The angiotensin type 2 receptor (AT2R) and the receptor Mas are components of the protective arms of the renin–angiotensin system (RAS), i.e. they both mediate tissue protective and regenerative actions. The spectrum of actions of these two receptors and their signalling mechanisms display striking similarities. Moreover, in some instances, antagonists for one receptor are able to inhibit the action of agonists for the respective other receptor. These observations suggest that there may be a functional or even physical interaction of both receptors. This article discusses potential mechanisms underlying the phenomenon of blockade of angiotensin-(1–7) [Ang-(1–7)] actions by AT2R antagonists and vice versa. Such mechanisms may comprise dimerization of the receptors or dimerization-independent mechanisms such as lack of specificity of the receptor ligands used in the experiments or involvement of the Ang-(1–7) metabolite alamandine and its receptor MrgD in the observed effects. We conclude that evidence for a functional interaction of both receptors is strong, but that such an interaction may be species- and/or tissue-specific and that elucidation of the precise nature of the interaction is only at the very beginning.
THE ANGIOTENSIN TYPE 2 RECEPTOR AND THE RECEPTOR MAS
The renin–angiotensin system (RAS) is usually associated with cardiovascular physiology and pathophysiology, in particular with hypertension . Some of the most commonly prescribed antihypertensive drugs, namely angiotensin-converting enzyme (ACE) inhibitors, angiotensin type 1 receptor (AT1R) blockers and direct renin inhibitors interfere with the RAS, i.e. they aim to prevent excessive stimulation of the AT1R by angiotensin II (AngII) [1–3]. Chronic activation of the AT1R, which is a classical seven-transmembrane-domain G-protein-coupled receptor (GPCR), contributes to the pathogenesis of hypertension by direct vasoconstriction, water- and sodium-retention (direct and through stimulation of aldosterone and vasopressin synthesis) and facilitation of sympathetic outflow . The AT1R further promotes hypertensive end-organ damage by pro-inflammatory and pro-fibrotic effects.
After years of scepticism, it is now widely acknowledged that apart from the AT1R, the RAS harbours further receptor subtypes, which mediate actions opposing those of the AT1R . The angiotensin type 2 receptor (AT2R) and the receptor Mas are the best characterized of these protective receptors within the RAS — the Mas-related G-protein-coupled receptor (MrgD) has been identified very recently and demonstrated to have similar properties when activated by alamandine (Figure 1) . Both receptors, AT2R and Mas, mediate a multitude of strikingly similar tissue protective and regenerating actions. These include anti-inflammatory, anti-fibrotic or neuroregenerative actions, vasodilatory effects and beneficial metabolic actions [6–10]. Deficiency of either AT2R or Mas does not cause a dramatic phenotype [11,12], but if the respective knockout animals are exposed to disease models such as myocardial infarction , stroke , chronic kidney disease  or atherosclerosis [16,17], symptoms are usually much more severe than in the wild-type controls, which again indicates that both receptors are crucial for tissue protection and repair.
Angiotensin peptides and their receptors
Despite very similar physiological and pathophysiological actions, the AT2R and Mas share only 31% sequence identity. Moreover, these receptors are localized on different chromosomes–the AT2R on the X-chromosome and the receptor Mas on chromosome 6–and their high-affinity, endogenous ligands/agonists are not identical.
Regarding Mas it has been well established that the main natural ligand is angiotensin-(1–7) [Ang-(1–7)] . Ang-(1–7) is generated from AngII by ACE2, which cleaves the amino acid phenylalanine from the C-terminal end of AngII. Ang-(1–7) can alternatively also be generated from angiotensin I (AngI) through cleavage of leucine from the C-terminal end by ACE2 generating angiotensin-(1–9) [Ang-(1–9)] and subsequently hydrolysis of Ang-(1–9) by ACE or neutral endopeptidase 24.11 (NEP) forming Ang-(1–7), but direct cleavage from AngII seems the more common and important way of Ang-(1–7) synthesis .
Regarding the AT2R, AngII itself is a ligand that binds with high affinity (IC50=0.5 nM) . However, since AngII binds with almost equal affinity to the AT1R and the AT2R, and since in most healthy tissues the AT1R is expressed in much higher density than the AT2R, stimulation with the endogenous ligand AngII usually elicits AT1R-mediated effects. Recent evidence supports the notion that at least in some organs such as the kidney angiotensin III (AngIII) may be the natural ligand for the AT2R [20,21].
Both receptors, AT2R and Mas, are GPCRs with seven transmembrane domains [18,22]. Strikingly, although their signalling mechanisms are quite unconventional for GPCRs and not fully elucidated, again major similarities have been described. For both receptors, signalling through activation of phosphatases, in particular Src homology 2 domain-containing protein tyrosine phosphatase (SHP)-1 and SHP-2, seems essential [23–26]. In both cases, activation of phosphatases has been shown to interfer in an inhibitory way with kinase driven, pro-inflammatory or pro-hypertrophic signalling cascades involving, for example, mitogen-activated protein kinases (MAPKs) or nuclear factor κB (NF-κB) [27,28]. Another shared signalling mechanism of importance is the increase in nitric oxide (NO) synthesis and subsequent accumulation of cGMP, which mediates the vasodilatory effects of both receptors [25,29–31]. Moreover, both receptors are able to form dimers with the AT1R resulting in a functional inhibition of the latter [32,33].
ARE AT2R/MAS LIGANDS SPECIFIC?
Numerous studies have reported the phenomenon that actions of Ang-(1–7) can be blocked by the AT2R antagonists PD123319 or PD123177. This observation was made in a variety of different experimental models, the majority of which looked at Ang-(1–7)-induced vasodilation or blood pressure lowering. For example, Ang-(1–7)-induced vasorelaxation in preparations of rat thoracic aortic rings  and porcine coronary arteries , or Ang-(1–7)-induced blood pressure reduction [36,37] could be blocked by PD123319. AT2R antagonists were also able to block Ang-(1–7)-induced synthesis of the vasorelaxant molecules NO , prostaglandin E2 (PGE2) or prostaglandin I2 (PGI2) , Ang-(1–7)-induced expression of endothelial and neuronal NO synthase [39,40] and Ang-(1–7)-induced release of the prostaglandin precursor arachidonic acid . Other experimental models, in which Ang-(1–7)-induced protective effects could be blocked by PD123319/PD123177 included models of atherosclerosis , salt-induced endothelial dysfunction [34,42], cortical Na+-ATPase activity [43,44], autophagy in the brain of spontaneously hypertensive rats (SHRs)  and in experimental model of ischaemic stroke.
In the latter case, we have demonstrated that PD123319 can block the cerebroprotective effects of centrally administered Ang-(1–7). Stroke was induced in male Sprague–Dawley rats by injection of endothelin-1 (ET-1; 3 μl of 80 μmol/l solution; 1 μl/min) into the vicinity of the right middle cerebral artery, causing transient occlusion [middle cerebral artery occlusion (MCAO)] of this vessel . The resultant cerebral infarct and neurological deficits, measured 3 days later, were significantly reduced by intracerebroventricular (icv) administration of Ang-(1–7) (1 μg/0.5 μl of artificial cerebrospinal fluid (aCSF)/h, icv infusion 5 days prior to and 3 days after MCAO; Figures 2A–2C). This protective effect of Ang-(1–7) was completely inhibited by simultaneous icv administration of the AT2R antagonist PD123319 (0.3 μg/0.5 μl of aCSF/h; Figures 2A–2C). Interestingly, we also observed the reverse: cerebral infarct size and neurological deficits were significantly ameliorated 3 days after MCAO by stimulation of the AT2R with the AT2R agonist Compound 21 [C21; 0.03 mg/kg intraperitoneally (i.p.) administered 4, 24 and 48 h post-ET-1-induced MCAO; Figures 2D–2F]. This effect was completely blocked by central administration of the Mas antagonist A779 (5 μg/0.5 μl of aCSF/h, icv infusion 5 days prior to and 3 days after MCAO; Figures 2D–2F).
Effects of Ang-(1–7) or the AT2R agonist C21 on infarct size and neurological deficits 3 days after MCAO in Sprague–Dawley rats and their inhibition by antagonists for AT2R or Mas, respectively
The blockade of Ang-(1–7) actions by AT2R antagonists and vice versa can be explained in several ways: (i) the respective ligands are unspecific meaning that Ang-(1–7) may stimulate the AT2R and AngII/C21 may activate the receptor Mas, or alternatively that the AT2R antagonists (PD123319/PD123177) also bind to and antagonize Mas and the Mas antagonist A779 blocks the AT2R; (ii) there is a functional and maybe physical interaction between the AT2R and Mas; and (iii) inhibition of Ang-(1–7) effects by PD123319 may actually be due to blockade of effects of the Ang-(1–7) metabolite alamandine by binding to its receptor MrgD.
These three possibilities are discussed in the following sections.
Binding characteristics of ligands for the AT2R and Mas
Regarding the specificity of Ang-(1–7)/A779 for Mas and AngII/C21/PD123319/PD123177 for AT2R, Widdop's group has previously published binding studies in which the affinity of AngII/Ang-(1–7)/A779/C21/PD123319 and some other ligands for the AT2R (and the AT1R) was determined in human embryonic kidney (HEK)-293 cells stably transfected with the AT2R (or the AT1R) . They demonstrated that the peptide AT2R agonist CGP42112A and AngII bind with the highest affinity to the AT2R (IC50=0.23 and 0.52 nM, respectively), followed by the non-peptide AT2R agonist C21 (IC50=2.29 nM) and PD123319 (IC50=5.6 nM). Interestingly, Ang-(1–7) also bound to the AT2R, but with lower affinity (IC50=0.25 μM). The IC50 of A779 or the Mas agonist AVE0991 was >1 μM and could not be determined, since concentrations higher than 1 μM were not tested.
In light of these data, it is plausible that Ang-(1–7) elicits at least part of its effects by unspecific binding to and activation of the AT2R. This would explain how the Ang-(1–7) effects can be blocked by an AT2R antagonist. However, it does not sufficiently explain the observation that in some studies the effects of Ang-(1–7) were completely and not just partially blocked by AT2R antagonists [30,34,36,37,39,40,42–44,47,48], although PD123319/123177 should not interfere with binding of Ang-(1–7) to Mas. This consideration leads to the question, whether PD123319/123177 are really specific and selective for the AT2R, or whether these molecules may bind to Mas as well. This critical question has not yet been addressed experimentally. However, there is indirect evidence for AT2R-independent effects of PD123319 from a study by Gorelik et al. , in which Ang-(1–7)-induced relaxation of preconstricted, isolated pig coronary arteries could be reduced by 70% by PD123319, but not by the unspecific AT1R/AT2R antagonists sarthran or saralasin.
It is also not known whether the AT2R agonists CGP42112A or C21 may bind to Mas. The latter is not unlikely, since the molecular structures of the AT2R agonist C21 and the Mas-agonist AVE0991 show striking similarities (Figure 3). The main structural features of C21 and related AT2R agonists are the imidazole ring, the acidic sulfonyl carbamate substituent, and the isobutyl chain all attached to a bicyclic scaffold . These elements are also found in AVE0991, although the sulfonyl carbamate is displaced by a sulfonyl urea . Structural modifications of both the sulfonyl carbamate substituent and the isobutyl group of C21 had in general a negative impact on AT2R affinity, but diverse substituents in the benzylic position were often accepted and bulkier groups such as aromatic rings (as found in AAV0991) can frequently be accepted . Notably, substitution of the sulfonyl carbamate of C21 for sulfonyl ureas also resulted in fairly potent AT2R ligands . The most characteristic feature of AVE0991 is the formyl group that is very seldom seen in drugs since this group is normally prone to undergo various types of reactions, primarily oxidations.
Chemical structures of the AT2R agonist C21 and the Mas agonist AVE0991
Nevertheless, structural similarity does not necessarily result in similar effects at the receptor, since even the slightest changes in the molecular structure can lead to pronounced changes in affinity or lead to a loss of intrinsic activity.
With regard to the Mas ligands, AVE0991 and A779, both of them may bind to the AT2R with low affinity (IC50 ≥10 μM). In Figure 1 of the publication by Bosnyak et al. , it can be noted that there seems to be an onset of replacement of 125I-[Sar1Ile8]AngII by AVE0991 and A779 in AT2R-transfected HEK-293 cells starting at a concentration of 1 μM. If one extrapolates these curves, an IC50 value of ~10 μM can be expected for AVE0991 and A779. However, since only concentrations up to 1 μM AVE0991/A779 were tested in this study, an IC50 value for binding of these ligands to the AT2R could not be determined.
Indications for a functional or physical interaction of the AT2R and Mas
Inhibition of signalling and function of one receptor by an antagonist for another receptor can be an indication of physical dimerization of these receptors. For example, Barki-Harrington's group has demonstrated that the β1-receptor antagonist propranolol can block AT1R-induced contraction of mouse cardiomyocytes, that AT1R blocker valsartan can attenuate isoproterenol-induced cardiomyocyte contraction and the AT1R and β1-receptor form complexes . As discussed and shown above, a similar phenomenon may occur for the AT2R and the receptor Mas: there are many examples for inhibition of Ang-(1–7) effects by the AT2R antagonist PD123319 [30,34–45,47,48,53], and we provide data in the present study (Figure 2) showing inhibition of C21-induced protective effects after ischaemic stroke by the Mas antagonist A779. This ‘cross-inhibition’ may in fact be an indication for AT2R–Mas dimerization.
It has been shown in the past that GPCRs including receptors of the RAS seem predestined to form homo- or hetero-dimers . Homodimerization of AT1Rs, which has been described by several groups using, for example, the BRET technique, seems to be constitutive and not influenced by binding of agonists or antagonists . Regarding any functional relevance, a so-called cross-inhibition has been observed for AT1Rs. For example, when mutated receptors that were either unable to bind the angiotensin receptor blocker candesartan or unable to signal through G-proteins were transfected into one cell, the inhibitory effect of candesartan on G-protein signalling was restored . Similar observations were made regarding transactivation of dimerizing AT1Rs [55,57]. AT1R dimers seem to be increased in cardiovascular disease such as hypertension and hypercholesterolaemia and reduced by ACE inhibitor treatment .
Homodimerization of AT2Rs has been reported by the groups of Saku and Quitterer to also be constitutive and ligand independent and to be crucial for basal, constitutive and agonist-induced signalling [32,58].
Heterodimerization within the RAS shows great diversity. AT2R and Mas have both been reported to bind to AT1Rs and to antagonize AT1R signalling and function, thereby being ‘physiological AT1R antagonists’ [32,33,59]. In addition, the AT1R has been demonstrated to bind to the bradykinin B2 receptor , the β1-adrenergic receptor , the dopamine D1 and D3 receptors [61,62] and also the endothelin B receptor .
Thus, dimerization among GPCRs including those of the RAS is multifarious, and it seems quite likely that there may also be dimerization between the AT2R and Mas.
Binding of PD123319 to the Mas-related receptor (MrgD)
It was recently reported by some of the authors of this article and colleagues that decarboxylation of the aspartate radical group of Ang-(1–7) results in another active hormone, alamandine . From what has been determined so far, alamandine seems to be another component of the ‘protective RAS’ and elicits effects resembling those of Ang-(1–7) such as endothelium- and NO-dependent vasodilation, lowering of blood pressure and anti-fibrosis . Interestingly, although sequence-wise alamandine differs only marginally from Ang-(1–7), its effects are mediated through the MrgD and its binding to Mas is weak or non-existent. These effects of alamandine cannot be blocked by the Mas-antagonist A779, but by D-Pro7-Ang-(1–7) and, surprisingly, also by PD123319 . Since alamandine still caused vasorelaxation in aortic rings isolated from AT2R-deficient mice, and this effect was blocked by PD123319, it has to be assumed that PD123319 is also an antagonist/ligand for MrgD. Consequently, in those studies, in which effects of Ang-(1–7) were seemingly blocked by PD123319, the actual underlying mechanism may have been inhibition of the effect of the Ang-(1–7) metabolite alamandine on the MrgD receptor.
OBSERVATIONS CONTRADICTING A MAS–AT2R INTERACTION
Although numerous studies have reported the phenomenon of blockade of Ang-(1–7) effects by the PD123319/PD123177 molecules [30,34–45,47,48,53], there is a considerable number of publications in which the inhibitory effect of the PD compounds on Ang-(1–7) actions was not observed, indicating that in these studies the effects of Ang-(1–7) were Mas-specific and did not involve the AT2R [66–70].
In addition, there are three publications in which an AT2R agonist elicited effects in tissues deficient in Mas or in which Ang-(1–7) elicited effects in tissues deficient in the AT2R. In one of these studies, the AT2R agonist CGP42112A was able to dilate aortic rings deficient of Mas , in a second study Ang-(1–7) was able to lower blood pressure in AT2R-knockout mice  and in a third study Ang-(1–7) prevented neointima formation in AT2R-knockout mice . These three studies suggest that, if any physical interaction between Mas and the AT2R should exist at all, this interaction is at least not required for the receptors to be functional.
There is currently no explanation for the controversy in these data, some of which strongly suggest a functional or even physical interaction of Mas and the AT2R, whereas others do not. However, it can be speculated that species or tissue-related differences may play a role as has been described for dimerization of other GPCRs, such as opioid receptors . Such differences may involve varying ratios of expression levels of Mas, MrgD and the AT2R leading to different dimerization/oligomerization patterns. Interestingly, in case of opioid receptors, certain agonists such as 6-guanidinonaltrindole only selectively activate receptor heterodimers, but not homomers. Whether such phenomena exist within the RAS still has to be explored but, if they do, this may lead to new approaches for the development of heterodimer-specific drugs.
IMPACT OF AN AT2R–MAS INTERACTION IN THE CONTEXT OF PHYSIOLOGICAL OR PHARMACOLOGICAL RECEPTOR STIMULATION
Provided that the AT2R and Mas actually interact functionally and/or physically, then such interactions may have an impact on their physiological action or on the action of drugs which bind to these receptors.
In the case of a physical interaction, i.e. dimerization, the most significant consequence would be that one receptor is only functional in the presence of the other receptor. For vascular tissue, this seems not to be the case, since the vasodilatory effect of Ang-(1–7) still occurred in the absence of the AT2R and vice versa [71–73]. However, such a functional dependence can be tissue-specific as has been shown for opioid receptors , meaning that at present a functional dependence of AT2R and Mas cannot generally be excluded. Consequently, there exists the possibility that in certain tissues endogenous or pharmacological AT2R/Mas agonists are only functional if both receptors are concomitantly expressed. In disease or tissue injury, both receptors are known to be up-regulated. Therefore, in the case of functional dependence, agonists for AT2R/Mas may only become fully functional if the expression of both receptors is increased simultaneously and in equal amounts.
A physical/functional interaction of both receptors could also mean that receptor agonists, which represent potential future drug classes to be used in the clinic, may be able to either activate only the single receptors or only the dimerized receptors, which again may result in a different spectrum of actions. Again, such a phenomenon is known to occur for other receptors that dimerize and may lead to the development of specific drugs that target either the individual or the dimerized receptors.
The AT2R and the receptor Mas belong to the protective arm of the RAS and have both been shown to ameliorate pathological changes and symptoms in various pre-clinical models including cardiovascular, renal, central nervous system (CNS) and metabolic diseases [6,75,76]. Both receptors exert their effects by strikingly similar signalling mechanisms and (patho-)-physiological actions, suggesting that they may functionally interact or even share downstream signalling pathways. This assumption must still be confirmed experimentally, but seems likely, since the AT2R and the receptor Mas are both known to homodimerize and to heterodimerize with the AT1R and with other GPCRs. Thus, functioning of the AT2R and Mas may be intertwined, but these functionally very similar receptors may also represent independent systems, which can compensate for each other in the event of loss or absence of one of them. Since agonistic and antagonistic ligands in most cases are only to a limited extent selective for either the AT2R or Mas, it is often difficult to truly distinguish between the AT2R and Mas actions, and the clarification of the nature of interaction between the AT2R and Mas will remain a challenging task.
artificial cerebrospinal fluid
- AngI etc.
angiotensin I etc. Ang-(1–7), angiotensin-(1–7)
- AT1R etc.
angiotensin type 1 receptor etc
human embryonic kidney
insulin regulated aminopeptidase
middle cerebral artery, causing transient occlusion
Mas-related G-protein-coupled receptor
Src homology 2 domain-containing protein tyrosine phosphatase
spontaneously hypertensive rat
U.M.S. has received modest research support from Vicore Pharma.