From the
Signaling cascades elicited by angiotensin II resemble those
characteristic of growth factor stimulation. In this report, we
demonstrate that angiotensin II converges with platelet-derived growth
factor (PDGF)
Angiotensin II elicits a hypertrophic phenotype in cultures of
smooth muscle cells and may be a critical mediator of vascular
hypertrophy and neointimal
hyperplasia
(1, 2, 3, 4) . Recent cloning
of the vascular type I angiotensin II receptor provided evidence for
including it in the G-protein-coupled receptor
superfamily
(5, 6) . Interestingly, certain aspects of
signal transduction characteristic of angiotensin II stimulation
resemble those evoked by platelet-derived growth factor. Activation of
phospholipase C-
Induction of mitogen-activated protein kinase activity
by platelet-derived and other growth factors appears subsequent to
activation of p21
In an effort to understand smooth
muscle cell responses to vascular growth factors, we examined signaling
proteins upstream of p21
We first measured tyrosine phosphorylation on Shc and
subsequent recruitment of GRB2 in response to angiotensin II. Shc was
immunoprecipitated from quiescent cultures of RASMC exposed to various
concentrations of angiotensin II for 10 min. Analysis of
tyrosine-phosphorylated proteins in Shc immunoprecipitates revealed
phosphorylation of 46-, 52-, and 66-kDa Shc isoforms
(Fig. 1A). Further analysis illustrated the recruitment
of GRB2 to Shc immune complexes in parallel with Shc phosphorylation
(Fig. 1B). GRB2 association with Shc correlated
quantitatively with angiotensin II. Complex formation was detectable at
1 nM angiotensin II and maximal by 100 nM. In similar
experiments, complex formation was detected within 5 min and plateaued
by 10 min following angiotensin II exposure. The complex was still
prominent at 120 min (data not shown). A similar pattern of Shc
phosphorylation (Fig. 1A) and GRB2 association
(Fig. 1B) was observed following treatment with PDGF.
Analysis of the blots with an antibody directed against Shc confirmed
the identity of these phosphorylated bands and assured equivalent
precipitation efficiency (Fig. 1C).
Angiotensin II elicits signaling responses that resemble PDGF
signaling in vascular smooth muscle cells. Our work substantiates these
observations by demonstrating that angiotensin II stimulates the
association of Shc and GRB2 in rat aortic vascular smooth muscle. In
addition to this phenomenon, we present a novel link between the
angiotensin II G-protein-coupled receptor and PDGF receptor signaling.
In this model, the PDGF receptor serves as a docking site for known
upstream activators of p21-ras in smooth muscle cells responding to
angiotensin II. Evidence supporting this hypothesis include: (i) Shc
immune complexes from angiotensin II and PDGF-treated cells contained a
180-kDa tyrosine phosphorylated protein that co-migrates with the PDGF
Tyrosine phosphorylation on Shc and
subsequent complex formation with GRB2 appear central to upstream
signaling strategies elicited by a wide variety of growth factors and
cytokines. Shc
Our results suggest that the
similarity between the upstream signaling events elicited by
angiotensin II and PDGF extend beyond Shc
The appearance of c-Src in Shc immune complexes
isolated from angiotensin II-stimulated cells is notable in that Src is
a putative activator of Shc. This was established by work demonstrating
that v-Src transformed rat-1 cells contain constitutively
phosphorylated Shc
(30) . Further c-Src can associate directly
with activated PDGF receptors via its SH2 domain
(29) , and c-Src
has been detected in Shc immune complexes isolated from PDGF-treated
A10 smooth muscle cells
(21) . Activation of Src by angiotensin
II may represent the key convergence point between the angiotensin II
and PDGF signaling pathways. Catalytically active Src, in close
association with Shc and the receptor, may account for the increased
tyrosine phosphorylation observed on Shc and the PDGF receptor in
response to angiotensin II. It is interesting that a pivotal role for
Src family tyrosine kinases has been suggested in smooth muscle
contractile responses elicited by angiotensin II and growth
factors
(31) .
Angiotensin II increases PDGF A chain,
transforming growth factor-
Media
transfer experiments did, however, suggest liberation of a factor
distinct from PDGF. The angiotensin II type I receptor antagonist,
losartan, fully blocked activation of the pathway when angiotensin II
was added directly to cells. In contrast, losartan only partially
blocked the activity present in angiotensin II-conditioned media. The
identity of this factor requires further investigation, but it may fall
in the lipid category given the profile of rapid production and the
inability of smooth muscle cells to rapidly secrete proteins. In this
regard, angiotensin II stimulates phospholipases A
We have demonstrated a
novel mechanism wherein angiotensin II receptor signal transduction
links with PDGF
-receptor signaling cascades, independent of PDGF.
Stimulation of smooth muscle cells with angiotensin II resulted in
tyrosine phosphorylation on Shc proteins and subsequent complex
formation between Shc and growth factor receptor binding protein-2
(GRB2). A 180-kDa protein co-precipitating with Shc
GRB2 complexes
also demonstrated increased phosphorylation in response to angiotensin
II. Immunoblot analyses and proteolytic digests failed to distinguish
this 180-kDa protein from authentic PDGF
-receptors. Corresponding
with Shc and PDGF receptor phosphorylation induced by angiotensin II
was the recruitment and phosphorylation of c-Src. Autocrine release of
platelet-derived growth factor failed to account for Shc complex
formation at the PDGF receptor following angiotensin II treatment, and
a specific angiotensin II type I receptor antagonist, losartan,
abolished the response. These results support a novel model for
cross-talk between the G-protein-linked angiotensin II receptor and the
PDGF receptor tyrosine kinase in vascular smooth muscle cells.
Communication with the PDGF receptor may account for the ability of
angiotensin II to elicit responses typical of growth factor signal
transduction.
(7) , tyrosine
kinases
(8, 9) , mitogen-activated protein
kinases
(10, 11) , and expression of early growth
response genes
(12) exemplify phenomena common to angiotensin II
and platelet-derived growth factor signal transduction. Since the
angiotensin II type I receptor is G-protein linked, the mechanism by
which it activates processes characteristic of growth factor signaling
is unclear.
. Proteins upstream of
p21
have recently been identified that sense
growth factor receptor activation and stimulate guanine nucleotide
exchange on p21
(13-18). Growth factor receptor
binding protein-2 (GRB2)
(
) senses activated
receptors by binding specific, phosphorylated tyrosine residues via its
SH2 domain. In addition to binding to phosphorylated tyrosine kinase
receptors, GRB2 also recognizes phosphorylated tyrosine residues on Shc
proteins. A number of growth factors, including PDGF and angiotensin
II, can stimulate tyrosine phosphorylation on Shc (19-23). Shc
can bind directly to PDGF
-receptors in stimulated cells and may,
through recruitment of GRB2, relay receptor activation to downstream
signaling proteins
(20, 21) . Salcini et al. (24) have recently demonstrated the requirement for Shc
GRB2
complexes in the transformation of fibroblasts by overexpression of Shc
proteins. The SH3 domains of GRB2 facilitate stable complex formation
with the nucleotide exchange factor SOS. This Shc
GRB2
SOS
complex, thereby, transmits receptor ligand binding to
p21
(25) .
in cultures of isolated, rat
aortic smooth muscle cells (RASMC) exposed to angiotensin II. In this
report, we demonstrate that angiotensin II induces phosphorylation on
Shc and recruitment of GRB2. Furthermore, Shc
GRB2 immune
complexes contained phosphorylated PDGF
-receptors and c-Src.
Assembly of this complex in response to angiotensin II was independent
of autocrine PDGF release. These observations illustrate an unexpected
link between angiotensin II type I receptor and PDGF
-receptor
signal transduction in vascular smooth muscle cells and suggest a
mechanism by which angiotensin II elicits signaling events
characteristic of mitogenic stimuli.
Materials
Recombinant human BB-PDGF and
antibodies specific for phosphotyrosine, Shc, GRB2, PDGF
-receptors and PDGF were from Upstate Biotechnology Incorporated,
Transduction Laboratories or Santa Cruz Biotechnology. A
radioimmunoassay for human PDGF was obtained fom Amersham Corp. Assays
were performed on media as directed and values were corrected for rat
PDGF cross-reactivity. Cell culture reagents and Protein G-agarose were
from Life Technologies, Inc. Secondary antibodies were purchased from
Jackson ImmunoResearch (West Grove, PA). ECL reagents were from
Amersham.
Cell Culture
Rat aortic smooth muscle cells were
isolated as described
(26) and maintained in Dulbecco's
modified Eagle's medium (DMEM) containing 15% fetal calf serum.
Cells were split at confluence and fed every third day. Cultures were
used between passages 5 and 10. A10 smooth muscle cells were obtained
from the American Type Culture Collection and cultured identically to
the RASMC.
Immunoprecipitations
Cells were grown to
confluence and serum starved in DMEM lacking fetal calf serum for 18 h.
Following stimulation, cell medium was removed, and cells were rinsed
twice with ice-cold phosphate-buffered saline (Ca,
Mg
-free). Cells were scraped into lysis buffer A (20
mM HEPES, pH 7.4, 1% Triton X-100, 50 mM sodium
chloride, 1 mM EGTA, 5 mM
-glycerophosphate, 30
mM sodium pyrophosphate, 100 µM sodium
orthovanadate, 1 mM phenylmethylsulfonyl fluoride, 10
µg/ml aprotinin, and 10 µg/ml leupeptin)
(27) . Cell
debris was removed by centrifugation at 12,000
g for
10 min. Supernatants containing approximately 350 µg of protein
were transferred to new tubes containing 2 µg of indicated antibody
and incubated at 4 °C for 2.5 h with mixing. Protein G-agarose was
added and lysates incubated for an additional 30 min. Precipitates were
washed three times in ice-cold lysis buffer A and resuspended finally
in SDS-PAGE (28) sample buffer. Complexes were boiled for 5 min and
electrophoresed through 10% SDS-PAGE gels. Proteins were transferred to
PVDF membranes (Immobilon, Millipore Corp., Bedford, MA) and processed
for immunoblot analysis.
Immunoblot Analysis
Nonspecific binding sites were
blocked in PBS containing 0.1% Tween 20 (PBS-T) and 1% bovine serum
albumin for 1 h at 20 °C. Primary antibodies were diluted in
blocking solution and incubated with the membranes for 1 h at room
temperature. Excess primary antibody was removed by washing the
membranes four times in PBS-T. The blots were incubated with
appropriate secondary antibodies in PBS-T containing 5% milk diluent
(Kirkegaard and Perry Laboratories, Inc.) for 1 h. Membranes were
washed as before and processed for ECL. In certain experiments the
filters were reprobed after stripping in 0.1 M Tris-HCl, pH
8.0, 2% SDS, and 100 mM -mercaptoethanol for 30 min at 52
°C. Filters were rinsed briefly in PBS-T and processed as above
with a different primary antibody.
V8 Digests
Serum-starved RASMC cultures were
labeled with 5 mCi/ml [P]orthophosphate in
phosphate-free DMEM. Cells were washed five times with ice-cold PBS to
remove unincorporated label, then stimulated with PDGF at 30 ng/ml or
angiotensin II at 1 µM for 15 min. Shc or PDGF receptors
were precipitated as described above and complexes resolved by
SDS-PAGE. Following exposure to film, gel fragments containing the
labeled 180-kDa protein were excised and transferred to a fresh gel.
SDS-PAGE sample buffer containing 2 µg of purified V8 protease was
added to indicated lanes and samples electrophoresed into a 4% stacking
gel (29). Electrophoresis was stopped for 1 h to allow digestion of the
proteins. Protein fragments were resolved through a 10% gel and exposed
to film for autoradiography.
Media Transfer Experiments
To examine PDGF
activity, media from RASMC exposed to angiotensin II at various
concentrations was transferred onto serum-starved A10 cells and
incubated at 37 °C for 10 min. Cells were lysed and 200 µg of
protein analyzed for phosphotyrosine incorporation by immunoblotting.
In certain experiments, media from RASMC treated with angiotensin II
was saturated with an antibody capable of neutralizing PDGF for 15 min
at 37 °C. Neutralized media was transferred onto fresh RASMC and
incubated for 10 min prior to analysis for Shc and receptor
phosphorylation. To examine autocrine release of factors other than
PDGF, media from RASMC treated with 10 nM angiotensin II was
saturated with 10 µM losartan and transferred onto fresh
RASMC that had been preincubated with 10 µM losartan.
Cells were incubated for 15 min at 37 °C and Shc protein complexes
analyzed as above.
Figure 1:
Angiotensin II stimulates Shc
phosphorylation and association with GRB2 and additional proteins in
vascular smooth muscle cells. Quiescent, confluent monolayers of RASMC
were stimulated with various concentrations of angiotensin II
(lanes 1-12) or PDGF (lane 13) for 10 min.
A, Shc immunoprecipitates were analyzed for phosphotyrosine
containing proteins and GRB2 (B). The blot in A was
stripped and reprobed with an antibody specific for Shc
(C).
Additional
tyrosine-phosphorylated proteins appeared in Shc immune complexes in
response to angiotensin II with a profile strikingly similar to that
observed from cells treated with PDGF (Fig. 1A). Two
such proteins at 60 and 180 kDa are similar in size to the protein
tyrosine kinase, Src, and the PDGF -receptor, respectively.
Tyrosine phosphorylation on these proteins correlated with
Shc
GRB2 complex formation both temporally and with respect to
angiotensin II concentration. This warranted additional investigation
because Src and PDGF
-receptors are putative activators of
Shc
(20, 30) . Analysis of the 180 kDa region with an
antibody specific for the PDGF
-receptor demonstrated
cross-reactivity with this protein and suggested that the receptor,
Shc, and GRB2 occur in a single complex in response to angiotensin II
(Fig. 2A). In agreement with the presence of the PDGF
receptor, a c-Src antibody recognized the phosphorylated 60-kDa protein
that co-precipitated with Shc in angiotensin II- and PDGF-stimulated
cells. This demonstrated recruitment of c-Src to the complex in
response to angiotensin II or PDGF (Fig. 2B).
Figure 2:
Association of PDGF receptors and c-Src
with Shc immune complexes isolated from cells treated with angiotensin
II or PDGF. Serum-deprived RASMC were stimulated with vehicle, 1
µM angiotensin II, or 30 ng PDGF/ml for 10 min. Shc
immunoprecipitates were analyzed with antibodies directed against the
PDGF -receptor (A) and c-Src
(B).
To
explore the possibility that PDGF -receptors contribute to
angiotensin II signaling, we next examined receptor phosphorylation in
immune complexes precipitated by two additional antibodies.
Fig. 3A (upper exposure) illustrates that
antibodies directed against Shc, GRB2, or phosphotyrosine
co-precipitated the 180-kDa protein and confirmed that tyrosine
phosphorylation of this protein was enhanced by angiotensin II or PDGF.
Again, identity of the 180-kDa protein as the PDGF
-receptor was
suggested by cross-reactivity with a PDGF
-receptor-specific
antibody (Fig. 3A, lower exposure).
Phosphorylation of the 180-kDa protein was further demonstrated by
incorporation of radiolabeled phosphate. PDGF
-receptors and Shc
were immunoprecipitated from angiotensin II- or PDGF-stimulated RASMC
that had been labeled with [
P]orthophosphate.
Label incorporation into precipitated PDGF
-receptors and the
180-kDa protein increased approximately 2-fold in PDGF and angiotensin
II-stimulated cells and confirmed agonist-dependent phosphorylation
(Fig. 3B).
Figure 3:
Phosphorylation of a 180-kDa protein
co-precipitating with Shc from angiotensin II- or PDGF-stimulated
cells. Serum-deprived, confluent monolayers of RASMC were stimulated
with 1 µM angiotensin II or 30 ng/ml PDGF. A.
tyrosine-phosphorylated PDGF receptors were co-precipitated from RASMC
treated with angiotensin II or PDGF using antibodies directed against
Shc (lanes 1-3), GRB2 (lanes 4-6), and
phosphotyrosine (lanes 7-9). The membranes were stripped
and analyzed with an antibody specific for the PDGF -receptor
(lower panel). B, RASMC were incubated in
phosphate-free DMEM supplemented with 5 mCi/ml
[
P]orthophosphate. Cultures were then stimulated
with vehicle (lane 1) PDGF, (lanes 2 and 3),
or angiotensin II (lanes 4 and 5). PDGF
-receptor (lanes 1, 2, and 4) or Shc
(lanes 3 and 5) complexes were immunoprecipitated.
Precipitated proteins were fractionated by SDS-PAGE and exposed for
autoradiography.
Successful incorporation of radiolabel
into the 180-kDa protein following angiotensin II stimulation allowed
comparison of proteolytic maps of the candidate band to authentic PDGF
-receptor digestions. Radiolabeled protein co-migrating with
authentic PDGF
-receptor (Fig. 4A) was excised,
transferred to a new gel and digested with V8 protease.
Fig. 4B typifies the digestion patterns of authentic
PDGF
-receptors and the 180-kDa protein. Numerous
protease-dependent fragments characterized authentic PDGF
-receptor digests and mirrored the pattern liberated by digestion
of the 180-kDa protein. Proteolytic maps in conjunction with antibody
cross-reactivity confirmed the 180-kDa protein present in Shc immune
precipitations as the PDGF
-receptor.
Figure 4:
Angiotensin II stimulates the
incorporation of radiolabeled phosphate into PDGF receptors in vascular
smooth muscle cells. RASMC were incubated in phosphate-free DMEM
supplemented with 5 mCi/ml [P]orthophosphate.
A, cultures were then stimulated with PDGF (lanes
1-3) or angiotensin II (lane 4). PDGF
-receptor (lanes 1 and 2) or Shc (lanes 3
and 4) complexes were immunoprecipitated. Precipitated proteins
were fractionated by SDS-PAGE and exposed for autoradiography.
B, the 180-kDa proteins labeled in A were excised,
transferred to a fresh gel, and treated with buffer (lane 1)
or buffer containing 2 µg of V8 protease (lanes
2-4). Lanes 1 and 2 illustrate patterns of
undigested and digested PDGF
- receptors, respectively.
Digestion patterns of the labeled 180-kDa protein that
co-precipitates with Shc in response to PDGF or angiotensin II are
presented in lanes 3 and 4,
respectively.
We next examined whether
the link between angiotensin II type I receptor stimulation and
assembly of Shc signaling complexes at the PDGF receptor was induced by
autocrine release of PDGF. We have observed previously that the rat A10
smooth muscle cell line is non-responsive to angiotensin II with
respect to eliciting tyrosine phosphorylation, but fully responsive to
PDGF in this regard. This cell line, therefore, provided a sensitive
system for detection of PDGF release by RASMC responding to angiotensin
II. Stimulation of PDGF receptor tyrosine kinase activity in A10 cell
cultures is evident by immunoblotting following exposure to as little
as 100 pg of PDGF/ml. When media from RASMC conditioned with
angiotensin II was transferred onto A10 cell cultures, however,
tyrosine phosphorylation on PDGF receptors was undetectable
(Fig. 5A). Fig. 5B provides additional
evidence for the lack of PDGF activity in media from RASMC conditioned
with angiotensin II. In this approach, media from RASMC treated with
angiotensin II was saturated with an antibody that neutralizes PDGF and
then transferred onto fresh cultures. Control experiments demonstrated
that the antibody could neutralize at least 10 ng of PDGF/ml.
Consistent with the A10 cell assay, activity in the media from
angiotensin II-stimulated cells was fully effective in eliciting Shc
and receptor phosphorylation. Finally, media from cells exposed to
angiotensin II contained undetectable levels of PDGF as determined by a
sensitive radioimmunoassay (data not shown).
Figure 5:
Autocrine release of PDGF fails to account
for angiotensin II stimulation of PDGF receptor signaling cascades.
A, media from angiotensin II conditioned RASMC was transferred
to cultures of rat A10 vascular smooth muscle cells. PDGF receptor
phosphorylation in cell lysates was determined by immunoblot analysis.
B, Media from angiotensin II-conditioned RASMC was saturated
with a PDGF-neutralizing antibody and transferred onto fresh cells. Shc
complexes were isolated and analyzed as in Fig.
1.
Although PDGF release
failed to account for activation of the signaling pathway in response
to angiotensin II, this did not rule out autocrine release of a factor
different from PDGF. To examine this possibility, we tested whether
media transferred from RASMC stimulated with 10 nM angiotensin
II could activate the signaling cascade in the presence of losartan.
Fig. 6A illustrates that losartan pretreatment of the
cells abolished complex formation following direct addition of 10
nM angiotensin II. In contrast, angiotensin II-conditioned
media contained an activity that was not fully blocked by the
angiotensin II receptor antagonist (Fig. 6B), suggesting
liberation of a factor other than PDGF.
Figure 6:
Losartan
inhibits Shc phosphorylation following direct addition of angiotensin
II to RASMC, but fails to inhibit activity in angiotensin
II-conditioned media. A, RASMC were pretreated with or without
10 µM losartan then stimulated with 10 nM
angiotensin II. B, media from primary RASMC exposed to
angiotensin II was transferred to fresh cells, pretreated with 10
µM losartan. In each experiment, Shc was
immunoprecipitated and proteins analyzed for
phosphotyrosine.
-receptor; (ii) an antibody specific for the PDGF
-receptor
cross-reacted with the 180-kDa protein that co-precipitates with Shc in
response to angiotensin II; (iii) proteolytic maps of the 180-kDa
protein are indistinguishable from authentic PDGF receptors; and (iv)
autocrine release of PDGF failed to account for Shc complex formation
in response to angiotensin II.
GRB2 complex formation is critical to activation of
p21
in cells responding to insulin
(24) ,
EGF
(23) , and in the transformation of cells by over expression
of Shc
(24) . Our observation that Shc and GRB2 associate in
response to angiotensin II implicates Shc and GRB2 as important
mediators in the angiotensin II signaling response. This is in
agreement with work by Schorb et al.(22) demonstrating
the phosphorylation of Shc in cardiac fibroblasts following exposure to
angiotensin II. Although they did not evaluate GRB2 recruitment to Shc,
it is likely that both cardiac fibroblasts and vascular smooth muscle
cells utilize this pathway to transduce angiotensin II signals. Since
Shc
GRB2 complex formation contributes to PDGF signal transduction
in vascular smooth muscle cells, activation of these molecules may
provide a mechanism through which angiotensin II activates
mitogen-activated protein kinases.
GRB2 complex formation.
To this end, we have detected PDGF receptors and c-Src in Shc immune
complexes isolated from angiotensin II-treated vascular smooth muscle
cells
(21) . Both of these proteins co-precipitate with Shc from
cells responding to PDGF. The appearance of PDGF receptors in Shc
immune complexes is consistent with recent results demonstrating the
association of Shc and PDGF receptors in unstimulated vascular smooth
muscle cells. Support for this hypothesis is provided by Fig. 2.
Identification of the 180-kDa protein as the PDGF receptor by antibody
cross-reactivity also shows little change in the amount of receptor
co-precipitated following angiotensin II stimulation. The recruitment
of GRB2 and c-Src to the complex, however, is clearly increased and
agonist dependent. Fig. 3A adds further support to this
hypothesis. Precipitations performed with antibodies specific for GRB2
and phosphotyrosine contain increased amounts of PDGF receptor in
response to angiotensin II and PDGF that correspond with
phosphorylation on the receptor. In contrast, precipitation with
antibodies to Shc showed constant receptor levels even though
phosphorylation on the receptor increased. Since Shc can associate with
the receptor in unstimulated cells and angiotensin II causes tyrosine
phosphorylation on Shc, one might expect the complex to assemble in
response to angiotensin II by using the PDGF receptor as a docking
site. Whether the receptor is activated in this complex, as might by
suggested by enhanced tyrosine phosphorylation, requires further
investigation.
1 and basic fibroblast growth factor
expression in vascular smooth muscle cells
(32) . In view of
this, we evaluated whether autocrine release of PDGF accounted for PDGF
receptor involvement in angiotensin II signal transduction. Since
maximal stimulation of Shc
GRB2 complex formation in response to
angiotensin II corresponded with PDGF at 10 ng/ml, we anticipated that
release of PDGF in response to angiotensin II would approach a similar
value. Several assays capable of detecting PDGF at concentrations much
lower that 10 ng/ml ruled out secretion of PDGF as the mechanism for
activation of the signaling pathway by angiotensin II. First, media
from cells exposed to angiotensin II contained undetectable levels of
PDGF as determined by a sensitive radioimmunoassay (data not shown).
This agrees with previous reports that adult rat aortic smooth muscle
cells synthesize and release little PDGF
(33) . Second, media
from cultures of RASMC exposed to various concentrations of angiotensin
II contained no activity capable of inducing PDGF receptor
phosphorylation in the angiotensin II-insensitive rat A10 vascular
smooth muscle cell line. PDGF concentrations as low as 100 pg/ml
stimulated detectable phosphorylation on PDGF receptors in A10 cell
cultures and verified this system as a sensitive bioassay for PDGF.
Lack of activity in the angiotensin II-conditioned media, therefore,
indicated a PDGF concentration below 100 pg/ml. Finally, a PDGF
neutralizing antibody failed to block activation of the signaling
pathway when added to angiotensin II-conditioned media. Taken together,
these data eliminate PDGF release as the mechanism for PDGF
-receptor phosphorylation in response to angiotensin II.
, C, and
D activity in vascular smooth muscle cultures with a time course
similar to activation of the Shc/GRB2
pathway
(34, 35, 36) .
-receptor signaling cascades and results in
activation of proteins upstream of p21
. The direct
involvement of the PDGF receptor in this process presents a novel
paradigm for cross-talk between G-protein-linked and growth factor
receptor signaling pathways. The activation of this pathway by either
PDGF or angiotensin II is paradoxical in that angiotensin II does not
elicit a mitogenic response typical of PDGF. This suggests that
additional, PDGF-specific pathways are required for full mitogenic
potential or that divergence of the pathways, downstream of Shc/GRB2,
facilitates the specific biological responses characteristic of these
two growth factors.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.