Angiotensin Type 2 Receptor
The angiotensin II type 2 receptor (AT2R) shares a high degree of homology with its well-known sister gene, the angiotensin II type 1 receptor (AT1R), and is similarly a part of the renin-angiotensin II system (RAS). The RAS is best known for its powerful effects on regulation of blood pressure and body fluid volume. The AT2R is activated after specific binding of the eight amino acid peptide angiotensin II (AngII), which is generated from angiotensin I by the angiotensin-converting enzyme. The AT2R is a member of the family of seven transmembrane receptors, the largest group of cell surface receptors in the body. Like its family members, the AT2R traverses the cell membrane seven times creating an NH2-terminal and three loops on the extracellular side and a COOH-terminal plus three loops on the intracellular side. The AT2R is present on multiple different cell types, although its expression level is often lower than that of AT1R. The AT2R’s biological functions and intracellular signaling partners have been difficult to tease out before the arrival of AT2R-specific agonists and antagonists. In line with the widespread location of the AT2R, it has effects in most biological systems including the central and peripheral nervous system, the cardiovascular system, endocrine systems, renal function, and the reproductive system. Although AT2R expression is generally low in normal adult tissues, it is highly important in development and in injury/repair mechanisms. Thus, the AT2R is highly expressed in the developing fetus and upregulated after adult tissue injury as well as in several pathological conditions. Expression of the AT2R can be modulated by pathological states associated with tissue remodeling or inflammation such as hypertension, atherosclerosis, myocardial infarction, stroke, and diabetes. Interestingly, the biological effects are often opposite to the AT1R effects. The most characterized AT2R effect being vasodilatation as opposed to the well-characterized AT1R-mediated vasoconstriction. Hence, from a pharmacological perspective, it would be valuable to have specific AT2R agonists, as these could be potential blood pressure–lowering drugs. The AT2R also elicit cardioprotective-, anti-inflammatory-, anti-proliferative effects and it also regulates apoptosis. Accordingly, studies have been performed to understand and map the location and function of the ligand-binding site and the intracellular signaling systems activated by the AT2R. While the receptor does not seem to activate the canonical G proteins (except Gi in some systems), several different signaling molecules appear to bind to the AT2R COOH-terminal including different kinases, phosphatases, and scaffolding molecules.
Receptor Structure, Ligand Binding, and Antagonists
Seven transmembrane (G protein-coupled) receptors for angiotensin (Ang) peptides include the AT1Ra and AT1Rb subtypes and the AT2R, that all bind the octapeptide AngII with subnanomolar affinities. The AT2R also binds the AngIII and Ang-(1-7) peptides with high affinity. In addition, at least two other receptors have been described, the AT4R (that binds AngIII) and the Mas receptor, that binds Ang-(1-7). Although no AT1R crystals have been described, the AT1R is one of the best characterized seven transmembrane receptors. Computer models indicate that the structure is much like the recently crystallized β2-adrenergic receptor (Adrenergic Receptor). The AT2R receptor is much less characterized, but it is thought that it too possesses a similar three-dimensional structure. Regarding the angiotensin receptor ligands, several different peptides exist and there has been some discussion as to which of these peptides actually bind to the different angiotensin receptors in vivo. For example, both the AngII, Ang-(1-7), and AngIII peptides activate the AT2R. AngIII is almost as potent as AngII, at least in the coronary vascular bed. Finally, there has been some data showing that the AT2R can dimerize with itself and with other seven transmembrane receptors, but not with the AT1R.
The best characterized ligand peptide is AngII that in turn is metabolized into several bioactive peptides. Aminopeptidases cleave AngII to Ang-(2-8) (AngIII) and Ang-(3-8) (AngIV). In addition, carboxypeptidases generate Ang-(1-7) from AngI-(1-10) and AngII-(1-8).
Historically, it has been difficult to separate the AT2R actions from the other Ang receptors because of the common main ligand AngII. The advent of specific AT2R agonists and antagonists has made it possible to better study the receptor’s biological actions. At least three specific AT2R agonists have been described. The AT2R agonist CGP-42112A was the first non-peptide agonist with reasonably high affinity for the receptor, and it has been widely used. Recently, a novel agonist, referred to as compound 21 and which binds the AT2R with almost as high affinity as AngII, has been developed. Compound 21 exhibits a dissociation constant for AT2R of 0.4 nM, while the dissociation constant for the AT1R is >10,000 nM. Compound 21 reduced the mean blood pressure in spontaneously hypertensive rats and induced vasodilatation in isolated arteries via AT2R stimulation (Bosnyak et al. 2010). A third agonist candidate is compound 22 that stimulates neurite outgrowth, phosphorylates p42/p44 (ERK1/2 MAPK), enhances duodenal alkaline secretion in Sprague-Dawley rats, and also lowers the blood pressure in anesthetized rats (Alterman 2010).
Selective AT2R antagonists are equally important. The most widely used antagonist is PD-123319. Among others, this molecule has been reported to block the effects of Ang-(1-7) in many studies, suggesting that Ang-(1-7) also activates the AT2R.
AT2R Signal Transduction
The AT2R signal transduction has been controversial since the receptor does not activate canonical G proteins (except Gi in a reconstituted system). Instead, the AT2R activates a series of kinases, phosphatases, and scaffold proteins including the AT2R receptor-interacting protein (ATIP) family that binds to the intracellular COOH-terminal tail of the receptor. Somewhat indirectly, an array study in cardiac fibroblasts with AT2R blockade (with PD123319) ascribed 24 genes to be specific for AT2R activation and these genes were related to ten different signaling pathways including cAMP/PKA, Ca2+, PKC, PTK, ERK1/2 (MAPK), PI-3 K, NO/cGMP, Rho, NF-kappaΒ, and JAK-STAT pathways. Specifically, collagen I and tissue inhibitor of metalloproteinase (TIMP)-1 mRNA levels decreased after AT2R blockade as compared to an AngII-treated group (Jiang et al. 2007).
Activation of protein phosphatases causing protein dephosphorylation.
Bradykinin/nitric oxide (NO)/cyclic guanosine 3′,5′-monophosphate (cGMP) pathway leading to NO release and vasodilatation.
Stimulation of phospholipase A(2) and release of arachidonic acid.
Rho inhibition, which may link the AT2R to the pathogenesis of hypertension and vascular proliferative disorders (Guilluy et al. 2008).
Activation of scaffolding/adapter proteins like the ATIP family that associate with molecular motors on microtubules.
Increased VEGF secretion that could increase angiogenesis and play a role in cancer and kidney disease.
The ATIP interaction was first discovered using the yeast two-hybrid system. This protein family presently includes six members that all interact with the AT2R: ErbB3, PLZF, CNK1 Na+/H+ exchanger NHE6, TIMP-3, and ATIP/ATBP; for review, see (Rodrigues-Ferreira and Nahmias 2010). Interestingly, association with these angiotensin receptor-binding proteins may change the intracellular location of the AT2R and underlie its roles in neuronal differentiation, tumor growth, and vascular remodeling. Using ATIP1 overexpressing mice (ATIP1-Tg), it has been shown that the ATIP1 plays an inhibitory role in the AT2R-mediated vascular remodeling. The ATIP1-Tg mice exhibited reduced neointima formation of the femoral artery after injury as compared to WT mice, which is in line with the perceived reduced AT2R signaling (Fujita et al. 2009). Control experiments confirmed the result by showing reduced NO, ERK1/2 phosphorylation, and TGFβ levels in the overexpression mice.
Finally, an important notion regarding the effect of AngII stimulation: Since the two AT1R and AT2R subtypes compete for available ligand, the AngII in vivo effect depends on the relative expression of the two receptors on the cell surface in a given biological system. Thus, blocking the AT1R with losartan leaves more AngII for AT2R stimulation yielding a “dual effect” because the receptors often have opposite actions. In addition, losartan also inhibits the renin/angiotensin feedback loop that functions as a negative feedback loop; therefore, blocking AT1R increases the production of AngII peptide. For example, the activated AT1R stimulates fibrosis and the AT2R attenuates fibrosis in many models; hence, losartan would inhibit fibrosis both by blocking the AT1R and by more available AngII for binding to unoccupied AT2Rs. In line with this notion, studies on transgenic mice with or without AT2 receptor expression reveal that the effects of AngII on injury-induced pancreatic fibrosis are determined by the balance between AT1R and AT2R signaling (Ulmasov et al. 2009).
The AT2R Is Important in the Central Nervous System (CNS)
Possible AT2R roles in CNS function, which affect learning, memory, satiety, and behavior have been suggested in both humans and rodents. At2r, gene knockout mice (At2r (-/y)) showed significant defects in their spatial memory, exhibited abnormal dendritic spine morphology, and had lost the AngII reduced food-intake response (Ohinata et al. 2008; Pawlowski et al. 2009). In line with these data, intracerebro-ventricularly administered AngII and AngIII dose-dependently suppressed food intake in mice and their anorexigenic activities were inhibited by an AT2R-selective antagonist (Ohinata et al. 2008).
Interestingly, the AT2R may also play a role in neurological disorders such as Alzheimer’s disease, depression, and Parkinson’s disease and possess a neuroprotective effect in response to brain injury such as stroke. The AT2R can attenuate inflammation and oxidative stress that have been suggested to be key factors in the pathogenesis and progression of several, if not all, of the here mentioned diseases. It should be noted that the exact molecular dysfunctions behind the progressive neurodegenerative disorder in Parkinson’s and Alzheimer’s diseases remain unidentified. It has therefore been suggested that AT2R agonists may protect neurons against cell death, and thus would prove beneficial in the treatment of these diseases. In the case of Parkinson’s disease, AngII may attenuate dopaminergic cell death. The AT2R agonist can reduce damage after an ischemic insult to the brain. Indeed, centrally administered CGP42112 (an AT2R agonist) exhibits a neuroprotective effect, which is independent of blood pressure in conscious rat models of stroke. This effect is likely enhanced by AT2R-induced vasodilatation and an increase in blood flow to the ischemic brain zone improving the brain function and limiting the infarct extension.
Furthermore, it is believed that the neuroprotective AT2R effect on stroke plays a role in the beneficial effects of AT1R blockade in stroke. Thus, losartan is superior in stroke treatment to other antihypertensive drugs that are equipotent with respect to their blood pressure–lowering effect. Losartan’s effect appears to be blood pressure independent and mediated through a dual action: selective AT1R blockade and indirect AT2R stimulation by leaving AngII for the unoccupied AT2Rs. This dual action is unique to AT1R blockers.
At the cellular level, AT2R signaling and phenotypic effects on nerve cell differentiation, apoptosis, and morphology have been studied extensively. In NG108-15 cells, the AT2R induces neural outgrowth, a process that requires parallel activation of ERK1/2 and Fyn, a member of the Src family kinases (Guimond et al. 2010).
The ATR2 Elicits Vasodilatation and Cardioprotection in the Cardiovascular System
The AT2R’s actions in the cardiovascular system are well characterized and include relaxation of cardiac and vascular smooth muscle tone most likely through increased NO production and bradykinin release. This is the classical AT2R effect; however, the AT2R also affects cellular apoptosis, inhibits proliferation, diminishes atherosclerotic lesions in humans, and limits the actions of the immune system after cardiac injury. Accordingly, AT2R agonists are under development for potential therapeutic use in hypertension. In addition, AT2R activation suppresses renin biosynthesis and release in renal juxtaglomerular cells, potentially further adding to the blood pressure–lowering effect.
Treatment of Wistar rats with the AT2R agonist compound 21, 24 h after an experimentally induced myocardial infarction, significantly reduced scar size and improved systolic and diastolic ventricular function (Kaschina et al. 2008). At the molecular level, compound 21 exerted anti-apoptotic effects (diminished myocardial infarction-induced Fas-ligand and caspase-3 expression in the peri-infarct zone) and anti-inflammatory effects (decreased serum monocyte chemoattractant protein-1 and myeloperoxidase as well as reduced cardiac interleukin-6, interleukin-1beta, and interleukin-2 expression). Finally, compound 21 treatment reduced phosphorylation of the ERK1/2 and p38 mitogen-activated protein kinases that are both involved in cell survival after myocardial infarction. Along with similar results from other groups, these data strongly suggest that AT2R agonists should be investigated for beneficial effects on systolic and diastolic function after myocardial infarction.
Since the AT1R and AT2R have opposite effects on vascular smooth muscle tone, and yet bind the same ligand, there are several possible explanations for a given experimental result. An interesting variation over this theme may exist, as spontaneous hypertensive rats may have lost the AT2R vasodilatory effect. In the heart, coronary constriction induced by Ang II, Ang III, and Ang-(1-7) is enhanced in spontaneous hypertensive rats as compared to Wistar rats that have normal blood pressure. The defect in the spontaneous hypertensive rats could be explained either by absence of counter-regulatory AT2R-mediated relaxation and/or by a change of the AT2R phenotype from relaxant to constrictor.
Caution is warranted in interpretation of the literature on AT2R effects on the cardiovascular system, since there have been conflicting reports concerning the in vivo effects of AT2R activation on cardiomyocyte apoptosis and hypertrophy. In some studies, the AT2R appears to be required for hypertrophic growth whereas in the work of others there are no effects on cardiac hypertrophy. Similar controversial findings have been reported in atherosclerosis with regard to AT2R effects on plaque morphology and neointima formation.
In an attempt to tease out the cardiovascular phenotype of the different Ang receptors, triple knockout mice lacking the AT1Ra, AT1b, and AT2R have been generated. As expected, ATR1 deletion alone reduced the heart rate and impaired the in vivo pressor response to an AngII bolus injection. In the triple knockout mice, AngII had no effect on blood pressure suggesting that there are no other AngII-sensitive receptors involved in blood pressure regulation (Gembardt et al. 2008).
Interestingly, the AT2R is expressed in human and rodent atherosclerotic lesions, and a series of papers suggest an atheroprotective action of these receptors. Chronic Ang-(1-7) administration inhibits the progression of atherosclerosis and improves endothelial function in a mouse model of atherosclerosis (apolipoprotein E-deficient (ApoE(-/-)) mice). These effects were reversed with either AT2R or Mas receptor blockade. At the molecular level, Ang-(1-7) decreased superoxide production and increased endothelial nitric oxide synthase immunoreactivity, thus improving NO bioavailability as a result of activation of AT2R and/or Mas receptors (Tesanovic et al. 2010). In agreement with these data, AT2R overexpression decreases collagen accumulation in atherosclerotic plaques.
Increasing the complexity of the RAS system, sex differences in blood pressure regulation exists. In females, a low AngII dose decreased blood pressure while it had no effect in men. Surprisingly, a high dose increased mean blood pressure more in men than in women. This could be attributed to an enhancement of the AT2R vasodilatory effects in women as underlined by increased AT2R mRNA levels. These data are consistent with the notion that estrogen upregulates AT2R expression. In turn, this could contribute to the lower blood pressure during pregnancy. In this condition, an enhanced AT2R-mediated vascular relaxation pathway involving increased expression/activity of endothelial AT2Rs and increased postreceptor-activated phospho-eNOS have been reported.
Although controversial because of contradictory results, there is accumulation of evidence suggesting that genetic polymorphisms in the AT2R gene can modify disease risk in both cancer and cardiovascular diseases. This is an important field to follow in the future because better understanding of the response in different patients can lead to more individually tailored medicine. Several different genetic polymorphisms have been described, although these mutations do often not appear to influence AT2R function. However, one of these, A1675G in the AT2R gene possibly decreases cardiovascular risk and the severity of atherosclerosis by modifying systemic inflammation especially in hypertensive males (Tousoulis et al. 2010).
The AT2R Affects Cancer and Inhibits Inflammation
While the AT1R proinflammatory effects are rather well established, most current evidence suggests the AT2R instead interferes with and inhibits proinflammatory pathways, though the AT2R data in inflammation are still rather preliminary and somewhat controversial. However, the principal mechanisms of AT2R putative anti-inflammatory actions support new possibilities in AT2R research and potential utilization of AT2R stimulation as a novel pharmacological concept in anti-inflammation as reviewed in (Rompe et al. 2010). Expression of AT2R protein is generally upregulated in injury animal models such as experimentally induced stroke, myocardial infarction, and neointima lesions, and AT2R activation interferes with multiple inflammatory pathways. The agonist compound 21 inhibits key proinflammatory cytokines and nuclear factor kappaB. Compound 21 dose-dependently (1 nM–1 μM) reduces tumor necrosis factor-alpha-induced interleukin 6 levels in primary human and murine dermal fibroblasts. After myocardial infarction in the mouse, compound 21 also decreases serum MCP-1 and myeloperoxidase as well as cardiac interleukin expression (IL-6, IL-1beta, and IL-2).
In general, the AT2R has effects that theoretically could play a role in cancer development including anti-proliferative, anti-angiogenic, and pro-apoptotic effects. In fact, AT2R expression seems to be upregulated in different types of tumor tissues, including mammary, prostate, and pancreatic cancer. However, the literature contains conflicting reports about the AT2R effect in cancer, so at this stage, it is not entirely clear if blocking or stimulating the receptor would inhibit tumor development.
Regarding prostate cancer, several reports show that AT2R activation induces apoptosis of cancer cells. AT2R overexpression in prostate cancer cells using an adenoviral vector induced apoptosis even without addition of AngII, suggesting that AT2R is constitutively active in this situation. The cell death–signaling pathway was evidenced by increased terminal deoxynucleotidyl-transferase-mediated dUTP nick-end-labeling staining and was dependent on activation of p38 mitogen-activated protein kinase, caspase-8, caspase-3, and p53 activation. AT2R overexpression also induced inhibition of proliferation, a significant reduction of S-phase cells, and an enrichment of G1-phase cells indicating that AT2R is a promising novel target gene for prostate cancer therapy (Li et al. 2009). In other experiments using LL/2 and 3-MCA tumor cells, the AT2R induced the release of vascular endothelial growth factor (VEGF), a soluble pro-angiogenic factor. Blocking this effect revealed a significant decrease in angiogenesis after antagonist (PD123,319) treatment or in AT2R-KO mice suggesting AT2R inhibition would impair tumor development by blocking malignant cell proliferation and tumor angiogenesis (Clere et al. 2010).
These studies thus imply that the effect of AT2R could be different depending on the cancer type and biological setting.
Roles of AT2R in Kidney Disease and Diabetes
It has been appreciated for some time that the RAS acts as a local paracrine system in the kidney with presence of all the known molecular components including angiotensinogen, renin, AngI, angiotensin-converting enzymes, AngII, the AT1R, and the AT2R. Overall, the AT2R is perceived to increase renal blood flow (likely by increasing local NO production), inhibit growth, increase sodium excretion, as well as to participate in fibrosis and remodeling after renal injury. The natriuretic effect is also strongly stimulated by AngIII, further underscoring its role as an AT2R ligand. Interestingly, the AT2R is also important for normal renal development. Inhibition of the AT2R using PD123319 during pregnancy induces malformations in the developing rat kidney.
The AT2R kidney effects on blood pressure and natriuresis add further evidence in favor of using AT2R agonists as antihypertensive agents. However, when reviewing AT2R effects on kidney remodeling and fibrosis, conflicting data appears (Wenzel et al. 2010). One line of data points to a role for the receptor in nephroprotection. AT2R-knockout mice exhibit increased renal injury and mortality in chronic kidney disease suggesting that pharmacological stimulation of the AT2R may positively influence renal pathologies. Other data support the notion that the AT2 receptor mediates pro-inflammatory effects and promotes renal fibrosis and hypertrophy, and consequently blocking the receptor would be the choice of therapeutic intervention.
At the molecular level, the AT2R upregulates slit diaphragm-associated molecules such as nephrin, that is, a key molecule for preventing proteinuria. In a mouse model for proteinuria, AT2R activation improves kidney function by regulating the cell cycle and principal molecular components in the podocyte. Podocytes have important functions in sealing of the glomerular permeability barrier, and therefore the AT2R is considered an interesting pharmacologic target for preventing proteinuria (Kawachi et al. 2009).
One of the most interesting roles of Ang receptors is their involvement in diabetes. Diabetics have more cardiovascular and kidney disease than the background population, and the RAS is considered a highly important pathophysiological component in both diseases. Not only does blocking of the AT1R reduce the development and progression of diabetic nephropathy. This treatment also delays onset of diabetes as compared to other antihypertensive agents even though these reduce the blood pressure to the same extent as AT1R blockers. It has been shown that the beneficial effects on glomerular injury achieved with AT1R blockers are contributed to, not only by blockade of the AT1R, but also by increasing AngII effects mediated through the AT2R (Naito et al. 2010).
The big question is: What is the quantitative significance of AT2R in the establishment of diabetic vascular dysfunction? Although, we do not have the answer to this, it is at least known that the enhanced AT2R and iNOS-induced, NO-mediated vasodilation, impairs AngII-induced contraction in an endothelium-independent manner at the early stage of type 2 diabetes. It is also known that insulin activation of the phosphatidylinositol 3-kinase (PI3K) pathway stimulates glucose uptake in peripheral tissues and NO synthesis in the endothelium. Since AT2R activation increases NO availability in the endothelium, the AT2R activation would contribute to better glucose uptake and delay insulin resistance. Shared insulin and AT2R signaling pathways could underlie the beneficial AT2R effects in metabolic and hypertensive cardiovascular diseases.
Diabetic conditions increase expression of the AT2R in the proximal tubule with a beneficial effect in kidney function and blood pressure regulation. In the hyperglycemic kidney, the AT2R is suggested to regulate key signaling pathways including Akt-mTOR-p70(S6K) and VEGF signaling.
Another interesting mechanism is that the AT2R mediates AngII and high glucose induced decreases in renal prorenin/renin receptor expression, which would protect the kidney in diabetic conditions where increased AngII is harmful. Low renin levels controlled by the AT2R also play a role in nephroprotection in obese Zucker rats.
Gene polymorphisms in the AT2R additionally suggest a link to diabetes, as high RAS activity and the A-allele of the AT2R G1675A polymorphism associate with high risk of severe hypoglycemia in type 1 diabetes. A potential preventive effect of RAS-blocking drugs in patients with recurrent severe hypoglycemia could be interesting to examine (Pedersen-Bjergaard et al. 2008).
The investigation of AT2R signal transduction and biological effects has increased considerably after the advent of specific, high affinity, and non-peptide AT2R agonists and antagonists that can now be used as tools to identify AT2R-specific effects in the scenario of multiple different Ang receptors. It has become clear that this receptor does not activate classical heterotrimeric G proteins, but rather signals through a series of small G proteins, kinases, phosphatases, and scaffolding molecules that mostly bind to the receptor COOH-terminal. A novel family of scaffolding proteins including ATIP that are especially attached to the AT2R have been identified. These events have lead to increased interest in the AT2R signaling and biology and especially in its putative pathophysiological roles. A long line of recent evidence suggests that pharmacological manipulation of AT2R activity could be clinically applicable in a wide range of diseases especially in hypertension, myocardial infarction, stroke, and diabetes. It has also become clear that the canonical notion that the AT2R antagonizes the effects of the AT1R probably is an oversimplification. Especially since the AT2R also possesses high affinity for Ang-(1-7) and AngIII that do not activate the AT1R. Nevertheless, many of the AT2R effects are counterbalanced by AT1R activation. Solid experimental evidence has demonstrated that AT2R elicits vasodilatation through NO release, and has anti-inflammatory and anti-proliferative effects, and lastly regulates apoptosis in many cell systems. A lot of interest centered on possible shared signaling pathways between insulin and AT2R signaling suggests that AT2R agonist could have beneficial and synergistic effects both in metabolic and cardiovascular disease by a dual mechanism of action including vasodilatation and NO metabolism.
However, effects still remain that we need to understand in more detail including clarification of contradictory results such as the dual roles of AT2R to both inhibit and enhance inflammation, fibrosis, and apoptosis, and we have only begun to investigate the AT2R’s role in malignant cells. Furthermore, it will be interesting to follow clinical trials of AT2R agonists and get a preliminary picture of the benefits these drugs may play in future medicine, especially, since we do not yet know which unwanted side effects might turn up.
- Bosnyak S, Welungoda IK, Hallberg A, Alterman M, Widdop RE, Jones ES. Stimulation of angiotensin AT2 receptors by the non-peptide agonist, compound 21, evokes vasodepressor effects in conscious spontaneously hypertensive rats. Br J Pharmacol. 2010;159(3):709–16. Epub 2010 Jan 28.PubMedCentralCrossRefPubMedGoogle Scholar
- Tousoulis D, Koumallos N, Antoniades C, Antonopoulos AS, Bakogiannis C, Milliou A, et al. Genetic polymorphism on type 2 receptor of angiotensin II, modifies cardiovascular risk and systemic inflammation in hypertensive males. Am J Hypertens. 2010;23:237–42. Epub 2009 Dec 3.CrossRefPubMedGoogle Scholar