Functional Trans-inactivation of Insulin Receptor Kinase by Growth-Inhibitory Angiotensin II AT2 Receptor
Nathalie Elbaz,
Katarina Bedecs,
Maryline Masson,
Malène Sutren,
A. Donny Strosberg and
Clara Nahmias
Institut Cochin de Génétique Moléculaire
Centre Nationale de la Recherche Scientifique UPR 0415 75014
Paris, France
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ABSTRACT
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The present study demonstrates negative
intracellular cross-talk between angiotensin II type 2
(AT2) and insulin receptors.
AT2 receptor stimulation leads to inhibition of
insulin-induced extracellular signal-regulated protein kinase (ERK2)
activity and cell proliferation in transfected Chinese hamster ovary
(CHO-hAT2) cells. We show that AT2 receptor
interferes at the initial step of insulin signaling cascade, by
impairing tyrosine phosphorylation of the insulin receptor (IR)
ß-chain. AT2-mediated inhibition of IR
phosphorylation is insensitive to pertussis toxin and is also detected
in neuroblastoma N1E-115 and pancreatic acinar AR42J cells that express
endogenous receptors. We present evidence that
AT2 receptor inhibits the
autophosphorylating tyrosine kinase activity of IR, with no
significant effect on insulin binding properties.
AT2-mediated inactivation of IR does not mainly
involve tyrosine dephosphorylation by vanadate-sensitive
tyrosine phosphatases nor serine/threonine phosphorylation by protein
kinase C. As a consequence of IR inactivation,
AT2 receptor inhibits tyrosine phosphorylation
of insulin receptor substrate-1 (IRS-1) and signal-regulatory protein
(SIRP
1) and prevents subsequent association of both IRS-1 and
SIRP
1 with Src homology 2 (SH2)-containing tyrosine phosphatase
SHP-2. Our results thus demonstrate functional trans-inactivation of IR
kinase by G protein-coupled AT2 receptor,
illustrating a novel mode of negative communication between two
families of membrane receptors.
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INTRODUCTION
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The octapeptide angiotensin II (Ang II), a major regulator of
blood pressure, is also involved in the control of cell proliferation
and hypertrophy. This peptide binds with similar affinity to two
subtypes of G protein-coupled receptors, AT1 and
AT2, that differ in their signaling pathways and
physiological functions (1, 2, 3, 4). The AT1 receptor
mediates most of the cardiovascular effects of Ang II and promotes cell
proliferation. Increasing evidence indicates that the
AT2 subtype may attenuate the effects of
AT1 on blood pressure regulation (5, 6, 7, 8, 9), cardiac
and vascular cell growth (10, 11), and tissue regeneration after injury
(12, 13, 14). In addition, the AT2 subtype plays a
critical role in the ontogeny of the kidney (15) and exerts antigrowth,
antifibrotic, and proapoptotic effects in vivo (4). In
cultured cells, the AT2 receptor promotes
neuronal differentiation (16, 17, 18) and apoptosis (19, 20, 21) and inhibits
cell proliferation induced by growth factors (22, 23, 24).
The AT2 receptor thus provides an
interesting model for investigating intracellular pathways involved in
the attenuation of cell growth. The question can be raised of whether
the growth-inhibitory AT2 receptor may act on the
same signaling molecules as those used by the mitogenic
AT1 subtype. Like other growth factor receptors,
AT1 mediates activation of the
Ras/Raf/extracellular signal-regulated kinase (ERK) cascade (25, 26),
whereas the AT2 receptor has been shown to
inhibit ERK activity (19, 27, 28, 29) and to activate protein tyrosine
phosphatases (19, 27, 30, 31, 32, 33) in different cell types. An interesting
feature recently described for mitogenic G protein-coupled receptors
including AT1, is their ability to trigger
autophosphorylation of growth factor receptor tyrosine kinases (RTK), a
phenomenon referred to as RTK transactivation (34, 35, 36).
AT1 thus transactivates the receptors for
platelet-derived growth factor (37), epidermal growth factor (38), and
insulin-like growth factor (39). In addition, the
AT1 subtype is able to mimic intracellular
effects of the insulin receptor in promoting tyrosine phosphorylation
of insulin receptor substrate-1 (IRS-1) (39, 40, 41, 42) and subsequent
association of IRS-1 with the SH2 domain-containing tyrosine
phosphatase SHP-2 (40). Whether the AT2 receptor
is also able to regulate the kinase activity of RTKs and
phosphorylation of downstream effectors has not yet been
investigated.
In the present study, we have examined the regulatory effect of
AT2 receptor stimulation on the insulin
receptor-signaling pathway. We show that in Chinese Hamster Ovary (CHO)
cells, AT2 receptor interferes with the
insulin-induced intracellular cascade leading to ERK activation and
cell proliferation. We present evidence that AT2
receptor inhibits autophosphorylation of insulin receptor ß-subunit
(IRß) with no major alteration of insulin binding properties.
Furthermore, AT2 receptor impairs insulin-induced
tyrosine phosphorylation of the insulin receptor substrates IRS-1 and
signal-regulatory protein SIRP
1. This, in turn, leads to reduced
association of IRS-1 and SIRP
1 with tyrosine phosphatase SHP-2 and
may thus account for the inhibitory effect of AT2 on
insulin-induced ERK activity and cell growth.
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RESULTS
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AT2 Receptor Mediates Inhibition of
Insulin-Induced ERK2 Activity and Cell Growth
Intracellular cross-talk between Ang II type 2
(AT2) and insulin receptors was analyzed in
CHO-hAT2 cells, stably transfected with the human
AT2 receptor gene and expressing endogenous
insulin receptors. Activation of ERK2 in these cells was visualized by
the characteristic decrease in electrophoretic mobility of the enzyme,
known to reflect its phosphorylation and activation. Insulin-induced
ERK2 activity was barely detectable at 1 min, maximal at 5 min, and
almost disappearing at 10 min, whereas Ang II and the
AT2-selective agonist CGP 42112 added alone had
no significant effect on basal ERK2 activity at every time tested (data
not shown). The inhibitory effect of CGP 42112 on ERK2 activity was
examined at time (5 min) of maximal stimulation of the enzyme by
insulin. Insulin induced a dose-dependent activation of ERK2 that
was consistently inhibited in the presence of CGP 42112 (Fig. 1A
). Inhibition by CGP 42112 was more
easily detectable at a submaximal dose of insulin (Fig. 1A
) and was
quantified as 44 ± 6% (n = 4) for a dose of 0.05 µg/ml (9
nM) insulin. Similar inhibition of insulin-induced
activation of ERK2 was observed for concentrations of Ang II and CGP
42112 ranging from 0.1 nM to 100 nM (data not
shown).

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Figure 1. Inhibitory Effect of CGP 42112 on Insulin-Induced
ERK2 Activity and Cellular Growth
A, CHO-hAT2 cells were left untreated (0) or treated for 5 min with the
indicated dose of insulin in the presence (+) or absence (-) of CGP
42112 (100 nM). Total cell lysates were analyzed by
immunoblotting with anti-ERK2 antibodies. The activated, slower
migrating form of ERK2 is indicated by a star, and both
forms are pointed out by arrows. Shown is one
representative experiment out of four. Lower panel shows
the corresponding quantification of ERK2 activation in this experiment
and is expressed as percent of slower migrating form of ERK2 relative
to total amount of ERK2. B, CHO-hAT2 cells (left panel)
or nontransfected CHO-WT cells (right panel) were
treated for 5 min with (+) or without (-) insulin (0.05 µg/ml), Ang
II (100 nM), and Sarile (1 µM), and total
cell lysates were analyzed for ERK2 activation as in panel A. Shown is
one representative experiment out of three. C, CHO-hAT2 cells were
grown in the presence (+) or absence (-) of insulin and CGP 42112 as
indicated, and numbered as described in Materials and
Methods. Results shown represent mean ± SEM
of three separate experiments performed in quadruplicate. *,
P = 0.056; n = 3.
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Treatment of CHO-hAT2 cells with an excess of
(Sar1, Ile8)-Ang II
(Sarile), an antagonist of AT1 and
AT2 receptors, completely reversed the inhibition
of ERK2 induced by Ang II, with no modification of either basal or
insulin-induced ERK2 activity (Fig. 1B
). Interestingly, CHO-WT cells
that had not been transfected with the AT2
receptor gene were more sensitive to low doses of insulin as revealed
by the complete shift from basal to phosphorylated form of ERK2 induced
by 0.05 µg/ml insulin (Fig. 1B
). Moreover, Ang II, as well as CGP
42112, was unable to inhibit ERK2 activity induced by insulin in
nontransfected cells (Fig. 1B
), further indicating that inhibition of
insulin-induced ERK2 activity in CHO-hAT2 cells is mediated by the
AT2 receptor subtype.
To evaluate whether AT2 receptor activation may
also interfere with insulin-induced cell proliferation, CHO-hAT2 cells
were treated for 48 h with insulin (0.01 µg/ml) in the presence
or absence of CGP 42112 (1 µM). Under these conditions,
insulin increased the number of cells by a factor of 2.2, and this
growth stimulation was inhibited by 38 ± 3% (n = 3) in the
presence of CGP 42112 (Fig. 1C
). CGP 42112 added alone had no
detectable effect on cell number (data not shown).
AT2 Receptor Mediates Inhibition of Insulin
Receptor Tyrosine Phosphorylation
To identify target proteins of the insulin signaling cascade
that may be affected by AT2 receptor stimulation,
we analyzed the effect of CGP 42112 on the overall profile of cellular
protein tyrosine phosphorylation induced by insulin. As shown in Fig. 2A
, insulin induced hyperphosphorylation
of a 97-kDa polypeptide that was strongly reduced in the presence of
CGP 42112. Insulin additionally induced a modest but consistent
increase in tyrosine phosphorylation of cellular polypeptides of
apparent molecular mass 120 kDa that was detectable at basal levels. Of
interest, phosphorylation of 120-kDa polypeptides was significantly
reduced upon activation of the AT2 receptor (Fig. 2A
).

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Figure 2. Inhibitory Effect of CGP 42112 on RTK
Autophosphorylation
A, CHO-hAT2 cells were treated as indicated in Fig. 1A , and total
cell lysates were analyzed by immunoblotting with antiphosphotyrosine
antibodies. Molecular mass markers are indicated in kilodaltons (kDa)
on the right. Shown is one representative experiment out
of four. B, CHO-hAT2 cells were treated as in panel A, and then
lectin-Sepharose precipitates were prepared and analyzed by successive
immunoblotting with antiphosphotyrosine (PY) and anti-IRß antibodies.
Apparent molecular mass is indicated in kilodaltons (kDa) on the
right. Shown is one representative experiment out of
eight. C, N1E-115 or AR42J cells were treated with insulin (0.1
µg/ml) in the presence or absence of CGP 42112 (100 nM),
and lectin-Sepharose precipitates were analyzed as in panel B. Shown is
one representative experiment out of three. D, N1E-115 or COS-hAT2
cells were treated with EGF (10 ng/ml) in the presence or absence of
Ang II (100 nM) or CGP 42112 (100 nM).
Anti-EGFR immunoprecipitates prepared from N1E-115 cells or total cell
lysates extracted from COS-hAT2 cells were analyzed by successive
immunoblotting with antiphosphotyrosine (PY) and sheep anti-EGFR
antibodies. Shown is one representative experiment out of three.
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We investigated the possibility that the 97-kDa polypeptide undergoing
hyperphosphorylation in response to insulin may correspond to the
insulin receptor ß chain (IRß). IRß purified on wheat-germ
lectin-Sepharose indeed migrated as a 97-kDa polypeptide and was
tyrosine phosphorylated in response to insulin in a dose-dependent
manner. As shown in Fig. 2B
, insulin-induced phosphorylation of IRß
was consistently inhibited in the presence of either Ang II or CGP
42112. CGP 42112 inhibited by 64 ± 4% (n = 7) tyrosine
phosphorylation of IRß induced by 0.01 µg/ml insulin within 5 min.
A similar inhibitory effect was observed with doses of CGP 42112
ranging from 0.01 nM to 100 nM (data not
shown). These results thus indicate that AT2
receptor interferes at the initial and essential step of the insulin
cascade, i.e. autophosphorylation of the insulin
receptor.
Pretreatment of CHO-hAT2 cells with pertussis toxin (100 ng/ml for
16 h) did not prevent the inhibitory effect of
AT2 receptor on insulin-induced tyrosine
phosphorylation of IRß (data not shown), indicating that this
AT2 signaling pathway does not involve coupling
to regulatory heterotrimeric Gi/Go proteins.
AT2-mediated inhibition of insulin-induced IRß
phosphorylation was also detected in other cell types that express
endogenous insulin and AT2 receptors, such as
neuroblastoma N1E-115 cells and pancreatic acinar AR42J cells (Fig. 2C
). In addition, AT2 receptor stimulation
impaired epidermal growth factor (EGF)-induced tyrosine phosphorylation
of endogenous EGF receptors in N1E-115 cells and in COS-hAT2 cells
(Fig. 2D
), indicating that the inhibitory effect of
AT2 is not solely restricted to the insulin
receptor expressed in CHO-hAT2 cells.
AT2 Receptors Mediate Inhibition of Insulin Receptor Kinase
Activity
To get further insight into the mechanism by which the
AT2 receptor inhibits IRß autophosphorylation,
we investigated whether AT2 receptor activation
modifies the binding properties or tyrosine kinase activity of the
insulin receptor or whether its main action is to contribute to
tyrosine dephosphorylation of IRß after its activation. Specific
binding of radiolabeled insulin to intact CHO-hAT2 cells remained
unaffected (102 ± 8%, n = 3) after treatment with CGP 42112
in conditions (100 nM for 5 min) that allowed maximal
inhibition of IRß phosphorylation.
Tyrosine kinase activity of the insulin receptor was then analyzed in
an in vitro kinase assay using the IRß chain as a
substrate. Incorporation of 32P to IRß was
measured after purification of insulin receptors from cells treated
with insulin in the presence or absence of CGP 42112. As shown in Fig. 3A
, autophosphorylating activity of IRß
was consistently reduced (43 ± 5% inhibition, n = 3) upon
AT2 receptor stimulation. Altogether, these
results indicate that AT2 receptor stimulation
leads to significant inactivation of the insulin receptor tyrosine
kinase, with no major alteration in insulin binding properties.

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Figure 3. Inhibitory Effect of CGP 42112 on IR Kinase
Activity and Insensitivity to Sodium Orthovanadate
A, CHO-hAT2 cells were treated as indicated in Fig. 2B , and then IRß
was purified on lectin column, and IR kinase autophosphorylating
activity was analyzed by autoradiography after separation on 10%
reducing SDS-PAGE. Shown is one representative experiment out of three.
B, Cells were pretreated or not for 16 h with 0.1
mM sodium orthovanadate before treatment for 5 min at 37 C
with the indicated dose of insulin in the presence (+) or absence (-)
of CGP 42112 (100 nM). Lectin-Sepharose precipitates were
analyzed as in Fig. 2B by immunoblotting with antiphosphotyrosine (PY)
and anti-IRß antibodies. Shown is one representative experiment out
of four.
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To investigate whether a tyrosine phosphatase may be involved in the
AT2/IRß negative cross-talk, regulation of
IRß phosphorylation was analyzed after treatment of CHO-hAT2 cells
with sodium orthovanadate, a potent tyrosine phosphatase inhibitor. As
a result of inhibiting endogenous tyrosine phosphatase activity, a
large increase in phosphotyrosine-containing proteins was observed at
basal levels, and sensitivity of the cells to insulin was increased by
a factor of 10 (Fig. 3B
). Indeed, in the presence of orthovanadate, a
10 times lower concentration of insulin (0.01 instead of 0.1 µg/ml)
was sufficient to induce high levels of phosphorylation of IRß, while
IRß phosphorylation reached saturating levels at 0.1 µg/ml insulin
(Fig. 3B
). Under these conditions of tyrosine phosphatase inhibition,
the AT2 agonist CGP 42112 was still able to
impair insulin-induced phosphorylation of IRß [47 ± 5%
inhibition (n = 4) for a dose of 0.01 µg/ml insulin]. These
results therefore indicate that AT2-mediated
IRß inactivation is not mainly due to dephosphorylation by a
vanadate-sensitive tyrosine phosphatase.
It is of interest to note that in addition to IRß, another
glycoprotein of apparent molecular mass 120 kDa was also retained on
lectin columns. Phosphorylation of this glycoprotein was detectable at
basal levels, increased in the presence of insulin, and significantly
decreased in the presence of CGP 42112 (Fig. 3B
). Analysis of this
120-kDa phosphoprotein, which may correspond to the 120-kDa polypeptide
depicted in whole-cell lysates (Fig. 2A
), will be addressed below in
more detail (Fig. 4
).

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Figure 4. Inhibitory Effect of CGP 42112 on Insulin-Induced
IRS-1 and SIRP 1 Phosphorylation and Association with SHP-2
A, CHO-hAT2 cells were left untreated (0) or treated as in Fig. 2A with
insulin (0.01 µg/ml) in the presence (+) or absence (-) of CGP 42112
(100 nM), and anti-IRS-1 immunoprecipitates were
immunoblotted successively with antiphosphotyrosine (PY) and anti-IRS-1
antibodies. Shown is one out of two independent experiments. B,
CHO-hAT2 cells were treated as in panel A, and lectin-Sepharose
precipitates were analyzed by successive immunoblotting with
antiphosphotyrosine (PY) and anti-SIRP 1 antibodies. Shown is one
representative experiment out of five. C,
CHO-hAT2 cells were treated as in panel A, and anti-SIRP 1
immunoprecipitates were analyzed by successive immunoblotting with
antiphosphotyrosine (PY) and anti-SIRP 1 antibodies. Shown is one
representative experiment out of three. D, CHO-hAT2 cells were either
left untreated, or treated for 1 min at 37 C with insulin (0.05
µg/ml) in the absence or presence of CGP 42112 (100 nM),
and anti-SHP-2 immunoprecipitates were submitted to successive
immunoblotting with antiphosphotyrosine (PY), anti-IRS-1,
anti-SIRP 1, and anti-SHP-2 antibodies. Shown is one representative
experiment out of six.
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We also examined the possibility that
AT2-mediated inhibition of IRß
autophosphorylation may be due to serine/threonine phosphorylation by
protein kinase C, as several protein kinase C isoforms have been shown
to cause significant reduction of insulin receptor autophosphorylation
and kinase activity (43, 44). Long-term treatment of CHO-hAT2 cells
with phorbol-12-myristate-13-acetate (100 ng/ml for 16 h),
allowing depletion of intracellular pools of protein kinase C, had no
effect on AT2-mediated inhibition of IRß
autophosphorylation (data not shown), therefore ruling out a major
involvement of phorbol ester-sensitive protein kinase C in the
AT2 inhibitory pathway.
AT2 Receptor Mediates Inhibition of Insulin Receptor Substrate
Phosphorylation
To investigate whether AT2-mediated
inactivation of IRß was functionally relevant, we analyzed the
consequence of AT2 receptor stimulation on the
phosphorylation of two IRß substrates, IRS-1 and SHC, which function
as major transducers of the insulin-induced ERK pathway. In CHO-hAT2
cells, SHC was poorly phosphorylated on tyrosine upon insulin
stimulation, and therefore the effect of AT2
receptor on the phosphorylation of SHC could not be analyzed properly
(data not shown). In contrast, analysis of anti-IRS-1
immunoprecipitates revealed that tyrosine phosphorylation of IRS-1 was
increased by insulin and was indeed inhibited in the presence of CGP
42112 (Fig. 4A
).
We also examined the effect of AT2 receptor
stimulation on a more recently characterized substrate of insulin
receptor, known as SIRP
1. SIRP
1 is a glycosylated transmembrane
protein of 115120 kDa, which undergoes tyrosine phosphorylation upon
mitogen stimulation and cell adhesion. Phosphorylated SIRP
1
interacts with the SH2 domain-containing tyrosine phosphatase SHP-2 and
contributes to regulating the ERK pathway (45, 46). To investigate the
possibility that SIRP
1 may correspond to the 120-kDa phosphoprotein
retained on lectin columns (Fig. 3B
), immunoblots were incubated with
specific anti-SIRP
1 antibodies. As shown in Fig. 4B
, endogenous
SIRP
1 expressed in CHO-hAT2 cells was purified on wheat-germ lectin
column as a protein of apparent molecular mass 120 kDa (p120). Tyrosine
phosphorylation of p120/SIRP
1 was detectable in unstimulated cells,
increased in response to insulin, and consistently decreased in the
presence of CGP 42112 (Figs. 3B
and 4B
). Phosphorylation of
p120/SIRP
1 induced by 0.01 µg/ml insulin within 5 min was
inhibited by 48 ± 7% (n = 7) in the presence of 100
nM CGP 42112. Immunoprecipitation experiments using
specific anti-SIRP
1 antibodies further confirmed that basal levels
of SIRP
1 tyrosine phosphorylation are increased after treatment with
insulin and inhibited upon addition of CGP 42112 (Fig. 4C
).
AT2 Receptor Mediates Reduced Association of IRS-1 and SIRP
1
with SHP-2
The SH2 domain-containing tyrosine phosphatase SHP-2 is a
positive transducer of the insulin-induced ERK cascade (47, 48, 49, 50)
that interacts with phosphorylated IRS-1 (48, 51, 52) and SIRP
1 (45, 46) after insulin receptor activation. We therefore analyzed the
consequence of AT2 receptor stimulation on the
association of both IRS-1 and SIRP
1 with SHP-2. CHO-hAT2 cells
treated with insulin in the presence or absence of CGP 42112 were
submitted to immunoprecipitation using anti-SHP-2 antibodies, and the
presence of phosphoproteins in the immunocomplexes was revealed by
immunoblotting with antiphosphotyrosine antibodies. As seen in Fig. 4D
(upper panel), two major phosphoproteins of apparent
molecular mass 185 kDa and 120 kDa, respectively, were detected in
anti-SHP-2 immunoprecipitates after insulin receptor stimulation.
Reblotting with anti-IRS-1 antibodies confirmed that p185 corresponds
to IRS-1. Association between p185/IRS-1 and SHP-2 was detected only
after treatment with insulin and was significantly reduced (59 ±
17%, n = 4) in the presence of CGP 42112 (Fig. 4D
), in good
correlation with previously observed regulation of IRS-1 tyrosine
phosphorylation (Fig. 4A
).
Reblotting the same membranes with specific anti-SIRP
1 antibodies
(Fig. 4D
) also confirmed that p120 corresponds to SIRP
1. Association
of phosphorylated p120/SIRP
1 with SHP-2 preexisted at basal level,
was increased upon insulin receptor stimulation, and consistently
inhibited (56 ± 5%, n = 3) in the presence of CGP 42112.
Good correlation was observed between p120/SIRP
1 tyrosine
phosphorylation (Fig. 4
, B and C) and association with SHP-2 (Fig. 4D
),
supporting a previous report that phosphorylated tyrosines of SIRP
1
associate with the SH2 domains of SHP-2 (53). Immunoprecipitated SHP-2
itself did not undergo increased tyrosine phosphorylation upon
treatment with insulin in the presence or absence of CGP 42112 (Fig. 4D
), in agreement with previous findings that SHP-2 is not a substrate
of IRß (51, 52).
Phosphorylated SIRP
1 has also been reported to bind to the SH2
domain-containing tyrosine phosphatase SHP-1 both in vitro
and in vivo (45, 54, 55), leading us to examine whether
AT2 receptor stimulation may also regulate the
association of SIRP
1 with SHP-1. Previous results from our group
have shown that SHP-1 is expressed and functionally activated by the
AT2 receptor in CHO-hAT2 cells (27). However, the
presence of SIRP
1 was never detected in anti-SHP-1
immunoprecipitates from CHO-hAT2 cells left untreated or treated with
insulin and/or CGP 42112 (data not shown), indicating that in these
cells, endogenous SIRP
1 preferentially associates with SHP-2 upon
insulin receptor stimulation.
Altogether, these data indicate that AT2-mediated
inactivation of IRß leads to reduced phosphorylation of its main
effector IRS-1 and its substrate SIRP
1. This, in turn, correlates
with decreased association of both IRS-1 and SIRP
1 with tyrosine
phosphatase SHP-2 and may account for
AT2-mediated decrease in ERK2 activation and cell
proliferation induced by insulin.
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DISCUSSION
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We have previously shown that AT2 receptor
mediates inhibition of EGF-induced ERK1 and ERK2 activity in N1E-115
neuroblastoma cells (27). The present study extends these data and
demonstrates functional negative cross-talk between
AT2 and insulin receptors, leading to inhibition
of insulin-induced ERK2 activity and cell proliferation in transfected
CHO cells. Negative cross-talk between AT2 and
insulin receptor (IR) targets the initial step of the insulin receptor
cascade, i.e. autophosphorylation of the insulin receptor
ß chain (IRß). Negative regulation of IRß phosphorylation upon
AT2 stimulation also occurs in cell lines of
neuronal (N1E-115 cells) and pancreatic (AR42J cells) origin expressing
endogenous AT2 and insulin receptors. In
addition, AT2 receptor stimulation impairs
autophosphorylation of endogenous EGF receptors in N1E-115 cells and in
stably transfected COS-hAT2 cells, indicating that the
AT2 inhibitory effect is not restricted to the
insulin receptor.
We show here that AT2 receptor inhibits the
autophosphorylating activity of insulin receptor kinase, without
affecting insulin binding properties. The AT2
receptor signaling pathway leading to IR kinase inactivation is
insensitive to pertussis-toxin and does not mainly involve
dephosphorylation of IRß by vanadate-sensitive tyrosine phosphatases.
AT2 receptor thus uses a novel intracellular
mechanism for inactivation of receptor tyrosine kinases. This mechanism
differs from that recently reported for the growth-inhibitory
somatostatin sst2 receptor, which mediates dephosphorylation of IRß
through activation of tyrosine phosphatase SHP-1 (56) via coupling to
pertussis toxin-sensitive Gi protein (57). In addition, our data
indicate that AT2-mediated inhibition of IR
kinase activity does not involve serine/threonine phosphorylation by
phorbol ester-sensitive protein kinase C. Although the mechanisms for
IR activation are not fully elucidated, it is now clear that the
2ß2 IR tetramer undergoes conformational changes upon ligand
binding, and several reports indicate that oxidation and reduction of
IR ß-subunit thiols may be involved in the transition between
inactive and active states of the receptor (58, 59, 60). However,
AT2-mediated inhibition of IRß
autophosphorylation was not significantly affected by treating the
cells either with thiol reducing agents, such as
N-acetyl-cysteine and butylated hydroxyanisole, or with
the potent oxidizing agent hydrogen peroxide (our unpublished
observations). It is therefore unlikely that the inhibitory effect of
AT2 receptor involves thiol-sensitive
conformational changes of the IR.
As a consequence of IRß inactivation, insulin-induced tyrosine
phosphorylation of the major insulin receptor substrate IRS-1 is
significantly reduced upon AT2 receptor
stimulation. This, in turn, causes a reduction of IRS-1 association
with the SH2 domain-containing tyrosine phosphatase SHP-2, which
plays a positive role in the IR signaling pathway leading to ERK
activation and cell proliferation (47, 48, 49, 50). AT2
receptor activation also impairs tyrosine phosphorylation of a recently
characterized substrate of the IR, i.e. signal-regulatory
protein SIRP
1, also designated as SHPS-1. SIRP
1 is a
receptor-like glycoprotein that plays a pivotal role in the regulation
of the ERK pathway, in response to mitogens (45, 46, 61, 62) and cell
adhesion (63, 64), through tyrosine phosphorylation and interaction
with tyrosine phosphatase SHP-2. However, whether association of
SIRP
1 with SHP-2 results in positive (53, 64) or negative (45)
modulation of ERK activity remains controversial. Our data, showing
that AT2-mediated growth inhibition correlates
with reduced association of SIRP
1 with SHP-2, support the hypothesis
that formation of the SIRP
1/SHP-2 complex may positively regulate
the ERK pathway. AT2-mediated inactivation of
IRß may thus account for negative regulation of downstream events
leading to ERK activation and cell proliferation. In addition, by
inhibiting an early step of insulin signaling, the
AT2 receptor might also interfere with other
intracellular pathways leading to metabolic and/or survival effects of
insulin. Preliminary results from our laboratory indicate that
AT2 receptor stimulation also causes inhibition
of insulin-induced phosphorylation of protein kinase B (our unpublished
observations).
Physiological relevance of the cross-talk between Ang II and
insulin is illustrated by the complex relationship between insulin
resistance and hypertension (65, 66). Ang II and insulin also interact
in regulating cell growth of human neuroblastomas (67) and ciliary
artery smooth muscle cells (68). In vascular smooth muscle cells,
AT1 receptor stimulation leads to IRS-1 tyrosine
phosphorylation and association with SHP-2 (40) as well as tyrosine
phosphorylation of insulin-like growth factor-1 receptor (39).
AT1 receptors also induce IRS-1 phosphorylation
and association with PI3 kinase in rat heart in vivo
(41, 42). Our data indicate that the same intracellular signaling
cascades (i.e. involving phosphorylation of IR and IRS-1)
can be activated upon AT1 stimulation and
attenuated after AT2 receptor activation,
providing molecular support to the opposite effects of
AT1 and AT2 receptors on
cell growth (3). Furthermore, it has recently been shown that insulin
and insulin-like growth factors IGF-I and IGF-II are able to
up-regulate AT2 receptor mRNA expression in
vascular smooth muscle cells (69), in A10 smooth muscle cells (70), and
in pheochromocytoma PC12W cells (71). Taken together, these results may
suggest that insulin induces a regulatory loop involving up-regulation
of the AT2 receptor for further modulation of its
effects on growth and/or metabolism. These data may imply a possible
role for AT2 receptors in insulin-sensitive
tissues and/or in pathophysiological situations characterized by local
or massive insulin release such as that encountered in insulinomas.
While examples of transactivation of receptor tyrosine kinases by
mitogenic G protein-coupled receptors have been reported previously
(34, 35, 36, 37, 38, 39), this is one of the first demonstrations of functional
trans-inhibition of receptor tyrosine kinase activity by G
protein-coupled receptor. The discovery of a novel type of
communication between two subfamilies of membrane receptors, and the
identification of receptor tyrosine kinases as cellular targets of the
growth-inhibitory effect of AT2 receptor, may
open new perspectives for the understanding of cell growth attenuation
and pathology of proliferative diseases. These data may also have
potential relevance to pharmacotherapy of important medical conditions
such as hypertension and diabetes.
 |
MATERIALS AND METHODS
|
---|
Reagents
FCS was purchased from Boehringer Ingelheim GmbH
Bioproducts (Ingelheim, Germany). CGP 42112 was from Neosystem
(Strasbourg, France). Protein G-Sepharose and
wheat-germ-lectin-Sepharose were from Pharmacia Biotech
(Piscataway, NJ). HAMs F12 and DMEM were from Life Technologies, Inc. (Gaithersburg, MD). All other chemicals, if
not specified, were from Sigma (St. Louis, MO). Mouse
monoclonal antiphosphotyrosine 4G10, rabbit polyclonal anti-IRS-1,
mouse monoclonal anti-ERK2, and sheep polyclonal anti-EGF receptor
antibodies were purchased from Upstate Biotechnology, Inc.
(Lake Placid, NY). Rabbit polyclonal anti-SHP-2 antibodies, and
anti-SIRP
1 (CT) antibodies directed against the C-terminal portion
of the molecule, were kindly provided by Dr. A. Ullrich (Max Planck
Institute, Munich, Germany) and were described elsewhere (45).
Monoclonal anti-IRß (CT1) antibodies, described previously (72), were
a generous gift of Dr. K. Siddle (Cambridge, UK). Rabbit polyclonal
anti-EGF receptor antibodies (raised against peptide 984996 of the
human EGF receptor) were generously given by Dr. K. Yannoukakos
(Athens, Greece). Secondary antibodies were from Amersham Pharmacia Biotech (Arlington Heights, IL).
Cell Lines and Culture Conditions
Chinese Hamster Ovary (CHO) cells, deficient in dihydrofolate
reductase, were transfected with a plasmid containing the coding region
of the human AT2 receptor gene as previously
described (27). A selected clone, CHO-hAT2, expressing 100 fmol
AT2 receptor/mg protein (
2.
103 recombinant AT2 and
103 endogenous IRs per cell), was grown in HAMs
F12 medium supplemented with 10% FCS and used at passages 1030.
COS-M6 cells were transfected with the coding region of the human
AT2 receptor gene inserted into the pcDNA3
expression vector (Invitrogen, San Diego, CA). One stable
transfectant, COS-hAT2, showing neomycin resistance and expressing 500
fmol AT2 receptor/mg protein, was grown in DMEM
supplemented with 10% FCS and used at passages 1525. Mouse
neuroblastoma N1E-115 cells, previously shown to express only the
AT2 receptor subtype, were grown as described
(73) and used at passages 4045. Rat pancreatic acinar AR42J cells,
shown to express both AT1 and
AT2 receptors (74), were kindly provided by Dr.
C. Susini (Toulouse, France). These cells were grown in DMEM
supplemented with 10% FCS and used at passages 2540.
Measurement of ERK2 Phosphorylation by Gel Shift Assay
CHO-hAT2 cells were seeded at a density of 2 x
105 cells per six-multiwell plates, and allowed
to recover for 24 h before they were growth arrested by serum
deprivation for 18 h, and treated at 37 C as indicated. Total cell
lysates were submitted to Western blotting using anti-ERK2 antibodies
as described before (27). ERK2 activation was quantified by
densitometry scanning using the NIH Image 1.44 software (NIH, Bethesda,
MD) and expressed as percent of slower migrating form of ERK2
relative to total amount of ERK2. Inhibition values are means ±
SEM of three to seven independent experiments.
Cell Counting
CHO-hAT2 cells were seeded at a density of 8000 cells per well
in 24-multiwell plates, grown for 55 h in HAMs F12 medium
supplemented with 10% FCS, and starved by total serum deprivation for
40 h. After 48 h treatment with insulin (0.01 µg/ml) in the
presence or absence of CGP 42112 (1 µM), cell number was
determined using a Coulter counter Z1. Statistical analysis was
performed using ANOVA.
Lectin Column Purification and Immunoprecipitation
Cells were seeded at a density of 3 x
106 cells per 15-cm plate, except for AR42J cells
that were seeded at 7 x 106 cells per 15-cm
plate. Cells were allowed to recover for 24 h and were rendered
quiescent by total serum deprivation for 18 h before appropriate
treatment. For lectin column purification, cells were solubilized in
lysis buffer (50 mM HEPES, pH 7.6, 1% Triton X-100, 150
mM NaCl, 20 mM EDTA, 30 mM sodium
pyrophosphate, 30 mM sodium fluoride, 2 mM
benzamidine, 1 mM sodium orthovanadate, 1
mM phenylmethylsulfonyl fluoride, and 1 µg/ml each of
aprotinin, pepstatin, antipain, and leupeptin) and incubated with wheat
germ lectin-Sepharose as previously described (72). For
immunoprecipitation, cells were solubilized in lysis buffer as
described (45). Lysates were clarified by centrifugation at 25,000
x g at 4 C for 10 min and precleared by incubation with 20
µl protein G-Sepharose beads for 1 h. Supernatants were
incubated with primary antibodies for 23 h at 4 C, and then for an
additional hour with 20 µl of protein G-Sepharose. After washing, the
pellets were resuspended in SDS sample buffer, submitted to 10%
SDS-PAGE, and immunoblotted with specified antibodies as described (27)
using the Renaissance Reagent Plus (NEN Life Science Products, Boston, MA) for immunodetection.
125I-Labeled Insulin Binding Assay
CHO-hAT2 cells were seeded at a density of 2 x
105 cells per well in six-multiwell plates and
allowed to grow for 24 h, before they were starved by serum
deprivation for 18 h and treated for 5 min at 37 C in HAMs F12
medium with or without CGP 42112 (100 nM). Cells were
washed once in binding buffer (50 mM HEPES, pH 7.5, 150
mM NaCl, 2 mM KCl, 2 mM
MgCl2, 2 mM
CaCl2, supplemented with 0.1% BSA, 0.01%
bacitracin, and 0.5 mM phenylmethylsulfonyl fluoride) and
incubated for 4 h at 4 C in 1 ml of the same buffer containing
200,000 cpm monoiodinated 125I-labeled insulin
(specific activity 371 µCi/µg, NEN Life Science Products). Cells were then washed three times in ice-cold PBS
and lysed in 1 N NaOH (1 ml). Bound radioactivity (
1500
cpm per well) was measured in a 1282 Compugamma counter
(LKB, Rockville, MD) Nonspecific binding was determined in
the presence of an excess of unlabeled insulin (1 µM) and
was less than 15% of total binding. Results presented are mean ±
SEM of three independent experiments performed in
triplicate.
IR Autophosphorylation Assay
CHO-hAT2 cells were seeded at a density of
106 cells per 10-cm plate, grown for 24 h,
and starved by serum deprivation for 18 h before treatment for 5
min at 37 C with insulin in the presence or absence of CGP 42112. IRs
were purified on lectin columns as described above, except that lysis
buffer contained no sodium orthovanadate, to allow complete
dephosphorylation of receptors before starting the in vitro
autophosphorylation assay. After washing, pellets of wheat germ
lectin-Sepharose containing IRs were incubated for 10 min on ice in
phosphorylation buffer (50 mM HEPES, pH 7.5, 150
mM NaCl, 12 mM
MnCl2, 12 mM
MgCl2, 2 mM sodium
orthovanadate, and 10 µg/ml aprotinin, leupeptin, pepstatin)
containing 1 µM ATP and 5 µCi
-32P-ATP (3000 Ci/mmol, NEN Life Science Products) per sample. The reaction was stopped by addition of
SDS sample buffer and heating to 100 C for 5 min. Samples were run on
10% SDS-PAGE and transferred to nitrocellulose, and phosphorylated
bands were visualized by autoradiography on hyperfilm-MP
(Amersham Pharmacia Biotech). The level of phosphorylation
was quantified by phosphorimager analysis (Amersham Pharmacia Biotech).
 |
ACKNOWLEDGMENTS
|
---|
We would like to thank Dr. A. Ullrich (Max Planck
Institute, Munich, Germany) for kindly providing anti-SIRP
1
and anti-SHP-2 antibodies, Dr. K. Siddle (Cambridge, UK) for the gift
of anti-IRß antibodies, and Dr. K. Yannoukakos (Athens, Greece) for
providing rabbit anti-EGFR antibodies. We acknowledge Drs. C. Susini
(Toulouse, France), S. Cazaubon (ICGM, Paris), A. F. Burnol
(Meudon, France) and T. Issad (ICGM, Paris) for fruitful discussions,
C. Federici for statistical analyses, and Dr. S. Louis for help in
finalizing the manuscript.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Dr. Clara Nahmias, ICGM-CNRS UPR 1415, 22, Rue Mechain, 75014 Paris, France.
This work was supported by grants from the Fondation pour la
Recherche Médicale, the Association pour le Développement
de la Recherche sur le Cancer, the Ligue Nationale contre le Cancer,
the Association pour la Recherche contre le Cancer, the Centre National
de la Recherche Scientifique, and the Institut National pour la
Santé et la Recherche Médicale. N.E was a fellow from the
Fondation pour la Recherche Médicale, and K.B. was supported by a
fellowship from the Swedish Natural Science Research Council.
Received for publication August 9, 1999.
Revision received March 3, 2000.
Accepted for publication March 22, 2000.
 |
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