review angiotensin ii signal transduction through the at1 ... · activates various intracellular...

Clinical Science (2007) 112, 417–428 (Printed in Great Britain) doi:10.1042/CS20060342 417

R E V I E W

Angiotensin II signal transduction through theAT1 receptor: novel insights into mechanisms

and pathophysiology

Sadaharu HIGUCHI∗1, Haruhiko OHTSU∗1, Hiroyuki SUZUKI∗, Heigoro SHIRAI∗,Gerald D. FRANK† and Satoru EGUCHI∗∗Cardiovascular Research Center, Department of Physiology, Temple University School of Medicine, Philadelphia, PA 19140,U.S.A., and †Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, TN 37232, U.S.A.

A B S T R A C T

The intracellular signal transduction of AngII (angiotensin II) has been implicated in cardiovasculardiseases, such as hypertension, atherosclerosis and restenosis after injury. AT1 receptor(AngII type-1 receptor), a G-protein-coupled receptor, mediates most of the physiological andpathophysiological actions of AngII, and this receptor is predominantly expressed in cardiovascularcells, such as VSMCs (vascular smooth muscle cells). AngII activates various signalling molecules,including G-protein-derived second messengers, protein kinases and small G-proteins (Ras, Rho,Rac etc), through the AT1 receptor leading to vascular remodelling. Growth factor receptors, suchas EGFR (epidermal growth factor receptor), have been demonstrated to be ‘trans’-activated by theAT1 receptor in VSMCs to mediate growth and migration. Rho and its effector Rho-kinase/ROCKare also implicated in the pathological cellular actions of AngII in VSMCs. Less is known aboutthe endothelial AngII signalling; however, recent studies suggest the endothelial AngII signallingpositively, as well as negatively, regulates the NO (nitric oxide) signalling pathway and, thereby,modulates endothelial dysfunction. Moreover, selective AT1-receptor-interacting proteins haverecently been identified that potentially regulate AngII signal transduction and their pathogenicfunctions in the target organs. In this review, we focus our discussion on the recent findings andconcepts that suggest the existence of the above-mentioned novel signalling mechanisms wherebyAngII mediates the formation of cardiovascular diseases.

Key words: angiotensin II, angiotensin II type 1 receptor (AT1 receptor), cardiovascular disease, signalling mechanism, vascularsmooth muscle cell.Abbreviations: ADAM, a disintegrin and metalloproteinase; AngII, angiotensin II; ARAP1, type 1 AngII-receptor-associatedprotein 1; AT1 receptor, AngII type-1 receptor; AT2 receptor, AngII type-2 receptor; ATRAP, AT1-receptor-associated protein; dn,dominant-negative; EC, endothelial cell; EGF, epidermal growth factor; EGFR, EGF receptor; eNOS, endothelial NO synthase;EP24.15, thimet oligopeptidase; ERK, extracellular-signal-regulated kinase; GEF, guanine nucleotide-exchange factor; GLP, GEF-like protein; GPCR, G-protein-coupled receptor; HB-EGF, heparin-binding EGF; HUVEC, human umbilical vein EC; IGF-1R,insulin-like growth factor-1 receptor; IRS-1, insulin receptor substrate-1; JAK2, Janus kinase 2; JNK, c-Jun N terminal kinase;MAPK, mitogen-activated protein kinase; MYPT, myosin phosphatase targeting subunit; NF-κB, nuclear factor κB; 3NT, 3-nitrotyrosine; p70S6K, p70 S6 kinase; PARP, poly(ADP-ribose) polymerase; PDGFR, platelet-derived growth factor receptor;PI3K, phosphoinositide 3-kinase; PKB, protein kinase B; PKC, protein kinase C; PLC, phospholipase C; PYK2, proline-richtyrosine kinase 2; ROCK, Rho-kinase; ROS, reactive oxygen species; S1P, sphingosine-1-phosphate; STAT, signal transducer andactivator of transcription; VSMC, vascular smooth muscle cell.1These authors contributed equally to this work.Correspondence: Dr Satoru Eguchi (email [email protected]).

C© 2007 The Biochemical Society

418 S. Higuchi and others

INTRODUCTION

AngII (angiotensin II), the major bioactive peptideof the renin–angiotensin system, plays a critical rolein controlling cardiovascular homoeostasis. It is alsostrongly implicated in various cardiovascular diseases,such as hypertension, atherosclerosis, restenosis afterangioplasty and heart failure. However, the literatureregarding the mechanistic insights by which AngIIcontributes to each of the pathophysiology of thesecardiovascular diseases remains obscure. This justifiesthe substantial amount of research ongoing towardsunderstanding the signal transduction network of AngIIwithin its target organs [1,2]. There are at least two seventransmembrane GPCRs (G-protein-coupled receptors)known to mediate AngII function, the AT1 receptor(AngII type-1 receptor) and AT2 receptor (AngII type-2receptor). The AT1 receptor has been shown to mediatemost of the physiological and pathophysiological actionsof AngII, and this subtype is predominantly expressedin cardiovascular cells, such as VSMCs (vascularsmooth muscle cells). Through this receptor, AngIIactivates a number of cytoplasmic signalling pathways,which may contribute to vascular remodelling, inducinghypertrophy, hyperplasia and migration of VSMCs. TheAT1 receptor interacts with multiple heterotrimeric G-proteins, including Gq/11, Gi, G12 and G13, and producessecond messengers, such as inositol trisphosphate,diacylglycerol and ROS (reactive oxygen species). It alsoactivates various intracellular protein kinases, such asreceptor and non-receptor tyrosine kinases and serine/threonine kinases, the MAPK (mitogen-activated pro-tein kinase) family [ERK (extracellular-signal-regulatedkinase), JNK (c-Jun N terminal kinase) and p38MAPK],p70S6K (p70 S6 kinase), Akt/PKB (protein kinase B) andvarious PKC (protein kinase C) isoforms [3–6].

Besides protein kinases, it has been recognized thatsmall GTP-binding proteins (G-proteins), such as Ras,Rho and Rac, are activated by AngII through the AT1 re-ceptor [7]. These small G-proteins appear to play import-ant roles in mediating cardiovascular remodelling inducedby AngII. Ras activation induced by AngII causesvascular hypertrophy and hyperplasia. AngII mainlyactivates Ras through the ‘transactivation’ of EGFR [EGF(epidermal growth factor) receptor]/ErbB1 with recruit-ment of a Ras GEF (guanine nucleotide-exchange factor),Sos, via adaptor proteins Shc and Grb2, and subsequentlyinduces the Raf/ERK and PI3K (phosphoinositide 3-kinase)/Akt pathways [7]. Moreover, recent findingshave demonstrated the upstream mechanisms as wellas downstream significances of growth-factor-receptortransactivation by AngII, which is a major focus ofthe present review. Rac is an important componentof the reduced NADPH oxidase complex to produceROS by AngII in VSMCs [8]. Rac is also implicatedin PAK1 (p21-activated kinase 1) activation by AngII

in VSMCs, which subsequently mediates JNK activationand hypertrophy [9,10]. Rho has been implicated inthe Ca2+ sensitization of smooth muscle contraction,whereas recent accumulating evidence further suggestthat the Rho/ROCK (Rho-kinase) pathway is critical forvascular remodelling induced by AngII [7].

Endothelial dysfunction is an early event in the patho-genesis of atherosclerosis and a feature of the insulin-resistant condition, including Type 2 diabetes, obesityand hypertension [11,12]. Not much is known aboutAngII signalling in ECs (endothelial cells); however, re-cent evidence suggests that AT1 receptor signalling inECs induce endothelial dysfunction, possibly through analternative of NO (nitric oxide) function and inductionof vascular insulin resistance [13]. Finally, the C-terminalcytoplasmic domain of the AT1 receptor appears toassociate with novel specific protein members with signaltransduction properties. These unique AT1-receptor-binding proteins may play an important role in AngIIsignal transduction pathways leading to cardiovascularremodelling.

On the basis of the above background information, inthis review we will focus our discussion on recent findingsthat suggest the existence of novel signalling conceptsof the AT1 receptor by which AngII mediates vascularand endothelial dysfunction, including the growth-factor-receptor transactivation, Rho/ROCK activation,endothelial signal cross-talk and AT1-receptor-bindingproteins. Recent review articles focusing on othersubjects of AngII signal transduction should also bereferenced along with this article, such as those describingthe general vascular signals including ROS [2], therelationship with hypertension [14], the link to kidneydiseases [15], caveolin-dependent signalling [16], arrestin-dependent signalling [17] and G-protein-independentAngII signalling [18]. In addition to the vasculature, theAT1 receptor is expressed in many other tissues, includingkidney, heart, adrenal gland, brain, lung and adiposetissues [19]. Although it may be outside of the scope ofthis review, it should be noted that some of the functionsof AT1 on the cardiovascular system are mediated throughother organs, such as through the kidney [20,21] orbrain [22], and not by direct action on the vasculature.Therefore tissue-selective signal transduction studies ofthe AT1 receptor beyond the cardiovascular system needto be developed further.

RECEPTOR TRANSACTIVATION CASCADES

The significance of the EGFR/ErbB1 transactivationpathway induced by AngII has been well documented incultured VSMCs [4]. In addition, AngII induces ErbB2/HER2, PDGFR (platelet-derived growth factor receptor)and IGF-1R (insulin-like growth factor-1 receptor) trans-activation, which may also contribute to vascular


Signal transduction of the AT1 receptor 419

dysfunction [23–25]. In this section, we will discuss thesignal transduction mechanism of receptor tyrosinekinase transactivation by AngII in relation to cardiovas-cular remodelling.

EGFR transactivation induced by AngIIPreviously, we have shown that a tyrosine kinase thatexists downstream of PLC (phospholipase C)/Ca2+

mediates ERK1/2 activation by AngII in VSMCs [26].Subsequently, we identified the tyrosine kinase asEGFR/ErbB1 [27]. Similar to EGF stimulation, AngIIrapidly transactivates the EGFR leading to activation ofthe Ras/ERK cascade. EGFR transactivation by AngIIis also required for activation of Akt/PKB, p70S6K andp38MAPK, induction of c-Fos and subsequent growthand migration of VSMCs [6]. In addition, there areinteresting reports that EGFR transactivation inducedby AngII leads to increases in intracellular Ca2+ [28] andROS [29].

The AngII-induced EGFR transactivation seems to beCa2+-sensitive or -insensitive [27,30,31], requires ROSand an upstream kinase, such as c-Src or c-Abl [29,32–34],and involves a metalloprotease-dependent production ofa EGFR ligand, such as HB-EGF (heparin-binding EGF),depending on the cell types [35–37]. HB-EGF sheddingand subsequent EGFR transactivation induced by AngIIappear to be mediated by ADAM17 (a disintegrin andmetalloproteinase 17) in ACHN tumour cells [38] andCOS7 cells [39]. Recently, we also identified ADAM17to mediate HB-EGF-dependent EGFR transactivationin VSMCs [40]. Alternatively, other mechanisms parti-cipate in AngII-induced EGFR transactivation pos-sibly through an intracellular-signal-dependent cascade[16,29,32]. In this regard, it was reported that phos-phorylation of the AT1 receptor at Tyr319 is requiredfor EGFR transactivation by promoting a recruitmentof EGFR [41]. This mechanism appears to be involved incardiac hypertrophy induced by AngII [42].

Although the possible presence of Gq-independentEGFR transactivation has been reported [43,44],HB-EGF shedding through ADAM17 induced by AngIIrequires Gq activation, since overexpression of a Gq-inhibitory mini-gene blocked HB-EGF shedding andEGFR transactivation induced by AngII. Also, HB-EGFshedding was not induced by an AT1 receptor mutantlacking a Gq coupling [39]. AngII-induced HB-EGFshedding through ADAM17 thus requires second mes-sengers, such as Ca2+ and ROS [39]. In addition tothese signalling molecules, ADAM-interacting proteinsappear to be involved in EGF transactivation induced byAngII [10]. Several distinct ADAM-interacting proteinshave been identified that include kinases, adaptors orsubstrates [45]. One of those proteins, Eve-1, associateswith ADAMs, including ADAM17, through its SH3domain and is required for HB-EGF shedding inducedby AngII in HT1080 cells [46]. An adaptor protein,

PACSIN3, also interacts with ADAMs through its SH3domain. PACSIN3 appears to be required for HB-EGFshedding induced by PMA and, in part, by AngII inHT1080 cells [47].

The phosphorylation of ADAMs may be an additionalmechanism by which AngII activates them. In responseto PMA, ERK phosphorylates and activates ADAM17via Thr735 phosphorylation in CHO (Chinese-hamsterovary) cells [48]. Similarly, p38MAPK appears to existupstream of ADAMs, leading to HB-EGF sheddingand EGFR transactivation induced by environmentalstress, including ROS [49]. However, we have shownthat ERK and p38MAPK exist downstream of EGFRtransactivation induced by AngII in VSMCs [35]. Inter-estingly, it was reported recently that PI3K activationby c-Src induces ADAM17 phosphorylation throughPDK1 (phosphoinositide-dependent kinase 1), leading toEGFR transactivation through amphiregulin shedding insquamous carcinoma cells [50]. Alternatively, upstreamtyrosine kinases (c-Src or Abl) might phosphorylateADAMs in response to AngII, thus leading to EGFRtransactivation (Figure 1).

AngII-induced EGFR transactivation regulates VSMCfunction, such as migration and hypertrophy. We foundthat metalloprotease inhibition by a pharmacologicalinhibitor not only attenuated the ERK activation byAngII, but also blocked growth and migration ofVSMCs stimulated by AngII [51]. Furthermore, wehave recently reported that dn (dominant-negative)ADAM17 markedly inhibited hypertrophy of VSMCsstimulated by AngII [40]. In addition, it has been shownthat AngII induces renal deterioration through EGFRtransactivation mediated through ADAM17 [52]. Takentogether, these data suggest that HB-EGF shedding byADAM17 and subsequent EGFR transactivation playcritical roles in cardiovascular remodelling induced byAngII.

PDGFR transactivationTransactivation of PDGFR can be induced through theAT1 receptor in some cells (including VSMCs [53]) andtissues that might have important functions, such ascell growth and migration [54,55]. Also, PDGFR trans-activation has been reported to mediate AngII-inducedERK activation in mesangial cells [56]. In the Ren2 rat,a model of AngII-driven hypertension and end-organdamage despite continuous hypertension and left ventri-cular hypertrophy, cardiac dysfunction was attenuatedby a PDGFR kinase inhibitor, imatinib. This tissue-protective effect of the PDGFR inhibitor was associatedwith decreased transactivation of PDGFR-β, activationof ERK, fibrosis and microvascular hypertrophy inducedby AngII [55]. Acute AngII infusion also leads to activ-ation of PDGFR as well as EGFR in the vasculature [57].Imatinib inhibits AngII-induced PDGFR transactivationand subsequent mesenteric arterial hypertrophy and



Figure 1 AT1 receptor signal transduction cascades mediated through growth factor receptor transactivation and theparallel Rho/ROCK pathwayAngII activates EGFR through proHB-EGF shedding by a metalloprotease ADAM17. ADAM activation by AngII requires second messengers, such as Ca2+ and ROS,and may involve ADAM-interacting proteins, such as Eve-1 and PACSIN3, and phosphorylation by putative ADAM kinases. EGFR transactivation leads to hypertrophy andmigration of VSMCs through the Ras/Raf/MEK (MAPK/ERK kinase)/ERK pathway and PI3K/Akt/mTOR (mammalian target of rapamycin)/p70S6K/eIF4E (eukaryotic translationinitiation factor 4E) pathway. PDGFR transactivation induced by AngII may require ROS, thus leading to hypertrophy and fibrosis. The Rho/ROCK pathway activated byAngII is in parallel with the EGFR transactivation pathway. AngII activates Rho/ROCK through RhoGEF (PDZ, Vav), leading to vascular contraction, migration, hypertrophy,and inflammation. MLC, myosin-light chain; MLC-p, phosphorylated MLC; MLCP, MLC phosphatase; Mnk-1, MAPK signal-integrating kinase; PHAS-1, phosphorylated, heatand acid stable regulated by insulin protein-1.

matrix expansion in the rats regardless of sustained hy-pertension, suggesting the role of PDGFR transactivationin mediating vascular remodelling [58].

Little is known regarding the mechanism of thePDGFR transactivation. AngII-induced PDGFR trans-activation in VSMCs is ligand-independent, but requires aROS-sensitive tyrosine kinase distinct from Src or JAK2(Janus kinase 2) [53]. However, H2O2-induced PDGFRtransactivation appears to require PYK2 (proline-richtyrosine kinase 2) and Src as well as PKCδ [59]. Inter-estingly, it has been recently reported that AngII inducesphosphorylation of ectodomain-truncated PDGFR-β atTyr751 and Tyr1021 and recruits the p85 subunit of PI3Kwithout the enhancement of PDGFR kinase activity inVSMCs [60].

IGF-1R transactivationThe significance of IGF-1R transactivation by AngIIhas been less clear. It was reported that AngII inducedtyrosine phosphorylation of IGF-1R as well as IRS-

1 (insulin receptor substrate-1) in VSMCs [25]. A Src-dependent IGF-1R intracellular transactivation pathwayinduced by AngII has been reported in VSMCs [61].By using the IGF-1R kinase inhibitor AG1024, it wasproposed that IGF-1R transactivation is required forPI3K and p70S6K activation by AngII, but not for thestimulation of ERK [61]. By using the same inhibitor,Touyz et al. [62] have suggested that AngII induces ROSproduction partially through IGF-1R transactivation,leading to p38MAPK and ERK5 activation in VSMCs.Obviously, further research beyond the application ofa pharmacological inhibitor is necessary to concludethe significance of IGF-1R transactivation by AngII inmediating vascular pathophysiology.

THE RHO/ROCK PATHWAYIn addition to its primary role in cytoskeletal re-organization and smooth muscle Ca2+ sensitization,Rho has been implicated in cardiovascular remodelling



associated with hypertension and other cardiovasculardiseases [63,64]. In VSMCs, AngII increases GTP-boundRhoA [65] as well as RhoA in the particulate fraction [66].The AT1 receptor is coupled to G12/13 as well as Gq inVSMCs [67,68], cardiac myocytes [69] and cardiacfibroblasts [70]. Therefore RhoGEFs sensitive to G12/13,such as p115RhoGEF, LARG (leukaemia-associatedRhoGEF) or PDZ-RhoGEF, may mediate Rho activ-ation [7,71,72]. Gq may also participate in the Rhoactivation through p63RhoGEF, a RhoGEF-sensing Gq,but not G12 or G13 [73], or tyrosine phosphorylation ofa RhoGEF, Vav, may be involved in RhoA activation byAngII in VSMCs [74]. In addition, β-arrestin 1 as wellas Gq are required to activate RhoA by AT1 receptorsexpressed in HEK293 cells (human embryonic kidney293 cells) [75].

Recently, we have shown [76] that AngII-inducedRho/ROCK pathway activation requires a tyrosinekinase, PYK2, and its upstream activation by PKCδ

in VSMCs, by using a ROCK substrate, MYPT(myosin phosphatase targeting subunit) phosphorylationat Thr696, as a marker of Rho activation. In addition, wefound that PYK2 may signal to Rho through phosphoryl-ation and activation of PDZ-RhoGEF, since PDZ-RhoGEF was co-immunoprecipitated with PYK2 andit was tyrosine-phosphorylated upon AngII stimulation[76]. Interestingly, an EGFR kinase inhibitor, AG1478,did not affect MYPT phosphorylation induced by AngII,whereas dnPYK2 did not affect EGFR transactivationinduced by AngII. Thus the Rho/ROCK pathway activ-ated by AngII is in parallel with EGFR transactivationpathways in VSMCs (Figure 1).

Rho and ROCK have been implicated in AngII-induced vascular remodelling [7]. Alternatively, JNK hasbeen shown to be indispensable for VSMC migrationstimulated by AngII [77]. In this regard, dnRho anda ROCK inhibitor, Y-27632, blocked AngII-inducedJNK activation [76]. dnRho, Y-27632 and dnJNKinhibited migration of VSMCs induced by AngII aswell. These data suggest that activation of Rho/ROCK isspecifically required for AngII-induced JNK activationand subsequent VSMC migration [76]. Interestingly,AngII induces phosphorylation of NF-κB (nuclear factorκB)/RelA at Ser536 through RhoA activation, leadingto NF-κB activation and subsequent induction of IL-6(interleukin-6) expression in VSMCs [78]. In addition, Y-27632 inhibits both AngII-induced MCP-1 (monocytechemoattractant protein-1) and PAI-1 (plasminogen-activator inhibitor-1) expression in VSMCs [79,80]. Thesedata suggest that ROCK has a critical role in theprogression of vascular inflammation.

Recent studies have demonstrated the participationof the Rho signalling pathway in several cardiovascularpathologies, including hypertension, atherosclerosis andrestenosis [63,64]. As expected by the role of Ca2+

sensitization, ROCK is reported to participate in AngII-

induced vasoconstriction through the AT1 receptor[81]. Also, it has been demonstrated that activationof the Rho/ROCK pathway by ROS is required forthe development of spontaneous tone in aorta fromAngII-infused rats [82]. AngII infusion increases theactivity of RhoA/ROCK, increases medial thickness andpromotes perivascular fibrosis in rat coronary arteries.Although oral treatment of fasudil, which is metabolizedto a specific ROCK inhibitor, did not prevent AngII-induced hypertension, these vascular alterations wereameliorated by its treatment [83]. In addition, fasudilinhibited the incidence of abdominal aortic aneurysminduced by AngII infusion in ApoE (apolipoproteinE)-deficient mice. Fasudil attenuated aortic caspase 3activity and DNA fragmentation as well as matrixmetalloprotease activity, indicating aortic wall apoptosisand proteolysis was suppressed by a ROCK inhibition[84]. Also, perivascular fibrosis induced by AngII wasdecreased in ROCK1+/− haploinsufficient mice [85].Thus, in addition to the EGFR transactivation pathway,the parallel Rho/ROCK pathway appears to be a criticalsignal transduction pathway induced by the AT1 receptorleading to vascular remodelling.

ENDOTHELIAL SIGNALLING OFTHE AT1 RECEPTOR

Although much less is known regarding the endothelialAngII signal transduction and function than those inVSMCs, an increasing body of evidence suggests novelroles of the endothelial AT1 receptor signalling in regu-lating the balance between NO and ROS in ECs [86].Dysfunctional endothelium, characterized by less pro-duction of NO as well as NO bioavailability, leads toaccelerated vasoconstriction, smooth muscle cell prolifer-ation and a prothrombotic state as well as inflammation.A decline in NO availability may be caused by decreasedexpression of eNOS (endothelial NO synthase), a lackof substrate or cofactors for eNOS, alterations in cellularsignalling such that eNOS is not appropriately activated(eNOS uncoupling) and accelerated NO degradation byROS [87].

In this regard, oxidative stress plays a major role in thepathogenesis of endothelial dysfunction and that AngII/AT1 receptor activation is critical in this process [88].In fact, AngII inhibition [ACE (angiotensin-convertingenzyme) inhibitors or AT1 receptor antagonists], anti-oxidants or genetic disruption of ROS-producing en-zymes have been shown to improve endothelial functionin human and animal studies [89–91]. NO reacts withsuperoxide anion, resulting in the formation of peroxy-nitrite (ONOO−) [92], which possess a high affinity tonitrate tyrosine residues forming 3NT (3-nitrotyrosine)and toxicity. AngII induces 3NT formation in vascularendothelium and inhibits endothelium-dependent vaso-relaxation [93].



Figure 2 Signalling mechanism by which the AT1 receptor modulates endothelial dysfunctionAngII induces ROS production through NADPH oxidase or eNOS uncoupling which mediates endothelial dysfunction. NO reacts with superoxide anion, resulting in theformation of peroxynitrite (ONOO−). Peroxynitrite induces endothelial dysfunction through the production of 3NT or PARP. Alternatively, the AT1 receptor stimulatesendothelial NO production through eNOS Ser1179 phosphorylation to protect endothelial function. In addition, AngII induces ICAM-1 and VCAM-1 through NF-κB inECs, and this cascade involves ROS and p38MAPK activation. BH4, tetrahydrobiopterin; CaM, calmodulin; DHFR, dihydrofolate reductase; ET-1, endothelin-1.

Stimulation of NO production by AngII through theendothelial AT1 receptor is also reported in culturedECs as well as in vivo studies, which is thought to bea vascular protection mechanism [94–97]. It has beendemonstrated that endogenous H2O2, derived fromNADPH oxidase, mediates endothelial NO productionby AngII [98]. We have recently reported that AngII-induced NO production requires Gq-dependent eNOSSer1179 phosphorylation and that eNOS gene transferinhibits AngII-induced VSMCs hypertrophy [95]. Alter-natively, AngII stimulates formation of S1P (sphingosine-1-phosphate) via activation of sphingosine kinase, whichthen activates eNOS through the S1P receptor [99].

On the other hand, eNOS may become uncoupledby AngII infusion, causing superoxide productionrather than NO production [94,100]. Also, endothelialNADPH-oxidase-derived H2O2 induced by AngII was

reported to mediate tetrahydrobiopterin deficiency anduncoupling of eNOS [101]. In addition, AngII has beenshown to activate PARP [poly(ADP-ribose) polymerase]via the AT1 receptor through NADPH in ECs [102].PARP is an energy-consuming enzyme which transfersADP ribose units to nuclear proteins. As a result ofthis process, intracellular NAD+ and ATP levels decreaseremarkably, resulting in EC dysfunction. On the basisof these findings, the balance between the generation ofROS, NO and peroxynitrite in ECs and VSMCscould be an important determinant of the pathologicalfunction of AngII in mediating endothelial dysfunction.In addition, a study has shown that AngII, through theAT1 receptor, inhibits insulin-induced NO productionin HUVECs (human umbilical vein ECs) by increasedsite-specific serine phosphorylation on IRS-1 [103]. InHUVECs, AngII induces phosphorylation of IRS-1



Table 1 AT1 receptor C-terminal-tail-specific-associated proteins

Protein identified Screened cDNA library Size Tissue distribution Cellular distribution Reference

ATRAP Yeast two-hybrid mouse kidney 18 kDa Kidney, heart and testis Three transmembrane protein [114]ARAP1 Yeast two-hybrid mouse embryo 57 kDa Ubiquitous Cytosolic protein [120]GLP Yeast two-hybrid mouse embryo 58 kDa Kidney, pancreas and heart Cytosolic protein [121]EP24.15 Yeast two-hybrid embryonic kidney 75 kDa Brain, pituitary and testis Cytosolic protein [123]

Figure 3 Specific AT1 receptor C-terminal tail-associated proteins and their functionsATRAP acts as a negative regulator of AT1-receptor-induced signal transduction, whereas ARAP1 and GLP appear to be positive regulators of AT1 . The function ofEP24.15 remains unknown.

at Ser312 and Ser616 by JNK and ERK respectively.Thus inhibition of JNK and ERK activity reversedthe negative effects of AngII on insulin-stimulatedNO production [103]. Furthermore, AngII induces theexpression of ICAM-1 (intercellular adhesion molecule-1) and VCAM-1 (vascular cell adhesion molecule-1)through NF-κB in ECs, and this cascade involves ROSand p38MAPK activation [104]. Although the AT2

receptor is not the focus of this review, it should benoted that AT2 receptors expressed in the endotheliummay counteract the effects of AT1 to prevent endothelialdysfunction, possibly through NO production [86,105].eNOS phosphorylation at Ser1179 through AT2 receptorswas also reported in aortic ECs [106]. The roles of ROSand protein kinases in mediating endothelial dysfunctioninduced by AngII are illustrated in Figure 2.

AT1 RECEPTOR C-TERMINALTAIL-ASSOCIATED PROTEINS

GPCRs interact with different classes of intracellularproteins, including heterotrimeric G-proteins, GRKs(GPCR kinases) and arrestins in general [107,108].

Although the intracellular third loop of the AT1 receptoris a key structural determinant in coupling the recep-tor to heterotrimeric G-proteins, a number of studieshave highlighted the functional importance of the C-terminal cytoplasmic domain in AT1 receptor signallingand desensitization and internalization [6,17,18,109]. TheC-terminal cytoplasmic domain of the AT1 receptor hasbeen reported to directly associate with several non-G-protein signalling molecules, such as JAK2 and PLCγ 1[110,111]. The C-terminal tail also provides a binding siteto form homo- and hetero-dimers of the AT1 receptor[112,113]. Therefore, in addition to desensitization andinternalization, the C-terminal cytoplasmic domain ofthe AT1 receptor appears to have critical roles in signaltransduction and other receptor functions [6], and alsosuggests a possibility that the C-terminal cytoplasmicdomain of the AT1 receptor might interact with pre-viously unrecognized cellular proteins that are uniqueto the AT1 receptor, which may be a key determinantin the efficacy and/or specificity of receptor function(Table 1).

ATRAP (AT1-receptor-associated protein) was iden-tified as a specific binding protein to the C-terminalcytoplasmic domain of the AT1 receptor by using a



yeast two-hybrid analysis [114]. ATRAP is a three-trans-membrane protein and may function as a negativeregulator of AT1-receptor-induced signal transduction[115,116]. Overexpression of ATRAP in VSMCspotentiated AT1 receptor internalization upon AngIIstimulation [117]. AngII-induced DNA synthesis wasmarkedly inhibited in these VSMCs and was associatedwith the inhibition of STAT3 (signal transducer andactivator of transcription 3) and Akt activation. In cardiacmyocytes, overexpression of ATRAP also decreases AT1

receptors on the cell surface, and decreases subsequentp38MAPK activation, c-fos promoter activation andprotein synthesis induced by AngII [118]. Although AT-RAP transgenic mice had no significant phenotype, neo-intimal formation after vascular injury was attenuated andthis was associated with reduced activity of ERK, STAT1and STAT3 [119].

Two other AT1 C-terminal tail-associated proteins,ARAP1 (type 1 AngII receptor-associated protein 1)and GLP (GEF-like protein), have been identified bythe yeast two-hybrid system [120,121]. ARAP1 appearsto bind and promote the AT1 receptor recycling tothe plasma membrane. Interestingly, ARAP1 transgenicmice that overexpress ARAP1 specifically in proximaltubules have hypertension [122], suggesting that renalARAP1 increases blood pressure through enhancementof the intrarenal AT1 signalling. GLP, a cytosolic protein,contains a motif of seven tandem repeats similar toa kind of GEF for the small G-protein Ran [121].Overexpression of GLP induced hypertrophy in VSMCsand renal proximal tubular cells. The hypertrophic effectis, at least in part, mediated through Akt activation andinhibition of p27kip1 protein expression [121]. Takentogether, these data suggest that the AngII-inducedhypertension and hypertrophy might be positively reg-ulated by these AT1 receptor C-terminal tail-associatedproteins (Figure 3). EP24.15 (thimet oligopeptidase; EC3.4.24.15) associates with the C-terminal cytoplasmicdomain of the AT1 receptor and the B2 bradykininreceptor [123]; however, the exact function of EP24.15remains unclear.

CONCLUSIONS

As described above, select findings of the AngII/AT1

signal transduction pathway in the cardiovascular systemdemonstrate progress in this area and are updated in thepresent review. Each of the AngII signalling componentscould play a specific role in mediating vascular re-modelling or endothelial dysfunction, leading to cardio-vascular diseases, such as hypertension, atherosclerosisand restenosis. However, since most of the findingsdiscussed in this review are from experiments at thecellular level, it is still unclear whether these cascadesare sufficiently applicable to explain human diseases.Therefore, in order to expand these research areas, the

confirmation of the findings by animal models or humansamples will definitely be necessary. In addition, furtherprogress on AngII signal transduction research by usinggenomic and proteomic approaches will help to betterunderstand the mechanism of cardiovascular diseases andits complications.

ACKNOWLEDGMENTS

This work was supported by National Institute of HealthGrants HL076770 (S. E.) and HL076575 (G. D. F.), byan American Heart Association Established InvestigatorAward 0740042N (S. E.), and by W. W. Smith CharitableTrust Grant H0605 (S. E.).

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Received 1 December 2006/3 January 2007; accepted 5 January 2007Published on the Internet 13 March 2007, doi:10.1042/CS20060342


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