Review article

Regulation of angiotensin II receptors beyond the classical pathway

Masatsugu Horiuchi, Jun Iwanami, Masaki Mogi


The RAS (renin–angiotensin system) plays a role not only in the cardiovascular system, including blood pressure regulation, but also in the central nervous system. AngII (angiotensin II) binds two major receptors: the AT1 receptor (AngII type 1 receptor) and AT2 receptor (AngII type 2 receptor). It has been recognized that AT2 receptor activation not only opposes AT1 receptor actions, but also has unique effects beyond inhibitory cross-talk with AT1 receptor signalling. Novel pathways beyond the classical actions of RAS, the ACE (angiotensin-converting enzyme)/AngII/AT1 receptor axis, have been highlighted: the ACE2/Ang-(1–7) [angiotensin-(1–7)]/Mas receptor axis as a new opposing axis against the ACE/AngII/AT1 receptor axis, novel AngII-receptor-interacting proteins and various AngII-receptor-activation mechanisms including dimer formation. ATRAP (AT1-receptor-associated protein) and ATIP (AT2-receptor-interacting protein) are well-characterized AngII-receptor-associated proteins. These proteins could regulate the functions of AngII receptors and thereby influence various pathophysiological states. Moreover, the possible cross-talk between PPAR (peroxisome-proliferator-activated receptor)-γ and AngII receptor subtypes is an intriguing issue to be addressed in order to understand the roles of RAS in the metabolic syndrome, and interestingly some ARBs (AT1-receptor blockers) have been reported to have an AT1-receptor-blocking action with a partial PPAR-γ agonistic effect. These emerging concepts concerning the regulation of AngII receptors are discussed in the present review.

  • angiotensin II receptor
  • angiotensin-receptor-interacting protein
  • angiotensin II type 1 receptor blocker
  • cardiovascular disease
  • peroxisome-proliferator-activated receptor
  • renin–angiotensin system


The RAS (renin–angiotensin system) plays a role not only in the cardiovascular system, including BP (blood pressure) regulation, but also in the central nervous system [1]. AngII (angiotensin II) binds two major receptors: the AT1 receptor (AngII type 1 receptor) and AT2 receptor (AngII type 2 receptor). The majority of well-known AngII actions are mediated via AT1 receptor stimulation, and ARBs (AT1 receptor blockers) are widely used as antihypertensive drugs, with the expectation of inhibition of the onset and progression of cardiovascular disease, and AT2 receptor stimulation by unbound AngII could also be expected during treatment with ARBs. There is recent accumulating evidence suggesting that the AT2 receptor not only opposes the AT1 receptor, but also has unique effects beyond an interaction with AT1 receptor signalling. These results point to the pathophysiological importance of the AT2 receptor in the clinical application of ARBs [2]. From this point of view, AT2 receptor agonists, such as compound 21, have been newly developed and are expected to be useful agents for improving pathological disorders [3]. Synthesis of this compound also enables one to examine AT2 receptor actions in vitro and in vivo by direct receptor stimulation, and principally offers the possibility to use AT2 receptor stimulation as a therapeutic tool [4].

Experimental studies have also demonstrated the existence of novel pathways beyond the classical actions of RAS. For example, studies have established a new regulatory axis in RAS and, in this axis, Ang-(1–7) [angiotensin-(1–7)] is finally produced from AngI (angiotensin I) or AngII by the catalytic activity of ACE (angiotensin-converting enzyme) 2. The discovery that Ang-(1–7) opposes the pressor, proliferative, profibrotic and prothrombotic actions mediated by AngII via the AT1 receptor has contributed to the realization that the RAS is composed of two opposing arms [5,6]. A new axis of RAS, the ACE2/Ang-(1–7)/Mas axis, has been highlighted as the counteracting partner of the ACE/AngII/AT1 receptor [7]. There are an increasing number of reports on the role of ACE2, Ang-(1–7) and Mas in the cardiovascular system. Moreover, it is reported that Ang-(1–7) can act as an endogenous ligand with AT2 receptor selectivity over the AT1 receptor [8]. These results support the concept that the interruption of cross-talk of various AngII receptors could determine and orient pathological states, resulting in the onset of cardiovascular diseases (Figure 1). Moreover, possible proteins interacting with AngII receptors have been screened and their functions revealed [9]. Experimental studies have also demonstrated other possible mechanisms regulating RAS, such as mechanical-stress-induced activation of the AT1 receptor, modification of AngII receptors involving dimerization and auto-antibodies against the AT1 receptor.

Figure 1 New regulatory axis in the RAS

AngII binds two major receptors, AT1 receptor and AT2 receptor, and AT2 receptor activation antagonizes AT1 receptor actions. The ACE2/Ang-(1–7)/Mas axis acts as a counteracting partner of the ACE/AngII/AT1 receptor. Interruption of the cross-talk of the various AngII receptors could determine and orientate pathological states, resulting in the onset of cardiovascular disease. DM, diabetes mellitus

AngII and PPAR (peroxisome-proliferator-activated receptor)-γ play an important role in not only the pathogenesis of insulin resistance, but also in vascular lesion formation. Consistent with this, we have reported that AT1 receptor deletion in ApoEKO (apolipoprotein E-knockout) mice increased PPAR-γ expression in adipose tissue, with adipocyte differentiation and improvement of insulin resistance, whereas AT2 receptor deletion in ApoEKO mice decreased PPAR-γ expression [10,11]. Some ARBs, such as telmisartan and irbesartan, have been reported to have an AT1-receptor-blocking action, with a partial PPAR-γ agonistic effect [12,13], suggesting that the possible cross-talk between PPAR-γ and AngII receptor subtypes is an intriguing issue to be addressed in order to understand the roles of RAS in the metabolic syndrome. In the present review, we have summarized these emerging concepts concerning the regulation of the functions of AngII receptors, mainly focusing on AngII-receptor-associated proteins and the interaction of AngII receptors and PPAR-γ, and discuss new potent therapeutic targets in cardiovascular and cerebrovascular disease.


AngII produced from AngI by ACE is a strong bioactive substance. AngII is a well-known bioactive substance in the regulation of BP (blood pressure) and is involved in the exaggeration of cardiovascular disease [1]. The AT1 receptor was cloned in 1991 and this finding accelerated the basic and clinical research into the classical RAS. AT1 receptor stimulation mediates the classical major actions of AngII, and the distribution of AT1 receptor covers most organs. Stimulation of AT1 receptors induces various signalling mechanisms. For example, AT1 receptor stimulation increases the influx of extracellular Ca2+, and mobilization of intracellular Ca2+ activates acute responses, such as vascular smooth muscle contraction, and also activates various kinase pathways to induce cell proliferation and hypertrophy signalling. AT1 receptor stimulation is well known to induce hypertension, stroke, cardiovascular events and renal diseases. These findings are also closely related to the development of RAS inhibitors, such as ACE inhibitors and ARBs, promoting the basic and clinical research in the RAS. Accordingly, large clinical trials and basic research have established the important roles of the ACE/AngII/AT1 receptor axis in the cardiovascular system, including BP regulation.

Studies have also indicated that AngII plays a role in the onset of diabetes and metabolic disorders. Moreover, clinical trials have demonstrated that blockade of RAS by an ACE inhibitor or ARB could result in a reduction in the onset of stroke, probably independent of its BP-lowering effect, and possible beneficial effects of RAS blockade on cognitive function are also becoming highlighted in the clinical field [2]. AT2 receptor stimulation appears to antagonize the signalling activated by AT1 receptor stimulation in various tissues, since the signalling mediated by AT2 receptor stimulation is transferred mainly by phosphatases and NO production. Therefore the action of AT2 receptor stimulation would be antagonistic to AT1-receptor-mediated signalling. The binding affinity of AngII for the AT2 receptor does not differ from that for the AT1 receptor. When the AT1 receptor is blocked and unbound, AngII can act on the AT2 receptor, stimulation of AT2 receptor might be involved in the effects of ARBs. Therefore it is conceivable that AT2 receptor stimulation could contribute to the effects of ARBs in various aspects.


AngII mediates various effects through complex signalling pathways on binding to its GPCRs (G-protein-coupled receptors), the AT1 receptor and AT2 receptor. These receptors are regulated by GPCR-interacting proteins such as ATRAP (AT1-receptor-associated protein), ARAP1 and ATIP (AT2-receptor-interacting protein) [14].

AT1-receptor-associated proteins

Previous studies have shown that the C-terminal cytoplasmic domain of the AT1 receptor directly associates with its downstream effectors, including Jak2 (Janus kinase 2), SHP (Src homology 2 domain-containing protein tyrosine phosphatase)-2, PLCγ1 (phospholipase Cγ1), eNOS (endothelial NO synthase) and RhoA, and is involved in AT1 receptor internalization, indicating the functional importance of the C-terminal cytoplasmic domain in AT1 receptor signalling. We have cloned ATRAP using a yeast two-hybrid screening system [15]. ATRAP has three transmembrane domains and interacts with the intracellular C-terminal domain of the AT1 receptor, but it does not interact with the AT2 receptor, m3 muscarinic receptor, bradykinin B2 receptor, endothelin ETB receptor or β2-adrenergic receptor. ATRAP is expressed in various tissues, such as the aorta, heart and lung, and especially the kidney, and co-localizes with the AT1 receptor in the mouse renal tubules [16,17]. ATRAP is reported to enhance AT1 receptor internalization with a consequent reduction in AT1 receptor signalling via a specific interaction with the C-terminal cytoplasmic domain of the AT1 receptor [18,19]. However, it is possible that ATRAP could directly influence various AT1-receptor-mediated signalling events, and it has been reported that, in the calcineurin/NFAT (nuclear factor of activated T-cells) signalling pathway, ATRAP partners with the N-terminus of CAML (calcium-modulating cyclophilin ligand) to negatively regulate AngII-induced NFAT activation, thereby leading to down-regulation of cell signalling in cardiac hypertrophy [20]. This negative calcineurin/NFAT regulation by ATRAP has been proposed, at least in part, to be independent of AT1 receptor signalling associated with AT1 receptor internalization. Consistent with this result, we have demonstrated that ATRAP negatively regulated VSMC (vascular smooth muscle cell) senescence by reducing AT1 receptor signalling, and that ATRAP-mediated inactivation of the calcineurin/NFAT pathway could be, at least partly, involved in prevention of VSMC senescence, irrespective of AT1 receptor blockade in some conditions [21]. To examine further the pathophysiological roles of ATRAP, we generated ATRAP-Tg (transgenic) mice, which exhibit decreases in neointimal formation, inflammatory response and NADPH oxidase activity involving a membrane-associated NADPH oxidase subunit, p22phox in the injured artery compared with those in wild-type mice [16]. Moreover, in ATRAP-Tg mice, the increase in heart/body weight ratio after treatment with AngII infusion or aortic banding was smaller than that in wild-type mice. These results indicate that ATRAP plays an inhibitory role in cardiovascular remodelling via the regulation of AT1 receptor signalling. Oppermann et al. [22] generated ATRAP-deficient (ATRAP−/−) mice and observed that they showed increased arterial pressure and plasma volume, suggesting that ATRAP probably modulated volume status by acting as a negative regulator of AT1 receptors in the renal tubules. Masuda et al. [23] investigated the expression and distribution of human ATRAP in the normal kidney and renal biopsy specimens from patients with IgA nephropathy, and suggested that ATRAP might play a role in balancing the renal RAS synergistically with the AT1 receptor by counter-regulatory effects in IgA nephropathy, and proposed an antagonistic effect of tubular ATRAP on AT1 receptor signalling. An increase in recycling of the AT1 receptor may have an influence on the therapeutic benefit of ARBs, and ATRAP could be a physiological negative regulator of AT1 receptor signalling. Therefore activation of ATRAP may be more specific and more physiological for inhibition of AT1 receptor signalling [9].

In addition to ATRAP, Inagami and co-workers [24] cloned another AT1-receptor-associated protein, named ARAP1, using a yeast two-hybrid screening approach, and reported that ARAP1 promoted recycling of AT1 receptors to the plasma membrane in HEK (human embryonic kidney)-293 cells, indicating a presumed role in concomitant recovery of receptor signal functions. They reported that kidney-specific ARAP1-Tg mice exhibit hypertension and renal hypertrophy with a decrease in urine volume [25]. Therefore their findings showed that proximal-tubule-specific overexpression of ARAP1 leads to hypertension, suggesting that renal ARAP1 plays an important role in the regulation of BP and renal function via activation of the intrarenal RAS. ARAP may have diverse functions compared with those of ATRAP, with an opposite effect on the trafficking of AT1 receptors. Cook et al. [26] screened a yeast two-hybrid mouse brain library with the rat AT1 receptor C-terminus and identified GABARAP [GABA (γ-aminobutyric acid)-receptor-associated protein], a protein involved in intracellular trafficking of the GABAA receptor, as a binding partner for the AT1 receptor and concluded that GABARAP binds to and promotes trafficking of the AT1 receptor to the plasma membrane. Possible pathophysiological roles of various AT1-receptor-associated proteins are shown in Figure 2. Further investigations are necessary to assess the regulation of these various AT1-receptor-associated proteins in physiological homoeostasis, as well as in pathological conditions.

Figure 2 Possible roles of ATAP, ARAP1 and GABARAP as proteins interacting with the C-terminal tail of the AT1 receptor

ATIP could inhibit AT1-receptor-mediated growth-promoting signals, whereas ARAP1 and GABARAP could enhance recycling and trafficking of the AT1 receptor. MAPK, mitogen-activated protein kinase; PLC, phospholipase C; STAT, signal transducer and activator of transcription.

AT2-receptor-associated proteins

The AT2 receptor does not couple in a typical manner to classical heterotrimeric G-proteins and elicits unusual signalling cascades involving the activation of protein phosphatases, inhibition of protein kinases and RhoA GTPase, and/or production of NO. The recent development of selective non-peptidic AT2 receptor agonists has provided new important tools for further evaluation of the roles of AT2 receptor stimulation in pathophysiological conditions and should relaunch interest in studying the intracellular effects and regulation of this receptor in various tissues [2729].

Yeast two-hybrid screens performed by different groups have identified several intracellular AT2-receptor-interacting proteins, such as ErbB3 [30], PLZF (promyelocytic leukaemia zinc finger) [31], CNK1 (connector enhancer of kinase suppressor of ras 1) [32], the Na+/H+ exchanger NHE6 [33], TIMP-3 (tissue inhibitor of metalloproteinases-3) [34] and ATIP/ATBP (AT2-receptor-binding protein) [35,36], which interact with the same intracellular C-terminal region of the AT2 receptor. The presence of proteins interacting with the AT2 receptor has been highlighted as factors regulating this unique receptor, suggesting the possibility that AT2-receptor-signalling-interacting proteins play key roles in these diverse mechanisms of AT2 receptor signalling. Accordingly, we have cloned ATIP as a protein interacting with the C-terminal tail of the AT2 receptor using a yeast two-hybrid system [35]. It has been shown to co-operate with the AT2 receptor to transinactivate receptor tyrosine kinases independent of G-proteins, and, in Chinese-hamster ovary cells expressing the human AT2 receptor, ATIP inhibits growth-factor-induced ERK (extracellular-signal-regulated kinase) 2 activation and DNA synthesis, and attenuates insulin receptor autophosphorylation in the same way as the AT2 receptor. In contrast, ATBP50 (ATBP of 50 kDa), which is identical with ATIP, has been reported by Wruck et al. [36] to potentially act as a membrane-associated Golgi protein that dictates the delivery of the AT2 receptor to the cell surface.

ATIP was found to be identical with a ubiquitously expressed tumour suppressor protein localized in mitochondria [37]. ATIP is also named MTUS1 (mitochondrial tumour suppressor gene 1), for which mutation or copy number variants are found in human malignant tumours [3739]. ATIP has three major transcripts: ATIP1, ATIP3 and ATIP4 [40]. ATIP3 is the major transcript in tissues; however, ATIP1 and ATIP4 are mainly expressed in the brain, indicating that ATIP plays biological roles in brain function. Moreover, recent in vivo studies indicate that AT2 receptor expression and/or agonist-induced activation could indeed attenuate tumour growth, vascularization and/or metastasis progression in different models of cancer. In this context, it should be noted that most AT2-receptor-binding proteins identified to date, including ErbB3, CNK1, PLZF, TIMP-3 and ATIPs, are related to cancer suppression [27].

We have reported that neointimal formation and the proliferation of VSMCs induced by polyethylene cuff placement on the femoral artery were greater in AT2-receptor-null mice than in wild-type mice [41], indicating that AT2 receptor stimulation attenuates vascular remodelling. Furthermore, AT2 receptor deletion from ApoEKO mice enhances atherosclerosis mainly through the inhibition of oxidative stress, indicating that AT2 receptor stimulation also attenuates atherosclerosis [42]. We have also demonstrated that AT2 receptor mRNA expression was increased in injured femoral arteries and ATIP mRNA expression was up-regulated after cuff placement, suggesting that interaction of the AT2 receptor and ATIP plays a role in vascular remodelling. Therefore we have investigated the vascular effects of ATIP1 in vivo using Tg mice overexpressing ATIP1 and have observed that the neointimal formation of the femoral artery after cuff placement was significantly smaller than that in wild-type mice, with decreases in superoxide anion production and the expression of pro-inflammatory cytokines [43].

On the other hand, the AT2 receptor is also reported to interact with a transcription factor, PLZF, in yeast two-hybrid studies. In cardiomyocytes, PLZF enhances protein synthesis and induces cardiac hypertrophy [31]. This apparent controversy between ATIP1-mediated anti-proliferative effects and PLZF-related trophic effects of the AT2 receptor in cardiac tissue could be explained by the difference in preferential coupling of the AT2 receptor to either ATIP1 or PLZF in different pathological conditions or the possible involvement of other AT2-receptor-binding molecules. However, AT2 receptor activation is well known to directly oppose the effects mediated by the AT1 receptor that enhance cardiac hypertrophy. Moreover, large clinical trials support the notion that ARB treatment, which causes relative stimulation of the AT2 receptor, could prevent cardiac hypertrophy and heart failure. Most studies addressing the involvement of the AT2 receptor in left ventricular hypertrophy have been performed in genetically altered mice, either AT2-receptor-deficient or AT2-receptor-overexpressing mice, and unfortunately this experimental approach has yielded highly controversial results. However, interestingly, the results of in vivo studies in wild-type animals using the AT2 receptor antagonist PD123319 are less controversial and have mainly revealed anti-growth effects of the AT2 receptor; therefore it can be expected that the novel non-peptide AT2 receptor agonist compound 21 will allow the effects of the AT2 receptor in cardiac hypertrophy to be studied by direct selective AT2 receptor stimulation, a novel in vivo approach which will hopefully help to overcome current controversies [44]. Consistent with this, Unger and co-workers [45] have reported that treatment with compound 21 significantly improved systolic and diastolic ventricular function in the rat after myocardial infarction, suggesting that direct AT2 receptor stimulation may be a novel therapeutic approach to improve systolic and diastolic function after myocardial infarction through anti-apoptotic and anti-inflammatory mechanisms. A human (pro)renin receptor has been cloned [46] that might also represent a novel pharmacological target. It has been proposed that there is also a putative regulatory interplay between the AT2 receptor and the (pro)renin receptor, since both share the adapter protein PLZF, and this interaction might inhibit the detrimental cardiovascular effect of (pro)renin receptor activation [29].

SHP-1 is one of the tyrosine phosphatases activated by AT2 receptor stimulation and is differentially phosphorylated, and SHP-1 is also a pivotal effector in the signal transduction pathway of the AT2 receptor in VSMCs [47]. We have demonstrated previously that SHP-1 binds with the intermediate portion of the intracellular third loop of the AT2 receptor and is an upstream effector of the AT2 receptor [48]. Interestingly, AT2-receptor-mediated SHP-1 activation is independent of G-protein activation [49], indicating that SHP-1 also acts as an inhibitory factor in pathological vascular remodelling beyond the effects of a typical GPCR as well as ATIP. Therefore we have examined the possible interaction of SHP-1 and ATIP [50]. We observed that, after AT2 receptor stimulation in rat neural cells, ATIP and SHP-1 were translocated into the nucleus after formation of their complex, resulting in the transactivation of MMS2 (methyl methanesulfonate-sensitive 2), a ubiquitin ligase variant involved in neuronal differentiation, expression and mediating the effects of the inhibitor of DNA-binding 1 proteolysis, with promotion of neural differentiation and repair (Figure 3). We have demonstrated that AT2 receptor signalling attenuates DNA damage and consequent vascular senescence, at least in part, through MMS2 transactivation [51]. These results provide a new insight into the contribution of AT2 receptor stimulation to neural differentiation via transactivation of MMS2 expression involving the association of ATIP and SHP-1. Therefore ATIP could be a more specific downstream target of the AT2 receptor and lead not only to enhance neural differentiation, but also to suppress tumour progression. However, it is still not clear whether AT2 receptor stimulation enhances the binding of ATIP and PLZF to the AT2 receptor, and whether ATIP and PLZF compete for AT2 receptor binding. Moreover, there is no evidence to support the notion that compound 21 actually utilizes AT2 receptor signalling through ATIP and or PLZF. These important issues have to be addressed to facilitate the more detailed functional aspects of AT2-receptor-associated proteins.

Figure 3 Possible roles of ATIP as a protein interacting with the C-terminal tail of the AT2 receptor

ATIP could enhance neural differentiation and protect neural cells from damage, improve vascular remodelling and attenuate tumour growth in concert with AT2 receptor stimulation.

Interaction of AngII receptors with PPAR-γ

Some ARBs, such as telmisartan and irbesartan, have been reported to have an AT1-receptor-blocking action, with a partial PPAR-γ agonistic effect [52,53]. We have reported that telmisartan exerts protective effects against ischaemic brain damage through synergistic effects of AT1 receptor blockade and PPAR-γ stimulation, and has a preventive effect on cognitive impairment in a mouse model of Alzheimer's disease with intracerebroventricular injection of amyloid β [54,55]. Consistent with our results, Washida et al. [56] have demonstrated that the anti-inflammatory and anti-oxidative effects of telmisartan were exerted in part by PPAR-γ activation, producing protective effects against cognitive impairment and white matter damage after chronic cerebral hypoperfusion in mice. Bähr et al. [57] recently reported in a clinical study that telmisartan activated PPAR-γ in circulating monocytes of patients with the metabolic syndrome.

These results suggest the possibility that an AT1-receptor-blocking action and PPAR-γ agonistic effect could act synergistically, leading to beneficial effects of organ protection. It has been reported that PPAR-γ activators suppressed AT1 receptor promoter activity measured by luciferase assay, but did not affect AT1 receptor mRNA stability in cultured VSMCs, suggesting that suppression occurs at the transcriptional level [58]. Sunagawa et al. [59] observed that endogenous AT1 receptor expression in VSMCs was significantly decreased by PPAR-γ stimulation at the mRNA and protein levels, with a decrease in VSMC proliferation. It has also been reported that AT1 receptor blockade decreases NF-κB (nuclear factor κB) activation, with PPAR-γ activation in the vasculature [60]. AT1 receptor stimulation activates ERK, and PPAR-γ stimulation inhibits this ERK activation in VSMCs [61]. On the other hand, it is known that AngII induces PPAR-γ activation in PC12W cells via AT2 receptor activation [62]. Taken together, these results support the notion that some ARBs with a PPAR-γ-agonistic effect could further enhance PPAR-γ stimulation and inhibit AT1 receptor action with AT2 receptor stimulation (Figure 4).

Figure 4 ARBs with a partial PPAR-γ agonistic effect could further enhance PPAR-γ stimulation and inhibit AT1 receptor action with AT2 receptor stimulation


Complex formation of AngII receptors and other GPCRs

GPCRs may form dimers as part of their normal trafficking and function, although GPCRs have traditionally been thought to act as monomers [63]. In this context, dimer formation of AngII receptors, such as an AT1 receptor homodimer, an AT2 receptor homodimer and a heterodimer of AT1 receptor with AT2 receptor have been reported [6466]. AbdAlla et al. [67] have reported that amyloid β induces the formation of cross-linked AT2 receptor oligomers that contribute to the dysfunction of Gαq/11 in an animal model of Alzheimer's disease [67], supporting the idea that the AT2 receptor plays a role in cognitive function. Moreover, it has also been reported that aldosterone produces a non-genomic endothelium-independent vasoconstrictor effect by enhancing intracellular transglutaminase activity and presumably inducing AT1 receptor dimer formation in mesenteric arterioles [68]. These results suggest that cross-talk of AngII receptors via possible dimerization could contribute to the regulation of a variety of functions of AngII.

AbdAlla et al. [69,70] also showed that the AT1 receptor undergoes heterodimerization with the brady-kinin B2 receptor in women with pre-eclampsia, and the AT1 receptor and bradykinin B2 receptor heterodimer contributes to AngII hypersensitivity. In contrast with these previous observations, Hansen et al. [71] collectively suggested that AT1 receptor–bradykinin B2 receptor heterodimerization does not occur as a natural consequence of their simultaneous expression in the same cell nor does the bradykinin B2 receptor influence AT1 receptor signalling. Other heterodimerization with the AT1 receptor has been reported, such as the AT1 receptor–EGFR (epidermal growth factor receptor), AT1 receptor–dopamine receptor, AT1 receptor–endothelin ETB receptor and AT1 receptor–Mas receptor [7278]. These results propose the intriguing possibility that AngII could regulate various pathophysiological states, such as cardiovascular remodelling, by directly interacting at the receptor level, and more mechanistic analysis of dimerization of these receptors could possibly reveal more detailed pathophysiological roles of AngII.

AngII receptor activation without AngII

Ligand-independent activation of GPCRs has been highlighted especially in the potential new discovery of drug targets [79]. AngII receptors are also reported to be activated via ligand-independent mechanisms. For example, mechanical stress activates the AT1 receptor independently of AngII [80]. On the other hand, overexpression of the AT2 receptor in COS1 cells enhances apoptotic signalling without AngII stimulation [81]. The detailed mechanisms of ligand-independent activation of AngII receptors have yet to be revealed. Moreover, the concept of mechanical activation of AT1 receptors or mechanical stimulation of AngII generation is still controversial. Therefore it is mandatory that we elucidate how this concept is physiologically and/or clinically relevant in cardiovascular and BP regulation.


AngII receptors play central roles in the regulation of BP and the cardiovascular system. Further elucidation of the regulatory mechanisms of the functions of AngII receptors beyond the classical ACE/AngII/AT1 receptor axis could provide us with possibilities for the development of novel drugs that regulate RAS in a more sophisticated manner, thereby treating hypertensive patients and achieving cardiovascular risk reduction more efficiently (Figure 5). For example, an AT2 receptor agonist, an ACE2 activator and a Mas receptor agonist have been developed. Receptor modification has also become a consideration beyond the traditional view of drug discovery. The roles of associated or interacting proteins with AngII receptors, such as ATRAP and ATIP, have been recently been focused as drug discovery targets in hypertensive patients. Therefore further elucidation of the functional regulation of these AngII-receptor-associated proteins, including investigating phosphorylation and dephosphorylation, transcriptional control and finding possible ligands, should be addressed. Moreover, the beneficial effects of some ARBs with PPAR-γ agonistic action with interaction of the AT1 receptor and AT2 receptor in clinical settings could be expected, since AngII also plays a central role in the pathogenesis of the metabolic syndrome. Receptor dimerization and ligand-independent pathways could also be new targets for drug discovery in hypertensive patients. Future drugs against downstream targets of AngII receptor signalling as more specific drug targets are expected for the prevention of the AngII-induced increase in BP and cardiovascular disease. Analysis of the detailed mechanisms in the regulation of AngII receptors may also clarify the difference between responders and non-responders to modification of the RAS in patients with hypertension and could lead to personalized medical treatment for cardiovascular disease.

Figure 5 Elucidation of the functional regulation of the AngII-receptor-associated proteins

Elucidation of the functional regulation of the AngII-receptor-associated proteins, including investigating phosphorylation and dephosphorylation, transcriptional control and finding possible ligands, would contribute to new drug discovery in hypertensive patients.


Our own work was partially supported by the Ministry of Education, Science, Sports, and Culture of Japan (grants to M.H. and M.M.).

Abbreviations: ACE, angiotensin-converting enzyme; Ang-(1–7), angiotensin-(1–7); AngI, angiotensin I; AngII, angiotensin II; ApoEKO, apolipoprotein E-knockout; AT1 receptor, AngII type 1 receptor; ARB, AT1 receptor blocker; AT2 receptor, AngII type 2 receptor; ATBP, AT2-receptor-binding protein; ATIP, AT2-receptor-interacting protein; ATRAP, AT1-receptor-associated protein; BP, blood pressure; CNK1, connector enhancer of kinase suppressor of ras 1; ERK, extracellular-signal-regulated kinase; GABA, γ-aminobutyric acid; GABARAP, GABA-receptor-associated protein; GPCR, G-protein-coupled receptor; MMS2, methyl methanesulfonate-sensitive 2; NFAT, nuclear factor of activated T-cells; NF-κB, nuclear factor κB; PLZF, promyelocytic leukaemia zinc finger; PPAR, peroxisome-proliferator-activated receptor; RAS, renin–angiotensin system; SHP, Src homology 2 domain-containing protein tyrosine phosphatase; Tg, transgenic; TIMP-3, tissue inhibitor of metalloproteinases-3; VSMC, vascular smooth muscle cell


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