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(Circulation Research. 2000;87:753.)
© 2000 American Heart Association, Inc.

Molecular Medicine

Functional Reconstitution of the Angiotensin II Type 2 Receptor and G_i Activation

Jakob Lerche Hansen, Guy Servant, Thomas J. Baranski, Toshiro Fujita, Taroh Iiri, Søren P. Sheikh

From the Laboratory for Molecular Cardiology and the Department of Medicine B, University of Copenhagen (J.L.H., S.H., S.P.S.), Denmark; the Department of Cellular and Molecular Pharmacology and The Cardiovascular Research Institute, University of California (G.S.), San Francisco, Calif; the Departments of Internal Medicine and Molecular Biology and Pharmacology, Division of Endocrinology, Diabetes and Metabolism, Washington University School of Medicine (T.J.B.), St. Louis, Mo; and the Department of Endocrinology and Nephrology and University of Tokyo School of Medicine (T.I.), Tokyo, Japan.

Correspondence to Søren P. Sheikh, Laboratory of Molecular Cardiology, Rigshospitalet 9312, University of Copenhagen, Juliane Mariesvej 20, DK-2100 Copenhagen, Denmark. E-mail sheikh{at}molheart.dk\\ © 2000 American Heart Association, Inc.

	Abstract

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Abstract—On the basis of the patterns of conserved amino acid sequence, the angiotensin II type 2 (AT₂) receptor belongs to the family of serpentine receptors, which relay signals from extracellular stimuli to heterotrimeric G proteins. However, the AT₂ receptor signal transduction mechanisms are poorly understood. We have measured AT₂-triggered activation of purified heterotrimeric proteins in urea-extracted membranes from cultured COS-7 cells expressing the recombinant receptor. This procedure removes contaminating GTP-binding proteins without inactivating the serpentine receptor. Binding studies using [¹²⁵I] angiotensin (Ang) II revealed a single binding site with a K_d=0.45 and a capacity of 627 fmol/mg protein in the extracted membranes. The AT₂ receptor caused a rapid activation of _i and _o but not of _q and _s, as measured by radioactive guanosine 5'-3-O-(thio)triphosphate (GTPS) binding. Activation required the presence of activated receptors, ß, and subunits. As a first step aimed at developing an in vitro assay to examine AT₂ receptor pharmacology, we tested a battery of Ang II–related ligands for their ability to promote AT₁ or AT₂ receptor–catalyzed G_i activation. Two proteolytic fragments of Ang II, Ang III and Ang1–7, also promoted activation of _i through the AT₂ receptor. Furthermore, we found that [Sar¹,Ala⁸]Ang II is an antagonist for both AT₁ and AT₂ receptors and that CPG42112 behaves as a partial agonist for the AT₂ receptor. In combination with previous observations, these results show that the AT₂ receptor is fully capable of activating G_i and provides a new tool for exploring AT₂ receptor pharmacology and interactions with G-protein trimers.

Key Words: AT₂ • angiotensin II type 2 receptor • G_i activation

	Introduction

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Angiotensin II (Ang II) is the primary effector of the renin/angiotensin system. This 8-amino acid peptide is a key regulator of blood pressure and body fluid homeostasis and plays a critical role in the pathophysiology of several cardiovascular diseases such as hypertension, hypertrophy, and congestive heart failure.¹ In particular, blocking the effects of Ang II with the use of angiotensin-converting enzyme inhibitors or losartan (a competitive AT₁ antagonist) is used in treatment of cardiovascular diseases. To understand the Ang II signal transduction pathways, it will be important to examine the individual components of the system.

Two Ang II receptor subtypes, type 1 (AT₁)² and type 2 (AT₂) receptors,³ have been identified. The AT₁ receptor has a widespread tissue distribution and mediates most of the known cardiovascular Ang II functions including vasoconstriction and cardiac and vascular hypertrophy.⁴ The expression pattern of the AT₂ receptor is more restricted and suggests that this receptor plays a role in growth, development, and differentiation. The AT₂ is highly expressed in fetal tissues, whereas in adults, it is confined to organs such as heart, vascular smooth muscle, brain, adrenal cortex, uterus, and ovarian follicles.⁴ AT₂ activation dilates blood vessels, inhibits growth, and induces apoptosis.¹ In addition, the AT₂ receptor plays a role in neuronal differentiation and regeneration⁵ and is upregulated in skin wounds, blood vessel neointima on injury, and during cardiac remodeling after a myocardial infaction.⁴

The AT₂ receptor, like the AT₁ receptor, contains seven stretches of hydrophobic amino acids, a hallmark of the family of serpentine receptors. This large family of surface receptors relays extracellular signals from hormones and sensory stimuli to heterotrimeric G proteins at the cytoplasmic face of the plasma membrane. The serpentine receptors activate G proteins by promoting exchange of GTP for GDP bound to the subunit of the heterotrimer, causing liberation of both -GTP and free ß complexes, which in turn activate effector enzymes and ion channels.⁶ On the basis of sequence homology and intracellular effector regulation, 16 distinct mammalian subunits have been subclassified into four subfamilies, _i1/2/3, _t, _z, that (except _z) are pertussis toxin (PTX) substrates, _s and _olf, that stimulate adenylyl cyclases, the _q, ₁₁, ₁₄, and _15/16, that activate phospholipase C-ßs (PLCß), and the _12/13 family, that can transform different cell lines.

Which signaling proteins or G proteins does the AT₂ receptor activate to exert its biological functions? It has proven difficult to ascertain whether AT₂ receptors activate heterotrimeric G proteins in vivo.⁷ Several reports indicate that the AT₂ receptor may couple to G_i. Thus, in rat hippocampal neurons and a few other cell types, blocking _i with PTX or antibodies directed against _i inhibits the AT₂ receptor effects.^{8 9 10} In two studies, a direct interaction between AT₂ receptors and _i was inferred from coimmunoprecipitation experiments.¹⁰ In contrast, in neuroblastoma cells and NG108-15 cells bearing AT₂ receptors, PTX does not block Ang II effects.¹¹ Moreover, in PC-12 cells bearing endogenous AT₂ receptors and in HEK293 or COS-7 cells overexpressing AT₂ receptors, Ang II does not apparently change intracellular concentrations of cAMP, cGMP, or inositol phosphates.³ These results suggest that the AT₂ receptor does not activate _q, _i, or _s and raise the question whether this receptor uses G proteins as signaling partners.

To examine whether the AT₂ receptor can activate G_i, we have assessed in vitro AT₂-G_i interactions in a solution of urea-washed membranes from AT₂-transfected COS-7 cells containing purified G-protein components.^{12 13} The AT₂ receptor triggered activation of G-protein subunits (_i and ß), as ascertained by guanosine 5'-3-O-(thio)triphosphate (GTPS) binding. Ligand-activated AT₂ stimulated GTPS binding to G_i and G₀, but not G_s or G_q. We infer from our results that AT₂ receptors directly activate _i and _o. The fidelity and degree of resolution of the interaction allow structure function studies and further AT₂ receptor pharmacological characterization.

	Materials and Methods

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Materials
Angiotensin-related peptides [Sar¹,Ala⁸]Ang II, [1–7]Ang II, Ang II, and Ang III were obtained from Bachem. Isoproterenol and Ang IV were obtained from Sigma and parathyroid hormone (PTH) peptide from Bachem. The radiolabeled [¹²⁵I]Ang II was obtained from Amersham Pharmacia Biotech and [³⁵S]GTPS from NEN Life Science Products. Losartan, PD123319, and CGP42112 were generous gifts from Dr Gaetan Guillemette (University of Sherbrooke, Canada). The Elk1 luciferase reporter was obtained from Stratagene.

Construction of the R142A-AT₂ Receptor
The point mutation was generated by polymerase chain reaction in two steps with Pfu polymerase with the wild-type receptor cDNA as a probe, as described.¹³

Cell Culture and Transfection
COS-7 cells were maintained in DME-H21 medium containing 10% FCS, 2.5 µg/mL Fungizone, and 10 µg/mL gentamycin. Transient transfections with wild-type serpentine receptors were performed using a DEAE-dextran/adenovirus method as described.¹³

Membrane Preparation
Membranes from COS-7 cells transfected with cDNAs encoding human AT₂, human PTH, or human ß₂ receptors were prepared as described.¹³ Membrane microsomal fractions, obtained by centrifugation, were stripped of GTP-binding proteins as described¹² by incubation in 6 mol/L urea.

G-Protein Purification
Alpha subunits were purified from Sf9 cell membranes infected with baculovirus encoding the wild-type protein, His₆-tagged ₂, and wild-type ß₁ as described.¹⁴

Ligand Binding
Binding of [¹²⁵I]Ang II was determined as described.¹⁵ Binding was initiated by suspending urea-extracted membranes in a mixture of [¹²⁵I]Ang II (100 pmol/L), cold Ang II, and a buffer consisting of 25 mmol/L Tris-HCl, pH 7.3, 100 mmol/L NaCl, 5 mmol/L MgCl₂, 1 mmol/L EDTA, 2 mg/mL BSA, 0.1 mg/mL bacitracin, and 50 µg/mL soybean trypsin inhibitor.

G Activation
Exchange of GTPS for GDP bound to Gs was measured using a modification of a previously described procedure.¹⁶ Briefly, membranes containing receptors (5 nmol/L) were preincubated with purified s (50 nmol/L) and ß (100 nmol/L) for 15 minutes on ice in a buffer containing 20 mmol/L Na-HEPES, pH 7.6, 1 mmol/L Tris-HCl, pH 7.6, 100 mmol/L NaCl, 2 mmol/L MgCl₂, 1 µmol/L GDP, and 1 mmol/L ß-mercaptoethanol. Assays were initiated by addition of agonist and [³⁵S]GTPS (10⁵ cpm per tube) in a total volume of 20 µL. After incubation for the indicated times at 30°C, reactions were terminated by adding a stop solution containing 20 mmol/L Tris-HCl, pH 8, 100 mmol/L NaCl, and 10 mmol/L MgCl₂ and filtered over nitrocellulose membranes on a vacuum manifold. Radioactivity was quantitated by liquid scintillation in a ß counter. Nonspecific binding (binding to the filter in the absence of membranes) was <10% of total binding.

Mitogen-Activated Protein Kinase (MAPK) Phosphorylation
COS-7 cells were cotransfected with plasmids encoding the AT₂ receptor, _i2, ß₂, and ₁. After transfection, the cells were incubated in DMEM supplemented with 10% FCS overnight followed by 16 hours of serum starvation. Next, the cells were stimulated with 10% FCS for 12 minutes in the presence and absence of Ang II. Cell lysates and quantification of MAPK phosphorylation was performed as described.¹⁷

Elk1 Activation
Transcriptional activation of Elk1 luciferase expression by MAPK was assessed by cotransfecting pFA2-Elk1, plasmids encoding pFR-luc, ß-galactosidase (ß-Gal), and the AT₂ receptor, _i2, ß₂, and ₁ expression plasmids in COS-7 cells. After transfection, the cells were incubated in DMEM supplemented with 10% FCS overnight. Next, the cells were serum-starved for 16 hours followed by a 24-hour incubation with 10% serum in the presence and absence of 100 nmol/L Ang II. Luciferase and ß-Gal activities were measured as described by the manufacturer.

	Results

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To examine the AT₂-receptor/G-protein coupling, we used urea-extracted COS-7 cell membranes transiently expressing AT₂ receptors and compared the results with those previously reported for other receptors: rhodopsin, gastrin-releasing peptide, PTHR, or ß₂AR.^{12 13} The addition of purified G-protein subunits (both _i and ß) allowed us to examine the selectivity of G-protein activation and assess the potency of Ang II–related receptor ligands.

Ligand Binding Studies
Urea-treated COS-7 cell membranes bearing the wild-type AT₂ receptor or a specific mutant R142A (a mutation that uncouples receptor–G-protein activation)¹⁸ were analyzed for Ang II binding. Figure 1 depicts representative curves showing the effects of increasing concentrations of Ang II and analogues on [¹²⁵I]Ang II binding. The rank order of binding affinities was Ang III>CGP42112A>Ang II>Ang1–7>Ang IV. The apparent K_d values for Ang II were 0.45 and 0.78 nmol/L, and the calculated B_max was 627 and 280 fmol/mg protein for AT₂ wild-type and R142A-AT₂ receptors, respectively.

View larger version (27K): [in this window] [in a new window]   Figure 1. Figure 1. Competition between Ang II–like peptides and [125I]Ang II for binding to AT2 and AT2 (R142A) mutant receptors. Urea-washed membranes of COS-7 cells expressing wild-type AT2 or R142A-AT2 () receptors were incubated as described15 with radiolabeled [125I]Ang II (100 pmol/L) and the indicated concentrations of Ang II (• and ), Ang III (), Ang1–7 (), Ang IV (), and CGP42112A ().

Receptor Activation Assay
To assess AT₂-mediated activation of _i, we measured ligand-dependent binding of radioactive GTPS in a mixture containing pure _i and ß and urea-washed membranes from COS-7 cells expressing recombinant receptors and NIH3T3 cells bearing native AT₂ receptors. In both of these systems, Ang II induced significant GTPS binding only in the presence of receptors _i and ß (Figures 2B and 2D). In urea-treated COS-7 membranes, Ang II increased GTPS binding 3- to 10-fold in different experiments. At maximal stimulation, radioactive GTPS bound to 10% to 30% of the total _i present in the assay. The effect of Ang II was complete within 3 minutes (not shown). This shows that the AT₂ receptor can catalyze the exchange of GDP for GTPS on _i. To explore the effects of urea treatment, we compared GTPS binding in P2 membranes and urea-washed membranes from COS-7 and NIH3T3 cells (Figure 2). Ang II induced a 7% and 19% increase in GTPS binding in COS-7 and NIH3T3 P2 membranes, respectively. Coexpression of _i and ß did not improve the Ang II effect (Figure 2). These results show that with native complement of G proteins, there is a trend toward AT₂ activation–enhanced GTP binding, although the signal-to-noise is hampered by the high level of AT₂-uncoupled GTP-binding proteins. The data also show that urea treatment drastically reduces endogenous GTP binding without inactivating the recombinant serpentine receptors consistent with previous findings.¹²

View larger version (38K): [in this window] [in a new window]   Figure 2. Figure 2. AT2-dependent activation of Gi in a reconstituted system. COS-7 membranes expressing recombinant AT2 receptors were treated without urea (A) or with (B) urea and Ang II in the presence of Gi subunits as indicated, and binding of [35S]GTPS to i was measured as described in Materials and Methods. Similar experiments were conducted using NIH3T3 membranes without (C) or with (D) urea treatment. Binding of [35S]GTPS was measured after incubating membranes for 10 minutes at 30°C in the presence or absence of Ang II (100 nmol/L), i/s (50 nmol/L), and/or ß (100 nmol/L). AT2 receptor concentration was 5 nmol/L in panels A and B. Bars represent means of triplicate determinations from 3 to 5 experiments using at least 2 different membrane preparations.

Figure 3 depicts the concentration dependence of the individual components of the system. The kinetics of G-protein activation would be expected to be saturable with each of the components. This prediction was fulfilled: GTPS binding was saturable with increasing concentrations of _i while keeping the ß concentrations fixed at 250 nmol/L (Figures 3A and 3B). The K_0.5 for _i was 24 nmol/L. Similarly, the GTPS binding was saturable in the converse experiment, increasing the ß concentrations at fixed concentrations of _i (Figure 3B). In addition, the GTPS binding was ligand-dependent (Figure 4A), with an EC₅₀ for Ang II of 24 nmol/L, which is comparable to values obtained in other assays.¹⁹

View larger version (23K): [in this window] [in a new window]   Figure 3. Figure 3. i and ß saturation of AT2-catalyzed exchange of GTPS for GDP. The GTPS binding was measured as described in Figure 2 legend, except that incubations were conducted at different concentrations of i and a fixed ß concentration (250 nmol/L) (A) or at different ß concentrations with a fixed i concentration (150 nmol/L) (B). The concentration of Ang II was 100 nmol/L. Values are means of duplicate determinations from 1 of 3 experiments.

View larger version (40K): [in this window] [in a new window]   Figure 4. Figure 4. Ang II saturation and receptor Gi coupling specificity. A, Relative i activation by AT2 receptor. Values represent means of duplicate determinations representative of 3 experiments. B and C, Urea-washed membranes containing recombinant receptors as indicated were mixed with i (50 nmol/L), s (50 nmol/L), o (50 nmol/L), q (70 nmol/L), and ß (100 nmol/L), and GTPS binding was measured as described. The concentrations of s were determined in GTPS loading experiments. Values are the mean±SEM of triplicate determinations representative of at least 3 experiments.

To test the specificity of _i activation, we examined the ability of AT₂ receptors to activate three additional subunits, ₀, _q, and _s. G₀ is a brain _i-like subunit. G_q activates PLC, and _s is the stimulatory regulator of adenylyl cyclase. Both of these subunits have no resemblance to _i in primary structure. The AT₂ activated ₀ and _i but failed to promote GTPS binding on _q and _s (Figure 4B). Two other serpentine receptors, PTHR and ß₂AR, activated _s, and the AT₁ receptor activated _q, confirming the functionality of these added subunits (Figure 4C). Furthermore, a point mutation of arginine (R142A-AT₂) in the conserved DRY sequence at the COOH-terminal part of ic2 produced a receptor that could not activate G_i, suggesting that AT₂ receptors activate G proteins by similar mechanisms as other receptors.¹⁸ These experiments show that AT₂ receptors discriminate between G_i, G_q, and G_s in this system. Another important question is whether the added _i might be activated by a serpentine receptor. We examined this using membranes expressing PTHR. The activated PTHR did not promote GDP exchange for GTPS on _i (Figure 4B). Thus, both the recombinant AT₂ and the purified _i have retained specificity in this system.

AT₂ Receptor Pharmacology
We examined the effect of different Ang II analogues on AT₁ and AT₂ receptor–induced _i activation. Figure 5 shows that in addition to Ang II, one of its proteolytic fragments, Ang III (desAsp¹-Ang II), behaved as an agonist on both AT₁ and AT₂ receptors. Ang1–7 selectively activated AT₂ receptors albeit with a much lower affinity than Ang II. Ang1–7 is another Ang II proteolytic fragment found in plasma. This peptide induces bradykinin-mediated hypotensive responses and reduces smooth muscle growth after vascular injury.^{20 21} Thus, AT₂ receptors could be responsible for these actions. CGP42112A, a pseudo-peptide that binds the AT₂ receptor with high affinity and selectivity, was initially considered an antagonist.⁷ Our results using this compound alone suggest that CGP42112A is a selective AT₂ agonist. A similar conclusion was previously made.¹⁷ However, in the presence of 100 nmol/L Ang II, a high concentration of CGP42112A works antagonistically and thus inhibits the effect of Ang II (Figure 5B). These data suggest that CGP42112A is a partial agonist. Losartan and PD123319, two nonpeptidic Ang II analogues, bind with high affinity and selectivity to AT₁ and AT₂ receptors, respectively.²² Accordingly, losartan selectively blocked AT₁ activity in the described assay (Figure 5). Olmesartan and candesartan also selectively blocked AT₁ receptor activity (data not shown). In almost all reported AT₂-mediated effects, PD123319 is a selective competitive antagonist (Table). In our study, this compound selectively blocked AT₂ receptor activity. In addition, an antagonist [Sar¹,Ala⁸]-Ang II, thought to block both receptors, did so in our assays (Figure 5).

View larger version (36K): [in this window] [in a new window]   Figure 5. Figure 5. Pharmacological profiles of AT1 and AT2 receptors. Urea-washed membranes expressing AT1 or AT2 receptors were incubated with purified i and ß. In addition, Ang II–related agents were added: Ang II (100 nmol/L), [Sar1,Ala8]Ang II (1 µmol/L), PD123319 (PD) (10 µmol/L), and CGP42112, Ang1–7, and Ang III as indicated. Values are the mean±SEM of triplicate determinations from at least 3 experiments. Results were analyzed by paired Student’s t test. *P<0.01, **P<0.001.

View this table: [in this window] [in a new window]   Table 1. AT2 Receptor Signal Transduction in Different Cellular Environments

AT₂ Receptor Effects in Intact COS-7 Cells
Given that our assay is a reconstitution of purified G proteins with urea-washed COS-7 membranes, we wanted to analyze whether the transfected AT₂ receptor could activate signaling pathways in intact cells. The AT₂ receptor has been reported to couple to ERK1/2 MAPK phosphorylation. To explore this concept, we investigated MAPK (ERK1/2) phosphorylation by Western blotting and MAPK transcriptional activation of Elk1 using Elk1 luciferase reporter plasmid. AT₂ receptor activation induced both MAPK phosphorylation and Elk1 luciferase expression in the presence of serum compared with the effect of serum alone (Figure 6). AT₂ receptor activation did not affect MAPK phosphorylation or activation in the absence of serum (not shown). These data show that AT₂ receptor activation in COS-7 cells is functionally coupled to MAPK.

View larger version (33K): [in this window] [in a new window]   Figure 6. Figure 6. AT2-induced MAPK activation in COS-7 cells. The ability of the AT2 receptor to induce MAPK phosphorylation (A) and luciferase expressed under control of the Elk1 promoter (B) was assessed in the absence and presence of 10% serum. Luciferase activity was normalized to ß-Gal activity to correct for transfection efficiency. Results are expressed as fold increase over values without serum or Ang II. Values are the mean±SEM of duplicate determinations from at least 3 experiments. *P<0.05, **P<0.01.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
We have set up a system to assay AT₂ receptor activation of G_i to understand the G-protein signaling properties of this receptor. The AT₂ receptor has diverse biological effects in cell growth, differentiation, and control of blood vessel tone (Table). However, the molecular events behind these effects have not been clearly defined. In particular, it has not been possible to document G protein–regulated activation of classical second messengers such as cAMP and the phospho-inositide metabolism.³ Almost all other serpentine receptors regulate at least one of these systems. Can the AT₂ receptor activate G proteins? To answer this question, we tested the ability of the AT₂ receptor to activate the subunit of G_i in a reconstituted system. In this system, the AT₂ receptor activated _i in a manner dependent on the concentrations of ligand, receptor, and G_i subunits. These results suggest that the AT₂ receptor activates G_i in much the same way as other receptors activate G proteins. In similar assays, rhodopsin activates transducin, and the gastrin-releasing peptide receptor activates G_q.^{12 18} Mutations can produce promiscuous receptors, which activate classes of G proteins not activated by the parent wild-type receptors.²³ Could the described assay simply unmask an inherent promiscuity of AT₂ receptors? To address this question, we performed two experiments, the results of which suggested that the assay does exhibit specificity. First, the AT₂ receptor was unable to induce GTPS binding to purified G_q and G_s, two different G proteins, and vice versa, the G_i was not activated by PTHR.

Data from ligand binding studies, use of PTX, which inactivates _i, and coimmunoprecipitation experiments using anti–G-protein antibodies, support the inference that the AT₂ receptor activates G proteins. First, GTPS reduces AT₂ receptor binding affinity in certain cell membranes and in HEK293 cells with overexpressed AT₂ receptors.^{3 11} Second, at least three AT₂-mediated biological effects are blocked by PTX: a delayed rectified K⁺ current in neurons,⁸ inhibition of NIH3T3 cell growth,⁹ and DNA synthesis in vascular smooth muscle cells.¹⁰ Third, AT₂ receptor protein has been coimmunoprecipitated with _i from fetal rat cells²⁴ and vascular smooth muscle cells,¹⁰ suggesting that a physical association exists. AT₂ receptor activation also promotes PTX-insensitive cellular events (Table). Our results show that the AT₂ receptor can support activation of G_i and G₀ but not G_q and G_s.

If the AT₂ receptor activates G_i, why does it not inhibit cAMP production or robustly activate MAPK, effects attributed to activation of _i and liberation of ß?^{7 22} Our results do not unequivocally answer this question; however, possible explanations can be offered. First, in at least three reports, AT₂ receptor stimulation led to MAPK phosphatase-1 (MKP-1) activation, an effect that prevents MAPK activation (Table).¹⁰ Moreover, we (Figure 6) and others suggest that the AT₂ receptor can activate MAPK in COS-7 cells and NG108-15 cells, the latter after prolonged stimulation.¹⁷ It is possible that in these cells, MKP-1 is expressed at lower levels or that its regulation in NG108-15 cells is time-dependent, ie, an initial activation that inhibits MAPK followed by its inactivation, resulting in MAPK activation. Second, the AT₂ receptor could need an unknown protein to organize G proteins and second messengers in its vicinity. Third, the repertoire and concentration of resident G proteins in a given cell type may modify receptor coupling. It is possible that, in vivo, the AT₂ receptor couples to G_i with a low affinity or with a low rate of GDP-GTP exchange. Thus, only cells expressing high levels of AT₂ and G_i would produce detectable levels of second messengers. Fourth, cell-specific posttranslational modifications of the AT₂ receptor could play a role in determining G-protein specificity.

Our simple assay proved useful for the determination of AT₂ receptor pharmacology, something that has been difficult to evaluate because of the lack of reproducible and easily quantifiable effects. We compared different peptidic and nonpeptidic Ang II analogues on AT₂-induced G_i activation. Furthermore, to validate our observations, we tested the same battery of analogues on membranes bearing AT₁ receptors, for which responses to these ligands are well characterized.⁷ We found that Ang II and its proteolytic fragment Ang III (lacks the amino-terminal Asp) act as agonists on the AT₂ receptor. Similarly, and as expected,²² they also triggered AT₁-induced G_i activation.

In one study, Ang1–7, an Ang II proteolytic fragment in which the carboxyl-terminal residue is cleaved, was reported to induce prostaglandin synthesis in human astrocytes through AT₂ receptors.²⁵ Moreover, evidence suggests that Ang II proteolytic fragments other than Ang III and including Ang1–7 may have some biological activities.⁷ Accordingly, we found that this peptide selectively activated the AT₂ receptor. Taken together, these results suggest that, in vivo, both Ang II and Ang III could serve as agonists for the AT₂ receptor given that they have similar affinities.⁷

	Acknowledgments

 
This work was supported in part by grants from the National Institutes of Health, the Danish Heart Foundation, the Danish Medical Research Council, the Novo-Nordisk Foundation, the Villadsen Family Foundation, the Birthe and John Meyer Foundation, and the Foundation of 17.12.1981.

Received April 7, 2000; revision received September 20, 2000; accepted September 21, 2000.

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