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INTRODUCTION |
Growth factor receptors belong to a family of receptors that
contain an extracellular ligand binding domain, a single transmembrane portion, and a large intracellular tyrosine kinase catalytic domain. Ligand-induced receptor autophosphorylation promotes the interaction of
the intracellular domains of the receptors with a number of downstream
effector proteins or enzymes. Typically, these proteins contain one or
more domains known as Src homology 2 (SH2)1 domains. Among these
SH2 domain-containing proteins are phosphoinositide-specific phospholipase C
(PLC
), the 85-kDa subunit of
phosphatidylinositol 3-kinase, GTPase-activating proteins, growth
factor receptor binding protein 2, the phosphotyrosine phosphatase
SHP-2, and members of the nonreceptor Src family of tyrosine kinases
(1, 2). Autophosphorylation of growth factor receptors occurs on
defined tyrosine residues. These phosphorylated residues function to
initiate cellular signaling cascades by acting as high affinity binding sites for the SH2 domains of various effector proteins. The selectivity of the receptor-effector interaction is determined, not only by the
phosphorylated tyrosine residue in the receptor but also by the three
amino acids C-terminal to the phosphorylated tyrosine and by the
structure of the SH2 domain of the interacting protein. For example,
one of the identified sites for binding of the SH2 domains of PLC
1
to the platelet-derived growth factor
and
receptors is a YIPP
motif present in the receptors at residues 1018-1021 and 1021-1024,
respectively. Phosphorylation of tyrosines 1018 and 1021 in these
motifs promotes binding of PLC
1 to the platelet-derived growth
factor receptor and tyrosine phosphorylation and activation of the
enzyme (3, 4).
Another family of cell surface receptors are the G-protein-coupled
receptors that contain seven membrane-spanning
-helices. These
receptors lack intrinsic tyrosine kinase activity. However, we have
previously shown that the G-protein-coupled angiotensin II (Ang II)
AT1 receptor in vascular smooth muscle cells (VSMC) activates the inositol 1,4,5-trisphosphate (IP3) and
diacylglycerol-generating enzyme, PLC
1, in a manner similar to that
observed for growth factor receptors. PLC
1 is transiently
tyrosine-phosphorylated in Ang II-stimulated VSMC with a time course
that parallels that of IP3 formation (5). Tyrosine
phosphorylation of PLC
1 appears to lie downstream from activation of
the c-Src tyrosine kinase because electroporation of neutralizing
anti-c-Src antibodies into VSMC virtually eliminates Ang II-induced
tyrosine phosphorylation of PLC
1 and blocks Ang II stimulation of
IP3 production (6). Furthermore, other G-protein-coupled
receptors, including those for platelet activating factor, thrombin,
and ATP, have also been shown to signal through the tyrosine
phosphorylation and activation of PLC
1 (7-9). In none of these
instances, however, is it known whether PLC
1 phosphorylation and
activation involves physical association of the SH2 domains of the
enzyme with the receptor.
AT1 post-receptor signaling in VSMC also involves
activation of the janus kinase (JAK)/signal transducers and activators
of transcription (STAT) pathway. Ang II stimulation of the
AT1 receptor activates the JAK/STAT pathway by inducing
rapid tyrosine phosphorylation, activation, and association of JAK2
with the receptor (10). JAK2-receptor association appears to depend on
a YIPP motif in the C-terminal intracellular domain of the
AT1 receptor that is identical to the PLC
1 SH2 domain
binding site identified in the platelet-derived growth factor receptor
(11). Because JAK2 does not contain any SH2 domains, the finding that
JAK2 associates with this motif in the AT1 receptor was
initially puzzling. Recently, however, we have found that JAK2
associates with the receptor as a consequence of the SH2
domain-containing SHP-2 phosphotyrosine phosphatase acting as an
adaptor or linker protein for JAK2
association.2 In the present
study, we have examined whether Ang II-induced tyrosine phosphorylation
and activation of PLC
1 in VSMC involves binding of PLC
1 to the
AT1 receptor in an Ang II- and tyrosine phosphorylation-dependent manner. In addition, we have
identified the interacting domains in the two proteins.
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EXPERIMENTAL PROCEDURES |
Materials--
Anti-AT1 receptor polyclonal
antibodies (N-10 and 306) and glutathione S-transferase
(GST)-PLC
1 fusion proteins (sc-4019, sc-4051, sc-4052, sc-4053, and
sc-4054) were purchased from Santa Cruz Biotechnology Inc. Anti-PLC
1
monoclonal antibody (clone D-7-3) and anti-phosphotyrosine monoclonal
antibody (clone 4G10) were obtained from Upstate Biotechnology.
Anti-SHP-2 monoclonal antibody was purchased from Transduction
Laboratories. Purified human recombinant c-Src enzyme and PP1 came from
Calbiochem. Affi-Gel 10, Muta-Gene Phagemid kit, and
detergent-compatible protein assay kit were purchased from Bio-Rad.
[3H]PIP2, GST-agarose, and monoclonal
anti-GST antibody were obtained from Amersham Pharmacia Biotech. All
other chemicals were purchased from Sigma.
Cell Culture--
VSMC from 200-300 g male Sprague-Dawley rat
aortas were cultured to near confluence at 37 °C under 5%
CO2 in Dulbecco's modified Eagle medium containing 10%
fetal bovine serum and supplemented with antibiotics (5, 6). Cells were
growth-arrested by incubation in serum-free Dulbecco's modified Eagle
medium for 36-48 h before Ang II exposure.
Immunoprecipitation and Immunoblotting--
VSMC were stimulated
with Ang II (10
7 M) for various times, and
cells were lysed and subjected to immunoprecipitation with anti-AT1 receptor antibody as described previously (10).
Immunoprecipitated proteins were separated by SDS-polyacrylamide gel
electrophoresis, transferred to nitrocellulose by electroblotting, and
probed with anti-PLC
1 or anti-phosphotyrosine antibody as described
previously (5, 6).
Preparation of VSMC Cell Lysates--
Growth-arrested VSMC were
stimulated with Ang II (10
7 M) for various
times, washed two times with ice-cold phosphate-buffered saline
containing 1 mM Na3VO4 and then
lysed in 1.0 ml of lysis buffer (25 mM Tris-HCl, pH 7.6, 0.15 M NaCl, 1% Triton X-100, 10% glycerol, 50 mM NaF, 1 mM Na3VO4, 1 mM phenylmethylsulfonyl fluoride, and 1 µg/ml aprotinin).
Cells were scraped off the plates and gently sonicated. Lysates were
cleared by centrifugation at 7,500 × g for 15 min, and
the protein concentration of the cleared lysates was determined by the
Bio-Rad detergent-compatible protein assay. In some experiments, SHP-2
was quantitatively removed (as confirmed by immunoblotting) from VSMC
lysates by immunoprecipitation with anti-SHP-2 antibody before use of
the lysates in in vitro binding assays.
Preparation of DNA Constructs Encoding GST-AT1
Receptor Fusion Proteins--
A 166-base pair fragment of the Ca18b
cDNA encoding the rat AT1A receptor was amplified by
the polymerase chain reaction and cloned into the pGEX-KG vector via
XbaI and HindIII restriction sites (12). Point
mutations and deletional mutations were introduced into the constructs
as described previously (11). The sequences of all DNA constructs were
verified by DNA sequence analysis.
In Vitro Binding Assays--
GST-AT1 fusion proteins
were expressed in DH5
Escherichia coli and purified by
affinity chromatography using immobilized glutathione-Sepharose 4B
beads. Five µg of fusion protein or GST alone prebound to beads was
incubated with 1.0 ml of VSMC cell lysate (0.9-1.0 mg of protein) for
2 h at 4 °C. The beads were then washed four times with
ice-cold lysis buffer containing 1 M NaCl. Bound proteins
were eluted by boiling in SDS sample buffer. Eluted proteins were
separated on 7.5% SDS-polyacrylamide gels, transferred to
nitrocellulose by electroblotting, and immunoblotted with anti-PLC
1
antibody. In some experiments GST fusion proteins were covalently
linked to Affi-Gel 10 according to the manufacturer's instructions for
use in binding competition experiments. In other experiments, GST
fusion proteins were phosphorylated in vitro by c-Src as
described previously (13), before use of the fusion proteins in
in vitro binding assays. For studies of in vitro
binding of the full-length AT1 receptor to PLC
1, GST
fusion proteins containing the various SH2 and SH3 domains of PLC
1
were utilized.
Assay of PLC Activity--
PLC activity was assayed using
[3H]PIP2-containing liposomes as substrate as
described previously by Goldschmidt-Clermont et al.
(14).
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RESULTS AND DISCUSSION |
To determine whether PLC
1 associates with the AT1
receptor in a ligand- and tyrosine
phosphorylation-dependent manner, we utilized a rabbit
polyclonal anti-AT1 receptor antibody directed against the
C-terminal 54 amino acid residues (306-359) of the rat
AT1A receptor (12). Cultured VSMC were stimulated with Ang II (10
7 M) for various times, cells were
lysed, and the AT1 receptor was immunoprecipitated from the
lysates with anti-AT1 receptor antibody. Immunoprecipitated
proteins were separated by gel electrophoresis, transferred to
nitrocellulose, and immunoblotted with anti-PLC
1 antibody. As shown
in Fig. 1, Ang II induced a rapid and
transient association of PLC
1 (140 kDa) with the AT1
receptor that was maximal within 30 s to 1 min. The time course of
Ang II-stimulated PLC
1-AT1 receptor association is thus
similar to that reported previously for Ang II-stimulated PLC
1
tyrosine phosphorylation and activation(5). Identical results were also
obtained when the experiments were repeated using a different rabbit
polyclonal anti-AT1 receptor antibody that recognizes
residues 15-24 in the N terminus of the rat AT1A receptor
(data not shown). However, in negative control experiments using rabbit
preimmune serum or an irrelevant rabbit polyclonal anti-GST antibody,
no PLC
1 was immunoprecipitated for any of the time points. To
investigate whether phosphorylation by an Src family tyrosine kinase is
required for the Ang II-induced association of PLC
1 with the
AT1 receptor, we also carried out coimmunoprecipitation
experiments in which cells were pretreated with the Src family
kinase-selective inhibitor, PP1 (10
6 M for 30 min) before Ang II stimulation. PP1, which has been shown previously to
be highly selective for Src family kinases relative to other known
tyrosine kinases (15), completely prevented AT1
receptor-PLC
1 association (data not shown), suggesting that tyrosine
phosphorylation of either PLC
1 or the receptor (or both) by a Src
family tyrosine kinase may be required for the association. This
possibility appears plausible because both PLC
1 and the AT1 receptor have been shown previously to be excellent
substrates for Src kinases in vitro (13, 16). Furthermore,
PLC
1 has been shown to form a complex with c-Src in several other
cell types (7, 17, 18). An alternative explanation for the inhibitory effect of PP1 is that Src kinase activity may be required for a
phosphorylation event that is upstream from either PLC
1 or AT1 receptor phosphorylation in a tyrosine phosphorylation
cascade.

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Fig. 1.
Time course of Ang II-stimulated association
of PLC 1 with the AT1 receptor in VSMC. VSMC were
stimulated for the times shown with Ang II (10 7
M). Cells were lysed, and cleared supernatants were
immunoprecipitated with anti-AT1 receptor antibody.
Immunoprecipitated proteins were separated by SDS-polyacrylamide gel
electrophoresis, transferred to nitrocellulose, and immunoblotted with
anti-PLC 1 antibody. Shown is a single blot (inset) and
densitometric analysis of blots from four separate experiments
(mean ± S.E.). In the three blots not shown, significant binding
of PLC 1 to the receptor was detected at 3 min.
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To confirm the results of the coimmunoprecipitation experiments and to
determine whether PLC
1 binds to the AT1 receptor
C-terminal intracellular domain, we utilized a GST-AT1
fusion protein (GST-AT1-(306-359)) containing the
C-terminal 54 amino acids of the rat AT1A receptor. The
GST-AT1 fusion and GST alone were expressed in E. coli and purified to homogeneity on a glutathione-agarose affinity
column. VSMC were treated with Ang II (10
7 M)
for various times, and cell lysates were prepared and used in in
vitro binding assays with the GST-AT1-(306-359)
fusion protein prebound to agarose beads. In control experiments,
lysates were also incubated with GST alone prebound to agarose beads.
After a 2-h incubation at 4 °C, the beads were washed extensively in buffer containing 1 M NaCl, and bound proteins were eluted.
The amount of PLC
1 eluted (and therefore bound by the fusion
protein) was then quantitated by immunoblotting with anti-PLC
1
antibody. As shown in Fig. 2, lysates
from Ang II-treated VSMC induced the binding of PLC
1 to the
GST-AT1-(306-359) fusion protein with a time course that
was similar to that observed for PLC
1 binding to the AT1
receptor in intact cells. No binding was detected for the GST alone
negative control (data not shown). Furthermore, when the cells from
which lysates were prepared were pretreated with PP1 (10
6
M for 30 min) before Ang II stimulation, PLC
1 binding to
the fusion protein was completely blocked (data not shown).

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Fig. 2.
Time course of Ang II-stimulated association
of PLC 1 in VSMC lysates with the GST-AT1-(306-359)
fusion protein. VSMC were stimulated with Ang II
(10 7 M) for the times indicated and then
lysed. Lysates were used in in vitro binding assays with the
GST-AT1-(306-359) fusion protein. Lysates were incubated
with the fusion protein prebound to agarose beads for 2 h at
4 °C. Incubation of the fusion protein with Ang II-treated lysates
resulted in tyrosine phosphorylation of the protein, as confirmed by
immunoblotting with anti-phosphotyrosine. Beads were washed extensively
with buffer containing 1 M NaCl, and bound proteins were
eluted. Binding of PLC 1 to the fusion protein was quantitated by
immunoblotting with anti-PLC 1 antibody. Shown is a representative
blot (inset) and densitometric analysis of blots from four
separate experiments (mean ± S.E.).
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Loss of binding of PLC
1 to the GST-AT1-(306-359) fusion
protein as a consequence of PP1 pretreatment suggests that binding requires the activity of c-Src or other Src family tyrosine kinases. One possibility is that Src kinase activity in the VSMC lysates is
required for direct tyrosine phosphorylation of the AT1
receptor C-terminal intracellular domain. This phosphorylation event
may, in turn, be required for PLC
1 binding to the receptor. To
determine whether the GST-AT1 fusion protein becomes
tyrosine phosphorylated when incubated with lysates from Ang II-treated
VSMC, cells were either treated or not treated with Ang II
(10
7 M for 30 s), and lysates were
prepared and then incubated with the GST-AT1-(306-359)
fusion protein prebound to agarose beads. Experiments were also carried
out in which cells were pretreated with PP1 (10
6
M for 30 min) prior to Ang II stimulation. After a 2-h
incubation at 4 °C, the beads were washed extensively, and bound
proteins were eluted. The relative phosphotyrosine content of the
eluted fusion protein was then assessed by immunoblotting with
anti-phosphotyrosine antibody. As shown in Fig.
3, lysates from Ang II-treated VSMC induced the tyrosine phosphorylation of the GST-AT1 fusion
protein. Ang II-stimulated phosphorylation, however, was completely
blocked when cells were pretreated with PP1. Furthermore,
phosphorylation was restricted to the AT1 receptor portion
of the fusion protein, as no tyrosine phosphorylation of the GST alone
negative control was detected (data not shown).

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Fig. 3.
Tyrosine phosphorylation of the
GST-AT1-(306-359) fusion protein by Ang II-treated VSMC
lysates. VSMC were either treated or not treated with Ang II
(10 7 M for 30 s) following either
pretreatment or no pretreatment with PP1 (10 6
M for 30 min). Cells were lysed and incubated with the
GST-AT1-(306-359) fusion protein prebound to agarose beads
for 2 h at 4 °C. Beads were then washed extensively with buffer
containing 1 M NaCl, and bound proteins were eluted. Bound
proteins were separated on SDS-polyacrylamide gels, transferred to
nitrocellulose by electroblotting, and immunoblotted with
anti-phosphotyrosine. Similar results were obtained in two separate
experiments.
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Recently we have shown that JAK2 association with the AT1
receptor involves the SH2 domain-containing SHP-2 phosphotyrosine phosphatase acting as an adaptor protein for JAK2 association. This
conclusion is based on in vitro binding assays with Ang
II-treated VSMC lysates and the GST-AT1-(306-359) fusion
protein in which lysates were quantitatively depleted of SHP-2 by
immunoprecipitation with anti-SHP-2 antibody before determining the
extent of JAK2 binding to the fusion protein. Immunodepletion of
lysates with anti-SHP-2 completely blocks JAK2 association with the
GST-AT1-(306-359) fusion protein.2 In the
present study, we have tested whether quantitative depletion of SHP-2
from VSMC lysates also alters PLC
1 binding to the
GST-AT1-(306-359) fusion protein. Lysates were prepared
from Ang II-treated (10
7 M for 30 s)
VSMC and then depleted of SHP-2 by immunoprecipitation with anti-SHP-2
antibody. Quantitative depletion of SHP-2 from lysates was confirmed by
immunoblotting with anti-SHP-2 antibody. Nondepleted (control) lysates
were also prepared. Immunodepleted and nondepleted lysates were then
used in in vitro binding assays with the
GST-AT1-(306-359) fusion protein prebound to beads.
PLC
1 binding to the fusion protein and to GST alone in the two
conditions was quantitated by immunoblotting with anti-PLC
1 antibody
as before. As shown in Fig. 4, PLC
1
bound to the fusion protein to approximately the same extent whether
from SHP-2-depleted or nondepleted lysates. No binding was detected for
the GST alone negative control. Therefore, we conclude that PLC
1
association with the AT1 receptor, unlike that of the JAK2
tyrosine kinase, does not depend on SHP-2 acting as an adaptor protein
for PLC
1 binding.

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Fig. 4.
Effect of immunodepletion of SHP-2 on binding
of PLC 1 in Ang II-treated VSMC lysates to the
GST-AT1-(306-359) fusion protein. VSMC were treated
with Ang II (10 7 M for 30 s), cells were
lysed, and lysates were either immunoprecipitated or not
immunoprecipitated (control) with anti-SHP-2 antibody. Quantitative
removal of SHP-2 from lysates by immunoprecipitation was confirmed by
immunoblotting. Immunodepleted and nonimmunodepleted lysates were then
used in in vitro assays with the
GST-AT1-(306-359) fusion protein or GST alone prebound to
agarose beads. After a 2-h incubation at 4 °C, beads were washed
extensively with buffer containing 1 M NaCl, and bound
proteins were eluted. Binding of PLC 1 to the fusion protein was
quantitated by immunoblotting with anti-PLC 1 antibody. Similar
results were obtained in two separate experiments.
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The hypothesis that c-Src or other Src family tyrosine kinase
modulates the PLC
1-AT1 receptor association is supported
further by the results of binding competition experiments with a
GST-AT1-(306-359) fusion protein phosphorylated in
vitro by c-Src. In these experiments, the
GST-AT1-(306-359) fusion protein was first covalently
linked to an agarose matrix and then allowed to bind PLC
1 in VSMC
lysates prepared from cells exposed to Ang II (10
7
M for 30 s). In addition, the purified free GST fusion
protein was either treated or not treated with purified human
recombinant c-Src and MgATP to obtain phosphorylated and
nonphosphorylated forms of the protein. Tyrosine phosphorylation of the
fusion protein by c-Src in vitro was confirmed by
immunoblotting of anti-GST immunoprecipitates with anti-phosphotyrosine
antibody. Free nonphosphorylated and phosphorylated forms of the
GST-AT1-(306-359) fusion protein were then used to compete
with the immobilized GST fusion protein for binding of PLC
1. The
amount of PLC
1 remaining bound to the immobilized fusion protein
after incubation with the competitor proteins was quantitated by
immunoblotting of glutathione-eluted proteins with anti-PLC
1
antibody. As shown in Fig. 5, no
competition was observed with the nonphosphorylated protein. However,
increasing concentrations of free tyrosine-phosphorylated
GST-AT1 fusion protein effectively competed with the
GST-AT1 receptor fusion protein agarose matrix for PLC
1
binding, suggesting that direct phosphorylation of the AT1
receptor C-terminal tail by c-Src may increase its binding affinity for
PLC
1.

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Fig. 5.
Competition of free phosphorylated and
nonphosphorylated GST-AT1-(306-359) fusion proteins with
an immobilized GST-AT1-(306-359) fusion protein for
binding by PLC 1. VSMC were treated with Ang II
(10 7 M for 30 s), and cell lysates were
prepared. Proteins in lysates were allowed to bind to the
GST-AT1-(306-359) fusion protein immobilized on Affi-Gel
10. Nonphosphorylated and c-Src-phosphorylated free GST-AT1-(306-359) fusion proteins at various
concentrations were then incubated for 30 min at 4 °C with the
immobilized GST fusion protein prebound by PLC 1. The amount of
PLC 1 that remained bound to the immobilized
GST-AT1-(306-359) was quantitated by immunoblotting with
anti-PLC 1 antibody. Similar results were obtained in three separate
experiments.
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Inhibition of PLC
1 binding to the immobilized GST-AT1
receptor fusion protein by the free phosphorylated but not the
nonphosphorylated fusion protein could also be due to an indirect,
allosteric interference rather than to competition for the binding
site. Therefore, to more directly demonstrate a role for receptor
phosphorylation in PLC
1 binding to the AT1 receptor, we
carried out in vitro binding assays with
GST-AT1-(306-359) fusion proteins that were either
phosphorylated or not phosphorylated in vitro by c-Src. The
AT1 receptor C-terminal cytoplasmic tail (residues
306-359) contains tyrosine residues at positions 312, 319, and 339. To determine whether phosphorylation of one or more of these residues is
required for binding of PLC
1, we individually mutated each tyrosine
residue in the GST-AT1-(306-359) fusion protein to a phenylalanine. Wild-type GST-AT1-(306-359),
GST-AT1-(306-359) ( Tyr-312
Phe),
GST-AT1-(306-359) (Tyr-319
Phe), and
GST-AT1-(306-359) (Tyr-339
Phe) fusion proteins
prebound to agarose beads were each treated with purified c-Src
and MgATP in vitro to obtain phosphorylated forms of the
proteins. Phosphorylated fusion proteins and the nonphosphorylated
wild-type fusion protein were then used in in vitro binding
assays to detect possible binding by PLC
1 from untreated VSMC
lysates. Binding was quantitated by immunoblotting with anti-PLC
1
antibody as described earlier. As shown in Fig. 6, PLC
1 in untreated lysates bound to
the wild-type GST-AT1 receptor fusion protein only if it
had been phosphorylated in vitro by c-Src. PLC
1 also
bound to in vitro phosphorylated
GST-AT1-(306-359) (Tyr-312
Phe) and
GST-AT1-(306-359) (Tyr-339
Phe) fusion proteins but
not to the GST-AT1-(306-359) (Tyr-319
Phe) fusion
protein, demonstrating that it is phosphorylation of Tyr-319
specifically that is required for tyrosine
phosphorylation-dependent association of PLC
1 with the
AT1 receptor.

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Fig. 6.
Effect of phosphorylation of
GST-AT1 fusion proteins by c-Src in vitro on
binding of the fusion proteins by PLC 1. The wild-type
GST-AT1-(306-359) fusion protein prebound to agarose beads
was either phosphorylated or not phosphorylated in vitro by
purified recombinant human c-Src. GST-AT1 fusion proteins
containing mutated tyrosines (at 312, 319, and 339) were also
phosphorylated in vitro by c-Src. Nonphosphorylated and
phosphorylated proteins prebound to beads were washed extensively with
buffer containing 1 M NaCl before use in in
vitro binding assays with lysates from untreated VSMC. After a 2-h
incubation at 4 °C, beads were again washed extensively with buffer
containing 1 M NaCl, and bound proteins were eluted.
Binding of PLC 1 to the fusion proteins was quantitated by
immunoblotting with anti-PLC 1 antibody. Similar results were
obtained in two separate experiments. Y, Tyr; F, Phe.
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In order for tyrosine phosphorylation of the AT1 receptor
C-terminal intracellular domain to have a role in mediating PLC
1 binding to the receptor in VSMC, it must occur rapidly (within 30 s) in response to Ang II stimulation. To investigate whether Ang II
induces rapid tyrosine phosphorylation of the AT1 receptor in VSMC, untreated cells or cells treated with Ang II
(10
7 M for 30 s) were lysed and
immunoprecipitated with anti-AT1 receptor antibody.
Immunoprecipitates were then immunoblotted with anti-phosphotyrosine antibody. Experiments were also carried out in which VSMC were pretreated with either the tyrosine phosphatase inhibitor, sodium orthovanadate (10
4 M for 30 min), or PP1
(10
6 M for 30 min). Results shown in Fig.
7 demonstrate that Ang II induces a rapid
and significant increase in the phosphotyrosine content of the
AT1 receptor in VSMC. Pretreatment with sodium orthovanadate increased the phosphotyrosine content of the receptor even in the absence of Ang II stimulation. In contrast, pretreatment with PP1 completely abolished the Ang II-induced tyrosine
phosphorylation of the receptor, suggesting a requirement for Src
family kinase activity in receptor phosphorylation.

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Fig. 7.
Ang II-stimulated tyrosine phosphorylation of
the AT1 receptor in VSMC. VSMC were either treated or
not treated with Ang II (10 7 M for 30 s)
following either pretreatment or no pretreatment (control) with sodium
orthovanadate (10 4 M for 30 min) or PP1
(10 6 for 30 min). Cells were lysed and immunoprecipitated
with anti-AT1 receptor antibody, and immunoprecipitated
proteins were immunoblotted with anti-phosphotyrosine antibody. In the
experiment shown, two differentially glycosylated forms of the
AT1 receptor were not resolved. Similar results were
obtained in two separate experiments.
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To further map the region of the AT1 receptor C-terminal
tail that interacts with PLC
1, we expressed a series to
GST-AT1 fusion proteins containing various deletional or
point mutations in the AT1 portion of the fusion protein.
Proteins were expressed in E. coli and purified by affinity
chromatography on glutathione-agarose (Table
I). Each mutant protein was then
individually tested for its ability to bind PLC
1 in lysates from Ang
II-treated (10
7 M for 30 s) VSMC.
Binding was detected by immunoblotting with anti-PLC
1 as described
earlier. Fusion proteins of the AT1 receptor containing
residues 306-359, 306-348, 306-329, and 318-359 were each bound by
PLC
1. In contrast, fusion proteins containing AT1 receptor residues 336-359, 323-359, and 306-318 were not bound by
PLC
1 (Fig. 8A). Deletional
analysis thus identifies residues located between positions 318 and 323 as being essential for PLC
1 binding. The YIPP motif in the
AT1 receptor C-terminal tail, which has been shown
previously to bind the JAK/SHP-2 complex, is located at positions
318-322. Thus it is likely that this motif also functions as a binding
site for PLC
1 and that, as shown also in Fig. 6, phosphorylation of
tyrosine 319 within the motif enhances PLC
1 binding in a manner
similar to that shown previously for the platelet-derived growth factor
and
receptors. This conclusion is also supported by the results
of in vitro binding assays using VSMC lysates from Ang
II-treated cells (10
7 M for 30 s) and
the GST-AT1-(306-359) fusion proteins in which the
tyrosine residues at positions 312, 319, and 339 of the AT1 receptor were each individually mutated to phenylalanines. Assays were
carried out with Ang II-treated (10
7 M for
30 s) VSMC lysates (which as shown earlier contain activated Src
family tyrosine kinases that can phosphorylate the fusion proteins) and
either wild-type GST-AT1-(306-359),
GST-AT1-(306-359) (Tyr-312
Phe),
GST-AT1-(306-359) (Tyr-319
Phe), or
GST-AT1-(306-359) (Tyr-339
Phe) fusion proteins. As
shown in Fig. 8B, PLC
1 from VSMC lysates bound to each of
the fusion proteins with the exception of
GST-AT1-(306-359) (Tyr-319
Phe), again indicating an
essential role for tyrosine 319 in PLC
1 binding.

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Fig. 8.
Mutational analysis of AT1
receptor amino acids required for in vitro binding of
PLC 1 to GST-AT1 fusion proteins. VSMC were treated
with Ang II (10 7 M for 30 s) and cell
lysates were prepared and used in in vitro binding assays
with GST-AT1 fusion proteins containing various deletional
or point mutations in the AT1 receptor C-terminal
intracellular domain. Lysates were incubated with each of the different
fusion proteins (prebound to agarose beads) for 2 h. Beads were
then washed extensively with buffer containing 1 M NaCl,
and bound proteins were eluted. The amount of PLC 1 eluted (and
therefore bound by a given fusion protein) was quantitated by
immunoblotting with anti-PLC 1 antibody. Similar results were
obtained in three separate experiments. Y, Tyr;
F, Phe.
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The importance of tyrosine 319 in PLC
1 binding to the
AT1 receptor and in activation of the PLC
1 enzyme was
further confirmed in in vitro binding assays in which
PLC
1 binding to the receptor was quantitated by PLC activity. Ang
II-treated VSMC lysates (10
7 M for 30 s)
were incubated with wild-type GST-AT1-(306-359), GST-AT1-(329-359), GST-AT1-(306-359) (Tyr-319
Phe), GST-AT1-(306-359) (Tyr-312
Phe), and
GST-AT1-(306-359) (Tyr-339
Phe) fusion proteins
prebound to agarose beads. Beads were washed extensively, and proteins
were eluted with reduced glutathione. Eluates were then assayed for PLC
activity using [3H]PIP2-containing liposomes
as substrate. As shown in Fig. 9, a
deletional mutant (323-359) lacking the YIPP motif and the point mutant (Tyr-319
Phe) lacking tyrosine 319 bound very little PLC
activity, whereas other mutants lacking tyrosines 312 (Tyr-312
Phe)
and 339 (Tyr-339
Phe) bound significantly more PLC activity, equivalent to that bound by the wild-type fusion protein.

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Fig. 9.
Mutational analysis of AT1
receptor amino acids required for association of PLC activity with
GST-AT1 fusion proteins. VSMC were treated with Ang II
(10 7 M for 30 s), and cell lysates were
prepared and mixed with the indicated GST-AT1 fusion
proteins prebound to agarose beads. After extensive washing of the
beads, PLC activity was determined using [3H]PIP2-containing liposomes as substrate.
Results shown represent mean ± S.E. from three separate
experiments. Y, Tyr; F, Phe.
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Full-length PLC
1 contains two SH2 domains and a single SH3 domain.
To determine which of these domains, if any, are required for
interaction of PLC
1 with the AT1 receptor, we also
carried out in vitro binding assays with commercially
available GST fusion proteins containing the various SH2 and SH3
domains of PLC
1 (Fig. 10A). VSMC were exposed to
Ang II (10
7 M) for 0, 0.5, and 1 min and then
lysed. Lysates were incubated with GST-PLC
1 fusion proteins prebound
to agarose beads. Beads were washed extensively, and bound
proteins were eluted with reduced glutathione. The amount of
AT1 receptor eluted was then quantitated by immunoblotting
with anti-AT1 receptor antibody. As shown in Fig.
10B, only the C-terminal SH2 domain of PLC
1 (residues
663-760 of the rat PLC
1 sequence) was required for
ligand-dependent binding of the enzyme to the
AT1 receptor.

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Fig. 10.
Deletional analysis of Src homology domains
of PLC 1 required for binding to the AT1 receptor.
VSMC were treated with Ang II (10 7 M) for 0, 0.5, and 1 min and then lysed. Lysates were incubated with the various
GST-PLC 1 fusion proteins prebound to agarose beads. Beads were
washed extensively with buffer containing 1 M NaCl, and
bound proteins were eluted. The amount of AT1 receptor eluted was quantitated by immunoblotting with anti-AT1
receptor antibody. In the experiment shown, two differentially
glycosylated forms of the AT1 receptor were resolved.
Similar results were obtained in three separate experiments.
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In summary, the results of the present study show for the first time
that PLC
1 binds to the G-protein-coupled AT1 receptor in
an Ang II- and tyrosine phosphorylation-dependent manner.
The PLC
1-AT1 receptor interaction appears to depend on
phosphorylation of tyrosine 319 in a YIPP motif in the C-terminal
intracellular domain of the AT1 receptor and binding of the
phosphorylated receptor by the most C-terminal of two SH2 domains in
PLC
1. PLC
1 thus binds to the same site in the receptor previously
identified for binding of the SHP-2 phosphotyrosine phosphatase/JAK2
tyrosine kinase complex. A single site in the C-terminal tail of the
receptor can, therefore, be bound in a ligand-dependent
manner by two different downstream effector proteins. The data
presented here further demonstrates that G-protein-coupled receptors
can physically associate with intracellular proteins other than G
proteins, creating membrane-delimited signal transduction complexes
similar to those observed for classic growth factor receptors.
We wish to thank Dr. Kenneth E. Bernstein in whose laboratory the GST-AT1 receptor cDNA
constructs were originally prepared.