Circulation Research. 1997;80:607-616
(Circulation Research. 1997;80:607-616.)
© 1997 American Heart Association, Inc.
Angiotensin II Signal Transduction in Vascular Smooth Muscle
Role of Tyrosine Kinases
Bradford C. Berk,
,
Marshall A. Corson
From the University of Washington, Department of Medicine, Cardiology
Division, Seattle.
Correspondence to Bradford C. Berk, MD, PhD, University of Washington, Department of Medicine, Mail Stop 357710, Seattle, WA 98195. E-mail bcberk{at}u.washington.edu
 |
Abstract
|
|---|
Abstract In this review, the role of tyrosine kinases in
angiotensin
IImediated signal transduction pathways in
vascular smooth
muscle is discussed. Angiotensin II was
isolated by virtue of
its vasoconstrictor abilities and has long been
thought to play
a critical role in hypertension. However, recent
studies indicate
important roles for angiotensin II in
inflammation, atherosclerosis,
and congestive heart
failure. The expanding role of angiotensin
II indicates
that multiple signal transduction pathways are
likely to be
activated in a tissue-specific manner. Exciting
recent data
show that angiotensin II directly stimulates tyrosine
kinases,
including pp60
c-src kinase (c-Src), focal adhesion
kinase (FAK),
and Janus kinases (JAK2 and TYK2).
Angiotensin II may activate
receptor tyrosine
kinases, such as Axl and platelet-derived
growth factor, by
as-yet-undefined autocrine mechanisms. Finally,
unknown tyrosine
kinases may mediate tyrosine phosphorylation
of Shc,
Raf, and phospholipase C-

after angiotensin II
stimulation.
These angiotensin IIregulated tyrosine
kinases appear
to be required for angiotensin II effects,
such as vasoconstriction,
proto-oncogene expression, and protein
synthesis, on the basis
of studies with tyrosine kinase
inhibitors. Thus, understanding
angiotensin
IIstimulated signaling events, especially
those related to tyrosine
kinase activity, may form the basis
for the development of new
therapies for cardiovascular diseases.
Key Words: tyrosine kinase signal transduction Src focal adhesion kinase platelet-derived growth factor
 |
Introduction
|
|---|
Angiotensin II, the
dominant effector of the renin-angiotensin
system,
regulates numerous physiological responses,
including
salt and water balance, blood pressure, and vascular tone.
Angiotensin
II is produced both systemically and locally in
the vessel wall
by the actions of renin, which converts
angiotensinogen into
angiotensin I, and ACE,
which cleaves angiotensin I to form
angiotensin
II. Although the vasoactive effects of angiotensin
II have
been well described for >40 years,
1 it has recently
been
appreciated that angiotensin II has many other effects
on
cardiovascular function. For example,
angiotensin II has
been shown to stimulate VSMC
growth,
2 3 increase the expression
of enzymes that produce
mediators of inflammation, such as phospholipase
A
2 and
NAD(P)H oxidase,
4 5 stimulate the JAK/STAT
pathway,
6 and activate gene transcription of
proto-oncogenes, such as
c-
fos.
7 These data
suggest that angiotensin II plays an important
role in
various cardiovascular diseases associated with VSMC
growth
and vessel wall inflammation, such as hypertension,
atherosclerosis,
and restenosis following
interventional procedures. Clinical
trials with ACE
inhibitors demonstrating survival benefits in
congestive
heart failure
8 and myocardial infarction
9
support
the importance of angiotensin II in the
pathogenesis of cardiovascular
disease. In this review
we will focus on signal transduction
mechanisms by which
angiotensin II exerts these effects, emphasizing
the role
of tyrosine kinases.
 |
The AT1 Receptor: Binding of
Angiotensin II
|
|---|
Angiotensin II binds to at least two
high-affinity receptors,
designated AT
1
receptor
10 11 and AT
2 receptor.
12
To date,
the signal transduction pathways activated by the
AT
2 receptor
remain unknown, so this review will focus on
AT
1 signaling.
The signal transduction events stimulated by
angiotensin II
binding to the AT
1 receptor are
similar to those stimulated
by growth factors and cytokines and
include activation of PLC,
13 calcium
mobilization,
14 activation of PKC,
15
induction of
proto-oncogenes,
7 protein tyrosine
phosphorylation,
16 17 18 and activation of
the 42- and 44-kD MAP kinases.
19 20
Analysis of AT1 receptor domains required for
binding angiotensin II, stimulating G protein activity, and
mediating downstream signal transduction became possible when the
AT1 receptor was cloned by two laboratories in
1991.10 11 The structure of the AT1 receptor
shows that it is a member of the seven transmembranespanning, G
proteincoupled receptor family. Other receptors in this family
include the
- and ß-adrenergic receptors, the muscarinic
acetylcholine receptor, the various serotonin receptors,
and important vascular receptors such as the thrombin, bradykinin, and
endothelin receptors. These receptors share the property that they bind
to heterotrimeric G proteins and lack intrinsic tyrosine kinase
activity, in contrast to growth factor receptors, such as the PDGF,
FGF, and EGF receptors. Nonetheless, it has become clear that many
angiotensin II effects require tyrosine
phosphorylation, as shown by studies with tyrosine
kinase inhibitors. For example, genistein, a tyrosine
kinase inhibitor, blocks angiotensin
IIstimulated MAP kinase (D.-F. Liao, unpublished data, 1996) and JNK
activity21 ; vessel contraction in response to both EGF and
angiotensin II is inhibited by genistein22 ;
and angiotensin IIstimulated protein synthesis is blocked
by genistein and herbimycin A.23 Because of the general
importance of tyrosine phosphorylation in cell growth,
inflammation, and migration, recent studies have focused on defining
the tyrosine kinases activated by angiotensin
II.
Insights into the nature of angiotensin II signaling have
been gained from structure function analysis of receptor
mutations (both deletion and site-directed mutagenesis). Based on its
cDNA sequence, the AT1 receptor is composed of 359 amino
acids with seven transmembrane domains arranged as shown in Fig 1
. Interactions among these domains and the
corresponding intracellular loops are responsible for determining the
nature of angiotensin II binding and receptor activation
after binding. For example, the angiotensin II binding site
appears to be the result of interactions among several of these
domains, including the NH2 terminal extension, Tyr92 in
extracellular loop 1, extracellular loop 3 (ASP278 and
ASP281), Tyr292, and Tyr215 (Fig 1
).24 25 26 27 Several transmembrane domains are responsible for
G-protein interactions with the receptor, including domains present
in the second and third intracellular loops.27 28 29 Recent
modeling analysis suggests that an interaction between
Asp74 and Tyr292 is critical for
angiotensin II binding and AT1 receptor
activation.30 Site-directed mutagenesis has established
that Asp74, a conserved residue within the second
transmembrane domain, is important in G-protein binding and
activation,26 29 as are the conserved residues
Tyr29230 and
Tyr2l5.31 In addition, the motif
Asp125-Arg126-Tyr127 in the amino
terminal region of the second cytoplasmic loop is important in
G-protein activation.29 These same residues may also be
important in angiotensin IIstimulated
growth.26

View larger version (41K):
[in this window]
[in a new window]
|
Figure 1. Representation of amino acids and signaling
domains in the rat AT1a receptor. The helixes were
positioned on the basis of modeling of G proteincoupled receptors.
Depicted are Ser, Thr, and Tyr residues, as well as residues thought to
be involved in specific signal transduction events. Highlighted
residues in transmembrane domains may be important in ligand binding
even if they are not phosphorylated.
|
|
 |
The AT1 Receptor: Role of Receptor
Phosphorylation in Signal Transduction and
Desensitization
|
|---|
AT
1 receptor
phosphorylation may serve at least three functions:
desensitization
of receptor signaling,
32 receptor
internalization,
33 and binding
of signal transduction
molecules.
6 In this review, we will
use the following
terms: desensitization, for the decrease in
physiological
response to a constant agonist
stimulus that occurs over time;
uncoupling, for the rapid (seconds to
minutes) decrease in receptor
signal transduction despite persistent
ligand binding; sequestration
(or internalization), for the rapid
(minutes to hours) removal
of receptor from plasma membrane without
change in total cellular
receptor number; and downregulation, for the
decrease in total
receptor number present in cells, which requires
hours to days.
34 Phosphorylation appears
to be a critical event in the uncoupling
of G proteincoupled
receptors.
32 35 Phosphorylation is
also
likely important in sequestration of certain G proteincoupled
receptors,
such as the ß
2-adrenergic
receptor.
36 G proteincoupled
receptors do not contain
intrinsic kinase activities but are
nonetheless
phosphorylated on serine and threonine residues
by
members of the GRK family.
37
Phosphorylation of the ß
2-adrenergic
receptor
by ßARK-1 or ßARK-2 (now termed GRK-2 and GRK-3,
respectively)
is likely important for receptor
uncoupling
35 38 and sequestration.
36
It appears that the phosphorylation-dependent
mechanisms responsible for sequestration and downregulation of the
AT1 receptor will be different from the
ß2-adrenergic receptor.33 39 40 Recently our
group41 and others42 have shown that the
AT1 receptor in vascular smooth muscle is
phosphorylated basally and in response to
angiotensin II. The majority of
phosphorylation in vivo was found on serine, and there
was a small increase in total phosphoserine content after agonist
exposure. GRK phosphorylation domains in G
proteincoupled receptors include the third intracellular loop and the
carboxyl-terminal tail.37 Although multiple serines may be
phosphorylated by GRKs, it appears that only the
initial one or two phosphorylations are
physiologically relevant. These sites, in a
number of receptors, are characterized by a pair of acidic amino acids
located amino terminal to the initial site of
phosphorylation,43 such as
Asp/Glu-Asp/Glu-X(0-5)-Ser/Thr. Recently, it was shown in
transfected 293 cells that the AT1 receptor can be
phosphorylated by GRK2, GRK3, and GRK5.32
However, the actual role of the GRKs in phosphorylation
of the AT1 receptor in relevant tissues such as vascular
smooth muscle remains speculative. It has been shown that
AT1 receptor sequestration is regulated by a motif in the
cytoplasmic tail involving residues carboxyl to
Tyr31239 and to
Leu31433 (Fig 1
). This region lacks highly
acidic amino acids typical of the GRK phosphorylation
motif (especially Asp and Glu). However, one region,
Asp343-Asn344-Met345-Ser346-Ser347-Ser348,
has partial homology to the consensus GRK
phosphorylation motif. In summary, serine
phosphorylation may play a role in AT1
receptor desensitization, although the precise
phosphorylation sites remain to be determined.
Recent studies have suggested that tyrosine residues in the carboxyl
tail may also be important in G proteincoupled receptor
sequestration. Specifically, Ferguson et al36 identified a
highly conserved tyrosine (Y326) in the motif NPXXY, which is involved
in the sequestration of the ß2-adrenergic receptor,
although these investigators did not demonstrate tyrosine
phosphorylation at this site. This motif is present
in the AT1 receptor at Tyr302 (NPLFY). However,
AT1 receptors in which Tyr302 was mutated to
Ala or in which triple alanine replacements of Phe301,
Tyr302, and Phe304 were performed showed no
substantial changes in their internalization kinetics.40
More recently, an AT1 receptor truncated at
Leu314, which still contained the NPXXY motif, failed to
show normal receptor sequestration.33 These findings
demonstrate that the NPLFY sequence of the AT1 receptor is
not an important determinant of agonist-induced sequestration, unlike
the ß2-adrenergic receptor.
Tyrosine phosphorylation of the AT1
receptor has been shown to occur,41 42 suggesting a role
in other receptor-mediated events such as signal transduction. There is
controversy regarding the extent to which tyrosine
phosphorylation is regulated by exposure to
angiotensin II. Immunoprecipitation of the AT1
receptor and Western blot analysis using anti-phosphotyrosine
antibody showed a small increase in agonist-dependent tyrosine
phosphorylation up to 6 minutes after
angiotensin II treatment.44 In contrast, when
VSMCs were metabolically labeled with
32P-orthophosphate and stimulated with
angiotensin II and the AT1 receptor was
immunoprecipitated, a slow increase in phosphotyrosine content was
observed.42 It should be noted that both of these
techniques measure total phosphotyrosine content; increases and
decreases in phosphorylation of individual tyrosine
residues and the actual site(s) of AT1 receptor
phosphorylation have not been determined after
angiotensin II binding. Tyr302 appears to be
important in signal transduction, because two groups40 45
have shown that mutating this residue to Ala or Phe caused alterations
in G-protein activation and IP3 production.
Tyr319 is of interest because it is part of the motif
Tyr-Ile-Pro-Pro, which is analogous to SH2 binding motifs found within
the PDGF receptor (Tyr-Ile-Ile-Pro) and within the EGF receptor
(Tyr-Leu-Pro-Pro).46 47 In the PDGF and EGF receptors,
these motifs have been shown to be target sequences for signaling
mediators when the tyrosine is phosphorylated. SH2
binding domains contain a critical tyrosine residue that, when
phosphorylated, promotes interactions with signaling
proteins that contain the SH2 domain. For example, PLC-
contains an
SH2 domain that interacts with the Tyr-Ile-Ile-Pro and Tyr-Leu-Pro-Pro
sequences present in the PDGF and EGF receptors.47
Thus, tyrosine phosphorylation of the AT1
receptor may be important in signal transduction events mediated by
binding of SH2 domaincontaining proteins.
 |
Overview of Tyrosine Kinase Signal Transduction by the
AT1 Receptor
|
|---|
Recent findings demonstrate that the AT
1 receptor
couples to
many intracellular signal transduction events. Five major
tyrosine
kinaseregulated pathways will be discussed in this review
on
the basis of activation of specific kinases shown to be regulated
by
angiotensin II in VSMCs (Figs 2

and 3

): Janus kinases (JAK
and TYK), c-Src, FAK, receptor
tyrosine kinases (Axl, EGF, and
PDGF), and calcium-dependent tyrosine
kinases (eg, Pyk2) that
phosphorylate signal substrates
such as Shc, Raf, and PLC-

.
The purpose of these multiple
receptor-activated kinases is
to provide an integrated series of
regulated cellular events.
It is unlikely that all these effects occur
simultaneously in
vivo. Thus, an important area of future
research is to determine
the physiological
variables and tissue-specific factors that
determine the relative
extent to which these signal transduction
pathways are
activated.

View larger version (39K):
[in this window]
[in a new window]
|
Figure 2. Summary of tyrosine kinasemediated signal
transduction pathways stimulated by angiotensin II in
VSMCs. Depicted are several cytosolic and transmembrane protein kinases
potentially activated by angiotensin II in VSMCs.
Phosphorylation of the linker protein Shc is
hypothesized to be mediated by Src, FAK, and other unidentified
tyrosine kinases (TyrKs). Of note, it has recently been demonstrated
that Pyk2 and Src may form a complex that leads to Src
activation.80 These kinases are also candidates to
phosphorylate Raf. Shc may also be
phosphorylated in a calcium-dependent manner by Pyk2
(or Pyk2-related kinases). Transactivation of Axl, EGF, PDGF, and
receptor tyrosine kinases is postulated by unknown means. Note that
JAK2 and TYK2, which are directly activated at the receptor,
are excluded from this figure.
|
|

View larger version (52K):
[in this window]
[in a new window]
|
Figure 3. Role of tyrosine kinases in induction of
c-fos expression by angiotensin II.
Angiotensin II binding stimulates tyrosine
phosphorylation of the linker protein Shc, which then
interacts with the GRB2-Sos complex, causing activation of Ras. Ras/Raf
then stimulates ERK1/2, which phosphorylates substrates
such as the transcription factor p62TCF. p62TCF
translocates to the nucleus, where it forms a complex at the SRE of the
c-fos gene. The c-fos promoter also contains a
sis-inducing factor element (SIE), which interacts with
members of the STAT family of transcription factors.
Angiotensin II stimulates the
phosphorylation and activity of STAT91 and STAT113
through the action of JAK2, thus providing a direct link between the
AT1 receptor and the nucleus. In VSMCs,
angiotensin II increases production of
prostaglandins, thereby activating adenyl cyclase,
increasing cAMP, and stimulating protein kinase A (PKA). In the adrenal
gland, the AT1 receptor may be coupled to Gi,
thereby inhibiting adenyl cyclase. Finally, angiotensin II
stimulates increases in diacylglycerol and intracellular calcium,
thereby activating PKC.
|
|
JAK and TYK
Recent studies indicate that the AT1 receptor
shares properties with cytokine receptors, such as the
interleukin-2, IFN-
, and IFN-
receptors. Similar to these
classical cytokine receptors, the AT1 receptor
stimulates tyrosine phosphorylation, activates
the MAP kinase pathway, and induces c-fos mRNA expression
(Fig 3
).7 16 17 18 19 20 In the case of the cytokine
receptors, tyrosine phosphorylation is mediated by
protein tyrosine kinases that associate with the receptor, including
the Src-related kinases Lck and Fyn48 and the Janus family
kinases (JAK and TYK).49 The Janus family kinases are key
mediators of mRNA expression characterized as "early growth response
genes." Transcription of these genes, exemplified by
c-fos and c-jun, does not require prior synthesis
of any other new gene products. Recent studies have established
that JAK and TYK associate with membrane receptors and, when
activated, stimulate tyrosine phosphorylation
of a family of transcription factors termed STAT.50 The
STAT proteins translocate to the nucleus, where they bind to SIEs and
stimulate transcription of early growth response genes. We recently
found that angiotensin II rapidly activates JAK2
and TYK2 and stimulates tyrosine phosphorylation of
STAT113 in VSMCs,6 and Baker's group showed that
angiotensin II activates STAT91 in cultured
neonatal cardiac fibroblasts.51 These data support the
role of angiotensin II as a cytokine similar to
IFN-
and IFN-
(Table
).49 52 In
addition, JAK2 was found to immunoprecipitate with the AT1
receptor, suggesting a role for this kinase in mediating the earliest
events activated by angiotensin II.6
These observations and the recent demonstration that thrombin also
activates JAK253 indicate the great extent to
which G proteincoupled receptors share signal mechanisms
activated by cytokine and growth factor receptors.
Src Family Kinases
The Src family kinases are likely candidates to mediate
AT1 receptor signal events, on the basis of the recent
demonstration that angiotensin II stimulates tyrosine
phosphorylation of PLC-
,6 a Src
substrate.54 The 60-kD c-Src is the best characterized
member of a family of nine cytoplasmic protein tyrosine kinases that
participate in growth factor signal transduction. Tissue-specific
expression of alternatively spliced gene products yields at least
14 different Src-related kinases.55 Three family members
(c-Src, Fyn, and Yes) are expressed ubiquitously and appear to have
partially overlapping functions, on the basis of studies with
transgenic mice.46 Functional domains shared by Src family
kinases include an amino-terminal myristoylation sequence for membrane
targeting, SH2 and SH3 domains, a kinase domain, and a carboxy-terminal
noncatalytic domain. These regions participate in a complex tonic
inhibition of Src family kinases that can be overcome in cells exposed
to mitogens. One of the residues that appears to be critical for
regulation of c-Src is Tyr530, which is not present in
v-Src. Phosphorylation of Tyr530 by Csk
inhibits c-Src activity,56 whereas
dephosphorylation of this residue appears to be an
activating mechanism. Autophosphorylation of
Tyr419 in the catalytic domain may be an activating signal.
c-Src activity is also inhibited via intramolecular interactions of the
carboxy-terminal catalytic domains with both the SH2 and SH3
domains.57 These SH2 and SH3 domains probably also
stimulate c-Src activity through interactions with regulators and
downstream/kinase substrates.
Recent studies from several laboratories,58 59 including
our laboratory,60 suggest that c-Src plays an important
role in angiotensin II signal transduction. We demonstrated
that angiotensin II stimulation of VSMCs was associated
with a rapid activation of c-Src.60 Specifically,
angiotensin II stimulated a 2- to 3-fold increase in
activity of c-Src within 2 minutes, measured by either
autophosphorylation or kinase activity toward
enolase.60 Bernstein's laboratory59 studied
the functional consequences of inhibiting c-Src activity by
electroporating a monoclonal antic-Src antibody into cultured VSMCs.
They demonstrated significantly greater inhibition of PLC-
phosphorylation and IP3 formation by the
antic-Src antibody compared with purified murine IgG. Data from
Izumo's laboratory61 support the importance of Src family
kinases as mediators of angiotensin II function. They
showed in cardiac fibroblasts that angiotensin II
activated both c-Src and Fyn.53 These findings
demonstrate that activation of Src family kinases is one of the
earliest signal events stimulated by angiotensin II and
strongly suggest that c-Src is involved in angiotensin
IIstimulated tyrosine phosphorylation of PLC-
.
FAK
Because angiotensin II stimulates changes in smooth
muscle cell shape and volume and promotes migration, alterations in
cell-matrix interaction are likely to be critical to
angiotensin II signal transduction. It has become clear
that focal adhesion complexes, specialized sites of cell adhesion, act
as supramolecular structures for the assembly of signal transduction
mediators. The best characterized tyrosine kinase localized to focal
adhesion complexes is a 125-kD protein termed FAK. This protein was
originally isolated from v-Srctransformed chicken embryo fibroblasts
by Kanner et al.62 FAK exhibits protein tyrosine kinase
activity toward other proteins with which it colocalizes at these
sites, such as paxillin.63 FAK lacks modular domains such
as SH2 or SH3 and resembles known protein tyrosine kinases only in its
catalytic domain. FAK is autophosphorylated at
Tyr397 in resting substrate-attached cells, and it
possesses sites favored for phosphorylation by Src,
such as tyrosines 407, 576, and 577. Phosphorylation of
these tyrosines markedly increases the protein tyrosine kinase activity
of FAK.64 FAK appears to play an important role in events
leading to cell proliferation, as suggested by the finding in
v-Srctransformed fibroblasts that
hyperphosphorylation of FAK occurs in concert with the
loss of a requirement for cell attachment for
growth.65
Angiotensin II rapidly stimulates tyrosine
phosphorylation of FAK in cultured
VSMCs.66 67 Tyrosine phosphorylation of
FAK appears to be due to autophosphorylation and
activation by unclear mechanisms, although major roles for Src and Csk
have been proposed, with an initial event, possibly
attachment-dependent activation of Csk, enhancing FAK
autophosphorylation.68 The upstream
activators of Csk are unknown, but it has been proposed
that activation by G proteincoupled receptors may be mediated by the
Rho family of GTPases, as suggested by studies using the Rho
inhibitor C3 botulinum exoenzyme.69 These
events have in common the formation of a multimeric complex at
focal adhesion contacts, including adaptors such as paxillin and
SH2-rich p130Cas,70 GTPases such as Sos and
dynamin,71 72 and effectors, such as
PLC-
.73 Autophosphorylation of FAK on
tyrosine generates a site for binding of SH2 domaincontaining
proteins and leads to recruitment and activation of Src-like protein
kinases including c-Src itself, Fyn, and Csk.74 75 The
signal transduction particle consisting of FAK and a Src-like kinase
then phosphorylates paxillin.63 It has become
clear that phosphorylation of paxillin in response to
angiotensin II is one of the earliest and most prominent
tyrosine phosphorylation events in
VSMCs.66 67 76 77 The functional significance of this
activation and the alterations in integrin signal transduction remain
unknown.
Two laboratories have independently reported the cloning of a second
FAK family member, denoted Pyk278 or cell adhesion
kinase-ß.79 In cells of neural origin, Pyk2 was found to
be activated by G proteincoupled receptor agonists, PKC
stimulation, or increased cytoplasmic calcium levels (eg, potassium
depolarization or calcium ionophore). Pyk2 has been postulated as a
potential link between calcium-dependent signaling pathways and protein
tyrosine kinase pathways (Fig 2
). Recently, Dikic et al80
reported that activated Pyk2, like FAK, complexes with c-Src
and that this interaction is required for Pyk2-mediated ERK activation.
As such, Pyk2 is a candidate both to regulate c-Src and to link G
proteincoupled vasoconstrictor receptors with protein tyrosine
kinasemediated contractile, migratory, and growth responses.
 |
Interactions With Tyrosine KinaseCoupled Receptors
|
|---|
Cross talk between G proteincoupled receptors and tyrosine
kinase
receptors has been shown recently by two
groups
81 82 to include
rapid activation of tyrosine kinase
receptors by agonist binding
to G proteincoupled receptors. In one
study, stimulation
of Rat-1 cells with endothelin-1, lysophosphatidic
acid, or
thrombin stimulated a rapid increase in tyrosine
phosphorylation
of the EGF receptor and the
neu oncoprotein.
81 Specific inhibition
of EGF
receptor function by either the selective tyrphostin,
AG1478, or a
dominant-negative EGF receptor mutant suppressed
ERK1/2 activation and
strongly inhibited induction of
fos gene
expression and DNA
synthesis by endothelin-1 or thrombin. A
study by Rao et
al
83 reported that thrombin stimulated tyrosine
phosphorylation
of the IGF-1 receptor as well as IRS-1
and PLC-

. In a similar
series of experiments,
82
stimulation of smooth muscle cells
with angiotensin II
resulted in tyrosine phosphorylation of
Shc proteins,
complex formation between Shc and GRB2, and increased
tyrosine
phosphorylation of PDGF ß-receptors that
coprecipitated
with Shc-GRB2 complexes. Autocrine release of PDGF
failed to
account for Shc complex formation at the PDGF receptor after
angiotensin
II treatment, and a specific
angiotensin II type I receptor
antagonist,
losartan, abolished the response. These studies
suggest that
angiotensin II signal transduction is mediated
in part by
PDGF (EGF?) receptor transactivation. However, the
number of distinct
cytoplasmic and membrane tyrosine kinases
that become activated
after AT
1 receptor stimulation suggest
that a general
"autocrine" mechanism is unlikely and that specific
intracellular
events mediate the transactivation that has been
reported.
In addition to signal events mediated by rapid activation of tyrosine
kinase receptors, angiotensin II may exert long-term
autocrine growth effects through interactions with receptor tyrosine
kinases, such as Axl (also called UFO or Ark84 ). Axl was
originally cloned as a transforming gene from human myeloid leukemia
cells.85 Axl is a member of a family of cell adhesion
moleculerelated tyrosine kinase receptors that includes the Rse/Tyro3
proteins (also called Sky, Brt, or Tif86 ) and the Nyk/Mer
proteins.87 The ligand for Axl was recently shown to be
Gas6,88 a 70-kD
-carboxyglutamic acidcontaining
protein related to protein S. Analysis of the structure of Gas6
showed that only the tandem globular domains (related to domains used
by laminin and agrin for binding to the dystroglycan complex) were
required for receptor binding and
autophosphorylation.89 Gas6 protein was
originally isolated as a growth-potentiating factor from the
conditioned medium of VSMCs.88 Concomitant stimulation of
VSMCs with Gas6 (activation of Axl) and thrombin or endothelin
(activation of their respective G proteincoupled receptors) caused a
synergistic increase in DNA synthesis. More recently, signal
transduction events stimulated by Axl90 and the related
Nyk/Mer87 were shown to include PLC-
, p70 S6 kinase,
Shc, GRB2, Raf-1, and ERK1/2, demonstrating important similarities to
angiotensin II signal transduction.
 |
Tyrosine Phosphorylation of Signal
Mediators
|
|---|
PLC
Angiotensin II binding to the AT
1 receptor
stimulates the phosphoinositide-specific
PLC to
hydrolyze phosphatidylinositol 4,5-bisphosphate, thereby
generating the
second messengers IP
3 and diacylglycerol. PLC
is a family
of at least three related genes: PLC-ß, PLC-

,
and
PLC-

.
91 Isoforms of PLC-ß are differentially
regulated
by G-protein

and ß

subunits.
92 93 In
contrast, the PLC-
isoforms are regulated by tyrosine
phosphorylation.
94 Regulation
of PLC-

isoforms is unclear but may involve changes in intracellular
calcium.
For most G proteincoupled receptors, it appears
that PLC-ß is
critical for production of IP
3. Two different,
but
not mutually exclusive, mechanisms may account for IP
3
formation
by angiotensin II. Similar to G proteincoupled
receptors,
such as the
1-adrenergic receptor,
G
q activated by angiotensin
II may
stimulate PLC-ß. Alternatively, angiotensin II
may
activate PLC-

, via tyrosine phosphorylation.
This distinction
is important because activation of calcium-dependent
tyrosine
kinases, such as Pyk2/CAK-ß, would be "downstream"
from
PLC-

, if tyrosine phosphorylation of PLC-

is
the primary mechanism
by which angiotensin II increases
intracellular calcium. Alternatively,
if angiotensin II
increases calcium by stimulating PLC-ß
(via G
q), then
activation of Pyk2/CAK-ß would be an important
"upstream"
tyrosine kinase event.
Our group has made several observations suggesting that in VSMCs
(especially in culture or in neointima) activation of PLC
by angiotensin II is mediated by tyrosine
phosphorylation of PLC-
. First, we observed in
cultured rat aortic VSMCs that PLC-
, but not PLC-ß or PLC-
, was
expressed constitutively. Second, angiotensin II
activated the PLC-
isoform, as shown by rapid tyrosine
phosphorylation.95 Third, the time course
for tyrosine phosphorylation of PLC-
was very
similar to the time course for IP3 formation, suggesting
that PLC-
activation by tyrosine phosphorylation is
responsible for IP3 generation. Fourth, pretreating VSMCs
with Na2VO4, a potent inhibitor of
tyrosine phosphatases, increased subsequent angiotensin
IIinduced IP3 formation.44 Fifth, when c-Src
activity was inhibited by electroporation of antic-Src antibodies,
there was a corresponding inhibition of PLC-
phosphorylation and IP3 formation,
suggesting that PLC-
is the predominant pathway for IP3
formation in response to angiotensin II in cultured
VSMCs.59 All these responses were completely inhibited by
losartan, indicating that the AT1 receptor is the
receptor by which angiotensin II stimulates PLC-
. In
light of the antic-Src antibody electroporation studies discussed
above, these data support the concept that c-Src is likely to be the
angiotensin IIstimulated PLC-
tyrosine kinase in
VSMCs. However, we have been unable to coprecipitate c-Src and PLC-
(U. Schmitz, unpublished data, 1996), suggesting that another kinase
and/or linker protein may mediate c-Srcdependent activation of
PLC-
.
c-Raf-1
Historically, the first molecules to be identified as being
tyrosine-phosphorylated in response to
angiotensin II were the 44- and 42-kD MAP kinases, also
termed ERK1/2.19 20 Activation of ERK1/2 requires dual
phosphorylation on threonine and tyrosine residues
found within the motif Thr-Glu-Tyr, which is mediated by the MAP kinase
kinase or MEK. MEK is, in turn, regulated by serine (and probably
tyrosine) phosphorylation by the kinase c-Raf-1,
although Raf-independent pathways for ERK1/2 activation have also been
proposed.96 Angiotensin II has been shown to
stimulate serine/threonine phosphorylation of
Raf-1,18 97 98 although the significance of
serine/threonine phosphorylation for Raf-1 activity is
uncertain. We have recently found that PKC downregulation inhibits
angiotensin IIstimulated Raf-1 association with Ras but
does not inhibit ERK1/2 activation, further suggesting that Raf-1 may
not be required for MEK activation in VSMCs.99 More
recently, we have observed that PKC
may interact with Ras and
appears to be required for activation of ERK1/2 (Fig 2
).100 It is clear that recruitment of Raf-1 to the plasma
membrane and interaction with the low-molecular-weight guanine
nucleotidebinding protein Ras101 102 is
required for Raf activity. In addition, tyrosine
phosphorylation of Raf is an early signal event
associated with translocation and likely
activation.103 104 105 We have recently shown that
angiotensin II stimulates tyrosine
phosphorylation of Raf (T. Ishida, unpublished data,
1996), although the function of Raf tyrosine
phosphorylation and the identity of the putative Raf
tyrosine kinase remain undetermined.
Shc-GRB2-Sos-Ras
The role of Shc-GRB2-Sos and Ras in signal
transduction by Gq-coupled receptors, such as the
AT1 receptor, is unclear. Importantly, tyrosine
phosphorylation of Shc61 82 106 and
activation of Ras by angiotensin II were recently
demonstrated.107 Tyrosine phosphorylation
of a linker protein called Shc appears to be an important mechanism
used by all G proteincoupled receptors on the basis of studies to
date.108 For Gi-coupled receptors, a pathway
for activation of Ras and ERK1/2 has been proposed (Fig 2
)106 107 108 in which release of G-protein ß
subunits
from G proteincoupled receptors stimulates downstream events leading
to tyrosine phosphorylation of Shc.109
c-Src has been proposed as a likely candidate.108 110 111
Additional signal mediators that may be required include PI 3-K and a
protein tyrosine phosphatase.108 Once Shc is
tyrosine-phosphorylated, it now binds GRB2 via SH2
domain interactions (Fig 2
). GRB2 also binds to the guanine
nucleotide exchange factor Sos via SH3
domains.112 Sos stimulates the release of GDP for GTP on
Ras, which then recruits Raf to the plasma membrane, where it is then
activated (probably via tyrosine and serine
phosphorylation). Active Raf phosphorylates
and activates MEK, which can then phosphorylate and
activate ERK1/2. The evidence for this pathway is strong and
includes the fact that transient expression of G-protein ß
subunits in COS-7 and other cells stimulates ERK1/2
activity.113 114 115 Inhibiting G-protein ß
subunit
activity by expression of the ß
subunitbinding site of ßARK
blocks ERK1/2 activation by Gi-coupled
receptors.110 The stimulatory effects of G-protein
ß
subunits were shown to be due to Ras activation, as demonstrated
by increased Ras-GTP formation and the ability of dominant negative Ras
to block ERK1/2 activation.113
In contrast to the well-defined role of G-protein ß
subunits in
signal transduction by Gi-coupled receptors, their role in
signal events stimulated by Gq-coupled receptors, such as
the AT1 receptor, is much less clear. Crespo et
al114 showed that overexpression of the
subunit of
transducin, to inhibit G-protein ß
subunit function, significantly
decreased ERK1/2 activation by the Gq-coupled
m1 muscarinic receptor. However, Faure et
al115 showed that the
subunit of transducin failed to
block ERK1/2 activation by the Gq-coupled bombesin
receptor. Further evidence that G-protein ß
subunits do not
mediate ERK1/2 activation by Gq-coupled receptors was
demonstrated by failure of a truncated form of ßARK to inhibit ERK1/2
activation by the
1B-adrenergic and m1
muscarinic receptors.110 113 Thus, it appears unlikely
that G-protein ß
subunits are necessary for
angiotensin IImediated activation of ERK1/2 in VSMCs.
There may be convergence of Gi- and Gq-coupled
receptor signal transduction with regard to the tyrosine kinase(s) that
phosphorylates Shc. Recently, van Biesen et
al109 demonstrated that the G-protein ß
subunitdependent pathway by which Gi-coupled receptors
stimulated Shc phosphorylation and ERK1/2 activation
was inhibited by genistein, implying that a tyrosine kinase was
required. Because the Gi-coupled thrombin receptor was
shown to increase c-Src autophosphorylation and
activity,83 116 c-Src is a likely candidate to mediate
these effects. The results with thrombin are very relevant for
angiotensin IImediated signal transduction, because both
thrombin and angiotensin II stimulate genistein-dependent
tyrosine phosphorylation of phospholipase C-
1 in
VSMCs.59 83 Angiotensin II was recently shown
to stimulate tyrosine phosphorylation of Shc in cardiac
fibroblasts,106 cardiac myocytes,61 and
smooth muscle cells.82 More recently, Touhara et
al108 showed that overexpression of G-protein ß
subunits promoted Shc tyrosine phosphorylation, which
was inhibited by wortmannin, implying a role for PI 3-K. We have shown
similar results for angiotensin II signal transduction in
VSMCs, including activation of c-Src,60 c-Srcdependent
stimulation of ERK1/2 (M. Ishida, unpublished data, 1996), and
wortmannin-sensitive activation of ERK1/2 (D.-F. Liao, unpublished
data, 1996). It has been suggested that Fyn (a Src family member) is
the Shc tyrosine kinase in cardiac myocytes.61 Thus, a
putative signal transduction cascade for angiotensin II may
be proposed in which activation of c-Src (or Fyn) causes tyrosine
phosphorylation of Shc, activation of Ras, and
stimulation of ERK1/2 (Fig 2
).
However, it is possible that angiotensin IIstimulated
signal events may be mediated by tyrosine kinases other than c-Src. A
candidate kinase is Pyk2, a calcium-dependent tyrosine kinase that
phosphorylates Shc independent of G-protein ß
subunit
activation and thereby activates ERK1/2 in brain78
(Fig 2
). We have studied this pathway in VSMCs and can find no evidence
for a Pyk2-mediated pathway. Specifically, inhibiting calcium
mobilization in VSMCs failed to block angiotensin
IImediated ERK1/2 activation,117 and PCR screening
failed to identify Pyk2 or a closely related intracellular tyrosine
kinase (D. Wuthrich, unpublished data, 1996). Thus, the most likely
tyrosine kinase to mediate angiotensin IIdependent
activation of Ras is c-Src. Current studies with VSMCs from c-Src
knockout mice and with cells expressing dominant-negative c-Src
constructs should characterize the role of c-Src (and its dependence on
G-protein ß
subunits) in angiotensin IImediated
activation of Ras. However, the present findings suggest that
multiple, possibly cell typeand receptor-specific, mechanisms may
exist for Ras activation by Gq proteincoupled receptors,
such as the AT1 receptor. Questions that remain to be
answered regarding activation of Ras by angiotensin II
include the role of G-protein ß
subunits (characterizing other
calcium-activated tyrosine kinases, such as Pyk2, which may be
important in angiotensin IIresponsive tissues, such as
brain, adrenal gland, and liver21 ) and the identity of
tyrosine kinases in addition to c-Src that may
phosphorylate Shc as well as Raf and PLC-
1.
 |
Conclusion
|
|---|
The concept that the AT
1 receptor acts as a
multifunctional
receptor by activating multiple tyrosine kinases is
well illustrated
by regulation of c-
fos expression by
angiotensin II (Fig 3

).
Angiotensin II induces
c-
fos mRNA expression rapidly in a PKC-
and
calcium-dependent fashion,
7 requiring activation of
PLC-
by tyrosine phosphorylation. The
c-
fos promoter contains the
SRE. Induction of the SRE occurs
upon formation of a ternary
complex with serum response factor,
p62
TCF, and the SRE.
118 ERK1/2 was shown to
phosphorylate p62
TCF (also known as elk-1
or
SAP-1), resulting in enhanced ternary complex
formation.
119 Thus, the SRE present in the
c-
fos promoter may be regulated
by angiotensin
IIstimulated ERK1/2 phosphorylation of
p62
TCF,
as shown for EGF.
119 Activation of
ERK1/2 requires activation
of Ras and Raf, events that are regulated by
tyrosine phosphorylation.
Finally, an SIE is
present in the c-
fos promoter and interacts
with members
of the STAT family of transcription factors (including
STAT91 and
STAT113).
49 The diversity of signal transduction
pathways
stimulated by angiotensin II, which leads to the expression
of
c-
fos and other early growth response genes, demonstrates
the
ability of angiotensin II to act as a cytokine
and regulate
more general cellular functions.
Angiotensin II was initially identified by its
physiological role in regulation of salt and water
metabolism, blood pressure, and vascular tone. However, as
demonstrated in this review, angiotensin II may participate
in a diversity of cellular functions via its effects mediated by the
AT1 receptor. Exciting new findings point to functions of
the AT1 receptor related to growth and vasoconstriction
(mediated by c-Src or Fyn), transactivation by PDGF and Axl, and
functions related to inflammation and migration (mediated by JAK, TYK,
c-Src, and FAK). It is clear that future work will be required to
determine the nature of the interactions among these different kinase
pathways and their role in the pathogenesis of hypertension and
atherosclerosis.
 |
Selected Abbreviations and Acronyms
|
|---|
| ßARK |
= |
ß-adrenergic receptor kinase |
| ACE |
= |
angiotensin-converting enzyme |
| c-Src, v-Src |
= |
pp60c-src kinase, pp60v-src kinase |
| Csk |
= |
c-Src kinase |
| EGF, FGF, PDGF |
= |
epidermal, fibroblast, and platelet-derived growth factors |
| ERK |
= |
extracellular signalregulated kinase |
| FAK |
= |
focal adhesion kinase |
| GRK |
= |
G proteincoupled receptor kinase |
| IFN |
= |
interferon |
| IP3 |
= |
inositol 1,4,5-trisphosphate |
| IRS-1 |
= |
insulin receptor substrate-1 |
| JAK, TYK |
= |
Janus kinases |
| JNK |
= |
c-Jun N-terminal kinase |
| MAP |
= |
mitogen-activated protein |
| MEK |
= |
MAP kinase kinase or ERK kinase |
| p62TCF |
= |
ternary complex factor |
| PI 3-K |
= |
phosphatidylinositol 3-kinase |
| PKC |
= |
protein kinase C |
| PLC |
= |
phospholipase C |
| SH2 |
= |
src-homology 2 |
| SIE |
= |
sis-inducing factor element |
| SRE |
= |
serum response element |
| STAT |
= |
signal transducers and activators of transcription |
| VSMC |
= |
vascular smooth muscle cell |
|
 |
Acknowledgments
|
|---|
This work was supported by grants from the National
Institutes
of Health (Heart, Lung, and Blood Institute) to Drs Berk and
Corson.
Dr Berk is an Established Investigator of the American Heart
Association.
We gratefully acknowledge the scientific contributions of
our
colleagues and collaborators, including K. Bernstein, M. Marrero,
W.
Paxton, J. Abe, J. Duff, M. Ishida, T. Ishida, M. Kusuhara,
D.-F.
Liao, P. Lucchesi, D. Wuthrich, and U. Schmitz.
 |
Footnotes
|
|---|
This manuscript was sent to Robert J. Lefkowitz, Consulting
Editor, for review by expert referees, editorial decision, and
final disposition.
Received July 24, 1996;
accepted January 3, 1997.
 |
References
|
|---|
-
Skeggs LT Jr, Marsh WH, Kahn JR, Shumway
NP. The purification of hypertensin I. J Exp
Med.. 1954;100:363-370.[Abstract]
-
Berk BC, Vekshtein V, Gordon HM, Tsuda T.
Angiotensin IIstimulated protein synthesis in cultured
vascular smooth muscle cells. Hypertension.. 1989;13:305-314.[Abstract/Free Full Text]
-
Geisterfer AAT, Peach MJ, Owens GK.
Angiotensin II induces hypertrophy, not
hyperplasia, of cultured rat aortic smooth muscle cells.
Circ Res.. 1988;62:749-756.[Abstract/Free Full Text]
-
Schlondorff D, DeCandido S, Satriano JA.
Angiotensin II stimulates phospholipases C and
A2 in cultured rat mesangial cells.
Am J Physiol.. 1987;253:C113-C120.[Abstract/Free Full Text]
-
Griendling KK, Minieri CA, Ollerenshaw JD, Alexander
RW. Angiotensin II stimulates NADH and NADPH oxidase
activation in cultured vascular smooth muscle cells. Circ
Res.. 1994;74:1141-1148.[Abstract/Free Full Text]
-
Marrero MB, Schieffer B, Paxton WG, Heerdt L, Berk
BC, Delafontaine P, Bernstein KE. Direct stimulation of Jak/STAT
pathway by the angiotensin II AT1 receptor.
Nature.. 1995;375:247-250.[Medline]
[Order article via Infotrieve]
-
Taubman MB, Berk BC, Izumo S, Tsuda T, Alexander RW,
Nadal-Ginard B. Angiotensin II induces
c-fos mRNA in aortic smooth muscle: role of Ca2+
mobilization and protein kinase C activation. J Biol
Chem.. 1989;264:526-530.[Abstract/Free Full Text]
-
SOLVD Investigators. Effect of enalapril on survival
in patients with reduced left ventricular ejection
fractions and congestive heart failure. N Engl J
Med.. 1991;325:293-302.[Abstract]
-
Pfeffer MA, Braunwald E, Moye LA, Basta L, Brown EJ
Jr, Cuddy TE, Davis BR, Geltman EM, Goldman S, Flaker GC. Effect
of captopril on mortality and morbidity in patients with left
ventricular dysfunction after myocardial infarction:
results of the survival and ventricular enlargement
trial. N Engl J Med.. 1992;327:669-677.[Abstract]
-
Murphy TJ, Alexander RW, Griendling KK, Runge MS,
Bernstein KE. Isolation of a cDNA encoding the vascular type-1
angiotensin II receptor. Nature.. 1991;351:233-235.[Medline]
[Order article via Infotrieve]
-
Sasaki K, Yamamo Y, Bardham S, Iwai N, Murray JJ,
Hasegawa M, Matsuda Y, Inagami T. Cloning and expression of a
complementary DNA encoding a bovine adrenal angiotensin
type-1 receptor. Nature.. 1991;351:230-232.[Medline]
[Order article via Infotrieve]
-
Mukoyama M, Nakajima M, Horiuchi M, Sasamura H, Pratt
RE, Dzau VJ. Expression cloning of type 2
angiotensin II receptor reveals a unique class of
seven-transmembrane receptors. J Biol Chem.. 1993;268:24539-24542.[Abstract/Free Full Text]
-
Griendling KK, Rittenhouse SE, Brock TA, Ekstein LS,
Gimbrone MA Jr, Alexander RW. Sustained diacylglycerol formation
from inositol phospholipids in angiotensin II-stimulated
vascular smooth muscle cells. J Biol Chem.. 1986;261:5901-5906.[Abstract/Free Full Text]
-
Brock TA, Alexander RW, Ekstein LS, Atkinson WJ,
Gimbrone MA Jr. Angiotensin increases cytosolic free
calcium in cultured vascular smooth muscle cells.
Hypertension. 1985;7(suppl I):I-105-I-109.
-
Griendling KK, Tsuda T, Berk BC, Alexander RW.
Angiotensin II stimulation of vascular smooth muscle cells:
secondary signalling mechanisms. Am J
Hypertens.. 1989;2:659-665.[Medline]
[Order article via Infotrieve]
-
Huckle WR, Prokop CA, Dy RC, Herman B, Earp S.
Angiotensin II stimulates protein-tyrosine
phosphorylation in a calcium-dependent manner.
Mol Cell Biol.. 1990;10:6290-6298.[Abstract/Free Full Text]
-
Huckle WR, Dy RC, Earp HS. Calcium-dependent
increase in tyrosine kinase activity stimulated by
angiotensin II. Proc Natl Acad Sci
U S A.. 1992;89:8837-8841.[Abstract/Free Full Text]
-
Molloy CJ, Taylor DS, Weber H.
Angiotensin II stimulation of rapid protein tyrosine
phosphorylation and protein kinase activation in rat
aortic smooth muscle cells. J Biol Chem.. 1993;268:7338-7345.[Abstract/Free Full Text]
-
Duff JL, Berk BC, Corson MA.
Angiotensin II stimulates the pp44 and pp42
mitogen-activated protein kinases in cultured rat aortic smooth
muscle cells. Biochem Biophys Res Commun.. 1992;188:257-264.[Medline]
[Order article via Infotrieve]
-
Tsuda T, Kawahara Y, Ishida Y, Koide M, Shii K,
Yokoyama M. Angiotensin II stimulates two myelin
basic protein/microtubuleassociated protein 2 kinases in cultured
vascular smooth muscle cells. Circ Res.. 1992;71:620-630.[Abstract/Free Full Text]
-
Zohn IE, Yu H, Li X, Cox AD, Earp HS.
Angiotensin II stimulates calcium-dependent activation of
c-Jun N-terminal kinase. Mol Cell Biol.. 1995;15:6160-6168.[Abstract]
-
Hollenberg MD. Tyrosine kinase pathways and
the regulation of smooth muscle contractility.
Trends Pharmacol Sci.. 1994;15:108-114.[Medline]
[Order article via Infotrieve]
-
Leduc I, Haddad P, Giasson E, Meloche S.
Involvement of a tyrosine kinase pathway in the growth-promoting
effects of angiotensin II on aortic smooth muscle
cells. Mol Pharmacol.. 1995;48:582-592.[Abstract]
-
Ji H, Leung M, Zhang Y, Catt KJ, Sandberg K.
Differential structural requirements for specific binding of nonpeptide
and peptide antagonists to the AT1 angiotensin
receptor. Identification of amino acid residues that determine binding
of the antihypertensive drug losartan. J
Biol Chem.. 1994;269:16533-16536.[Abstract/Free Full Text]
-
Noda K, Saad Y, Kinoshita A, Boyle TP, Graham RM,
Husain A, Karnik SS. Tetrazole and carboxylate groups of
angiotensin receptor antagonists bind to the
same subsite by different mechanisms. J Biol
Chem.. 1995;270:2284-2289.[Abstract/Free Full Text]
-
Bihoreau C, Monnot C, Davies E, Teutsch B, Bernstein
KE, Corvol P, Clauser E. Mutation of Asp74 of the rat
angiotensin II receptor confers changes in
antagonist affinities and abolishes G-protein
coupling. Proc Natl Acad Sci U S A.. 1993;90:5133-5137.[Abstract/Free Full Text]
-
Yamano Y, Ohyama K, Kikyo M, Sano T, Nakagomi Y,
Inoue Y, Nakamura N, Morishima I, Guo DF, Hamakubo T, et al.
Mutagenesis and the molecular modeling of the rat
angiotensin II receptor (AT1). J Biol Chem.. 1995;270:14024-14030.[Abstract/Free Full Text]
-
Shirai H, Takahashi K, Katada T, Inagami T.
Mapping of G protein coupling sites of the angiotensin II
type 1 receptor. Hypertension.. 1995;25:726-730.[Abstract/Free Full Text]
-
Ohyama K, Yamano Y, Chaki S, Kondo T, Inagami
T. Domains for G-protein coupling in angiotensin II
receptor type I: studies by site-directed mutagenesis.
Biochem Biophys Res Commun.. 1992;189:677-683.[Medline]
[Order article via Infotrieve]
-
Marie J, Maigret B, Joseph MP, Larguier R, Nouet S,
Lombard C, Bonnafous JC. Tyr292 in the seventh transmembrane
domain of the AT1A angiotensin II receptor is essential for
its coupling to phospholipase C. J Biol Chem.. 1994;269:20815-20818.[Abstract/Free Full Text]
-
Hunyady L, Bor M, Balla T, Catt KJ. Critical
role of a conserved intramembrane tyrosine residue in
angiotensin II receptor activation. J
Biol Chem.. 1995;270:9702-9705.[Abstract/Free Full Text]
-
Oppermann M, Freedman NJ, Alexander RW, Lefkowitz
RJ. Phosphorylation of the type 1A
angiotensin II receptor by G protein-coupled receptor
kinases and protein kinase C. J Biol Chem.. 1996;271:13266-13272.[Abstract/Free Full Text]
-
Thomas WG, Thekkumkara TJ, Motel TJ, Baker
KM. Stable expression of a truncated AT1A receptor in CHO-K1
cells: the carboxyl-terminal region directs agonist-induced
internalization but not receptor signaling or desensitization.
J Biol Chem.. 1995;270:207-213.[Abstract/Free Full Text]
-
Campbell PT, Hnatowich M, O'Dowd BF, Caron MG,
Lefkowitz RJ, Hausdorff WP. Mutations of the human beta
2-adrenergic receptor that impair coupling to Gs interfere with
receptor down-regulation but not sequestration. Mol
Pharmacol.. 1991;39:192-198.[Abstract]
-
Bouvier M, Hausdorff WP, de Blasi A, O'Dowd BF,
Kobilka BK, Caron MG, Lefkowitz RJ. Removal of
phosphorylation sites from the beta 2-adrenergic
receptor delays onset of agonist-promoted desensitization.
Nature.. 1988;333:370-373.[Medline]
[Order article via Infotrieve]
-
Ferguson SS, Ménard L, Barak LS, Koch WJ,
Colapietro AM, Caron MG. Role of phosphorylation
in agonist-promoted beta 2-adrenergic receptor sequestration: rescue of
a sequestration-defective mutant receptor by beta ARK1.
J Biol Chem.. 1995;270:24782-24789.[Abstract/Free Full Text]
-
Premont RT, Inglese J, Lefkowitz RJ. Protein
kinases that phosphorylate activated G
protein-coupled receptors. FASEB J.. 1995;9:175-182.[Abstract]
-
Hausdorff WP, Bouvier M, O'Dowd BF, Irons GP, Caron
MG, Lefkowitz RJ. Phosphorylation sites on two
domains of the beta 2-adrenergic receptor are involved in distinct
pathways of receptor desensitization. J Biol
Chem.. 1989;264:12657-12665.[Abstract/Free Full Text]
-
Hunyady L, Baukal AJ, Balla T, Catt KJ.
Independence of type I angiotensin II receptor endocytosis
from G protein coupling and signal transduction. J
Biol Chem.. 1994;269:24798-24804.[Abstract/Free Full Text]
-
Hunyady L, Bor M, Baukal AJ, Balla T, Catt KJ.
A conserved NPLFY sequence contributes to agonist binding and signal
transduction but is not an internalization signal for the type 1
angiotensin II receptor. J Biol
Chem.. 1995;270:16602-16609.[Abstract/Free Full Text]
-
Paxton WG, Marrero MB, Klein JD, Delafontaine P, Berk
BC, Bernstein KE. The angiotensin II AT1
receptor is tyrosine and serine phosphorylated can
serve as a substrate for the SRC family of tyrosine kinases.
Biochem Biophys Res Commun.. 1994;200:260-267.[Medline]
[Order article via Infotrieve]
-
Kai H, Griendling KK, Lassegue B, Ollerenshaw JD,
Runge MS, Alexander RW. Agonist-induced
phosphorylation of the vascular type 1
angiotensin receptor. Hypertension.. 1994;24:523-527.[Abstract/Free Full Text]
-
Fredericks ZL, Pitcher JA, Lefkowitz RJ.
Identification of the G protein-coupled receptor kinase
phosphorylation sites in the human ß2-adrenergic
receptor. J Biol Chem.. 1996;271:13796-13803.[Abstract/Free Full Text]
-
Marrero MB, Schieffer B, Paxton WG, Duff JL, Berk BC,
Bernstein KE. The role of tyrosine
phosphorylation in angiotensin II-mediated
intracellular signalling. Cardiovasc Res.. 1995;30:530-536.[Medline]
[Order article via Infotrieve]
-
Laporte SA, Servant G, Richard DE, Escher E,
Guillemette G, Leduc R. The tyrosine within the NPXnY motif of
the human angiotensin II type 1 receptor is involved in
mediating signal transduction but is not essential for
internalization. Mol Pharmacol.. 1996;49:89-95.[Abstract]
-
Pascal SM, Singer AU, Gish G, Yamazaki T, Shoelson
SE, Pawson T, Kay LE, Forman K-JD. Nuclear magnetic resonance
structure of an SH2 domain of phospholipase C-gamma 1 complexed with a
high affinity binding peptide. Cell.. 1994;77:461-472.[Medline]
[Order article via Infotrieve]
-
Fantl WJ, Johnson DE, Williams LT. Signalling
by receptor tyrosine kinases. Annu Rev Biochem.. 1993;62:453-481.[Medline]
[Order article via Infotrieve]
-
Pleiman CM, Clark MR, Gauen LK, Winitz S, Coggeshall
KM, Johnson GL, Shaw AS, Cambier JC. Mapping of sites on the Src
family protein tyrosine kinases p55blk, p59fyn, and p561yn which
interact with the effector molecules phospholipase C-gamma 2,
microtubule-associated protein kinase, GTPase-activating protein, and
phosphatidylinositol 3-kinase. Mol Cell Biol.. 1993;13:5877-5887.[Abstract/Free Full Text]
-
Darnell JEJ, Kerr IM, Stark GR. Jak-STAT
pathways and transcriptional activation in response to IFNs and other
extracellular signaling proteins. Science.. 1994;264:1415-1421.[Abstract/Free Full Text]
-
Ihle JN, Witthuhn BA, Quelle FW, Yamamoto K,
Thierfelder WE, Kreider B, Silvennoinen