Human angiotensin II type-2 receptor inhibition of insulin-mediated ERK-2 activity via a G-protein coupled signaling pathway

Human angiotensin II type-2 receptor inhibition of insulin-mediated ERK-2 activity via a G-protein coupled signaling pathway

Molecular Brain Research 124 (2004) 62 – 69 www.elsevier.com/locate/molbrainres Research report Human angiotensin II type-2 receptor inhibition of i...

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Molecular Brain Research 124 (2004) 62 – 69 www.elsevier.com/locate/molbrainres

Research report

Human angiotensin II type-2 receptor inhibition of insulin-mediated ERK-2 activity via a G-protein coupled signaling pathway Steven A. Moore, Nancy Huang, Olivia Hinthong, Robert D. Andres, Tom N. Grammatopoulos, James A. Weyhenmeyer * Department of Cell and Structural Biology, University of Illinois, B107 Chemical and Life Science Building, 601 S. Goodwin Ave., Urbana, IL 61801, USA Accepted 23 February 2004

Abstract While it has been shown that the angiotensin type-2 (AT2) receptor plays an important role in the development and differentiation of many tissues, the second messengers involved in its signaling pathways are just beginning to be understood. To further determine the signaling pathways for the AT2 receptor, we have investigated whether human angiotensin type-2 receptor transfected into Chinese hamster ovary (CHO) cells can modulate insulin-induced extracellular signal-related protein kinase (ERK-2) phosphorylation via a G-protein coupled mechanism. Our results indicate that the human AT2 receptor decreases insulin-induced ERK-2 phosphorylation through a G-protein mediated pathway since inhibition was attenuated by pertussis toxin (a Gi/G0 inhibitor). Our findings further indicate that the inhibitory response was insensitive to sodium orthovanadate (a PTPase inhibitor), but sensitive (attenuated) to okadaic acid, suggesting an important role for protein phosphatase 2A (PP2A). We have also shown that alanine substitution of the putative G-protein coupling DRY141 – 143 motif of the second intracellular loop significantly decreases the human AT2 receptor’s ability to inhibit insulin-induced ERK-2 phosphorylation. Our results support the hypothesis that the AT2 receptor inhibits insulin-induced ERK-2 activity via a G-protein coupled pathway involving the up-regulation of PP2A. D 2004 Elsevier B.V. All rights reserved. Theme: Neurotransmitters, modulators, transporters, and receptors Topic: Peptide Receptor Structure and Function Keywords: Angiotensin; G-protein coupled receptor; Insulin; ERK; PP2A; PTPase

1. Introduction Angiotensin II (Ang II), the most potent effector molecule of the renin –angiotensin system, is best known for its role in blood pressure regulation, body fluid homeostasis and electrolyte balance [16,21]. Recent studies suggest that Ang II can affect a number of cell processes including growth [4], proliferation [25,26], differentiation [23] and death [7,10,22,24,28]. The major angiotensin (AT) receptor subtypes, AT1 and AT2, have been identified on the basis of their high affinity binding to isoform-specific non-peptide antagonists losartan and PD123319, respectively [27]. Both PD123319 and losartan have been used extensively to * Corresponding author. Office of the Vice President, University of Illinois, 346 Henry Administration Building, 506 South Wright Street, Urbana, IL 61801, USA. Tel.: +1-217-333-8075; fax: +1-217-265-5444. E-mail address: [email protected] (J.A. Weyhenmeyer). 0169-328X/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.molbrainres.2004.02.006

characterize the distribution and function of their respective AT receptor subtypes in the nervous system [8,17]. Indeed, it appears that Ang II can influence such diverse cellular functions by the expression ratio and distribution of these specific receptor subtypes. Several groups have reported that AT1 and AT2 receptors can functionally antagonize each other. For example, studies have shown that the AT1 receptor can upregulate mitogen-activated protein kinase (MAPK) activity, and while the AT2 receptor can decrease AT1 receptor-mediated MAPK activity [12,19]. MAPK has been shown to increase cell growth and proliferation, and effect differentiation and apoptosis. Therefore, identifying AT1/AT2 receptor expression ratios is important to further understanding their role in many cellular functions. While the intracellular signaling cascades and physiological roles of the AT1 receptor have been well documented, relatively little information is known about the AT2 receptor. Unlike the AT1 receptor, which appears to signal mainly

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through G-proteins, the AT2 receptor is thought to have both G-protein-dependent [9,11,13,18,28] and -independent signaling mechanisms [1– 3,5]. Recently, several groups have reported G-protein independent signaling pathways for the AT2 receptor. In PC12W cells, which only express AT2 receptors, Brechler et al. [3] found that Ang II stimulates protein tyrosine phosphatase activity via a pertussis toxin (PTX) insensitive G-protein independent pathway. In addition, Akashita et al. [1] demonstrated that AT2 receptors inhibit ERK-1 and -2 activity in vascular smooth muscle cells. The inhibition was attenuated by orthovanadate, but not okadaic acid, suggesting the inhibition was mediated through a PTPase rather than protein phosphatase 2A (PP2A). Using a neuronal cell line, Bedecs et al. [2] found that Ang II specifically decreased serum-induced ERK-1/2 phosphorylation compared to control. The receptor-mediated inhibition was rapid, transient and insensitive to both PTX and okadaic acid. These investigators also suggested that the pathway involves a vanadate-sensitive PTPase. Elbaz et al. [5] have shown that the human AT2 receptor can inhibit insulin receptor-induced ERK-2 phosphorylation in Chinese hamster ovary (CHO) cells via a G-protein independent mechanism. In these studies, stably transfected CHO-hAT2 cells were stimulated for 5 min with insulin in the presence of CGP-42112 (an AT2 receptor agonist), which resulted in a rapid, but transient decrease in ERK-2 activity compared to control. The inhibition pathway was PTX insensitive, suggesting that it was not due to coupling of regulatory hetero-trimeric Gi/Go proteins. They also reported that the inhibition was vanadate and okadaic acid insensitive, suggesting that the AT2-mediated ERK-2 inactivation was not due to dephosphorylation by a vanadatesensitive PTPase or PP2A. Further, they demonstrated that the negative cross-talk between the AT2 receptor and insulin receptor targets the initial steps in the insulin receptor signaling pathway by directly inhibiting both the autophosphorylation of the insulin receptor h chain and phosphorylation of IRS-1 tyrosine residues. While there is growing evidence indicating that the AT2 receptor signals, at least in part, through a G-protein independent mechanism, several reports have shown that the neuronal AT2 receptor signaling may be G-protein mediated [11 – 15]. Using primary cultured hypothalamus – thalamus –septum – midbrain (HTSM) neurons, these investigators demonstrated a PTX sensitive AT2 signaling pathway that could stimulate K+ currents, inhibit AT1 receptor mediated MAPK activation, and facilitate UV-induced apoptosis. The pathway was okadaic acid sensitive, suggesting a serine/threonine phosphatase (PP2A) mediated pathway. They also found that PP2A activity persisted with continuous Ang II stimulation over 24 h. In addition, this G-protein coupled pathway was orthovanadate-insensitive, indicating an absence of PTPase up-regulation. Using adult rat ventricular myocytes and cardiac microvascular endothelial cells, Fischer et al. [6] demonstrated that AT2 receptors decrease the levels of phosphorylated MAPK via an okadaic

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acid sensitive pathway, suggesting that PP2A does play a role in AT2 receptor signaling. There are several possibilities regarding the regulatory mechanism(s) that determine the signaling pathway(s) (Gprotein dependent and independent) being up-regulated by the AT2 receptor in a given cell system. Emerging evidence suggests that the two distinct signaling mechanisms for the AT2 receptor are, at least in part, dependent on cell type and cell state. There is also the possibility that AT2 receptor subtypes, or splice variants, may be responsible for the existence of the two distinct signaling pathways. Alternatively, G-protein coupled receptor signaling mechanisms may be regulated by site-specific phosphorylation of the receptor and/or recruitment of adapter proteins [20]. The most recent findings suggest that AT2 receptor signaling may be temporally regulated, so that the two pathways are activated sequentially with their maximal activity falling at different points following agonist stimulation. This temporal regulatory strategy would presumably allow the receptor to maintain the inhibition of downstream effectors over a longer time course [20]. Our previous studies have shown that angiotensin II binding of the transiently expressed human AT2 receptor in CHO-K1 cells is GTPgS sensitive, suggesting that the receptor is G-protein coupled [18]. We have found that this effect was significantly attenuated with mutation of the Gprotein coupling DRY141 – 143 motif. Our findings have shown that mutation of the highly conserved DRY141 – 143 motif resulted in a decrease in GTPgS-induced Ang II affinity shift compared to wild-type receptor, suggesting that mutations in the DRY motif partially decoupled the receptor from G-proteins, further suggesting that the human AT2 receptor is G-protein coupled when expressed in CHO-K1 cells. In the present study, we have examined whether the human AT2 receptor can inhibit insulin-induced ERK-2 phosphorylation via a G-protein mediated signaling pathway and have begun to identify downstream effectors in the pathway.

2. Materials and methods 2.1. Experimental materials CHO-K1 cells were obtained from ATCC (Manassas, VA, USA). F12 media was purchased from BioWhittaker (Walkersville, MD, USA). Antibiotics, LipofectAMINE 2000 and OptiMEM 1 media were purchased from Invitrogen (Carlsbad, CA, USA). Ang II, insulin, pertussis toxin, okadaic acid and sodium orthovanadate were purchased from Sigma (St. Louis, MO, USA). pCR3.0 plasmid (Invitrogen) containing the human AT2 receptor’s cDNA coding sequence was generously provided by Dr. Terry Elton (Brigham Young University, Provo, UT, USA). Phosphop44/42 MAP kinase (Thr202/Tyr204) mouse antibody was purchased from Cell Signaling Technology (Beverly, MA, USA), and HRP conjugated secondary anti-mouse IgG

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antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). ECL Western blotting detection reagents were obtained from Amersham/Pharmacia Biotech (Piscataway, NJ, USA). All other chemicals were purchased from Sigma unless otherwise indicated. 2.2. Cell culture CHO-K1 cells were grown in T75 flasks and maintained in 1:1 ratio of Dulbecco’s modified Eagles medium (DMEM) and Ham’s F12, containing 10% fetal bovine serum and antibiotics. The cells were maintained at 37jC in a humidified 10% CO2 incubator. Cells were grown to 90% confluence and detached from flasks with cell dissociation solution (Sigma), resuspended in DMEM and either plated in passing flasks or for transfections. 2.3. Mutagenesis protocol Human AT2 receptor cDNA in pCR3.0 was used for sitedirected mutagenesis. Substitute-specific nucleotides were made using a site-directed mutagenesis kit (Stratagene, La Jolla, CA, USA). Mutagenic oligonucleotides were designed and constructed by the University of Illinois Biotechnology Center Oligonucleotide Synthesis Lab. All mutagenic primers were designed to substitute alanine for the targeted amino acid(s). Mutations were confirmed by the University of Illinois Sequencing Laboratory. 2.4. Transfection protocol CHO-K1 cells were plated at a concentration of 7.5  106cells/T75 flask 24 h prior to transfection. Cells were transfected with either wild-type or mutant human AT2 receptor gene. DNA and LipofectAMINE 2000 mixtures were combined in OptiMEM 1 reduced serum media at room temperature for 20 min. The DNA-LipofectAMINE 2000 transfection mixture was added to growth medium (without antibiotics) and cells were incubated for an additional 5 h. The mixture was replaced with normal growth media and cells were stimulated approximately 40 h post transfection, allowing sufficient time for the expression of receptor. AT2 receptor expression level in CHO cell membrane preparations following transfection was f 300 fmol receptor/mg protein. 2.5. Cell stimulations CHO cells were incubated in serum-free media at 37 jC for 4 h prior to treatment to decrease basal levels of ERK-2 phosphorylation. To determine the time and concentration effect of insulin stimulation on our cultures, cells were stimulated with insulin (0.1 – 100 nM) in serum-free media for 0 to 20 min. To determine the effect of Ang II on insulininduced ERK-2 phosphorylation, cultures were pre-incubated with Ang II (0.001 – 1.0 AM) for 0 to 40 min. In these experiments, cells were stimulated with insulin (1 nM for 5

Fig. 1. Effect of insulin on ERK-2 activation in CHO cells. (A) Effect of insulin stimulation time on ERK-2 phosphorylation. CHO cells were treated with 1 nM insulin from 0 to 20 min. Positive control ( + C) was treated for 20 min with 5% FBS to determine maximal ERK-2 phosphorylation. Data were normalized to negative control ( C = no insulin) and presented as % ERK-2 phosphorylation. (B) Effect of insulin concentration on ERK-2 phosphorylation. CHO cells were treated for 5 min with insulin (0 – 100 nM). Positive control ( + C) was treated for 5 min with 5% FBS to determine maximal ERK-2 phosphorylation. Data were normalized to negative control ( C = no insulin) and presented as % ERK-2 phosphorylation. Stimulated cells were lysed and assayed by Western analysis using antibody specific for Phospho-p44/42 (ERK-1/ERK-2) MAP kinase (Thr202/Tyr204). Data are presented as the mean F SEM (nz4). *p < 0.05 vs. control.

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min) during the last 5 min of the Ang II treatment. To examine whether AT2 receptor inhibition of insulin-induced ERK-2 phosphorylation was G-protein dependent, cells were treated with pertussis toxin (300 ng/ml) for 18 to 20 h prior to Ang II/ insulin stimulation. To determine whether AT2 receptor mediation of insulin-induced ERK-2 phosphorylation was protein phosphatase dependent, cells were treated with okadaic acid (10 nM) for 3 h or sodium orthovanadate (10 AM) for 18 to 20 h prior to the addition of Ang II/insulin. Following stimulation, cells were lysed with Western lysis buffer (62.5 mM Tris –HCl, 2% SDS, 10% glycerol, 50 mM DTT, 0.01% bromophenol blue) and stored at 80 jC. 2.6. Western analysis Protein (20 to 30 Ag) was resolved by 12% SDS-PAGE and transferred to PVDF membranes. Nonspecific binding was blocked by incubating the membranes with blotto (10 mM Tris, 150 mM NaCl, 0.05% Tween-20, 5% dry milk) for 1 h at room temperature. Membranes were incubated overnight with a phospho-p44/42 (ERK-1/2) MAP kinase (thr202/tyr204) monoclonal antibody (1:2000), which recognizes dual phosphorylated (active) ERK-2. The membranes were washed with TBST and incubated with HRP-conjugated anti-mouse IgG (1:2000) for 1 h at room temperature. Bands were visualized by the exposure of film to chemoluminescence and quantitated via densitometry scanning using NIH image software (NIH, Bethesda, MD). Data were normalized to controls and presented as percentage above or below control values. 2.7. Statistical analysis Data were analyzed using SigmaPlot. Statistical significance was determined by one-way ANOVA followed by Fisher’s PLSD test. Differences in p-values were considered significant if p < 0.05. Data are represented as the mean F SEM.

3. Results 3.1. Effect of insulin on the activation of ERK-2 in CHO cells The time-dependent effect of insulin-induced ERK-2 phosphorylation is shown in Fig. 1A. Cells treated with 1 nM insulin revealed a f 2-fold transient increase in ERK-2 phosphorylation with a maximum between 5 and 10 min. Insulin-induced ERK-2 phosphorylation levels returned to control levels after 20 min. Insulin increases ERK-2 phosphorylation levels in CHO cells in a concentration-dependent manner (Fig. 1B). CHO cells treated with 100 nM insulin for 5 min resulted in a fourfold increase of ERK-2 phosphorylation. Indeed, cells treated with concentrations >1 nM insulin resulted in a

Fig. 2. Effect of AT2 receptor mediated Ang II signaling on insulin-induced ERK-2 phosphorylation levels in transfected CHO cells expressing hAT2 receptor. (A) Effect of Ang II (1 AM) stimulation time on insulin-induced ERK-2 phosphorylation. Negative control ( C) was treated with 1 AM Ang II to determine ERK-2 phosphorylation baseline. Positive control ( + C) was stimulated with 1 nM insulin, in the absence of Ang II, which confirms maximal ERK-2 phosphorylation following insulin stimulation. Data were normalized to positive control ( + C) and presented as % ERK-2 phosphorylation. (B) Effect of Ang II concentration (20 min stimulation) on insulin-induced ERK-2 phosphorylation. Negative control ( C) was treated with 1 AM Ang II to determine ERK-2 phosphorylation baseline. Positive control ( + C) was stimulated with 1 nM insulin, in the absence of Ang II, which confirms maximal ERK-2 phosphorylation levels. Data were normalized to positive control ( + C) and presented as % ERK-2 phosphorylation. Stimulated cells were lysed and assayed by Western analysis using antibody specific for Phospho-p44/42 (ERK-1/ERK-2) MAP kinase (Thr202/Tyr204). Data are presented as the mean F SEM (nz4). *p < 0.05 vs. control.

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insulin-induced ERK-2 phosphorylation at 40 min time point) for all remaining experiments. 3.3. Effect of pertussis toxin on the ability of the AT2 receptor to modulate insulin-induced ERK-2 activation Cultures treated with pertussis toxin (300 ng/ml), a potent Gi/G0 inhibitor, completely attenuated AT2 receptor-mediated inhibition of insulin-induced ERK-2 activation (Fig. 3). The effect was statistically significant for all time points examined (0 to 40 min). 3.4. Effect of okadaic acid and sodium orthovanadate on the modulation of insulin-induced ERK-2 activation by the AT2 receptor

Fig. 3. Effect of pertussis toxin on human AT2 receptor’s ability to inhibit insulin-induced ERK-2 phosphorylation. Transfected CHO-hAT2R cells were stimulated with 1 AM Ang II for 0 – 40 min and 1 nM insulin for 5 min in the presence ( w ) or absence (5) of 300 ng/ml PTX. Cell lysates were assayed by Western analysis using antibody specific for Phospho-p44/42 (ERK-1/ERK-2) MAP kinase (Thr202/Tyr204). Data are presented as the mean F SEM (nz4). *p < 0.05 vs. no PTX control.

statistically significant increase in ERK-2 phosphorylation levels when compared to control. Based on these findings, cells were treated with 1 nM insulin for 5 min (resulting in a f 2-fold increase in ERK-2 phosphorylation levels over control) for all remaining experiments.

Okadaic acid, a specific PP2A inhibitor at V 10 nM, completely attenuated AT2 receptor-mediated inhibition of insulin-induced ERK-2 phosphorylation (Fig. 4). The attenuation effect was statistically significant at 20, 30 and 40 min. Sodium orthovanadate (10 AM) had no significant effect on the ability of the AT2 receptor to inhibit insulin-induced ERK2 phosphorylation (Fig. 4). 3.5. Effect of DRY motif mutation on the AT2 receptor’s modulation of insulin-induced ERK-2 activation Site-directed mutagenesis of the AT2 receptor resulted in significant loss of function for several mutants when com-

3.2. Effect of AT2 receptor activation on insulin-induced ERK-2 phosphorylation levels A time-dependent effect of Ang II attenuation of insulininduced ERK-2 activation was also observed (Fig. 2A). Our results demonstrate that a 10-min pre-incubation with 1 AM Ang II significantly decreased insulin-induced ERK-2 phosphorylation (26.4 F 4.4%). Immunoblot analysis further demonstrated that pre-incubating cells with Ang II for 30 min resulted in a maximal 42.1 F 7.0% decrease in insulininduced ERK-2 phosphorylation. Activation of the AT2 receptor attenuated insulin-induced ERK-2 phosphorylation in a concentration-dependent manner (Fig. 2B). While the addition of 1 nM Ang II for 20 min resulted in a small decrease in insulin-activated ERK-2 levels, significant inhibition was only seen at levels z10 nM Ang II. Cells pre-incubated with 100 nM Ang II for 20 min resulted in a 30.4 F 8.1% decrease of insulin-induced ERK-2 phosphorylation. The presence of 1 AM Ang II did not increase the inhibition observed with 100 nM Ang II. Based on these findings, cells were treated with 1 AM Ang II (resulting in a maximum 42.3 F 6.0% inhibition of

Fig. 4. Effect of okadaic acid and sodium orthovanadate on human AT2 receptor’s ability to inhibit insulin-induced ERK-2 phosphorylation. Transfected CHO-hAT2R cells were pre-incubated with 1 AM Ang II (0 – 40 min), and 1 nM insulin for 5 min in the absence (5) or presence of 10 nM okadaic acid ( w ) or 10 mM sodium orthovanadate (o). Cell lysates were assayed by Western analysis using antibody specific for Phospho-p44/ 42 (ERK-1/ERK-2) MAP kinase (Thr202/Tyr204). Data are presented as the mean F SEM (nz4). *p < 0.05 vs. control.

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Fig. 5. Effect of alanine substitutions in the DRY141 – 143 motif on the AT2 receptor’s ability to inhibit insulin-induced ERK-2 phosphorylation. Sitedirected mutagenesis of human AT2 receptor was carried out and CHO cells were transfected with wild-type or mutant receptor. Transfected cells were pre-incubated with 1 AM Ang II (40 min) and 1 nM insulin for the last 5 min of Ang II treatment. Cell lysates were assayed by Western analysis using antibody specific for Phospho-p44/42 (ERK-1/ERK-2) MAP kinase (Thr202/Tyr204). Wt receptor is positive control (100% maximum inhibition of insulin-stimulated ERK-2 phosphorylation). Data are presented as the mean F SEM (nz4). *p < 0.05 vs. control.

pared to the wild-type receptor’s ability to inhibit insulininduced ERK-2 activation (Fig. 5). The ability of the D141-A mutant AT2 receptor to inhibit insulin-induced ERK-2 activation was decreased by 27.5 F 11.3%, which was significantly different from the wild-type receptor control. Further, the Y143-A mutant inhibition of insulin-induced ERK-2 phosphorylation was reduced by 24.0 F 15.5%, which was not significantly different from wild type. The most significant effect was seen with the R142-A and DRY-AAA mutations, which decreased AT2 receptor ability to inhibit insulin-induced ERK-2 phosphorylation by 66.3 F 12.1% and 44.7 F 12.4% from control, respectively.

4. Discussion In the present study, we have shown that the human AT2 receptor can inhibit insulin-induced ERK-2 phosphorylation in CHO cells via a G-protein mediated signaling pathway. We have also demonstrated that a serine/threonine phosphatase, which is most likely PP2A, plays an important role in the downstream signaling effects of the human AT2 receptor mediated G-protein coupled pathway. Using our insulin stimulation paradigm, we observed an ( f 85%) increase in ERK-2 phosphorylation from control following a 5-min treatment with 1 nM insulin. The effect

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was transient, with ERK-2 phosphorylation rapidly increasing for the 5 min following insulin stimulation, plateauing, and then decreasing to baseline within 20 min. A maximum fourfold increase of ERK-2 phosphorylation was observed with 5-min treatments of 100 nM insulin. Although our insulin response findings were more robust than those previously reported in CHO cells [5], they are generally consistent with previous studies. In the present study, we examined the time-dependent effect of AT2 receptor stimulation on insulin-induced ERK-2 phosphorylation. Using the CHO cell system we have previously described [18], we examined the AT2 receptor’s modulation of insulin-induced ERK-2 phosphorylation over time. Our findings suggest that pre-incubation with 1 AM Ang II for 40 min is essential for maximum inhibition of insulin induced ERK-2 phosphorylation. Our findings further suggest that AT2 receptor inhibition of ERK-2 phosphorylation is time-dependent and demonstrate that significant AT2 receptor-mediated inhibition of insulin-induced ERK-2 phosphorylation occurs after the 5-min AT2 receptor agonist stimulation time reported previously [5]. We also investigated the second messengers involved in this AT2 receptor mediated ERK-2 inhibition response. While the primary amino acid sequence of the AT2 receptor suggests that it is a G-protein coupled receptor (GPCR), it is thought to have both G-protein-dependent [9,11,13,18,28] and -independent signaling mechanisms [1 – 3,5]. Others have reported [3] that the AT2 receptor can induce protein tyrosine phosphatase (PTPase) activity through a G-protein independent mechanism, which is attenuated by the PTPase inhibitor sodium orthovanadate. In contrast, Huang et al. [11] reported that the ability of the AT2 receptor to up-regulate PP2A was sensitive to pertussis toxin (a potent Gi/G0 protein inhibitor) and okadaic acid, but not to sodium orthovanadate, suggesting that this G-protein mediated pathway up-regulates PP2A activity (not PTPase activity). Our previous studies have shown that the AT2 receptor is GTPgS sensitive [6,18], indicating that the receptor is functionally coupled to G-proteins. To determine whether the AT2 receptor-mediated inhibition of ERK-2 activation observed in our system was G-protein mediated, transiently transfected cells were treated with pertussis toxin prior to Ang II/insulin stimulation. Pertussis toxin completely attenuated AT2 receptor’s inhibition of insulin-induced ERK-2 phosphorylation. Together these findings suggest that the human AT2 receptor can inhibit insulin-induced ERK-2 phosphorylation via a Gprotein coupled mechanism. To further characterize this G-protein-coupled signaling pathway, okadaic acid and sodium orthovanadate were used to determine the putative role of PP2A or a PTPase. Our results demonstrate that okadaic acid attenuated the ability of the AT2 receptor to inhibit insulin-induced ERK2 activation, suggesting that PP2A plays a significant role in this G-protein mediated effect. Treatment with sodium orthovanadate did not effect the modulation of ERK-2 phosphorylation by the AT2 receptor, suggesting that

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PTPase does not play a significant role in this pathway. These results suggest that the AT2 receptor-mediated signaling pathway is consistent with that characterized in primary rat neuronal cultures [11– 13]. Previous studies by Huang et al. [11,13] reported that the AT2 receptor upregulates a pertussis toxin sensitive G-protein, which in turn up-regulates an okadaic acid sensitive serine/threonine phosphatase (PP2A). To further determine whether this inhibitory pathway is indeed G-protein coupled, we constructed mutant AT2 receptors with alanine substitutions in the putative G-protein coupling DRY motif. Our previous studies demonstrate that several of these mutants are partially decoupled from Gproteins [18]. In the present study, we used the DRY motif mutant receptors to determine what role, if any, the motif plays in the inhibition of insulin-induced ERK-2 activation by the AT2 receptor. All four mutant receptors had reduced ability to inhibit insulin-induced ERK-2 phosphorylation compared to wild-type receptor. The most significant decrease was seen with the R142-A mutant, followed by the DRY knockout mutant, and the D141-A and Y143-A mutants. These experiments provide further evidence to suggest that the AT2 receptor inhibits insulin-induced ERK-2 activation via a G-protein mediated pathway. As additional AT2 receptor signaling mechanisms are identified, it appears that up-regulation of serine/threonine and tyrosine phosphatases plays an important role (see Fig. 6). Nouet and Nahmias [20] hypothesized that the AT2 receptor up-regulates several phosphatases at differing time points following agonist stimulation. A temporally regu-

lated signaling strategy could lead to a more robust inhibition of the insulin-induced ERK-2 activity over time. Perhaps the transient non-G-protein mediated pathway described by Elbaz et al. [5] is the initial AT2 receptormediated ERK-2 inhibition pathway in CHO cells, since its effect appears to return to baseline by 10 min after agonist stimulation. Our results in CHO cells are consistent with those from another group in neurons, which show that AT2 receptor’s G-protein mediated signaling requires 20 to 30 min to reach significant levels [11]. It is possible that, as the transient non-G-protein mediated AT2 receptor inhibition pathway [5] returns to baseline the G-protein mediated pathway we have characterized in the present study becomes significantly up-regulated, prolonging the ability of the AT2 receptor to inhibit insulin receptor signaling. Additional studies will be needed to determine the mechanism of regulation of these two distinct AT2 receptor signaling pathways. Finally, our findings and others [5] have shown that CHO cells are one of the few cell types that display both G-proteindependent and G-protein-independent AT2 receptor signaling pathways. Therefore, CHO cells may be a valuable tool for further investigation of the mechanism of regulation of the two AT2 receptor signaling pathways.

Acknowledgements This work was supported by a grant from the National Science Foundation (IBN-9906442).

Fig. 6. Diagram of AT1 receptor, AT2 receptor and insulin receptor signaling in CHO cells. (A) Previously confirmed pathway of AT2 receptor inhibition of insulin receptor signaling in CHO cells [5]. (B) Mechanism of AT2 receptor inhibition of insulin receptor signaling in CHO cells characterized by this study.

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