Angiotensin II Receptors AT<sub>1</sub> and AT<sub>2</sub>: New Mechanisms of Signaling and Antagonistic Effects of AT<sub>1</sub> and AT<sub>2</sub>
Tadashi Inagami, Ph D; Satoru Eguchi, MD; Satoshi Tsuzuki, Ph D; Toshihiro Ichiki, MD
(Jpn Circ J 1997; 61: 807-813)
Key Words: Angiotensin II Receptors; Type 1 and Type 2; Signaling; Phosphotyrosine metabolism
Mailing address: Tadashi Inagami, Ph D, Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, TN 37232-0146, USA

The existence of 2 different angiotensin II (Ang II) receptors was first suggested by the differential stability of Ang II receptors to dithiothreitol. Subsequently, isoform-specific non-peptide (losartan, PD123177, PD123319) and peptide (CGP42112) antagonists were identified and the different isoforms of Ang II receptors were termed type 1 and type 2 angiotensin II receptors (AT<sub>1</sub> and AT<sub>2</sub>; for review see Refs 1 and 2; Fig 1).
   Further, AT<sub>1</sub> has itself been shown to consist of subtypes, which are designated AT<sub>1A</sub> and AT<sub>1B</sub>.<sup>3-6</sup> However, these AT<sub>1</sub> subtypes are limited to rodents (rat and mouse) - higher mammals exhibit only one type of AT<sub>1</sub> receptor. Studies have revealed that almost all of the effects traditionally ascribed to Ang II are mediated by AT<sub>1</sub> receptors, including contraction of smooth muscle, the inotropic effect on cardiac myocytes, stimulation of aldosterone release from the adrenal cortex, facilitation of  catecholamine release from nerve endings, the hypertensive action of centrally administered Ang II, hypertrophic actions on vascular smooth muscle cells (VSMCs), mitogenic effects on some fibroblast cells, and activation of the tyrosine kinase pathway and mitogen-activated protein kinase (MAPK) and renal tubular effect in sodium reabsorption. <sup>1, 2</sup> Thus, there seem to be few physiologic functions left for the AT<sub>2</sub> receptor to perform.
   The abundant expression of AT<sub>2</sub> in fetal mesenchymal, renal and brain tissues would seem to suggest a role for this receptor in fetal development and organ morphogenesis, but AT<sub>1</sub> is equally abundant in various fetal tissues. AT<sub>2</sub> expression declines rapidly after birth, and only a few types of adult tissue express AT<sub>2</sub> mRNA at a level detectable by in situ hybridization, Northern blot analysis, or RNAase protection assay. These are rat adrenal medulla and cortex, kidney, heart, uterine myometrium and vasculature, ovarian granulosa cells, pancreas, and certain brain nuclei such as the locus ceruleus, the inferior olivary nucleus, a few thalamic nuclei, and the cerebellum.<sup>7,8</sup> A few cell lines express AT<sub>2 exclusively, eg, mouse R3T3 fibroblast cells,<sup>9</sup> a subline of pheochromocytoma PC12W cells,<sup>10</sup> preadipocyte mouse T3-L 1 cells,<sup>11</sup> mouse fetal fibroblast cells, and the neuroblastoma-glioma hybrid cell line NG108-15.<sup>12</sup> These cells do not express the AT<sub>1</sub> receptor. However, other cell lines express both AT<sub>1</sub> and AT<sub>2</sub>, such as the neuroblastoma cell line N1E 115<sup>13</sup> and the transformed rat pancreatic acinar cell line AR42J.<sup>14</sup> In contrast, cultured VSMCs express AT<sub>1</sub> but not AT<sub>2</sub>.<sup>15</sup>
   In this paper, we will discuss the results of our current studies into the cross-talk between the AT<sub>1</sub> receptor and MAPK activation (which involves protein tyrosine kinase mechanisms, a pathway usually found in growth hormone-stimulated signaling pathways) as well as our findings in AT<sub>2</sub> and AT<sub>1</sub> gene-null mice regarding the roles of AT<sub>2</sub>, which have remained elusive.
   Activation of the tyrosine kinase system by 7 transmembrane domain receptors (G-protein-coupled receptors; GPCRs) has been observed, but the mechanism remains unclear. Because tyrosine kinase activation by Gi-coupled receptors seems to be different from that mediated by the Gq-coupled receptor,<sup>16</sup> we have focused our studies on the Gq-coupled angiotensin type 1 receptor AT<sub>1</sub> in VSMCs, which, in the absence of growth factors such as serum, platelet-derived growth factors (PDGF-BB), or basic fibroblast growth factor (bFGF), do not undergo proliferation. However, Ang II induces hypertrophic changes but not mitogenic changes (cell proliferation) despite the fact that it stimulates MAPK activation.
   AT<sub>1</sub> coupled to the heterotrimeric G-protein Gq activates phospholipase Cbeta (PLCbeta), which generates inositol trisphosphate (IP<sup>3</sup>) via a calcium-mediated pathway. In contrast, growth factor receptors such as PDGF-R and epidermal growth factor (EGF)-R do not utilize the heterotrimeric G-protein but instead activate PLCgammal directly through phosphorylation of a tyrosyl residue.
   Morrero et al<sup>17</sup> reported that, in rat aortic VSMCs, Ang II acting on AT<sub>1</sub> receptors causes activation of PLCgamma1 rather than PLC-beta.<sup>17</sup> Further, their results showed that this cascade, which leads to MAPK activation, is initiated or mediated by a low molecular weight cytosolic tyrosine kinase, Src60.<sup>18, 19</sup> The mechanism involved in the activation of Src60 remains unclear. The hypothesis that activation involves direct association of the protein tyrosine kinase Src60 with AT receptors was subsequently disproved.<sup>19</sup>
   Studies involving rat VSMCs containing abundant PLCbeta<sub>1 and beta<sub>3</sub> have revealed that an increase in cytosolic Ca<sup>2+</sup> is sufficient to activate Ras (Fig 2), Raf-1, and the MAPK system, as well as its upstream components, Grb2 and Sos. In addition, an increase in cytosolic Ca<sup>2+</sup> induced by the presence of PLCbeta or Ca<sup>2+</sup> ionophore has been found to elicit MAPK activation.<sup>20</sup> Interestingly, this process does not involve protein kinase C (PKC) and activation of PLCgamma is not necessary. Thus, the mechanism is more readily explained by the existing signaling system except for the link connecting elevated cytosolic Ca<sup>2+</sup> to a tyrosine kinase system. Ang II-stimulated (via AT<sub>1</sub>) recruitment of Grb2-Sos or Shc-Grb2-Sos suggests the presence of some tyrosine kinase to mediate the Ca-calmodulin-dependent tyrosine kinase system. Thus, in addition to the well-known Ca<sup>2+</sup> -mediated contractile response, the AT<sub>1</sub>-induced cytosolic Ca<sup>2+</sup> elevation seems to be intimately involved in tyrosine kinase and MAPK activation, via cross-talk between GPCRs and tyrosine kinase. How Ca<sup>2+</sup> activates a tyrosine kinase cascade is the subject of intensive investigation. It seems to be distinct from the Gi-coupled GPCR, which seems to involve Gbeta-Ggamma subunits of Gi proteins along with phosphatidyl inositol-3-kinase (PI3K).<sup>21</sup>
   Importantly, AT<sub>2</sub> function also seems to be involved in the signaling of the phosphotyrosine cascade or its turn-off. Bottari et al<sup>22</sup> showed that AT<sub>2</sub> in the pheochromocytoma cell line PC12W activates phosphotyrosine phosphatase (PTP). This cell line expresses AT<sub>2</sub> but not AT<sub>1</sub>. We<sup>23</sup> and Mukoyama et al<sup>24</sup> cloned AT<sub>2</sub> cDNA from rat cells and tissues and examined the PTP activity in PC12W cells and COS-7 cells expressing the cloned AT<sub>2</sub>. Cell membranes were rigorously freed from the plasma component by ultracentrifugation at 100,000xg. In this system, AT<sub>2</sub> inhibited PTP, presumably bound tightly by a transmembrane domain, by Ang II.<sup>23</sup> In contrast, when a membrane-associated fraction in the postnuclear fraction was rapidly isolated from mouse R3T3 cells (which express AT<sub>2</sub> but not AT<sub>1</sub> receptors), it caused activation of PTP<sup>24</sup> (Fig 3a and b). The PTP activity was detectable with both nitrophenylphosphate (pNPP) and the peptide substrate Raytide, which contains a phosphotyrosine residue. Further, this activity was inhibited by the PTP-specific inhibitor sodium orthovanadate.<sup>25</sup> These results suggest that cytosolic PTP, which is not part of a receptor, is activated by AT<sub>2</sub>. It is important to note that growth stimulation of the fibroblast R3T3 cells by bFGF was suppressed by Ang II in a dose-dependent manner (Fig 3c). The dose dependence (IC<sub>50</sub> approximately 0.3 nmol/L) showed a good correlation with the dose dependence (0.5 nmol/L) of PTP activation. These results indicate that AT<sub>2</sub> mediates activation of PTP, which affects the growth of R3T3 cells.<sup>24, 25</sup>
   In cells expressing AT<sub>1</sub>, Ang II activates MAPK, which leads to a mitogenic or hypertrophied response through activation of a tyrosine kinase system.<sup>26, 28</sup> In contrast, AT<sub>2</sub> activates PTP and inhibits cell growth, thus opposing the proliferative effect of AT<sub>1</sub> as shown in Fig 4. Nakajima et al<sup>29</sup> showed a similar mechanism in VSMCs expressing the AT<sub>2</sub> receptor.<sup>29</sup> As VSMCs in medium containing serum or growth factors do not express AT<sub>2</sub> receptors, cells were transfected with a chimeric AT<sub>2</sub> gene fused to the 5'-flanking region of smooth muscle myosin. Rat carotid artery expressing transfected AT<sub>2</sub> showed a marked reduction in neointima formation following balloon catheterization. Cultured VSMCs transfected with the same AT<sub>2</sub> expression vector showed a marked reduction in MAPK activity. These results indicate that AT<sub>2</sub> receptors may reduce MAPK activity, which, in turn, will reduce neointimal smooth muscle cell growth.<sup>29</sup> In these studies, AT<sub>2</sub> receptors were stimulated only by endogenous Ang II. Janiak et al<sup>30</sup> reported the selective activation of AT<sub>2</sub> receptors as an effective approach in the suppression of neointima formation following balloon catheterization. However, the expression of AT<sub>2</sub> receptors in the neointimal tissues in rat carotid artery or aorta is minimal.<sup>31</sup> Thus, transfection of AT<sub>2</sub> receptors may be required for their therapeutic use.
   Prolonged serum depletion elicits programmed cell death of R3T3 cells as evidenced by internucleosomal DNA degradation. This process of apoptosis is enhanced by 48 h treatment with Ang II.<sup>32</sup> PC12W cells also undergo apoptosis upon depletion of their specific growth factor, nerve growth factor (NGF) and this process is also accelerated by Ang II. Both R3T3 and PC12W cells express AT<sub>2</sub>, but not AT<sub>1</sub>, receptors. Thus, signals from AT<sub>2</sub> receptors may enhance the apoptotic process initiated by the withdrawal of growth factors.<sup>32</sup>
   The mechanism of the action of AT<sub>2</sub> receptors participating in this process seems to result from reduced MAPK activity caused by the activation of MAPK phosphatase 1 (MKP-1), a protein tyrosine phosphatase that inactivates MAPK by dephosphorylating the tyrosine phosphate group in MAPK. The inactivation of MAPK and accompanying increase in DNA fragmentation by Ang II in these AT<sub>2</sub>-bearing cells are reversed by pertussis toxin (PTX) and orthovanadate. Yamada et al<sup>32</sup> provided strong evidence that AT<sub>2</sub> signaling involves Gi or Go and activation of PTP. Recently, Hayashida et al<sup>33</sup> showed that a synthetic peptide containing a 22-residue sequence from the third cytosolic loop of rat AT<sub>2</sub>, when transferred into VSMCs by lipofectamine-liposome, suppresses the MAPK activity of the VSMCs and that this inhibition is reversed by PTX or orthovanadate. These results indicate that the third cytosolic loop may play a part in the activation of a Gi-mediated PTP that inhibits MAPK. Thus, these receptor studies show that the action of AT<sub>2</sub> receptors is to inactivate MAPK and that MAPK activity can be used as a sensitive index of AT<sub>2</sub> action, rather than directly determining PTP activity, which is difficult because of the high background contribution of several PTPs. These approaches seem to provide a positive answer to the longstanding question as to whether a G protein is involved in AT<sub>2</sub> action.
   The demonstration of the direct binding of AT<sub>2</sub> to immunoprecipitated Gialpha2 and Gialpha3 by Zhang and Pratt<sup>34</sup> provides a basis for answering this question.

Targeted Gene Deletion of the AT<sub>2</sub> Gene
   To determine the overall physiologic role of AT<sub>2</sub> receptors, Ichiki et al<sup>35</sup> and Hein et al<sup>36</sup> eliminated the gene encoding AT<sub>2</sub> receptors in mice by targeted deletion. Resultant AT<sub>2</sub>-null mice exhibited elevated pressor sensitivity in response to an intravenous infusion of Ang II and their basal diastolic and systolic blood pressure were increased by about 25 mmHg compared with the AT<sub>2</sub>-intact F<sub>2</sub> hybrid mice (129 OlaxC57BL/6). An increase in mean arterial basal blood pressure was not found by Hein et al,<sup>36</sup> possibly because of various technical problems, including heterogeneity of genetic background. (In the gene-deleted mice, the genome of embryonic stem cells from the 129 Ola strain is mixed with the genome of C57BL/6 mice.) It is interesting to note, however, the opposing effects of AT<sub>1</sub> and AT<sub>2</sub> receptors on blood pressure regulation. Targeted deletion of the gene encoding AT<sub>1</sub> receptors resulted in a reduction in blood pressure of about 45 mmHg,<sup>37, 38</sup> whereas deletion of the gene encoding AT<sub>2</sub> receptors increased blood pressure about 25 mmHg.<sup>35</sup> Angiotensinogen gene deletion resulted in a decrease in blood pressure of about 20-25  mmHg,<sup>39, 40</sup> which accounts for the opposing actions of AT<sub>1</sub> and AT<sub>2</sub> as the effect of angiotensin II is completely removed. Given the general observation arising from many targeted gene deletion experiments that receptor subtypes seldom undergo compensatory changes, the simple arithmetic of Fig 5 shows that AT<sub>1</sub> (AT<sub>1A</sub>) is the dominant pressor receptor, whereas AT<sub>2</sub> appears to be a depressor receptor that operates by an as yet unknown mechanism.
   AT<sub>2</sub> receptors are expressed in the kidney, and previous studies suggest that AT<sub>2</sub> antagonists have diuretic effects.<sup>41</sup> Lo et al<sup>42</sup> isolated tubular function from hemodynamic function by maintaining a constant
</sub>

Fig 5.   Positive effect of AT<sub>1</sub> receptors and negative effect of AT<sub>2</sub> receptors on blood pressure as determined using AT<sub>1A</sub> and AT<sub>2</sub> gene knockout mice. The angiotensinogen gene knockout mice reveal the effect of total loss of Ang II receptors (compiled from data in Refs 35, 37-40).


renal blood flow using the method of Roman et al,<sup>43</sup> and observed that intravenous infusion of the AT<sub>2</sub> blocker PD123319 markedly and rapidly increased diuresis and natriuresis from the rat kidney.<sup>43</sup> Conversely, the AT<sub>2</sub> agonist CGP42112A suppressed diuresis and natriuresis, indicating the very interesting function of renal tubular AT<sub>2</sub> in sodium retention. We were able to confirm this observation in rats and mice using AT<sub>2</sub> antagonists, and, further, using AT<sub>2</sub>-gene deleted mice to ascertain that the site of antagonist binding is indeed the AT<sub>2</sub> receptor because the AT<sub>2</sub> knockout mice did not show a diuretic response to PD123319. In recent studies, however, Siragy and Carey<sup>44</sup> did not observe a similar natriuretic effect of PD123319 in conscious rats. Only the AT<sub>1</sub> blocker losartan caused natriuresis. Furthermore, they reported increased cGMP levels in the renal interstitial fluid as renal AT<sub>2</sub> receptors were stimulated, a result that is opposite to that reported previously when stimulation of AT<sub>2</sub> receptors was found to reduce cGMP levels.<sup>22, 45</sup> These results are intriguing because they suggest that AT<sub>2</sub> receptor stimulation attenuates the pressor and mitogenic response of AT<sub>1</sub> receptors to Ang II, whereas AT<sub>1</sub> and AT<sub>2</sub> receptors work in concert to retain salt, albeit via different mechanisms. These results suggest a primary and intimate role of Ang II in the retention of salt.
   AT<sub>2</sub> receptor expression is also clearly seen is in the central nervous system, including the brain stem (locus ceruleus, inferior olivary nucleus), several thalamic nuclei, lateral septum, and amygdala (central amygdaloid nucleus and medial amygdaloid nucleus).<sup>46, 47</sup> AT<sub>2</sub>-null mice obtained by targeted gene deletion show markedly lowered exploratory ambulation in a new environment and markedly increased avoidance of light areas, indicating emotional instability or fearfulness.<sup>35</sup> Such behavioral changes may reflect an action on the amygdala and locus ceruleus, which sends off long projections to the cerebral cortex. They do not seem to reflect antagonism of the well-known central roles of AT<sub>1</sub> receptors in the control of blood pressure, water drinking, and vasopressin release through AT<sub>1</sub> receptors in circumventricular organs and hypothalamic nuclei.

Overview
   It is clear that there are 2 main categories of Ang II receptor, AT<sub>1</sub> and AT<sub>2</sub>. In recent years increased attention has focused on the AT<sub>2</sub> receptor in order to determine its structure, signaling mechanism, and function. Although much remains to be determined, it appears that the actions of Ang II via the AT<sub>2</sub> receptor are generally suppressive in nature, whereas functions via the AT<sub>1</sub> receptor are more commonly stimulatory. In so far as biologic activity is concerned, there is now evidence that Ang II acting via the AT<sub>2</sub> receptor can suppress cellular proliferation. Gene deletion studies in animals suggest that the AT<sub>2</sub> receptor may subserve a vasodepressor function, although the mechanism remains to be determined. AT<sub>2</sub> agonist and antagonist studies indicate that this receptor might subserve antidiuretic and antinatriuretic actions, at least in animals. Animals lacking AT<sub>2</sub> receptors exhibit impaired drinking responses and behavioral alterations.<sup>46, 47</sup>
   Details of the biologic importance of the AT<sub>2</sub> receptor remain unclear and further information is awaited. This matter is clearly potentially relevant to the therapeutics of cardiovascular disorders, especially heart failure and hypertension, but also coronary heart disease. For example, recent studies by Levy et al<sup>48</sup> indicate that aortic fibrosis elicited by chronic infusion of Ang II is prevented by the AT<sub>2</sub> antagonist PD123319 rather than by the AT<sub>1</sub> blocker losartan. This finding may imply another hitherto unknown role of AT<sub>2</sub> in cardiovascular tissue.


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