The total length and the size of the AT1 and AT2 receptors are similar. The binding sites for these two receptors are also similar. The third loop, which is considered to be the major signal transmitter region for both receptors, is the major difference between the two receptors. However, the signal homology is only about 33%. The AT2 receptor is the major focus of this lecture.

Cloning of the AT1 and AT2 receptors from the bovine and rat by this group showed a marked difference in the sequence between the receptors in the area related to the internalization of the receptor.

Two types of Ang II receptors exist: dithiothreitol-sensitive, and dithiothreitol-insensitive. The AT1 receptor is a specific AT1 blocker (losartan, candesartan, etc), and the AT2 receptor is a specific AT 2 blocker (PD123319, PD123177). The cloning and sequencing of these receptors confirms the differences between these two receptors. AT1 has a unique structure, while cloning showed that the AT2 receptor has 34% amino acid identity with AT1. However, the coupling of these receptors remains controversial. Whether the AT2 receptor is always Gi coupled and the AT1 receptor mainly Gq coupled is unsettled.

The physiological function and regulation of the AT1 and AT2 receptor differs, but further work must be done to fully elucidate this matter. The AT2 receptor is a growth inhibitor.
The regulation of AT2 is very complicated because the expression of AT2 in the fetus is very high, and it is mainly in the mesenchymal tissues. However, immediately after birth, AT2 is reduced precipitously and yet it remains at a finite level in certain tissues.

Data obtained from the vasculature cannot be transferred directly to the heart. The heart seems to be more susceptible to environmental changes. Both AT1 and AT2 are expressed in cardiac fibroblasts and mycocytes. AT2 in the myocytes is markedly reduced after birth, but it is re-expressed under pressure overload and in the failing heart, and probably Ang II is increased. There is an intracardiac Ang II generating system in cardiomyocytes, which is markedly stimulated by myocyte stretch.

Inotropic and chronotropic actions are the short-term effects of Ang II on the heart. The long-terms effects include ventricular hypertrophy and dilated cardiomyopathy, coronary artery remodeling, and cardiac ischemia.  AT1 is known to stimulate cellular growth. Many of the growth mechanisms have been published, but it remains unclear how these growth mechanisms are interrelated and how these are related to AT2- stimulated cell growth.

AT1 and AT2 are generally thought to work in opposite directions. For example, AT1 stimulates cell growth of fibroblasts, pheochromocytoma and neuroblastoma. AT2 seems to suppress cell growth.  In vivo, the renal interstitial fluid production of nitric oxide is stimulated by AT2, whereas it is not by AT1. In the resistance vessels, AT-1 stimulates vasocontraction and AT-2 stimulates vasorelaxation. Cell growth suppression by Ang II via AT2 is done through the activation of SHP-1, MKP-1, PP2A, and B2-NOS, among other factors.

Modulation of the AT2 receptor has been done in various ways by different investigators, such as AT deletion, AT overexpression using transgenic technology, or AT antagonism. This has yielded different results regarding myocyte growth by AT, some showing it enhances growth, some showing it suppresses growth, and some showing no effect. The different results may be due to the different techniques used, with newer techniques being more accurate. Also, in terms of vascular function, different results have been obtained by different investigators regarding the effect of AT on vascular smooth muscle cell (VSMC) hypertrophy. Again, different methodologies may give rise to different results.

However, AT1 and AT2 also have parallel actions. Aortic hypertrophy by Ang II in the rat aorta is blocked by PD123319 rather than by losartan. Tyrosine hydroxylase is stimulated by AT and AT2. Cardiac hypertrophy is blocked by an AT1 or an AT2 blocker.

Inagami and colleagues showed using the AT2 knockout left ventricular hypertrophy (LVH) model that aortic banding and infusion of Ang II did not cause LVH in the mice lacking the AT2 gene. Also, that the protein level of p70S6k was significantly decreased in the AT2 null mouse heart. All protein levels of the PI3 kinase-Akt/PKB-p70S6k pathway were decreased in the AT2 null mouse heart.

Systolic blood pressure is elevated in the AT2 knockout mouse, compared to the wild-type mouse. Interestingly, aortic banding or angiotensin infusion increases myocardial wall thickness in the wild-type mice, but in the knockout mice, posterior wall thickness does not increase. However, in terms of hemodynamic function, there was no difference between the wild-type and the knockout mice for LVDd and fractional shortening.

Wall thickness or cross sectional areas are determined by PI3kinase, as shown by a number of studies. Protein levels of the PI3kinase pathway were significantly decreased in the AT2 null mouse heart with aortic banding, whereas they are increased in the aortic-banded wild-type mouse heart. Also, the heart size increases in the wild-type mouse, to about the size seen in cardiomyopathy, compared to the AT2 null mouse. The myocyte cross-sectional area is increased about 2-fold in the wild-type mouse, but there is little change in the AT2 null mouse. Surprisingly, even collagen type 1 synthesis increases in the aortic-banded wild -type mice, but is inhibited in the aortic-banded AT2 knockout mice.
In the AT2 null mouse heart, E/A is maintained without a hypertrophic response with a 2-week infusion of Ang II, compared to the wild-type mice. In contrast, work by Harada showed that in AT1 knockout mice, Ang II does not change the cross sectional areas. Also, AT1 is a major factor in determining normal blood pressure. A drop of about 35 mm Hg was seen in the AT1 null mice. Interestingly, in the AT1 knockout mice, aortic banding itself increased the cross-sectional area. The increase was the same whether or not AT1a is present. Fibrosis around the perivascular area is present.

In sum, there are 2 major subtypes of the Ang II receptor, AT1 and AT2. Traditionally, AT2 receptor appear to have an antagonistic function against AT1. However, the function of AT2 remains controversial. Inagami and colleagues reported that pressure overload or Ang II stimulation did not induce LVH in AT2 knockout mice. However, recent studies reported that pressure overload still induced LVH in AT1a knockout mice.

Inagami and colleagues examined the binding protein through various segments of AT2 using the yeast two-hybrid screening system, by using the AT2 carboxyl terminal (C tail) as a bait to look for the DNA or protein that binds the C-tail protein. They identified what they named AT2CZ. Although AT2 C-tail has a binding protein, it did not bind to the third loop. AT2CZ is a specific binding protein, only binding to the AT2 carboxyl terminus. The specificity of this binding was confirmed using an in vitro binding assay.

Localization of AT2CZ shows its expression is tissue selective. In the heart it is expressed in a large amount, but it is not expressed in the large aorta. It is expressed in a small amount in brain tissue, in the colon it expresses a fairly large amount, perhaps due to a different method being used for testing, and in the kidney it is expressed in a limited amount. Fortunately, the immortalized cell H9C2 expresses AT2CZ, so perhaps this type of cell may be used more extensively.

AT2CZ translocates to the nucleus by Ang II stimulation via the AT2 receptor, but not by the AT1 receptor. AT2CZ is localized on the periphery of the nucleus after translocation.
AT2CZ does not seem to localize in any membranous structure, even with Ang II present. When both AT2 and AT2CZ are present, Ang II stimulation results in AT2CZ appearing in the perinuclear area within about 30 minutes and begins to appear in the nucleus in about 2 hours. In about 3 hours, AT2CZ is inside the nucleus while AT2 remains on the periphery of the nucleus.

Arrestin is not involved in the AT2-AT2CZ complex. Arrestin also binds the C tails of various G-protein coupled receptors, including AT1. So, using AT1-expressing cells and beta-2 arrestin-expressing cells, Inagami and colleagues showed there is binding about 15 minutes after stimulation by Ang II. In contrast, AT2-expressing cells do not bind in response to beta-2 arrestin. So, this is a rare receptor not bound by beta arrestin.
Interestingly, Ang II induces AT2 internalization in the cell membrane in the presence of AT2CZ. In contrast, if AT2CZ is present, Ang II stimulation results in a reduced amount of AT2 receptors on the cell membrane. This may indicate that AT2 has begun to be internalized into the cytosoles and nucleus.

In the nucleus, the DNA element in the PI3K 855”-flanking region binds to the AT2CZ zinc finger domain. This was shown by Inagami and colleagues in a consensus sequence in which various mutations were introduced. Ang II induces P13K 85expression mediated by AT2 in the presence of AT2CZ. Also, in R3T3 cells, Ang II greatly increases P13K 85expression. They also showed that in the cells containing the CZ factor that AT2 stimulates the nucleus to express PI3K. Shioi et al showed that this is the mechanism that activates many of the cardiac hypertrophic factors, including calcineurin. Thus, Inagami and colleagues think this is one of the mechanisms whereby AT2 induces cardiac hypertrophy. The change in the PI3K pathway is time dependent, as a consequence of internalizing the CZ gene.

Ang II increases leucine uptake via the AT2-AT2CZ complex, indicating a hypertrophic response. Leucine uptake is inhibited by PD compounds, but not by losartan, indicating a hypertrophic response.

Inagami and colleagues identified a new zinc finger protein, AT2CZ, which binds AT2. AT2CZ translocates to the nucleus by Ang II stimulation via AT2, but not AT1. AT2CZ increases P13K 85 expression level 1 hour after Ang II stimulation. AT2 may work as a growth promoter in the presence of AT2CZ. If both AT1 and AT2 receptors have hypertrophic effects, an ACE inhibitor is a better choice than an AT1-specific receptor blocker to prevent or treat cardiac hypertrophy. CZ was discovered as a switch for hypertrophic or growth inhibitory mechanisms by AT2. CZ and its related signaling mechanism provide a target for cell growth control.