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


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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{alpha}1) and prevents subsequent association of both IRS-1 and SIRP{alpha}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.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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{alpha}1. This, in turn, leads to reduced association of IRS-1 and SIRP{alpha}1 with tyrosine phosphatase SHP-2 and may thus account for the inhibitory effect of AT2 on insulin-induced ERK activity and cell growth.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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. 1AGo). Inhibition by CGP 42112 was more easily detectable at a submaximal dose of insulin (Fig. 1AGo) 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).



View larger version (25K):
[in this window]
[in a new window]
 
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.

 
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. 1BGo). 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. 1BGo). Moreover, Ang II, as well as CGP 42112, was unable to inhibit ERK2 activity induced by insulin in nontransfected cells (Fig. 1BGo), 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. 1CGo). 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. 2AGo, 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. 2AGo).



View larger version (39K):
[in this window]
[in a new window]
 
Figure 2. Inhibitory Effect of CGP 42112 on RTK Autophosphorylation

A, CHO-hAT2 cells were treated as indicated in Fig. 1AGo, 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.

 
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. 2BGo, 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. 2CGo). 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. 2DGo), 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. 3AGo, 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.



View larger version (49K):
[in this window]
[in a new window]
 
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. 2BGo, 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. 2BGo by immunoblotting with antiphosphotyrosine (PY) and anti-IRß antibodies. Shown is one representative experiment out of four.

 
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. 3BGo). 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. 3BGo). 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. 3BGo). Analysis of this 120-kDa phosphoprotein, which may correspond to the 120-kDa polypeptide depicted in whole-cell lysates (Fig. 2AGo), will be addressed below in more detail (Fig. 4Go).



View larger version (25K):
[in this window]
[in a new window]
 
Figure 4. Inhibitory Effect of CGP 42112 on Insulin-Induced IRS-1 and SIRP{alpha}1 Phosphorylation and Association with SHP-2

A, CHO-hAT2 cells were left untreated (0) or treated as in Fig. 2AGo 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{alpha}1 antibodies. Shown is one representative experiment out of five. C, CHO-hAT2 cells were treated as in panel A, and anti-SIRP{alpha}1 immunoprecipitates were analyzed by successive immunoblotting with antiphosphotyrosine (PY) and anti-SIRP{alpha}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{alpha}1, and anti-SHP-2 antibodies. Shown is one representative experiment out of six.

 
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. 4AGo).

We also examined the effect of AT2 receptor stimulation on a more recently characterized substrate of insulin receptor, known as SIRP{alpha}1. SIRP{alpha}1 is a glycosylated transmembrane protein of 115–120 kDa, which undergoes tyrosine phosphorylation upon mitogen stimulation and cell adhesion. Phosphorylated SIRP{alpha}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{alpha}1 may correspond to the 120-kDa phosphoprotein retained on lectin columns (Fig. 3BGo), immunoblots were incubated with specific anti-SIRP{alpha}1 antibodies. As shown in Fig. 4BGo, endogenous SIRP{alpha}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{alpha}1 was detectable in unstimulated cells, increased in response to insulin, and consistently decreased in the presence of CGP 42112 (Figs. 3BGo and 4BGo). Phosphorylation of p120/SIRP{alpha}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{alpha}1 antibodies further confirmed that basal levels of SIRP{alpha}1 tyrosine phosphorylation are increased after treatment with insulin and inhibited upon addition of CGP 42112 (Fig. 4CGo).

AT2 Receptor Mediates Reduced Association of IRS-1 and SIRP{alpha}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{alpha}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{alpha}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. 4DGo (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. 4DGo), in good correlation with previously observed regulation of IRS-1 tyrosine phosphorylation (Fig. 4AGo).

Reblotting the same membranes with specific anti-SIRP{alpha}1 antibodies (Fig. 4DGo) also confirmed that p120 corresponds to SIRP{alpha}1. Association of phosphorylated p120/SIRP{alpha}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{alpha}1 tyrosine phosphorylation (Fig. 4Go, B and C) and association with SHP-2 (Fig. 4DGo), supporting a previous report that phosphorylated tyrosines of SIRP{alpha}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. 4DGo), in agreement with previous findings that SHP-2 is not a substrate of IRß (51, 52).

Phosphorylated SIRP{alpha}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{alpha}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{alpha}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{alpha}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{alpha}1. This, in turn, correlates with decreased association of both IRS-1 and SIRP{alpha}1 with tyrosine phosphatase SHP-2 and may account for AT2-mediated decrease in ERK2 activation and cell proliferation induced by insulin.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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 {alpha}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{alpha}1, also designated as SHPS-1. SIRP{alpha}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{alpha}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{alpha}1 with SHP-2, support the hypothesis that formation of the SIRP{alpha}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
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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). HAM’s 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{alpha}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 984–996 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 HAM’s F12 medium supplemented with 10% FCS and used at passages 10–30. 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 15–25. Mouse neuroblastoma N1E-115 cells, previously shown to express only the AT2 receptor subtype, were grown as described (73) and used at passages 40–45. 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 25–40.

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 HAM’s 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 2–3 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 HAM’s 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 {gamma}-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{alpha}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.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Nahmias C, Strosberg AD 1995 The angiotensin AT2 receptor: searching for signal-transduction and physiological function. Trends Pharmacol Sci 16:223–225[CrossRef][Medline]
  2. Csikos T, Chung O, Unger T 1998 Receptors and their classification: focus on angiotensin II and the AT2 receptor. J Hum Hypertens 12:311–318[CrossRef][Medline]
  3. Horiuchi M, Lehtonen JYA, Daviet L 1999 Signaling mechanism of the AT2 angiotensin II receptor: cross-talk between AT1 and AT2 receptors in cell growth. Trends Endocrinol Metab 10:391–396[CrossRef][Medline]
  4. Nouet S, Nahmias C 2000 Signal transduction from the angiotensin II AT2 receptor. Trends Endocrinol Metab 11:1–6[CrossRef][Medline]
  5. Hein L, Barsh GS, Pratt RE, Dzau VJ, Kobilka BK 1995 Behavioural and cardiovascular effects of disrupting the angiotensin II type-2 receptor in mice. Nature 377:744–747[CrossRef][Medline]
  6. Ichiki T, Labosky PA, Shiota C, Okuyama S, Imagawa Y, Fogo A, Nikimura F, Ichikawa I, Hogan BL, Inagami T 1995 Effects on blood pressure and exploratory behaviour of mice lacking angiotensin II type-2 receptor. Nature 377:748–750[CrossRef][Medline]
  7. Masaki H, Kurihara T, Yamaki A, Inomata N, Nozawa Y, Mori Y, Murasawa S, Kizima K, Maruyama K, Horiuchi M, Dzau VJ, Takahashi H, Iwasaka T, Inada M, Matsubara H 1998 Cardiac-specific overexpression of angiotensin II AT2 receptor causes attenuated response to AT1 receptor-mediated pressor and chronotropic effects. J Clin Invest 101:527–535[Abstract/Free Full Text]
  8. Oliverio MI, Kim HS, Ito M, Le T, Audoly L, Best CF, Hiller S, Kluckman K, Maeda N, Smithies O, Coffman TM 1998 Reduced growth, abnormal kidney structure, and type 2 (AT2) angiotensin receptor-mediated blood pressure regulation in mice lacking both AT1A and AT1B receptors for angiotensin II. Proc Natl Acad Sci USA 95:15496–15501[Abstract/Free Full Text]
  9. Siragy H, Inagami T, Ichiki T, Carey R 1999 Sustained hypersensitivity to angiotensin II and its mechanism in mice lacking the subtype-2 (AT2) angiotensin receptor. Proc Natl Acad Sci USA 96:6506–6510[Abstract/Free Full Text]
  10. Bartunek J, Weinberg EO, Tajima M, Rohrbach S, Lorell BH 1999 Angiotensin II type 2 receptor blockade amplifies the early signals of cardiac growth response to angiotensin II in hypertrophied hearts. Circulation 99:22–25[Abstract/Free Full Text]
  11. Akishita M, Ito M, Lehtonen JY, Daviet L, Dzau VJ, Horiuchi M 1999 Expression of the AT2 receptor developmentally programs extracellular signal-regulated kinase activity and influences fetal vascular growth. J Clin Invest 103:63–71[Abstract/Free Full Text]
  12. Janiak P, Pillon A, Prost JF, Vilaine JP 1992 Role of angiotensin subtype 2 receptor in neointima formation after vascular injury. Hypertension 20:737–745[Abstract]
  13. Nakajima M, Hutchinson HG, Fujinaga M, Hayashida W, Morishita R, Zhang L, Horiuchi M, Pratt RE, Dzau VJ 1995 The angiotensin II type 2 (AT2) receptor antagonizes the growth effects of the AT1 receptor: gain-of-function study using gene transfer. Proc Natl Acad Sci USA 92:10663–10667[Abstract]
  14. Lucius R, Gallinat S, Rosenstiel P, Herdegen T, Sievers J, Unger T 1998 The angiotensin II type 2 (AT2) receptor promotes axonal regeneration in the optic nerve of adult rats. J Exp Med 188:661–670[Abstract/Free Full Text]
  15. Nishimura H, Yerkes E, Hohenfellner K, Miyazaki Y, Ma J, Hunley TE, Yoshida H, Ichiki T, Threadgill D, Phillips JA, Hogan BML, Fogo A, Brock JW, Inagami T, Ichikawa I 1999 Role of the angiotensin type 2 receptor gene in congenital anomalies of the kidney and urinary tract, CAKUT, of mice and men. Mol Cell 3:1–10[Medline]
  16. Laflamme L, de Gasparo M, Gallo JM, Payet MD, Gallo-Payet N 1996 Angiotensin II induction of neurite outgrowth by AT2 receptors in NG108–15 cells. Effect counteracted by the AT1 receptors. J Biol Chem 271:22729–22735[Abstract/Free Full Text]
  17. Stroth U, Meffert S, Gallinat S, Unger T 1998 Angiotensin II and NGF differentially influence microtubule proteins in PC12W cells: role of the AT2 receptor. Brain Res Mol Brain Res 53:187–195[Medline]
  18. Gendron L, Laflamme L, Rivard N, Asselin C, Payet MD, Gallo-Payet N 1999 Signals from the AT2 (angiotensin type 2) receptor of angiotensin II inhibit p21ras and activate MAPK (mitogen-activated protein kinase) to induce morphological neuronal differentiation in NG108–15 cells. Mol Endocrinol 13:1615–1626[Abstract/Free Full Text]
  19. Yamada T, Horiuchi M, Dzau VJ 1996 Angiotensin II type 2 receptor mediates programmed cell death. Proc Natl Acad Sci USA 93:156–160[Abstract/Free Full Text]
  20. Dimmeler S, Rippmann V, Weiland U, Haendeler J, Zeiher AM 1997 Angiotensin II induces apoptosis of human endothelial cells. Protective effect of nitric oxide. Circ Res 81:970–976[Abstract/Free Full Text]
  21. Shenoy UV, Richards EM, Huang XC, Sumners C 1999 Angiotensin II type 2 receptor-mediated apoptosis of cultured neurons from newborn rat brain. Endocrinology 140:500–509[Abstract/Free Full Text]
  22. Stoll M, Steckelings UM, Paul M, Bottari SP, Metzger R, Unger T 1995 The angiotensin AT2-receptor mediates inhibition of cell proliferation in coronary endothelial cells. J Clin Invest 95:651–657[Medline]
  23. Meffert S, Stoll M, Steckelings UM, Bottari SP, Unger T 1996 The angiotensin II AT2 receptor inhibits proliferation and promotes differentiation in PC12W cells. Mol Cell Endocrinol 122:59–67[CrossRef][Medline]
  24. Liakos P, Bourmeyster N, Defaye G, Chambaz EM, Bottari SP 1997 ANG II AT1 and AT2 receptors both inhibit bFGF-induced proliferation of bovine adrenocortical cells. Am J Physiol 273:C1324–C1334
  25. Berk BC 1999 Angiotensin II signal transduction in vascular smooth muscle : pathways activated by specific tyrosine kinases. J Am Soc Nephrol 10:S62–S68
  26. Inagami T, Eguchi S, Numaguchi K, Motley ED, Tang H, Matsumoto T, Yamakawa T 1999 Cross-talk between angiotensin II receptors and the tyrosine kinases and phosphatases. J Am Soc Nephrol 10:S57–S61
  27. Bedecs K, Elbaz N, Sutren M, Masson M, Susini C, Strosberg AD, Nahmias C 1997 Angiotensin II type 2 receptors mediate inhibition of mitogen-activated protein kinase cascade and functional activation of SHP-1 tyrosine phosphatase. Biochem J 325:449–454[Medline]
  28. Huang XC, Richards EM, Sumners C 1996 Mitogen-activated protein kinases in rat brain neuronal cultures are activated by angiotensin II type 1 receptors and inhibited by angiotensin II type 2 receptors. J Biol Chem 271:15635–15641[Abstract/Free Full Text]
  29. Fischer TA, Singh K, O’Hara DS, Kaye DM, Kelly RA 1998 Role of AT1 and AT2 receptors in regulation of MAPKs and MKP-1 by ANG II in adult cardiac myocytes. Am J Physiol 275:H906–H916
  30. Bottari SP, King IN, Reichlin S, Dahlstroem I, Lydon N, de Gasparo M 1992 The angiotensin AT2 receptor stimulates protein tyrosine phosphatase activity and mediates inhibition of particulate guanylate cyclase. Biochem Biophys Res Commun 183:206–211[Medline]
  31. Brechler V, Reichlin S, de Gasparo M, Bottari SP 1994 Angiotensin II stimulates protein tyrosine phosphatase activity through a G-protein independent activity. Receptors Channels 2:89–97[Medline]
  32. Buisson B, Laflamme L, Bottari SP, de Gasparo M, Gallo-Payet N, Payet MD 1995 A G protein is involved in the angiotensin AT2 receptor inhibition of the T-type calcium current in non-differentiated NG108–15 cells. J Biol Chem 270:1670–1674[Abstract/Free Full Text]
  33. Lehtonen JYA, Daviet L, Nahmias C, Horiuchi M, Dzau VJ 1999 Analysis of functional domains of angiotensin II type 2 receptor involved in apoptosis. Mol Endocrinol 13:1051–1060[Abstract/Free Full Text]
  34. Daub H, Weiss FU, Wallasch C, Ullrich A 1996 Role of transactivation of the EGF receptor in signalling by G-protein-coupled receptors. Nature 379:557–560[CrossRef][Medline]
  35. Daub H, Wallasch C, Lankenau A, Herrlich A, Ullrich A 1997 Signal characteristics of G protein-transactivated EGF receptor. EMBO J 16:7032–7044[Abstract/Free Full Text]
  36. Prenzel N, Zwick E, Daub H, Leserer M, Abraham R, Wallasch C, Ullrich A 1999 EGF receptor transactivation by G-protein-coupled receptors requires metalloproteinase cleavage of pro-HB-EGF. Nature 402:884–888[CrossRef][Medline]
  37. Linseman DA, Benjamin CW, Jones DA 1995 Convergence of angiotensin II and platelet-derived growth factor receptor signaling cascades in vascular smooth muscle cells. J Biol Chem 270:12563–12568[Abstract/Free Full Text]
  38. Eguchi S, Numaguchi K, Iwasaki H, Matsumoto T, Yamakawa T, Utsunomiya H, Motley ED, Kawakatsu H, Owada KM, Hirata Y, Marumo F, Inagami T 1998 Calcium-dependent epidermal growth factor receptor transactivation mediates the angiotensin II-induced mitogen-activated protein kinase activation in vascular smooth muscle cells. J Biol Chem 273:8890–8896[Abstract/Free Full Text]
  39. Du J, Sperling LS, Marrero MB, Phillips L, Delafontaine P 1996 G-protein and tyrosine kinase receptor cross-talk in rat aortic smooth muscle cells: thrombin and angiotensin II-induced tyrosine phosphorylation of insulin receptor substrate 1 and insulin-like growth factor receptor. Biochem Biophys Res Commun 218:934–939[CrossRef][Medline]
  40. Ali MS, Schieffer B, Delafontaine P, Bernstein KE, Ling BN, Marrero MB 1997 Angiotensin II stimulates tyrosine phosphorylation and activation of insulin receptor substrate 1 and protein tyrosine phosphatase 1D in vascular smooth muscle cells. J Biol Chem 272:12373–12379[Abstract/Free Full Text]
  41. Saad MJA, Velloso LA, Carvalho CRO 1995 Angiotensin II induces tyrosine phosphorylation of insulin receptor substrate 1 and its association with phosphatidylinositol 3- kinase in rat heart. Biochem J 310:741–744[Medline]
  42. Velloso L, Folli F, Sun X, White M, Saad M, Kahn C 1996 Cross-talk between the insulin and angiotensin signaling systems. Proc Natl Acad Sci USA 93:12490–12495[Abstract/Free Full Text]
  43. Bossenmaier B, Mosthaf L, Mischak H, Ullrich A, Häring HU 1997 Protein kinase C isoforms ß1 and ß2 inhibit the tyrosine kinase activity of the insulin receptor. Diabetologia 40:863–866[CrossRef][Medline]
  44. Kellerer M, Mushack J, Seffer E, Mischak H, Ullrich A, Häring HU 1998 Protein kinase C isoforms {alpha}, {delta} and {theta} require insulin receptor substrate-1 to inhibit the tyrosine kinase activity of the insulin receptor in human kidney embryonic cells (HEK 293). Diabetologia 41:833–838[CrossRef][Medline]
  45. Kharitonenkov A, Chen Z, Sures I, Wang H, Schilling J, Ullrich A 1997 A family of proteins that inhibit signalling through tyrosine kinase receptors. Nature 386:181–186[CrossRef][Medline]
  46. Fujioka Y, Matozaki T, Noguchi T, Iwamatsu A, Yamao T, Takahashi N, Tsuda M, Takada T, Kasuga M 1996 A novel membrane glycoprotein, SHPS-1, that binds the SH2-domain- containing protein tyrosine phosphatase SHP-2 in response to mitogens and cell adhesion. Mol Cell Biol 16:6887–6899[Abstract]
  47. Milarski KL, Saltiel AR 1994 Expression of catalytically inactive Syp phosphatase in 3T3 cells blocks stimulation of mitogen-activated protein kinase by insulin. J Biol Chem 269:21239–21243[Abstract/Free Full Text]
  48. Noguchi T, Matozaki T, Horita K, Fujioka Y, Kasuga M 1994 Role of SH-PTP2, a protein-tyrosine phosphatase with Src homology 2 domains, in insulin-stimulated Ras activation. Mol Cell Biol 14:6674–6682[Abstract]
  49. Xiao S, Rose DW, Sasaoka T, Maegawa H, Burke TR Jr., Roller PP, Shoelson SE, Olefsky JM 1994 Syp (SH-PTP2) is a positive mediator of growth factor-stimulated mitogenic signal transduction. J Biol Chem 269:21244–21248[Abstract/Free Full Text]
  50. Yamauchi K, Milarski KL, Saltiel AR, Pessin JE 1995 Protein-tyrosine-phosphatase SHPTP2 is a required positive effector for insulin downstream signaling. Proc Natl Acad Sci USA 92:664–668[Abstract]
  51. Kuhné MR, Pawson T, Lienhard GE, Feng G-S 1993 The insulin receptor substrate 1 associates with the SH2-containing phosphotyrosine phosphatase Syp. J Biol Chem 268:11479–11481[Abstract/Free Full Text]
  52. Kharitonenkov A, Schnekenburger J, Chen Z, Knyazev P, Ali S, Zwick E, White M, Ullrich A 1995 Adapter function of protein-tyrosine phosphatase 1D in insulin receptor/insulin receptor substrate-1 interaction. J Biol Chem 270:29189–29193[Abstract/Free Full Text]
  53. Takada T, Matozaki T, Takeda H, Fukunaga K, Noguchi T, Fujioka Y, Okazaki I, Tsuda M, Yamao T, Ochi F, Kasuga M 1998 Roles of the complex formation of SHPS-1 with SHP-2 in insulin-stimulated mitogen-activated protein kinase activation. J Biol Chem 273:9234–9242[Abstract/Free Full Text]
  54. Timms JF, Carlberg K, HG, Chen H, Kamatkar S, Nadler MJS, Rohrschneider LR, Neel BG 1998 Identification of major binding proteins and substrates for the SH2-containing protein phosphatase SHP-1 in macrophages. Mol Cell Biol 18:3838–3850[Abstract/Free Full Text]
  55. Veillette A, Thibaudeau E, Latour S 1998 High expression of inhibitory receptor SHPS-1 and its association with protein tyrosine phosphatase SHP-1 in macrophages. J Biol Chem 273:22719–22728[Abstract/Free Full Text]
  56. Bousquet C, Delesque N, Lopez F, Saint-Laurent N, Estève JP, Bedecs K, Buscail L, Vaysse N, Susini C 1998 sst2 Somatostatin receptor mediates negative regulation of insulin receptor signaling through the tyrosine phosphatase SHP-1. J Biol Chem 273:7099–7106[Abstract/Free Full Text]
  57. Lopez F, Esteve JP, Buscail L, Delesque N, Saint-Laurent N, Theveniau M, Nahmias C, Vaysse N, Susini C 1997 The tyrosine phosphatase SHP-1 associates with the sst2 somatostatin receptor and is an essential component of sst2-mediated inhibitory growth signaling. J Biol Chem 272:24448–24454[Abstract/Free Full Text]
  58. Wilden PA, Pessin JE 1987 Differential sensitivity of the insulin-receptor kinase to thiol and oxidizing agents in the absence and presence of insulin. Biochem J 245:325–331[Medline]
  59. Clark S, Konstantopoulos N 1993 Sulphydryl agents modulate insulin-and epidermal growth factor (EGF) receptor kinase via reaction with intracellular receptor domains: differential effects on basal versus activated receptors. Biochem J 292:217–223[Medline]
  60. Schmid E, El Benna J, Galter D, Klein G, Droge W 1998 Redox priming of the insulin receptor ß-chain associated with altered tyrosine kinase activity and insulin responsiveness in the absence of tyrosine autophosphorylation. FASEB J 12:863–870[Abstract/Free Full Text]
  61. Ochi F, Matozaki T, Noguchi T, Fujioka Y, Yamao T, Takada T, Tsuda M, Takeda H, Fukunaga K, Okabayashi Y, Kasuga M 1997 Epidermal growth factor stimulates the tyrosine phosphorylation of SHPS- 1 and association of SHPS-1 with SHP-2, a SH2 domain-containing protein tyrosine phosphatase. Biochem Biophys Res Commun 239:483–487[CrossRef][Medline]
  62. Takeda H, Matozaki T, Fujioka Y, Takada T, Noguchi T, Yamao T, Tsuda M, Ochi F, Fukunaga K, Narumiya S, Yamamoto T, Kasuga M 1998 Lysophosphatidic acid-induced association of SHP-2 with SHPS-1: roles of RHO, FAK, and a SRC family kinase. Oncogene 16:3019–3027[CrossRef][Medline]
  63. Tsuda M et al. 1998 Integrin-mediated tyrosine phosphorylation of SHPS-1 and its association with SHP-2. Roles of Fak and Src family kinases. J Biol Chem 273:13223–13229[Abstract/Free Full Text]
  64. Oh ES, Gu H, Saxton TM, Timms JF, Hausdorff S, Frevert EU, Kahn BB, Neel BG, Thomas SM 1999 Regulation of early events in integrin signaling by protein tyrosine phosphatase SHP-2. Mol Cell Biol 19:3205–3215[Abstract/Free Full Text]
  65. Hsueh WA 1992 Effect of the renin-angiotensin system in the vascular disease of type II diabetes mellitus. Am J Med 92:13S–19S
  66. Hall JE, Brands MW, Zappe DH, Alonso Galicia M 1995 Insulin resistance, hyperinsulinemia, and hypertension: causes, consequences or merely correlations? Proc Soc Exp Biol Med 208:317–329[Abstract]
  67. Chen LI, Prakash OM, Re RN 1993 The interaction of insulin and angiotensin on the regulation of human neuroblastoma cell growth. Mol Chem Neuropathol 18:189–196[Medline]
  68. Dubey RK, Flammer J, Luscher TF 1998 Angiotensin II and insulin induce growth of ciliary artery smooth muscle: effects of AT1/AT2 antagonists. Invest Ophthalmol Vis Sci 39:2067–2075[Abstract]
  69. Kambayashi Y, Nagata K, Ichiki T, Inagami T 1996 Insulin and insulin-like growth factors induce expression of angiotensin type-2 receptor in vascular smooth muscle cells. Eur J Biochem 239:558–565[Abstract]
  70. Saward L, Zahradka P 1996 Insulin is required for angiotensin II-mediated hypertrophy of smooth muscle cells. Mol Cell Endocrinol 122:93–100[CrossRef][Medline]
  71. Tamura M, Wanaka Y, Landon EJ, Inagami T 1999 Intracellular sodium modulates the expression of angiotensin II subtype 2 receptor in PC12W cells. Hypertension 33:626–632[Abstract/Free Full Text]
  72. Issad T, Combettes M, Ferre P 1995 Isoproterenol inhibits insulin-stimulated tyrosine phosphorylation of the insulin receptor without increasing its serine/threonine phosphorylation. Eur J Biochem 234:108–115[Abstract]
  73. Nahmias C, Cazaubon SM, Briend-Sutren MM, Lazard D, Villageois P, Strosberg AD 1995 Angiotensin II AT2 receptors are functionally coupled to protein tyrosine dephosphorylation in N1E-115 neuroblastoma cells. Biochem J 306:87–92[Medline]
  74. Chappell MC, Jacobsen DW, Tallant EA 1995 Characterization of angiotensin II receptor subtypes in pancreatic acinar AR42J cells. Peptides 16:741–747[CrossRef][Medline]