) in 293 cells (n=3). B,
293 cells were incubated with Ang II (100 nmol/L for every experiment
performed) in the presence or absence of losartan (1
µmol/L) for 5 minutes, and ERK1/ERK2 activation was measured (n=3;
upper panel). Equal loading of the blot was confirmed by reprobing with
an antibody against ERK1/ERK2 (lower panel). C, Time course
analysis for ERK1/ERK2 activation by Ang II in AT1Rwt- and
AT1R(Del221/222)-transfected cells. Cells were incubated with Ang II
for the indicated time points (upper panel). Equal loading was
confirmed with an antibody against ERK1/ERK2 (lower panel). D,
Densitometric analysis of ERK1/ERK2 activation by Ang II.
Extent of ERK1/ERK2 phosphorylation was quantified by
scanning densitometry using the NIH Image program and normalized
against ERK1/ERK2 protein content (n=3). IB indicates immunoblot.
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First, a time course was studied. Stimulation of both AT1Rwt and
AT1R(Del221/222) by Ang II caused maximal activation of ERK1/ERK2 at 3
minutes (Figures 1C
and 1D
). However, the ERK1/ERK2 activation
by Ang II in cells transfected with AT1R(Del221/222) was significantly
less than in cells transfected with AT1Rwt (3.0±0.5fold versus
19.4±3.2fold, P<0.01).
Effect of Cytoplasmic Ca2+ on Ang IIinduced
ERK1/ERK2 Activation
Cytoplasmic Ca2+ plays an important role in
Ang IImediated signal transduction.23 24 25 26 To determine
the calcium requirement for Ang IIinduced ERK1/ERK2 activation in
AT1Rwt- and AT1R(Del221/222)-transfected cells, we used BAPTA-AM, a
cytoplasmic Ca2+ chelator.24
ERK1/ERK2 activation by Ang II in AT1Rwt-transfected cells was almost
completely Ca2+ independent (Figure 2
). In contrast, ERK1/ERK2 activation in
AT1R(Del221/222)-transfected cells was completely
Ca2+ dependent. As a positive control, tumor
necrosis factor-
was used (Figure 2
). These results suggest
that 2 different pathways exist for Ang IIinduced ERK1/ERK2
activation by the AT1.
Effect of Ras and Rap1 on ERK1/ERK2 Activation
To gain further insight into the upstream mechanisms responsible
for Ca2+-dependent and -independent ERK1/ERK2
activation, we studied the ability of Ang II to stimulate Ras and Rap1.
Previously, it has been shown that Ca2+ seemed to
be required for activation of Rap1.27 Ras was
activated by Ang II in AT1Rwt-transfected cells (Figure 3A
) to a magnitude similar to that
observed in vascular smooth muscle cells.8 28 However, Ang
II failed to stimulate Ras activity in AT1R(Del221/222)-transfected
cells (Figure 3A
). In contrast, Rap1 was activated in
AT1R(Del221/222)-transfected cells, but not in AT1Rwt-transfected cells
(Figure 3B
). Chelation of Ca2+ by BAPTA-AM
completely abolished Rap1 activation by Ang II in
AT1R(Del221/222)-transfected cells (data not shown). Consistent
with these differences in activation of small G proteins,
cotransfection with RasN17, a dominant-negative mutant of Ras,
dramatically inhibited Ang IIinduced ERK1/ERK2 activation in AT1Rwt,
but not in AT1R(Del 221/222) (Figure 3C
). Furthermore,
cotransfection with Rap1N17, a dominant-negative mutant of Rap1, almost
completely abolished ERK1/ERK2 activation in AT1R(Del 221/222) (Figure 3D
). On the basis of these results, we speculate that the major
pathway for ERK1/ERK2 activation by AT1Rwt occurs via Ras activation
and, in addition, a minor pathway exists via Rap1 that is independent
of amino acids 221 and 222.

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Figure 3. Different effects of Ras and Rap1 on ERK1/ERK2
activation. A, Effect of Ras on Ang IIinduced ERK1/ERK2 activation in
AT1Rwt- or AT1R(Del221/222)-transfected cells. Ras activation was
determined using Raf1-RBD-GST (20 µg). Western blots were performed
with an antibody against Ras (upper panel). The extent of Ras
activation was quantified by scanning densitometry (lower panel) (n=3).
*P<0.01 vs control in AT1Rwt-transfected cells. B,
Effect of Rap1 on Ang IIinduced ERK1/ERK2 activation in AT1Rwt- or
AT1R(Del221/222)-transfected cells. Rap1 activation was determined
using RalGDSRBD-GST fusion protein (20 µg). Western blots were
performed with an antibody against Rap1 (upper panel). The extent of
Rap1 activation was quantified by scanning densitometry (lower panel)
(n=3). *P<0.01 vs control in
AT1R(Del221/222)-transfected cells. C, Effect of RasN17 on ERK1/ERK2
activation by Ang II. Cells were cotransfected with AT1Rwt or
AT1R(Del221/222) and LacZ or RasN17. Western blots were performed with
antibodies against phosphospecific ERK1/ERK2 (upper panel) or Ras
(lower panel). D, Effect of Rap1N17 on ERK1/ERK2 activation by Ang II.
IP indicates immunoprecipitation; IB, immunoblot.
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Effect of AT1R(Del221/222) on AKT Activation by Ang
II
It has previously been shown that Ang II binding to the
AT1 stimulated AKT, and AKT activity was required
for vascular smooth muscle cell growth.12 Therefore, the
effects of AT1R(Del221/222) on Ang IIinduced AKT-activation were
determined. Ang II activated AKT in cells transfected with
AT1Rwt, with peak at 10 minutes. No activation occurred in cells
transfected with AT1R(Del221/222) (Figure 4A
). Preincubation with the PI3K
antagonist, LY294002, completely inhibited Ang IIinduced
AKT activation in AT1Rwt-transfected cells (Figure 4B
). In
contrast, LY294002 did not show any effect on ERK1/ERK2 activation
by Ang II (Figure 4B
).

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Figure 4. Activation of AKT by Ang II. A, Effect on Ang
IIinduced AKT activation in AT1Rwt- or AT1R(Del221/222)-transfected
cells. Cells were incubated with Ang II for 10 minutes, and Western
blots were performed with an antibody against phosphospecific AKT
(p-AKT; residue 473). Membranes were reprobed with an antibody against
AKT (upper panel). The extent of AKT phosphorylation
was quantified by scanning densitometry (lower panel) (n=3).
*P<0.01 vs control in AT1Rwt-transfected cells. B,
Effect of LY294002 on Ang IIinduced AKT and ERK1/ERK2 activation.
Transfected cells were preincubated with LY294002 (10 µmol/L)
for 30 minutes, followed by treatment with Ang II for 10 minutes, and
Western blots were performed with an antibody against phosphospecific
AKT (residue 473). Membranes were then reprobed with
antiphospho-ERK1/ERK2 (n=3). IB indicates immunoblot.
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Effect of AT1R(Del221/222) on JNK Activation by Ang II
Although AKT and ERK1/ERK2 are involved in cell growth, JNK,
another kinase activated by Ang II, has been suggested to play
a role in apoptosis.15 25 Therefore, we examined
Ang IIinduced JNK activation in cells transfected with
AT1R(Del221/222). As expected, JNK was activated by Ang II in
AT1Rwt-transfected cells, with a maximal increase at 15 minutes (Figure 5A
). In AT1R(Del221/222)-transfected
cells, JNK was stimulated to a similar magnitude (Figure 5A
). To
determine whether activation of JNK via the AT1Rwt and the
AT1R(Del221/222) required the same upstream mediators, we studied the
role of CDC42, a known upstream activator of JNK. CDC42N17,
a dominant-negative mutant of CDC42, was cotransfected with AT1Rwt or
AT1R(Del221/222), and JNK activity was measured.18
Activation of JNK was completely abolished in 293 cells cotransfected
with CDC42N17 and AT1Rwt or AT1R(Del221222) (Figure 5B
). Cells
cotransfected with LacZ or RasN17 and AT1Rwt or AT1R(Del221/222) had no
effect on Ang IIinduced jun phosphorylation (Figure 5B
).

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Figure 5. Effect of Ang II and CDC42N17 on JNK activation.
A, Time course of JNK activation in AT1Rwt- and
AT1R(Del221/222)-transfected cells. Cells were incubated with Ang II
for the indicated times, and the JNK activation assay was performed
(upper panel). The extent of Jun phosphorylation was
quantified by scanning densitometry (lower panel; n=3). B, Effect of
CDC42N17 on Ang IIinduced JNK activation. Cells were cotransfected
with AT1Rwt or AT1R(Del221/222) and LacZ, CDC42N17, or RasN17 (upper
panel). The extent of Jun phosphorylation was
quantified by scanning (lower panel) (n=5). *P<0.01 vs
LacZ/control in AT1Rwt; **P<0.01 vs Ras/control in
AT1Rwt; #P<0.01 vs LacZ/control in AT1R(Del221/222);
##P<0.01 vs Ras/control in AT1R(Del221/222). IP
indicates immunoprecipitation; IB, immunoblot.
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Effect of Ang II on Serum DeprivationInduced
Apoptosis in AT1Rwt- and AT1R(Del221/222)-Transfected
Cells
To gain further insight into the physiological
role of amino acids 221 and 222 in the third cytoplasmic loop of the
AT1, we examined the effect of Ang II on serum
deprivationinduced apoptosis in 293 cells
transfected with AT1Rwt or AT1R(Del221/222. Apoptosis was
induced by removing serum for 24 hours in the presence or absence of
Ang II. Ang II significantly decreased apoptosis induced by
serum deprivation in cells transfected with AT1Rwt (Figures 6A
through 6C). However, in cells
transfected with AT1R(Del221/222), Ang II had no effect on
apoptosis induction (Figures 6A
through 6C). To ensure
that the morphological changes observed with DAPI staining were due to
apoptosis, we also measured apoptosis with TUNEL
staining and DNA laddering and obtained results similar to those with
DAPI stain (Figure 6B
and data not shown). To verify that the
effect of Ang II on serum deprivationinduced
apoptosis was not a nonspecific feature of
AT1R(Del221/222)-transfected cells, we determined the ability of
thrombin to prevent apoptosis. Serum
deprivationinduced apoptosis in AT1Rwt- and
AT1R(Del221/222)-transfected cells was significantly inhibited by
thrombin to the same extent (Figure 6C
). Furthermore, we
investigated H2O2-induced
apoptosis, which was also inhibited by Ang II in
AT1Rwt-transfected cells, but not in cells transfected with
AT1R(Del221/222) (data not shown).

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Figure 6. Signal transduction pathways involved in serum
deprivationinduced apoptosis. A, Effect of Ang II
on serum deprivationinduced apoptosis.
Apoptosis was induced in AT1Rwt- or
AT1R(Del221/222)-transfected cells by serum deprivation for
24 hours. Cells were coincubated with Ang II or PBS. After incubation,
cells were centrifuged to pellet detached cells and then
stained with DAPI. A representative experiment is
shown. B, Effect of Ang II on serum deprivationinduced
apoptosis. Cells were cultured on coverslips and treated as in
panel A. Cells were stained with TUNEL reagent
(Roche/Boehringer Mannheim). C, Effect of thrombin on serum
deprivationinduced apoptosis. Cells were
incubated with either thrombin (2 U/mL) or Ang II (n=5).
*P<0.01 vs serum deprivation in
AT1Rwt-transfected cells; #P<0.01 vs serum
deprivation in AT1R(Del221/222)-transfected cells. D,
Effect of PD98059 and LY294002 on serum
deprivationinduced apoptosis. Cells were
cotransfected with pcDNA3.1-LacZ and AT1Rwt or At1R(Del221/222). Cells
were coincubated with Ang II, PD98059 (10 µmol/L), and LY294002
(10 µmol/L) as indicated. Transfected living and
apoptotic cells were identified by ß-galactosidase staining
(n=5). *P<0.01 vs serum deprivation in
AT1Rwt-transfected cells; **P<0.05 vs serum
deprivation in AT1Rwt-transfected cells;
#P<0.01 vs serum deprivation+Ang II in
AT1Rwt-transfected cells.
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Because AKT and ERK1/ERK2 are both known to have antiapoptotic
functions, and our data demonstrate that AKT- and ERK1/ERK2-activation
occur via 2 independent pathways (Figure 4B
), we used
pharmacological inhibitors to determine their relative
roles in the antiapoptotic effect of Ang II. The PI3K
inhibitor LY294002 blocked the antiapoptotic effect
of Ang II in AT1Rwt-transfected cells to a greater extent than the
mitogen-activated protein kinase (MEK)/ERK1
inhibitor PD98059 (Figure 6D
). When used in
combination, PD98059 and LY294002 completely abolished the
antiapoptotic effect of Ang II in AT1Rwt-transfected cells
(Figure 6D
). In cells transfected with AT1R(Del221/222), the
inhibitors showed no effect (Figure 6D
).
Effect of Constitutively Active AKT on Serum
DeprivationInduced Apoptosis
We further investigated the role of Ang II in the signal
transduction pathways dependent on amino acids 221 and 222. We
cotransfected constitutively active AKT13 and
AT1R(Del221/222) to show that we could rescue cells from serum
deprivationinduced apoptosis. Indeed, as shown in
Figure 7
, overexpression of
constitutively active AKT significantly inhibited serum
deprivationinduced apoptosis in
AT1R(Del221/222)-transfected cells (Figure 7
).

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Figure 7. Effect of constitutive active AKT on serum
deprivationinduced apoptosis. Cells were
cotransfected with constitutively active AKT and AT1R(Del221/222).
After apoptosis induction, cells were stained with DAPI (n=4).
*P<0.01 vs serum deprivation in
AT1R(Del221/222)-transfected cells; #P<0.01 vs serum
deprivation+Ang II in AT1R(Del221/222)-transfected
cells.
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Discussion
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In the present study, we demonstrated that binding of Ang II
to
AT
1 activates ERK1/ERK2 via 2 pathways
and that activation of
ERK1/ERK2 and AKT by Ang II via the third
cytoplasmic loop of
the AT
1 is necessary for
serum deprivationinduced apoptosis
inhibition
(Figure 8

). The major pathway (as defined
by the
magnitude of ERK1/ERK2 activation) stimulates Ras, is
independent
of cytoplasmic Ca
2+, and requires the
presence of amino acids
221 and 222 in the third cytoplasmic loop,
whereas the minor
pathway activates Rap1 and is dependent on
cytoplasmic Ca
2+ and not on amino acids 221 and
222. In contrast, the third cytoplasmic
loop is not required for Ang
IImediated activation of
CDC42 and the downstream kinase, JNK.
Finally, the activation
of AKT and ERK1/ERK2 that is dependent on amino
acids 221 and
222 in the third cytoplasmic loop is required for Ang
IImediated
inhibition of serum deprivationinduced
apoptosis. These
data provide evidence for the first time that
ERK1/ERK2 activation
by Ang II occurs via 2 distinct pathways (via Ras
and via Rap1),
as well as a direct link between activation of several
kinases
by the third cytoplasmic loop of the AT
1
and inhibition of apoptosis
by Ang II.

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Figure 8. Model for the signaling events induced by Ang II
binding to the AT1. The major pathway for the ERK1/ERK2
activation and AKT activation requires amino acids 221 and 222 of the
third cytoplasmic loop of the AT1. These pathways are
necessary for the apoptosis inhibition by Ang II via the
AT1 (AT1R). In contrast, JNK activation via CDC42 and
activation of ERK1/ERK2 by Rap1 are independent of amino acids 221 and
222 and not involved in the inhibition of apoptosis.
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The "minor" pathway for ERK1/ERK2 activation by the
AT1, which requires Rap1 activity and cytoplasmic
Ca2+, was revealed by deletion of 2 amino acids
(221 and 222) in the third cytoplasmic loop. The fact that Rap1
activation was not observed in cells transfected with AT1Rwt has 2
possible explanations. The most likely explanation is that signals
stimulated by Ang II binding to the AT1Rwt inhibit Rap1 activity. A
less likely explanation is that the AT1R(Del 221/222) has acquired a
novel function (ie, a receptor metamorphosis) that results in altered
coupling to downstream mediators. Given that the deletion of amino
acids 221 and 222 in the AT1 abolishes
phosphoinositide turnover,21 it seems that
RAP1 activation requires an ambient level of intracellular calcium. The
fact that the 2 AT1-stimulated ERK1/ERK2 pathways
are characterized by a difference in their requirement for
Ca2+ may provide an explanation for reports that
have found varying degrees of calcium dependence for ERK1/ERK2
activation by Ang II in multiple cell types.23 26 29 30 It
is possible that differences in the relative expression of the
mediators responsible for the 2 pathways identified in the present
study may explain these findings. If such differences occurred in vivo,
there would be potentially significant
physiological effects in tissue responses to Ang
II.
Insight into the roles of the 2
AT1-dependent ERK1/ERK2 activation pathways may
be derived from consideration of the specific functions of Ras and
Rap1. Ras is activated by Grb2 adaptor proteins and the guanine
nucleotide exchange factor SOS, whereas Rap1 is
activated by CRK adaptor proteins and the
guanine-nucleotide-exchange factor C3G. Two 2
nonexclusive roles for Rap1 have been proposed, as
follows31 : (1) as a negative regulator of Ras-dependent
signal events and (2) as an upstream Ras-like small G protein that
couples to downstream kinases.30 32 In support of Rap1
inhibiting Ras are the data of Okada et al,33 which showed
a "competition" between Ras and Rap1 for binding to Raf1. These
authors showed that insulin stimulation of the human insulin receptor
inactivated Rap1 and decreased the amount of Rap1 bound to
Raf1, whereas association of Raf1 with Ras was increased. In contrast,
Franke et al27 showed in platelets that Rap1 was
activated by GPCRs, which required a rise in cytoplasmic
Ca2+ and was induced by agents that increase
cytoplasmic Ca2+. Finally, York et
al32 reported that activation of ERK1/ERK2 by nerve growth
factor in PC12 cells required both Ras and Rap1. Initial activation of
ERK1/ERK2 required Ras, but sustained ERK1/ERK2 activation required
Rap1 via stimulation of the downstream B-Raf/MEK/ERK cascade. Thus, our
data provide evidence that in cells expressing both B-Raf and Raf1, Ang
II may regulate ERK1/ERK2 via these parallel pathways.
The nature of the interaction between signal mediators and the
AT1 third cytoplasmic loop remains undefined.
However, it is clear that Ang II binding induces a conformational
change in the AT1 that, in the presence of
GTP-bound G proteins, confers an additional conformational change to
yield a high-affinity state of the receptor.34 Our
findings now indicate further that a critical conformational change in
the AT1 is mediated by the third cytoplasmic
loop. When this interaction is disrupted, alternative pathways such as
the Rap1-dependent pathway described in the present study are
revealed.
The signaling pathways that determine cell death versus cell
survival via the AT1 are poorly understood. It is
known that binding of Ang II to the AT1
activates AKT and ERK1/ERK2 and both kinases participate in
inhibition of apoptosis. An important
physiological property of Ang II is its ability to
regulate cell growth and apoptosis.19 35 Recently,
it has been found that Ang II can exert both antiapoptotic and
proapoptotic effects in a cell- and receptor-specific manner.
Binding of Ang II to the AT1 inhibits
apoptosis of vascular smooth muscle cells35 and
293 cells (as shown in the present study). In contrast, in
endothelial cells, the AT1
promotes apoptosis by activation of caspases via the
AT1 as well as via the
AT2.19 However, the regulatory
mechanisms by which Ang II inhibits apoptosis via the
AT1 in smooth muscle cells or other cell lines
are poorly defined. Pollman et al35 showed that Ang II
inhibited NO-induced apoptosis in vascular smooth muscle cells.
Furthermore, it is well known that PI3K, AKT, and ERK1/ERK2 are
involved in cell survival and in apoptosis
suppression.8 14 Thus, our data for the first time connect
the antiapoptotic functions of the AT1
with the activation of kinases AKT and ERK1/ERK2 because of amino acids
221 and 222 of the third cytoplasmic loop of the
AT1.
Consistent with our findings that the third cytoplasmic
loop is important for G protein coupling are recent studies
demonstrating that G proteins play a role in mitogenesis and cell
growth.36 Furthermore, JNK, which seems to have a
proapoptotic function, is not activated by events
dependent on amino acids 221 and 222. The effects of Ang II on growth
and apoptosis mediated by ERK1/ERK2, AKT, and JNK in vitro are
likely to be important in vivo, as shown by studies in the rat carotid
balloon injury model.37 Together with the present
study, these results suggest that the AT1 third
cytoplasmic loop is critical in determining the nature of Ang II
effects on cell growth and apoptosis. Moreover, mutations in
the third cytoplasmic loop of the AT1 or
alterations in G proteins that couple to the third cytoplasmic loop may
be pathogenic in vascular diseases such as hypertension,
atherosclerosis, and restenosis after balloon
angioplasty.
 |
Acknowledgments
|
|---|
This study was supported by NIH/National Heart, Lung, and
Blood
Institute Grants R01HL491921 and HL 59975 (to B.C.B.). J.H.
was
supported by Deutsche Forschungsgemeinschaft Grant HA 2868/1-1.
We
thank Dr Shalloway (Cornell University, Ithaca, NY) for providing
the
Raf-RBD, Dr Meinkoth (University of Pennsylvania, Philadelphia,
Pa) for
the RalGDS-RBD construct, and Dr S. Dimmeler (University
of Frankfurt,
Frankfurt, Germany) for the AKT construct.
Received October 13, 1999;
accepted January 27, 2000.
 |
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