Tyrosine Kinase Activation by the Angiotensin II Receptor in the
Absence of Calcium Signaling*
T. N.
Doan,
M. Showkat
Ali, and
K. E.
Bernstein
From the Department of Pathology and Laboratory Medicine,
Emory University School of Medicine, Atlanta, Georgia 30322
Received for publication, April 17, 2001
 |
ABSTRACT |
The angiotensin II type 1 (AT1) receptor signals via heterotrimeric G-proteins
and intracellular tyrosine kinases. Here, we investigate a modified
AT1 receptor, termed M5, where the last five tyrosines
(residues 292, 302, 312, 319, and 339) within the intracellular
carboxyl tail have been mutated to phenylalanine. This receptor did not
elevate cytosolic free calcium or inositol phosphate production in
response to angiotensin II, suggesting an uncoupling of the receptor
from G-protein activation. Despite this, the M5 receptor still
activated tyrosine kinases, induced STAT1 tyrosine phosphorylation, and
stimulated cell proliferation. We also studied another AT1
mutant receptor, D74E, stably expressed in Chinese hamster
ovarian cells and a fibroblast cell line from mice with a genetic
inactivation of G
q/11. Both cell lines have a
deficit in calcium signaling and in G-protein activation, and yet in
both cell lines, angiotensin II induced the time-dependent tyrosine phosphorylation of STAT1. These studies are the first to show
the ability of a seven-transmembrane receptor to activate intracellular
tyrosine kinase pathways in the absence of a G-protein-coupled rise in
intracellular calcium.
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INTRODUCTION |
The AT11 receptor is a seven-transmembrane
receptor that signals via heterotrimeric G-proteins (1, 2). Ligand
binding activates Gq, leading to the generation of inositol
1,4,5-trisphosphate (IP3) and
a rise in cytosolic free calcium. This, in turn, affects cell
contractility, secretion, gene transcription, and cell proliferation.
More recently, the AT1 receptor has also been shown to
signal by activating nonmembrane tyrosine kinases including Src, Fyn, and Pyk2 (3). In 1995, our group reported that the binding of
angiotensin II to the AT1 receptor activated the
intracellular tyrosine kinase Jak2 and that this led to the tyrosine
phosphorylation and nuclear translocation of the transcription factor
STAT1 (4). Although the activation of tyrosine kinases by
G-protein-coupled receptor has now been established in several systems
including vascular smooth muscle cells (5, 6), mesangial cells (7), zona glomerulosa cells (8), and cardiac cells (9, 10), the
interplay between G-protein activation and receptor-mediated kinase
activation is less clear. Structure-function studies of the
AT1 receptor have defined amino acids critical for coupling the receptor to G-proteins. For example, mutation of either
Asp74 or Tyr292 results in the loss of
G-protein coupling and blocks the ligand-dependent production of IP3 (11, 12). However, the effect of these
mutations on AT1 receptor-dependent tyrosine
kinase signaling is not known. In fact, dissecting the importance of
angiotensin II-dependent tyrosine phosphorylation signaling
from G-protein activation is difficult because of multiple conversion
points. For example, both the G-protein and tyrosine kinase pathways
activate mitogen-activated protein kinase (9, 10), and both also give
rise to an increase in cytosolic free calcium (5, 6, 7).
To separate the effects of AT1 receptor activation of
G-proteins from that of tyrosine kinases, we studied a modified
AT1 receptor termed M5. The M5 mutant varies from the
wild-type AT1 receptor in that the last five tyrosines
(positions 292, 302, 312, 319, and 339) within the intracellular
carboxyl tail of the AT1 receptor were mutated to
phenylalanine. We found that the M5 receptor cannot induce a
ligand-mediated calcium signal, suggesting an uncoupling of the mutant
receptor from G-protein activation. In contrast, the M5 receptor
retained its ability to activate the Jak-STAT pathway. We also studied
two other cell lines with defects in calcium signaling. One is a
Chinese hamster ovarian cell (CHO) cell line stably transfected
with an AT1 receptor containing a D74E mutation. This
modified AT1 receptor was previously shown to be uncoupled
from G-protein activation (11). In addition, we investigated
angiotensin II signaling in fibroblasts derived from mice with a
genetic inactivation of G
q/11 (13, 14). In both cell
lines, angiotensin II stimulated the tyrosine phosphorylation of STAT1.
These studies are the first to show the ability of a seven-transmembrane receptor to activate an intracellular tyrosine kinase pathway in the absence of a G-protein-coupled rise in
intracellular calcium.
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MATERIALS AND METHODS |
Chinese Hamster Ovarian Cell Clones--
The M5 construct
was made using dut
ung
mutagenesis
(Bio-Rad) and verified by DNA sequencing. Native CHO were
maintained in F-12 media (Life Technologies, Inc.) supplemented
with 10% fetal bovine serum (Summit Biotechnology), 0.1 units/ml penicillin-streptomycin, 2 mM
L-glutamine, and 1 mM sodium pyruvate. For the
generation of AT1 and M5 CHO clones, the cells were stably
transfected with either wild-type rat AT1A or M5 plasmid
DNA under the control of the SV40 promoter (vector pZeoSV; Invitrogen,
Carlsbad, CA) using Lipofectin (Life Technologies, Inc.). The
transfected cells were selected with 250 µg/ml of zeocin until
individual clones were prevalent. The cells were ring-cloned and
screened for binding of 125I-sarcosyl1,
Ile8-angiotensin II (125I-Sar-Ile). The cells
were maintained in 125 µg/ml of zeocin for all subsequent cultures.
CHO cells stably expressing the AT1 D74E mutant were a kind
gift of Dr. Eric Clauser, INSERM, Paris, France (11). Fibroblasts derived from knockout mice with a targeted mutation inactivating G
q/11 were a kind gift of Dr. Melvin Simon, California
Institute of Technology, Pasadena, CA (13, 14).
125I-Sar-Ile Binding Assay--
Ligand binding was
measured as described previously (4). Briefly, cells were seeded onto
24-well plates overnight. The cells were washed twice with
physiological saline (in mM): 120 NaCl, 5 KCl, 1.2 MgSO4, 10 NaHCO3, 1.2 KH2PO4, 1 CaCl2, 10 Hepes, and
0.25% bovine serum albumin, pH 7.4. Varying concentrations of
125I-Sar-Ile were then added in the absence or presence of
1 µM angiotensin II for 1 h at room temperature. The
plates were placed on ice and washed four times with physiological
saline without bovine serum albumin. NaOH (0.5 M) was added
to each well, and the lysed cells were counted with a
counter.
Protein content was determined for each plate using a Bio-Rad
Dc protein assay kit (Bio-Rad).
Calcium Measurements--
Cells were harvested using 0.05%
trypsin/EDTA (Life Technologies, Inc.). The cells were washed with
Hepes buffer (in mM): 140 NaCl, 5 KCl, 2 CaCl2,
10 glucose, 1 MgCl2, and 10 Hepes, pH 7.4. The cells were
loaded with 20 µM fura-2/AM (Molecular Probes) for
30 min at 37 °C and washed for 30 min in the absence of fura-2/AM. Cells were resuspended in 2 ml Hepes buffer, and fluorescence measurements were taken on a time-based scale with an SLM Aminco spectrofluorometer. The fluorophore was excited at 340 and 380 nm. The
emission was collected at 510 nm. The ratio (340/380) values were
converted to calcium concentrations, where the Kd for fura-2 was 224 nM at 37 °C (15). The relative
maximum and relative minimum ratio were acquired in the presence of
0.1% Triton X-100 and 12.5 mM EGTA, respectively.
Inositol-1,4,5 Trisphosphate Production--
IP3
production was measured as described previously (5). Briefly, cells
were serum-deprived for 12-16 h and then stimulated with 100 nM angiotensin II for various times in 5 ml F-12 media. Trichloroacetic acid was added to stop the reaction. The cells were
scraped and centrifuged at 6000 × g for 10 min. The
supernatant was extracted with 1,1,2-trichloro-trifluoroethane and
trioctylamine (3:1). The aqueous top layer was saved, and the amount of
IP3 was determined by radioreceptor assay according to the
manufacturer's guidelines (PerkinElmer Life Sciences).
Immunoprecipitation and Western Blot--
Cells were
serum-deprived for 16-20 h and stimulated with 100 nM
angiotensin II for various durations. The cells were washed twice with
cold phosphate-buffered saline and lysed with 1 ml of RIPA (20 mM Tris, pH 7.5, 10% glycerol, 1% Triton X-100, 1% deoxycholic acid, 0.1% SDS, 2.5 mM EDTA, 50 mM
NaF, 1 mM phenylmethylsulfonyl fluoride, 1 mM
Na3VO4, and 10 µg/ml aprotinin). The cells
were scraped, sonicated, and centrifuged at 10,000 × g
for 10 min at 4 °C. The supernatant was collected, and protein
concentration was determined using the Bio-Rad Dc protein
assay kit. Lysate was incubated with 1-5 µg of antibody and 20 µl
of a 50% slurry of protein A/G plus Sepharose beads (Santa Cruz
Biotechnology, Santa Cruz, CA) for 2-24 h at 4 °C. The
immunoprecipitant was washed two-four times with wash buffer (25 mM Tris, pH 7.5, 150 mM NaCl, and 0.1% Triton
X-100) and resuspended in SDS loading buffer. Proteins were separated
on 8% SDS-PAGE and transferred onto nitrocellulose membranes
(Schleicher & Schüll). For Cos-7 cells, the cells were
transiently transfected with 0.5 µg of pBOS/Jak2 (gift from
Dr. D. M. Wojchowski, Pennsylvania State University) and 10 µg of either pZEO/AT1 or of pZEO/M5 using Lipofectin
(Life Technologies, Inc.).
The nitrocellulose membranes were blocked with 5% dry skim milk in
TBST (20 mM Tris, pH 7.2, 150 mM NaCl, and
0.05% Tween 20) for 1 h at room temperature. The membrane was
incubated with 1 µg/ml of monoclonal anti-phosphotyrosine (PY99;
Santa Cruz Biotechnology, Santa Cruz, CA), 1:1000 polyclonal anti-Jak2
(Upstate Biotechnology, Inc.), or 1:1000 monoclonal anti-STAT1
(Transduction Laboratories, Lexington, KY) for 2 h. The membrane
was washed three times for 30 min and subsequently incubated with
1:4000 secondary anti-mouse Ig (Amersham Pharmacia Biotech) for 1 h. The membrane was then washed and exposed for enhanced
chemiluminescence (PerkinElmer Life Sciences) on X-Omat Blue film
(Eastman Kodak Co., Rochester, NY). Unless otherwise stated, all
membranes were stripped after blotting with anti-phosphotyrosine and
reblotted with anti-Jak2 or anti-STAT1 to ensure equal loading of the
immunoprecipitated protein.
Cellular Proliferation Assay--
CHO cells were plated onto
96-well plates at a density of 5 × 103 cells/well
with F-12 media containing no serum for 16 h. The cells
were then treated with vehicle or angiotensin II for 4 h. MTS tetrazolium (Owen's reagent) (CellTiter 96®
Aqueous; Promega, Madison, WI) was added at a ratio of 1:5
relative to the volume of the media. The reaction proceeded for 4 h at 37 °C and was terminated by the addition of 2% SDS. The
absorbance was recorded at 490 nm. For each condition, the samples were
done in triplicate and averaged for each experiment.
All data were presented as representative trace or mean ± S.E.
Statistical significance was determined when p < 0.05 using student's t test.
 |
RESULTS |
Using site-directed mutagenesis, the rat AT1A receptor
tyrosine residues 292, 302, 312, 319, and 339 were converted to
phenylalanine. This mutant AT1 receptor, termed M5, lacks
tyrosine residues in the intracellular carboxyl portion of the
molecule. The wild-type AT1 and M5 mutant were each
transfected into cloned Chinese hamster ovarian cells, which were
screened for receptor expression by the binding of the angiotensin II
antagonist 125I-Sar-Ile. For further study, we selected
clones with similar level of expressed receptor; Scatchard analysis
indicated that the Bmax for cells expressing the
wild-type AT1 receptor was 0.33 ± 0.07 pmol/µg of
protein (clone 1, n = 7) although the
Bmax for the clone expressing the M5 receptor
was 0.47 ± 0.09 pmol/µg of protein (clone 5, n = 8). Receptor expression in these stable clones was significantly
higher than the endogenous angiotensin II receptors in native CHO cells
(0.15 ± 0.05 pmol/µg of protein; n = 4). The
wild-type AT1 receptor had a Kd of
0.24 ± 0.09 nM (n = 6) whereas the M5
cells had a Kd of 0.18 ± 0.04 nM
(n = 7). Thus, the M5 mutant showed an affinity for 125I-Sar-Ile that was equivalent to that of the wild-type receptor.
In CHO cells, the AT1 receptor couples only to
Gq and not Gs, Gi, or
Go (16). The activation of Gq results in the
increase of cytosolic free calcium released from intracellular stores
(2). To test whether G-protein coupling occurs with the M5 receptor, we
measured angiotensin II-mediated intracellular calcium release (Fig.
1). Nontransfected CHO cells have little
to no change in intracellular calcium when treated with 100 nM angiotensin II. In contrast, the addition of 100 µM ATP activated the endogenous purinergic
receptors resulting in a brisk transient increase in cytosolic free
calcium. An equivalent protocol was performed on cells stably
transfected for wild-type AT1 receptor (Fig.
1B). Here, the addition of angiotensin II resulted in a
transient rise in cytosolic free calcium, indicating activation of
Gq. As expected, the subsequent addition of ATP also
induced a transient increase in cytosolic free calcium. We observed
that losartan, a selective AT1 receptor antagonist,
completely blocked the angiotensin II-mediated calcium response but had
no effect on the ATP-evoked calcium response (data not shown). In
contrast to the response of the wild-type AT1 receptor, CHO
cells expressing the M5 mutant showed no increase in cytosolic free
calcium in response to angiotensin II (Fig. 1C). This is not
because of an intrinsic defect in calcium signaling per se,
because these cells were responsive to ATP. We also evaluated the
signaling of a second CHO clone expressing the M5 receptor (clone 8).
Despite a nearly 10-fold greater expression of M5 receptor levels than
observed with clone 5 cells, clone 8 still showed no rise of
intracellular calcium in response to angiotensin II (data not shown).
These data suggested that mutation of the carboxyl-terminal five
tyrosines of the AT1 receptor disabled receptor activation of Gq.

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Fig. 1.
Calcium signaling. Fura-2/AM-loaded CHO
native (A)-, AT1 (B)-, or M5
(C)-expressing cells were stimulated with 100 nM
angiotensin II (Ang II) and 100 µM ATP at the
time indicated by the arrow. The traces were representative
of at least four separate experiments for each cell type.
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Angiotensin II-mediated calcium signaling is dependent on
IP3 formation. Therefore, we investigated whether the M5
cells were deficient in IP3 production. As shown in Fig.
2A, CHO cells expressing the
wild-type AT1 receptor responded to angiotensin II with a rapid increase in IP3 levels within 15 s of
stimulation. Quantitation showed a 4.0 ± 1.9-fold increase over
basal levels of IP3 after 15 s (Fig. 2B).
In contrast, cells expressing the M5 receptor showed no significant
change (1.1 ± 0.1-fold) in IP3 production after
angiotensin II addition. These data are consistent with the lack of a
ligand-mediated calcium response; taken together, they suggest a loss
of Gq activation by the M5 receptor.

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Fig. 2.
IP3 production.
A, CHO AT1 ( )- or M5 ( )-expressing cells
were stimulated with 100 nM angiotensin for 0, 0.25, 0.5, 3, or 5 min. IP3 production was measured by radioreceptor
assay. The data are from one experiment that is representative
of three independent experiments. B, the average-fold change
of IP3 generation in AT1 or M5 cells when
stimulated with 100 nM angiotensin II for 15 s
(n = 3; * depicts p < 0.05).
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In addition to IP3 and calcium signaling, the
AT1 receptor also activates nonmembrane tyrosine kinases,
such as Jak2. Initially, we chose to investigate M5 activation of the
Jak-STAT pathway in transiently transfected Cos-7 cells. When these
cells were transiently transfected with constructs encoding both the
wild-type AT1 receptor and Jak2, angiotensin II induced a
marked increase of Jak2 tyrosine phosphorylation within 3 min of ligand
addition (Fig. 3A).
Surprisingly, the M5 receptor also responded to angiotensin II with an
increase of Jak2 tyrosine phosphorylation. In fact, repetition of this
protocol showed that the time course and magnitude of Jak2 activation
were similar for both the wild-type AT1 and M5 receptors.

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Fig. 3.
Jak2 and STAT1 phosphorylation. Cos-7
cells were co-transfected with 0.5 µg of pBOS/Jak2 and 10 µg of
either pZEO/AT1A (left panel) or pZEO/M5
(right panel). The cells were serum-deprived for 16-24 h
and were stimulated with angiotensin II (Ang II) for 0, 3, 6, or 10 min. Jak2 (Fig. 3A, n = 8) or STAT1
(Fig. 3B, n = 6) was immunoprecipitated
(IP) and immunoblotted (IB) as indicated in the
figure. For the STAT1 experiments (Fig. 3B), the cells were
treated with or without 10 µM valsartan (Val)
for 30-60 min before angiotensin II stimulation.
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A consequence of Jak2 activation is the tyrosine phosphorylation of the
transcription factor STAT1. When Cos-7 cells were transiently
transfected with Jak2 and either the wild-type AT1 receptor
or the M5 receptor, STAT1 was phosphorylated in an equivalent time-dependent fashion upon ligand addition. The
phosphorylation of STAT1 by angiotensin II was inhibited when cells
expressing either receptor type were pretreated with 10 µM valsartan, a specific AT1 receptor
antagonist. Thus, the experiments in Fig. 3 show that the M5 receptor,
despite its inability to signal via calcium stimulation, remains
fully capable of activating the Jak-STAT system in response to
angiotensin II.
In addition to Cos-7 cells, angiotensin II also stimulates STAT1
tyrosine phosphorylation in CHO cells stably expressing the AT1 receptor (17). Therefore, we asked whether a CHO clone
expressing the M5 receptor would induce STAT1 phosphorylation.
Stimulation of native CHO cells with angiotensin II did not show a
time-dependent change in STAT1 tyrosine phosphorylation
(Fig. 4A). In contrast, both
AT1- and M5-expressing cell clones showed an angiotensin II-mediated increase of STAT1 tyrosine phosphorylation that peaked at
about 30 min after ligand addition. The difference in the time course
of STAT1 tyrosine phosphorylation in transiently transfected Cos-7
cells (peak phosphorylation at 5-10 min) and stably transfected CHO
cells (peak phosphorylation at ~30 min) was equivalent to that found
in previously published work (4, 17). In summary, these experiments and
those in Fig. 3 show that the M5 receptor remains capable of activating
tyrosine kinases despite the absence of calcium signaling.

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Fig. 4.
Tyrosine phosphorylation of STAT1 in CHO
cells stably expressing AT1 and M5 receptors. Native
CHO cells (A), AT1 cells (B), or M5
cells (C) were stimulated with 100 nM
angiotensin II (Ang II) for 0, 30, 60, and 120 min.
Whole-cell lysates were immunoprecipitated (IP) with mAb
STAT1 and separated on 8% SDS-PAGE. After transferring the proteins
onto nitrocellulose membranes, the membranes were blotted for
phosphotyrosine using mAb PY99 or mAb STAT1. The blots were
representative of at least seven different experiments.
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The exact mechanism of angiotensin II-mediated STAT1 phosphorylation in
CHO cells appears to be somewhat different from that observed in the
transiently transfected Cos-7 cells. In CHO cells stably expressing the
wild-type AT1 or the mutant M5 receptors, we were unable to
document a ligand-mediated increase of Jak2 tyrosine phosphorylation
(data not shown). In addition, the Jak-specific inhibitor AG490 was not
effective in inhibiting angiotensin II-mediated STAT1
phosphorylation in CHO cells but did block STAT1 phosphorylation in
smooth muscle cells, a system in which angiotensin II-mediated stimulation of Jak2 has been well established (data not shown). In
contrast to the negative results with AG490, pretreatment of CHO
AT1 or M5 cells with the broad spectrum tyrosine kinase
inhibitor genistein did block angiotensin II-mediated STAT1 tyrosine
phosphorylation (Fig. 5). Thus, although
the exact mechanism for STAT1 tyrosine phosphorylation in CHO cells is
not fully understood, these data establish that, despite an absence of
calcium signaling, the M5 receptor can stimulate intracellular tyrosine
kinases in response to angiotensin II.

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Fig. 5.
Genistein inhibited the angiotensin
II-mediated phosphorylation of STAT1. CHO cells stably expressing
AT1 or M5 receptors were stimulated with angiotensin II
(Ang II) for 0, 30, 60, and 120 min. As indicated, some
cells were pretreated with 100 µM genistein for 1 h.
Whole cell lysates were immunoprecipitated (IP) and
immunoblotted (IB) as noted in the figure.
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To further investigate the relationship between heterotrimeric
G-proteins and angiotensin II-dependent activation of
tyrosine kinases, we studied two additional lines of cells. Previously, it was reported that a CHO cell line stably transfected with an AT1 receptor bearing a D74E point mutation does not couple
to heterotrimeric G-proteins and, as a consequence, does not mobilize intracellular calcium in response to angiotensin II (11). When the CHO
D74E cell line was stimulated with angiotensin II and studied for the
time-dependent tyrosine phosphorylation of STAT1, we found
this receptor was similar to the M5 receptor in that tyrosine kinase
activity was preserved in the absence of calcium signaling (Fig.
6A).

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Fig. 6.
STAT1 tyrosine phosphorylation in CHO cells
stably expressing AT1 D74E and in fibroblasts with
inactivated G q/11. A, serum-deprived
CHO D74E cells were stimulated with 100 nM angiotensin II
(Ang II) for 0, 30, 60, and 120 min. Whole-cell lysates were
immunoprecipitated (IP) with anti-phosphotyrosine mAb PY20
(Transduction Laboratories) and separated on 8% SDS-PAGE.
Proteins were transferred to nitrocellulose membranes and blotted with
mAb STAT1. The data presented are representative of at least three
different experiments. B, serum-deprived fibroblasts,
isolated from mice expressing inactivated G q/11, were
stimulated with 100 nM angiotensin II for 0, 3, 6, and 10 min. Whole-cell lysates were immunoprecipitated with mAb STAT1 and
separated on 8% SDS-PAGE. Proteins were transferred to nitrocellulose
membranes and blotted with mAb PY99 or mAb STAT1. These data are
representative of at least five different experiments.
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We also investigated angiotensin II-dependent tyrosine
phosphorylation of STAT1 using fibroblasts isolated from
G
q/11-deficient mice (13, 14). These mice were prepared
using embryonic stem cell targeting and bear a mutation in both
G
q and G
11 rendering these proteins
functionally inactive. Again, angiotensin II resulted in the tyrosine
phosphorylation of STAT1 in a time-dependent fashion (Fig.
6B). The time course of STAT1 phosphorylation was more rapid than that observed with CHO cells but was similar to the time course
observed in Cos-7 cells (Fig. 3) and in rat aortic smooth muscle cells
(4). As anticipated, the effect of angiotensin II on STAT1
phosphorylation in G
q/11-deficient fibroblast was markedly blunted by the AT1 receptor inhibitor valsartan
(data not shown).
A known effect of angiotensin II is the rapid stimulation of early
response genes (19) and the onset of cellular proliferation (2, 16).
The onset of cell proliferation was evaluated in CHO cells expressing
either the wild-type AT1 or the M5 receptor mutant. These
cells were serum-starved and then treated for 4 h in the absence
or presence of 100 nM angiotensin II (Fig.
7). The addition of angiotensin II
stimulated cellular proliferation in both AT1 and M5 cells
as compared with cells not treated with the ligand (Fig. 7, Ang II
versus Basal). The mitogenic effect of angiotensin II was
ablated when the AT1 or M5 cells were pretreated with
genistein or herbimycin, both potent inhibitors of tyrosine kinases.
The native CHO cells did not show any significant differences in cell
proliferation with or without angiotensin II (data not presented).
These data suggest that the activation of tyrosine kinases by
angiotensin II appears necessary for the induction of CHO cell
proliferation by this ligand. Furthermore, because proliferation is
observed with the M5 mutant, G-protein-mediated calcium signaling
appears not to be necessary for the proliferation initiated by
angiotensin II.

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Fig. 7.
Cell proliferation. CHO cells stably
expressing AT1 and M5 receptors were serum-deprived for
12-16 h. Cell proliferation was measured after 4 h in the absence
or presence of angiotensin II (Ang II), 100 µM
genistein (1 h pre-incubation), or 50 µM herbimycin (1 h
pre-incubation). An asterisk denotes p < 0.01 for a comparison of basal versus angiotensin II
addition to AT1 and M5 cells. The data were representative
of three individual experiments.
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 |
DISCUSSION |
The classical signaling pathway for the AT1 receptor
is to couple and activate heterotrimeric G-proteins. Recently,
experiments established that this receptor can also activate
nonmembrane tyrosine kinases including the Jak-STAT pathway (4, 18). A
problem in defining the functional role of angiotensin II-mediated
tyrosine kinase activation is the difficulty of differentiating the
effects mediated by heterotrimeric G-proteins and calcium elevation
from the effects predominantly due to the kinase activation. To gain insight into this question, we studied the M5 angiotensin II receptor. This molecule contains five discrete point mutations converting the
carboxyl five tyrosine residues to phenylalanine. In response to
ligand, the M5 receptor showed no significant elevation of IP3 and cytosolic free calcium. This observation was
consistent with the work of Marie et al. (12) who identified
a single point mutation of the AT1 receptor, Y292F, that
hindered the production of total inositol phosphates. Marie et
al. (12) concluded that this mutant receptor was uncoupled from
its endogenous G-proteins. In our hands, the M5 mutation is not
functionally identical to the Y292F mutation; we find that an
AT1 receptor with a single Y292F mutation showed a partial
calcium signal in response to angiotensin II (data not shown). Thus, we
believe that the four additional Tyr to Phe mutations present in
the M5 receptor are necessary for full elimination of G-protein-coupled signaling.
Despite the inability of the M5 receptor to stimulate an increase of
intracellular calcium, the receptor is still able to activate tyrosine
kinases including Jak2. Previously, our laboratory identified the
AT1 motif 319YIPP as critical in
angiotensin II-mediated activation of Jak2 (18). This work suggested
that it was the proline residues within the YIPP motif that were
critical for Jak2 activation. Because the M5 mutant contains a Y319F
mutation, the ability of the M5 mutant to stimulate Jak2 reemphasizes
that mechanisms apart from AT1 receptor phosphotyrosine
residues are critical in the activation process. At present, the exact
biochemical pathway by which the AT1 receptor stimulates
intracellular tyrosine kinases is unknown.
Other groups have investigated the structural basis for AT1
receptor activation of heterotrimeric proteins. In particular, Bihoreau
et al. (11) found that an AT1 receptor with a
D74E mutation cannot induce a calcium transient. Using CHO cells stably transfected with the D74E receptor by Bihoreau et al. (11), we independently confirmed that the AT1 D74E receptor does
not stimulate calcium but does stimulate STAT1 tyrosine
phosphorylation, similar to our experience with M5. Finally, our
studies of cells with a genetic inactivation of G
q and
G
11 showed that the AT1 receptor can
activate tyrosine phosphorylation in the absence of these G-proteins.
Signaling by the AT1 receptor in the absence of
G
q/11 is similar to M5 receptor signaling in that both
appear to represent examples of tyrosine kinase activation in the
absence of G-protein coupling.
Normally, angiotensin II-mediated signaling is complex with many
conversion points between signaling pathways stimulated by elevated
intracellular calcium and pathways downstream of kinase activation. Our
work now establishes that in CHO cells, the onset of cell proliferation
observed with angiotensin II can be attributed in large measure to an
activation of tyrosine kinase pathways. The creation of mutant
receptors lacking the ability to stimulate specific intracellular
pathways is a powerful approach toward establishing the functional
cause and effects of individual signaling pathways.
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ACKNOWLEDGEMENTS |
We thank Dr. Laurie Dirksen for help in
constructing the M5 receptor. Dr. Eric Clauser provided the
CHO-AT1 D74E clone. Dr. Melvin Simon provided the
fibroblasts isolated from mice expressing the inactivated
G
q/11 mutation. Dr. D. M. Wojchowski provided the
pBOS/Jak2 construct.
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FOOTNOTES |
*
This work was supported in part by National
Institutes of Health Grants DK39777, DK44280, DK51445, and HL61710.
Dr. Doan is supported by National Institutes of Health Grant
F32 HL10269.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.
To whom correspondence should be addressed: Woodruff
Memorial Bldg., Rm. 7107A, 1639 Pierce Dr., Emory University, Atlanta, GA 30322. Tel.: 404-727-3134; Fax: 404-727-8540; E-mail:
kbernst@emory.edu.
Published, JBC Papers in Press, April 23, 2001, DOI 10.1074/jbc.C100199200
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ABBREVIATIONS |
The abbreviations used are:
AT1, angiotensin II type 1;
IP3, inositol 1,4,5-trisphosphate;
STAT, signal transducers and activators of transcription;
CHO, Chinese
hamster ovarian cell(s);
PAGE, polyacrylamide gel electrophoresis;
mAb, monoclonal antibody;
125I-Sar-Ile, 125I-sarcosyl1, Ile8-angiotensin II.
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