Functional Reconstitution of the Angiotensin II Type 2 Receptor and Gi Activation
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 Kd=0.45 and a capacity of 627 fmol/mg protein in the extracted membranes. The AT2 receptor caused a rapid activation of αi and αo but not of αq and αs, as measured by radioactive guanosine 5′-3-O-(thio)triphosphate (GTPγS) binding. Activation required the presence of activated receptors, βγ, and α 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 Gi activation. Two proteolytic fragments of Ang II, Ang III and Ang1–7, also promoted activation of αi 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 Gi and provides a new tool for exploring AT2 receptor pharmacology and interactions with G-protein trimers.
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 α subunit of the heterotrimer, causing liberation of both α-GTP and free βγ complexes, which in turn activate effector enzymes and ion channels.6 On the basis of sequence homology and intracellular effector regulation, 16 distinct mammalian α subunits have been subclassified into four subfamilies, αi1/2/3, αt, αz, that (except αz) are pertussis toxin (PTX) substrates, αs and αolf, that stimulate adenylyl cyclases, the αq, α11, α14, and α15/16, that activate phospholipase C-βs (PLCβ), and the α12/13 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 Gi. Thus, in rat hippocampal neurons and a few other cell types, blocking αi with PTX or antibodies directed against αi inhibits the AT2 receptor effects.8 9 10 In two studies, a direct interaction between AT2 receptors and αi 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 αq, αi, or αs and raise the question whether this receptor uses G proteins as signaling partners.
To examine whether the AT2 receptor can activate Gi, we have assessed in vitro AT2-Gi 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 (αi and βγ), as ascertained by guanosine 5′-3-O-(thio)triphosphate (GTPγS) binding. Ligand-activated AT2 stimulated GTPγS binding to Gi and G0, but not Gs or Gq. We infer from our results that AT2 receptors directly activate αi and αo. The fidelity and degree of resolution of the interaction allow structure function studies and further AT2 receptor pharmacological characterization.
Materials and Methods
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]GTPγS 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
Membranes from COS-7 cells transfected with cDNAs encoding human AT2, human PTH, or human β2 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.
Alpha subunits were purified from Sf9 cell membranes infected with baculovirus encoding the wild-type protein, His6-tagged γ2, and wild-type β1 as described.14
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.
Exchange of GTPγS for GDP bound to Gαs was measured using a modification of a previously described procedure.16 Briefly, membranes containing receptors (≈5 nmol/L) were preincubated with purified αs (50 nmol/L) and βγ (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 β-mercaptoethanol. Assays were initiated by addition of agonist and [35S]GTPγS (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 β 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, αi2, β2, and γ1. 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
Transcriptional activation of Elk1 luciferase expression by MAPK was assessed by cotransfecting pFA2-Elk1, plasmids encoding pFR-luc, β-galactosidase (β-Gal), and the AT2 receptor, αi2, β2, and γ1 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 β-Gal activities were measured as described by the manufacturer.
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 β2AR.12 13 The addition of purified G-protein subunits (both αi and βγ) 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 Kd values for Ang II were ≈0.45 and ≈0.78 nmol/L, and the calculated Bmax was 627 and 280 fmol/mg protein for AT2 wild-type and R142A-AT2 receptors, respectively.
Receptor Activation Assay
To assess AT2-mediated activation of αi, we measured ligand-dependent binding of radioactive GTPγS in a mixture containing pure αi and βγ 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 GTPγS binding only in the presence of receptors αi and βγ (Figures 2B⇓ and 2D⇓). In urea-treated COS-7 membranes, Ang II increased GTPγS binding 3- to 10-fold in different experiments. At maximal stimulation, radioactive GTPγS bound to 10% to 30% of the total αi 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 GTPγS on αi. To explore the effects of urea treatment, we compared GTPγS 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 GTPγS binding in COS-7 and NIH3T3 P2 membranes, respectively. Coexpression of αi and βγ 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
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: GTPγS binding was saturable with increasing concentrations of αi while keeping the βγ concentrations fixed at 250 nmol/L (Figures 3A⇓ and 3B⇓). The K0.5 for αi was ≈24 nmol/L. Similarly, the GTPγS binding was saturable in the converse experiment, increasing the βγ concentrations at fixed concentrations of αi (Figure 3B⇓). In addition, the GTPγS 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
To test the specificity of αi activation, we examined the ability of AT2 receptors to activate three additional α subunits, α0, αq, and αs. Gα0 is a brain αi-like subunit. Gαq activates PLC, and αs is the stimulatory regulator of adenylyl cyclase. Both of these α subunits have no resemblance to αi in primary structure. The AT2 activated α0 and αi but failed to promote GTPγS binding on αq and αs (Figure 4B⇑). Two other serpentine receptors, PTHR and β2AR, activated αs, and the AT1 receptor activated αq, confirming the functionality of these added α 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 Gi, suggesting that AT2 receptors activate G proteins by similar mechanisms as other receptors.18 These experiments show that AT2 receptors discriminate between Gi, Gq, and Gs in this system. Another important question is whether the added αi might be activated by a serpentine receptor. We examined this using membranes expressing PTHR. The activated PTHR did not promote GDP exchange for GTPγS on αi (Figure 4B⇑). Thus, both the recombinant AT2 and the purified αi have retained specificity in this system.
AT2 Receptor Pharmacology
We examined the effect of different Ang II analogues on AT1 and AT2 receptor–induced αi 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⇓).
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.
We have set up a system to assay AT2 receptor activation of Gi 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 α subunit of Gi in a reconstituted system. In this system, the AT2 receptor activated αi in a manner dependent on the concentrations of ligand, receptor, and Gi subunits. These results suggest that the AT2 receptor activates Gi in much the same way as other receptors activate G proteins. In similar assays, rhodopsin activates transducin, and the gastrin-releasing peptide receptor activates Gq.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 GTPγS binding to purified Gq and Gs, two different G proteins, and vice versa, the Gi was not activated by PTHR.
Data from ligand binding studies, use of PTX, which inactivates αi, and coimmunoprecipitation experiments using anti–G-protein antibodies, support the inference that the AT2 receptor activates G proteins. First, GTPγS 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 αi 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 Gi and G0 but not Gq and Gs.
If the AT2 receptor activates Gi, why does it not inhibit cAMP production or robustly activate MAPK, effects attributed to activation of αi and liberation of βγ?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 Gαi with a low affinity or with a low rate of GDP-GTP exchange. Thus, only cells expressing high levels of AT2 and Gαi 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 Gαi 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 Gαi 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
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.
- © 2000 American Heart Association, Inc.
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