Published online before print June 5, 2006, doi: 10.1161/01.HYP.0000228997.88162.a8
(Hypertension. 2006;48:316.)
© 2006 American Heart Association, Inc.
Original Articles |
Angiotensin II Type 2 Receptor–Bradykinin B2 Receptor Functional Heterodimerization
From the Division of Endocrinology and Metabolism, Department of Medicine and W. M. Keck Center for Cell Imaging, University of Virginia, Charlottesville.
Correspondence to Helmy M. Siragy, PO Box 801409, University of Virginia Health System, Charlottesville, VA 22908-1409. E-mail hms7a{at}virginia.edu
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Abstract |
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Angiotensin II type 2 (AT2R) or bradykinin B2 (B2R) receptor activation enhances NO production. Recently, we demonstrated enhancement of NO production when AT2R and B2R are simultaneously activated in vivo. However, the mechanism involved in this enhancement is unknown. Using confocal fluorescence resonance energy transfer microscopy, we report the distance between the AT2R and B2R in PC12W cell membranes to be 50±5 Å, providing evidence and quantification of receptor heterodimerization as the mechanism for enhancing NO production. The rate of AT2R–B2R heterodimer formation is largely a function of the degree of AT2R–B2R expression. The physical association between the dimerized receptors initiates changes in intracellular phosphoprotein signaling activities leading to phosphorylation of c-Jun terminal kinase, phosphotyrosine phosphatase, inhibitory protein
B
, and activating transcription factor 2; dephosphorylation of p38 and p42/44 mitogen-activated protein kinase and signal transducer inhibitor of transcription 3; and enhancing production of NO and cGMP. Controlling the expression of AT2R–B2R, consequently influencing their biologically active dimerization, presents a potential therapeutic target for the treatment of hypertension and other cardiovascular and renal disorders.
Key Words: receptors, bradykinin • nitric oxide • angiotensin • bradykinin
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Introduction |
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The angiotensin II type 2 receptor (AT2R) mediates a vasodilator1–3 cascade that includes bradykinin (BK), NO, and cGMP. BK, the major effector hormone of the kallikrein–kinin system, acts mainly through the BK B2 receptor (B2R) to mediate most of its cardiovascular and renal actions. Our previous data demonstrated that, compared with individual receptor contributions, simultaneous activation of AT2R and B2R led to a 70% increase in NO production,1 suggesting interaction between these 2 receptors. However, the molecular mechanism involving the AT2R–B2R–NO pathway is unknown.
Receptor–receptor crosstalk is an essential process for plasma membrane–localized receptors, including those involving the superfamily of the 7 trans-membrane G protein–coupled receptors (GPCRs).4 It is well established that a variety of cell surface receptors interact with each other to form dimers and that this is essential for their activation. Recent studies provided evidence for the existence of GPCR homodimers and heterodimers.5 Heterodimerization has effects on ligand binding, receptor activation, desensitization, and trafficking, as well as receptor signaling different from those of the homodimer or oligomer.6,7 Heterodimerization provides a newly recognized means of modulation of receptor function, as well as cross-talk between GPCRs. We hypothesized that AT2R and B2R heterodimerize to enhance NO production. In this study, fluorescence resonance energy transfer (FRET) microscopy was used to measure the molecular proximity between AT2R and B2R in PC12W in vivo and to provide evidence for and quantification of receptor heterodimerization. To evaluate the regulation of this dimer unit, we incubated PC12W cells with agonists and/or antagonists to either AT2R or B2R. Immunoaffinity chromatography followed by immunoblotting detection was used to quantify the changes in heterodimer formation. To demonstrate functional consequences of AT2R–B2R heterodimerization, we monitored changes in intracellular phosphoprotein signaling activities that have been linked previously to classical functions of AT2R and B2R and NO and cGMP production in response to different pharmacological agents.
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Methods |
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Cell Culture
PC12W cells (rat pheochromocytoma cells) were obtained from the American Type Culture Collection, cultured in DMEM (GIBCO) with 10% horse serum, 5% FCS, 100 U/mL penicillin, and 1 mg/mL streptomycin in a humidified atmosphere of 95% air and 5% CO2.
Laser Scanning Confocal FRET Microscopy and Image Analysis
For the protein localization using FRET microscopy techniques, we used the laser scanning confocal FRET (C-FRET) microscopy.7,8 We configured the Biorad Radiance 2100 laser scanning confocal/multiphoton microscopy system as a C-FRET system as described.9 The system consists of a Nikon TE300 epifluorescent microscope, a Plan Fluor 100X NA 1.4 oil immersion objective lens, argon ion laser (457,488,514), HeNe 543 nm, and 633 nm (www.cellscience.bio-rad. com). This has 3 fluorescence and 1 transmission channels. Nondescanned detectors, which can be used for multiphoton microscopy imaging, were used for both C-FRET and 2-photon excitation fluorescence resonance energy transfer (2p-FRET) image acquisition. For C-FRET, an Argon laser emitting at 488 nm was used to excite the donor fluorophore, whereas a HeNe Green laser emitting at 543 nm was used to excite the acceptor fluorophore. Emissions from the fluorophores were split using a 560-nm dichroic mirror and filtered using an HQ528/30 nm filter for the donor emission channel and an HQ590/70 nm filter for the acceptor channel (www.chromatech.com). The images from donor and acceptor channels were acquired for single-labeled (control) cells and double-labeled cells for further FRET data processing as described in the literature.10–14The anti-receptor antibodies for AT2R or B2R reacted specifically with either the AT2R15–17 or the B2R,18 respectively. To confirm the absence of cross-reactivity of these receptor antibodies, we studied Chinese hamster ovary cells transfected with AT2R or B2R. Using a PIERCE immunoaffinity matrix with subsequent immunoblot detection, we were able to detect the AT2R but not the B2R with the AT2R antibody and the B2R but not the AT2R with B2R antibody (data not shown) in Chinese hamster ovary cells transfected with AT2R or B2R, respectively. It is noteworthy to state that there were no immunoblots detected at 110 Kd, suggesting an absence of dimerization artifacts.
Serum-starved PC12W cells expressing both AT2R and B2R (in the absence of the AT1 receptor) were used. We determined AT2R and B2R protein levels in PC12W cells. AT2R and B2R antibodies were labeled with Alexa fluorophores 488 and 555 to demonstrate colocalization and interaction between AT2R and B2R using C-FRET microscopy as an accurate measure of molecular proximity at angstrom distances (10 to 100 Å) with higher spatial resolution beyond the limits of conventional microscopy.
Functional Assays
Bio-Plex Phosphoprotein Cellular Signaling Assays
Bead-based multiplex Luminex xMAP technology assays (Bio-Rad Laboratories) that directly detect phosphorylated proteins, c-Jun terminal kinase (JNK), phosphotyrosine phosphatase (PTP), extracellular signal–regulated kinases (1 and 2), p38 mitogen–activated protein kinases (MAPKs), activating transcription factor 2 (ATF2), signal transducer and activator of transcription 3 (STAT3), and inhibitory protein B (IB), were used in lysates derived from cell culture using highly specific antibodies exclusively developed and validated by Cell Signaling Technology, Inc. PC12W cells were incubated with AT2R and B2R agonists or antagonists individually or combined for 24 hours. The cells were then lysed and centrifuged. Using these 96-well plate-format assays, we profiled the specific phosphorylation state of multiple proteins simultaneously in a single sample. Data from the reaction were acquired using the Bio-Plex suspension array system, a dual-laser, flow-based microplate reader system that can discriminate 
100 different bead based assays. The contents of the wells are drawn up into the reader.
Protein Tyrosine Phosphatase
PC12W cells were incubated with AT2R and B2R agonists or antagonists individually or combined for 24 hours. The cells were then lysed and centrifuged. Protein tyrosine phosphatase activity was measured in the supernatant by using an assay kit (Takara).19 The sensitivity is 0.125x10–5 units/µL, and the specificity is 100%.
Coimmunoprecipitation and Immunoblotting of AT2R and B2R
PC12W cell groups were divided into 2 sets. The first set of cells was treated with agonists and/or antagonists to the AT2R and/or B2R for 30 minutes to test the effect of receptor activation on heterodimer formation. The second set of cells was treated with agonists and/or antagonists to the AT2R and/or B2R for 24 hours to test the effect of altering the expression of these receptors on heterodimer formation. After washing 3 times with PBS, immunoaffinity chromatography was used by anti-B2R antibodies or by anti-AT2R antibodies using a PIERCE immunoaffinity matrix. Anti-AT2R and anti-B2R antibodies raised against the third and second extracellular loops, respectively, were used for immunoaffinity purification and subsequent immunoblot detection. Proteins were dissolved and separated by 10% SDS-PAGE under reducing conditions. B2R and AT2R were identified in immunoblot using anti-receptor antibodies.
cGMP and NO Measurements
Nitrate/nitrite and cGMP levels in cell lysate samples were measured by using an enzyme immunoassay kit.1 The sensitivity was 2.0 µmol/L and 0.09 pmol/mL for nitrate/nitrite1 and cGMP,3 respectively, and the specificity was 100% for both. The intra-assay and interassay cross-reactivity with other cyclic nucleotides was <0.01%.
Statistical Analysis
Data are expressed as mean±SE. Differences between mean values of agonists and/or antagonists to the AT2R and/or B2R and vehicle were analyzed by ANOVA, with a subsequent Tukey honestly significant difference multiple-comparisons test. Differences of P<0.05 were considered significant.
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Results |
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C-FRET Microscopy Study
We characterized the cell membrane site-specific distribution of AT2R (Figure 1A) and B2R (Figure 1B) proteins. Our results demonstrated that the average distance between AT2R–B2R-dimerized receptors is 50±5 Å, allowing the signal to be transferred from the excited molecular donor fluorophore to the acceptor fluorophore by means of intermolecular long-range dipole–dipole coupling (Figure 1C), thus confirming their physical association (Figure 1D and 1E). AT2R–B2R heterodimers were detected both on the cell membrane, as well as intracellularly mainly in the perinuclear region.
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Coimmunoprecipitations and Immunoblotting
AT2R and B2R monomers and dimers were detected by their specific antibodies. In the 110 Kd dimer band, both AT2R and B2R were detected in the same blot. Treatment with agonists and/or antagonists to either AT2R or B2R for 30 minutes to influence the receptor activation state without changing the receptor expression level did not affect the level of heterodimer formation (data not shown).
The percentage change in AT2R and B2R expression and dimerization after administration of different pharmacological agents for 24 hours (Figure 2A through 2C) is shown in Table 1. Our results demonstrate that an increase in the expression of AT2R and/or B2R was consistently accompanied by an increase in heterodimer formation. The maximum increase in AT2R–B2R heterodimer formation was observed as a result of combined treatment of an AT2R agonist and a B2R antagonist, each individually increasing both receptors’ expression and inducing a 250% increase in heterodimer formation.
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Regulation of Activity of JNK, p42/44MAPK, p38MAPK, ATF2, STAT3, IB, and PTP in Response to Changes of Dimer Formation
Simultaneous measurement (Bio-Plex, Bio-Rad Laboratories) of JNK, p38MAPK, p42/44MAPK, STAT3, IB, ATF2, and PTP in response to manipulation of AT2R and B2R demonstrated that the receptor heterodimers are functional (Figure 3). Our data (Table 2) demonstrate that pharmacological agents producing a maximum number of AT2R–B2R heterodimers selectively led to phosphorylation of JNK, PTP, IB, and ATF2 and dephosphorylation of p38 and p42/44 MAPK and STAT3.
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) or CGP42112A (CGP, 100 nmol, 
) individually or combined (
) for 24 hours. The cells were then lysed and centrifuged. The specific phosphorylation state of multiple proteins was measured simultaneously in the same treatment sample. Data acquired using the Bio-Plex suspension array system, a dual-laser, and flow-based microplate reader system. Bar graph represents percentage change from control. Data shown are average of 3 experiments.
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Intracellular NO and cGMP Production in Response to Changes of Dimer Formation
As shown in Figure 4, both AT2R and B2R agonists individually and combined increased NO and cGMP levels, whereas their respective antagonists decreased NO and cGMP. AT2R agonist CGP 42112A (Ciba Geigy) and B2R agonist BK increased NO and cGMP from (3.461±0.25 µmol/L, 41.17+3.25 pg/mL) at baseline to (9.321±1.25 µmol/L; P<0.05, 69.5±5.7pg/mL; P<0.05) and (7.46±0.5 µmol/L; P<0.05, 55.6+1.8pg/mL; P<0.05). Antagonists to the AT2R (PD123319 [PD]) and B2R (icatibant) had no effect on the basal production of NO and cGMP. The combination of AT2R antagonist with either the B2R agonist or antagonist did not change the level of NO or cGMP as compared with the basal production level. In contrast, the addition of the AT2R agonist CGP to either the B2R agonist or antagonist increased NO production. CGP and BK combination increased NO and cGMP (9.64±0.80 µmol/L; P<0.05, 77.3± 2.6 pg/mL; P<0.001). The maximum increase in NO and cGMP production (12.4±1.7 µmol/L; P<0.001, 90.9±6.8; P<0.001) was persistently associated with the combination of AT2R stimulation and B2R inhibition that also led to a maximum increase in AT2R–B2R heterodimer formation with AT2R agonist CGP and B2R antagonist icatibant, respectively.
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Discussion |
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In this study, we report the formation of a stable functional AT2R–B2R heterodimer unit. The distance between the third extracellular loop in the AT2R and the second extracellular loop in the B2R is 50±5 Å, which indicates the close molecular proximity between AT2R and B2R, providing evidence of receptor heterodimerization as the mechanism to enhance NO production. This result was supported and strengthened by the coimmunoprecipitation results, indicating that active high-affinity AT2R and B2R heterodimers were stable with no preceding cross-linking, which demonstrates the strength of the physical association between the 2 dimerized receptors. The C-FRET data demonstrated the physical association between the 2 dimerized receptors without any stimulation and suggests the presence of constitutive heterodimers. It is possible that homodimers or even heterotetramers of AT2R, B2R, and angiotensin II type 1 (AT1R) are formed. Because the aim of the current study was to prove the presence AT2R and B2R heterodimers, such possibilities were not studied. In addition, C-FRET demonstrated that AT2R–B2R heterodimers were detected at the plasma membrane and in the perinuclear region. This is in agreement with a recent report of AT2R internalization.20 Previous studies suggested formation of dimers in the endoplasmic reticulum.21,22 Our study suggests active transport of the bound antibody–dimer complex from the cell surface to the intracellular compartment, because the cells were not permeabilized, and the fluorescent-labeled antibody is unable to penetrate the cell membrane.
The rate of formation of the AT2R–B2R heterodimer seems to be regulated in large part by the status of AT2R and B2R expression. Treatment with agonists and/or antagonists to either AT2R or B2R for 30 minutes to influence the receptor activity state without changing the receptor expression level did not affect the level of heterodimer formation, suggesting that receptor activation does not contribute principally to the level of heterodimer formation. Our results demonstrate that an increase in the expression of AT2 and/or B2 receptors was consistently accompanied by an increase in heterodimer formation after administration of different pharmacological agents for 24 hours. It is noted that pharmacological agents that increased 1 type of receptor similarly affected the expression or dimer formation of the other receptor, or both, at different levels. This finding is consistent with recent studies examining the effects angiotensin II on the regulation of B2 receptors.23 The reverse was also observed between AT1R and kallikrein gene therapy.24
The maximum increase in AT2R–B2R heterodimer formation was observed as a result of combined treatment of an AT2R agonist and a B2R antagonist, each individually increasing the expression of both receptors and inducing a 250% increase in heterodimer formation. It is possible that the increase in dimer formation with PD treatment could be because of increased conversion of monomer to dimer. However, the monomer did not increase with PD treatment. Furthermore, we observed a decrease in B2R dimer and monomer in response to PD. Although B2R blockade substantially increased the AT2R and B2R expression, it only slightly increased AT2R–B2R heterodimer formation. At present, the mechanisms underlying the difference between B2R antagonist versus AT2R agonist with or without B2R antagonist on dimer formation observed in the current study are unknown. It is noteworthy that AT2R stimulation was always associated with increased dimer formation. Thus, factors that influence AT2R expression and activity may play a more important role in dimer formation than factors affecting B2R expression. In the present study, we could not determine whether all dimers present in vivo are coprecipitated in vitro. However, it is unlikely that coimmunoprecipitation promoted the partial dissociation of dimers, because all of the cell groups were exposed to same experimental conditions. Dimers were still visible under reducing conditions and boiling in SDS buffer, demonstrating the strength of physical association between the 2 dimerized receptors. The combination of FRET with immunoprecipitation studies provides an additional evidence for dimerization.
To demonstrate functional consequences of AT2R–B2R heterodimerization, we monitored changes in intracellular phosphoprotein signaling activities that have been linked previously to classical functions of AT2R and B2R, such as NO production, antiproliferative properties, and apoptosis. Simultaneous measurement of JNK, MAPK, p38 and p42/44 MAPK, STAT3, IB, ATF2, and PTP in response to manipulation of AT2R and B2R demonstrated that the receptor heterodimers are functional. Our data demonstrated that pharmacological agents producing the maximum number of AT2R–B2R heterodimers selectively led to phosphorylation of JNK, PTP, IB, and ATF2 and dephosphorylation of p38 and p42/44 MAPK and STAT3. Taken together, these receptor manipulations were associated with a 258% and 80% increase in NO and cGMP production, respectively, in agreement with recent studies on the role of these signaling pathways in the production of NO.24–37 JNK and ATF2 phosphorylation were highly associated with NO production when the effects of other pathways were statistically excluded (partial correlation coefficient: 0.08 and 0.31, respectively). Similarly, dephosphorylation of STAT3 was associated with NO production. It is noted that the correlation of JNK with NO production remained constant in presence or absence of contributions from other pathways, suggesting little correlation between JNK and other signaling molecules studied. This represents the first study to our knowledge to conduct an extensive signaling pathway analysis comparing molecules mediated by AT2R and B2R to influence NO production.
The maximum increase in NO and cGMP production was persistently associated with the combination of AT2R stimulation and B2R inhibition that also led to maximum increase in AT2R–B2R heterodimer formation with CGP and icatibant, respectively. This is in agreement with a recent report that B2R blockade further increased, rather than decreased, the effect of activated AT2R on cGMP and NO production.38 Both B2R agonist and antagonist were observed to have a similar influence on NO production when combined with AT2R agonist. However, the mechanism of NO production because of B2R agonist is different from that of the antagonist. Icatibant and CGP combined administration increased the expression of both B2R and AT2R. When the B2R is blocked by icatibant, unopposed stimulation of the upregulated and dimerized AT2R via CGP is facilitated, driving NO production via the AT2R. Similarly, both B2R and AT2R are involved in the production of NO in the BK+CGP combined treatment group. However, the negative influence of BK on AT2R and B2R expression and dimer formation reduced the production level of NO as compared with the icatibant+CGP–treated group.
In conclusion, for functional enhancement of NO production, receptors must first be expressed. Dimerization occurs as a function of receptor number but also requires AT2R activation. This explains why icatibant alone increases B2R and AT2R expression but neither dimer formation nor NO or cGMP production.
Perspectives
In the light of our present study and recent reports of the heterodimerization between AT1R–AT2R39 and AT1R–B2R,40–42 it is clear that each of these receptors may heterodimerize with 1 of the 2 other receptors. Determination of which receptors should form a heterodimer likely depends on the distribution and availability of the individual receptors. Selective induction of the AT2R and B2R expression and dimer formation presents a new therapeutic intervention target for the treatment of hypertension and other cardiovascular and renal disorders.
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Acknowledgments |
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Sources of Funding
This study was supported by grants DK-61400 and HL-57503 to H.M.S. and HL-65659 to R.M.C. from the National Institutes of Health. H.M.S. was the recipient of Research Career Development Award K04-HL-03006 from the National Institutes of Health.
Disclosures
None.
Received January 12, 2006; first decision January 30, 2006; accepted May 16, 2006.
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