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(Circulation Research. 1997;80:607-616.)
© 1997 American Heart Association, Inc.

Articles

Angiotensin II Signal Transduction in Vascular Smooth Muscle

Role of Tyrosine Kinases

Bradford C. Berk, , Marshall A. Corson

From the University of Washington, Department of Medicine, Cardiology Division, Seattle.

Correspondence to Bradford C. Berk, MD, PhD, University of Washington, Department of Medicine, Mail Stop 357710, Seattle, WA 98195. E-mail bcberk{at}u.washington.edu

	Abstract

Top Abstract Introduction The AT1 Receptor:... The AT1 Receptor:... Overview of Tyrosine Kinase... Interactions With Tyrosine... Tyrosine Phosphorylation of... Conclusion References

 
Abstract In this review, the role of tyrosine kinases in angiotensin II–mediated signal transduction pathways in vascular smooth muscle is discussed. Angiotensin II was isolated by virtue of its vasoconstrictor abilities and has long been thought to play a critical role in hypertension. However, recent studies indicate important roles for angiotensin II in inflammation, atherosclerosis, and congestive heart failure. The expanding role of angiotensin II indicates that multiple signal transduction pathways are likely to be activated in a tissue-specific manner. Exciting recent data show that angiotensin II directly stimulates tyrosine kinases, including pp60^c-src kinase (c-Src), focal adhesion kinase (FAK), and Janus kinases (JAK2 and TYK2). Angiotensin II may activate receptor tyrosine kinases, such as Axl and platelet-derived growth factor, by as-yet-undefined autocrine mechanisms. Finally, unknown tyrosine kinases may mediate tyrosine phosphorylation of Shc, Raf, and phospholipase C- after angiotensin II stimulation. These angiotensin II–regulated tyrosine kinases appear to be required for angiotensin II effects, such as vasoconstriction, proto-oncogene expression, and protein synthesis, on the basis of studies with tyrosine kinase inhibitors. Thus, understanding angiotensin II–stimulated signaling events, especially those related to tyrosine kinase activity, may form the basis for the development of new therapies for cardiovascular diseases.

Key Words: tyrosine kinase • signal transduction • Src • focal adhesion kinase • platelet-derived growth factor

	Introduction

Top Abstract Introduction The AT1 Receptor:... The AT1 Receptor:... Overview of Tyrosine Kinase... Interactions With Tyrosine... Tyrosine Phosphorylation of... Conclusion References

 
Angiotensin II, the dominant effector of the renin-angiotensin system, regulates numerous physiological responses, including salt and water balance, blood pressure, and vascular tone. Angiotensin II is produced both systemically and locally in the vessel wall by the actions of renin, which converts angiotensinogen into angiotensin I, and ACE, which cleaves angiotensin I to form angiotensin II. Although the vasoactive effects of angiotensin II have been well described for >40 years,¹ it has recently been appreciated that angiotensin II has many other effects on cardiovascular function. For example, angiotensin II has been shown to stimulate VSMC growth,^{2 3} increase the expression of enzymes that produce mediators of inflammation, such as phospholipase A₂ and NAD(P)H oxidase,^{4 5} stimulate the JAK/STAT pathway,⁶ and activate gene transcription of proto-oncogenes, such as c-fos.⁷ These data suggest that angiotensin II plays an important role in various cardiovascular diseases associated with VSMC growth and vessel wall inflammation, such as hypertension, atherosclerosis, and restenosis following interventional procedures. Clinical trials with ACE inhibitors demonstrating survival benefits in congestive heart failure⁸ and myocardial infarction⁹ support the importance of angiotensin II in the pathogenesis of cardiovascular disease. In this review we will focus on signal transduction mechanisms by which angiotensin II exerts these effects, emphasizing the role of tyrosine kinases.

	The AT1 Receptor: Binding of Angiotensin II

Top Abstract Introduction The AT1 Receptor:... The AT1 Receptor:... Overview of Tyrosine Kinase... Interactions With Tyrosine... Tyrosine Phosphorylation of... Conclusion References

 
Angiotensin II binds to at least two high-affinity receptors, designated AT₁ receptor^{10 11} and AT₂ receptor.¹² To date, the signal transduction pathways activated by the AT₂ receptor remain unknown, so this review will focus on AT₁ signaling. The signal transduction events stimulated by angiotensin II binding to the AT₁ receptor are similar to those stimulated by growth factors and cytokines and include activation of PLC,¹³ calcium mobilization,¹⁴ activation of PKC,¹⁵ induction of proto-oncogenes,⁷ protein tyrosine phosphorylation,^{16 17 18} and activation of the 42- and 44-kD MAP kinases.^{19 20}

Analysis of AT₁ receptor domains required for binding angiotensin II, stimulating G protein activity, and mediating downstream signal transduction became possible when the AT₁ receptor was cloned by two laboratories in 1991.^{10 11} The structure of the AT₁ receptor shows that it is a member of the seven transmembrane–spanning, G protein–coupled receptor family. Other receptors in this family include the - and ß-adrenergic receptors, the muscarinic acetylcholine receptor, the various serotonin receptors, and important vascular receptors such as the thrombin, bradykinin, and endothelin receptors. These receptors share the property that they bind to heterotrimeric G proteins and lack intrinsic tyrosine kinase activity, in contrast to growth factor receptors, such as the PDGF, FGF, and EGF receptors. Nonetheless, it has become clear that many angiotensin II effects require tyrosine phosphorylation, as shown by studies with tyrosine kinase inhibitors. For example, genistein, a tyrosine kinase inhibitor, blocks angiotensin II–stimulated MAP kinase (D.-F. Liao, unpublished data, 1996) and JNK activity²¹ ; vessel contraction in response to both EGF and angiotensin II is inhibited by genistein²² ; and angiotensin II–stimulated protein synthesis is blocked by genistein and herbimycin A.²³ Because of the general importance of tyrosine phosphorylation in cell growth, inflammation, and migration, recent studies have focused on defining the tyrosine kinases activated by angiotensin II.

Insights into the nature of angiotensin II signaling have been gained from structure function analysis of receptor mutations (both deletion and site-directed mutagenesis). Based on its cDNA sequence, the AT₁ receptor is composed of 359 amino acids with seven transmembrane domains arranged as shown in Fig 1. Interactions among these domains and the corresponding intracellular loops are responsible for determining the nature of angiotensin II binding and receptor activation after binding. For example, the angiotensin II binding site appears to be the result of interactions among several of these domains, including the NH2 terminal extension, Tyr⁹² in extracellular loop 1, extracellular loop 3 (ASP²⁷⁸ and ASP²⁸¹), Tyr²⁹², and Tyr²¹⁵ (Fig 1).^{24 25 26 27} Several transmembrane domains are responsible for G-protein interactions with the receptor, including domains present in the second and third intracellular loops.^{27 28 29} Recent modeling analysis suggests that an interaction between Asp⁷⁴ and Tyr²⁹² is critical for angiotensin II binding and AT₁ receptor activation.³⁰ Site-directed mutagenesis has established that Asp⁷⁴, a conserved residue within the second transmembrane domain, is important in G-protein binding and activation,^{26 29} as are the conserved residues Tyr²⁹²³⁰ and Tyr^2l5.³¹ In addition, the motif Asp¹²⁵-Arg¹²⁶-Tyr¹²⁷ in the amino terminal region of the second cytoplasmic loop is important in G-protein activation.²⁹ These same residues may also be important in angiotensin II–stimulated growth.²⁶

View larger version (41K): [in this window] [in a new window]   Figure 1. Representation of amino acids and signaling domains in the rat AT1a receptor. The helixes were positioned on the basis of modeling of G protein–coupled receptors. Depicted are Ser, Thr, and Tyr residues, as well as residues thought to be involved in specific signal transduction events. Highlighted residues in transmembrane domains may be important in ligand binding even if they are not phosphorylated.

	The AT1 Receptor: Role of Receptor Phosphorylation in Signal Transduction and Desensitization

Top Abstract Introduction The AT1 Receptor:... The AT1 Receptor:... Overview of Tyrosine Kinase... Interactions With Tyrosine... Tyrosine Phosphorylation of... Conclusion References

 
AT₁ receptor phosphorylation may serve at least three functions: desensitization of receptor signaling,³² receptor internalization,³³ and binding of signal transduction molecules.⁶ In this review, we will use the following terms: desensitization, for the decrease in physiological response to a constant agonist stimulus that occurs over time; uncoupling, for the rapid (seconds to minutes) decrease in receptor signal transduction despite persistent ligand binding; sequestration (or internalization), for the rapid (minutes to hours) removal of receptor from plasma membrane without change in total cellular receptor number; and downregulation, for the decrease in total receptor number present in cells, which requires hours to days.³⁴ Phosphorylation appears to be a critical event in the uncoupling of G protein–coupled receptors.^{32 35} Phosphorylation is also likely important in sequestration of certain G protein–coupled receptors, such as the ß₂-adrenergic receptor.³⁶ G protein–coupled receptors do not contain intrinsic kinase activities but are nonetheless phosphorylated on serine and threonine residues by members of the GRK family.³⁷ Phosphorylation of the ß₂-adrenergic receptor by ßARK-1 or ßARK-2 (now termed GRK-2 and GRK-3, respectively) is likely important for receptor uncoupling^{35 38} and sequestration.³⁶

It appears that the phosphorylation-dependent mechanisms responsible for sequestration and downregulation of the AT₁ receptor will be different from the ß₂-adrenergic receptor.^{33 39 40} Recently our group⁴¹ and others⁴² have shown that the AT₁ receptor in vascular smooth muscle is phosphorylated basally and in response to angiotensin II. The majority of phosphorylation in vivo was found on serine, and there was a small increase in total phosphoserine content after agonist exposure. GRK phosphorylation domains in G protein–coupled receptors include the third intracellular loop and the carboxyl-terminal tail.³⁷ Although multiple serines may be phosphorylated by GRKs, it appears that only the initial one or two phosphorylations are physiologically relevant. These sites, in a number of receptors, are characterized by a pair of acidic amino acids located amino terminal to the initial site of phosphorylation,⁴³ such as Asp/Glu-Asp/Glu-X_(0-5)-Ser/Thr. Recently, it was shown in transfected 293 cells that the AT₁ receptor can be phosphorylated by GRK2, GRK3, and GRK5.³² However, the actual role of the GRKs in phosphorylation of the AT₁ receptor in relevant tissues such as vascular smooth muscle remains speculative. It has been shown that AT₁ receptor sequestration is regulated by a motif in the cytoplasmic tail involving residues carboxyl to Tyr³¹²³⁹ and to Leu³¹⁴³³ (Fig 1). This region lacks highly acidic amino acids typical of the GRK phosphorylation motif (especially Asp and Glu). However, one region, Asp³⁴³-Asn³⁴⁴-Met³⁴⁵-Ser³⁴⁶-Ser³⁴⁷-Ser³⁴⁸, has partial homology to the consensus GRK phosphorylation motif. In summary, serine phosphorylation may play a role in AT₁ receptor desensitization, although the precise phosphorylation sites remain to be determined.

Recent studies have suggested that tyrosine residues in the carboxyl tail may also be important in G protein–coupled receptor sequestration. Specifically, Ferguson et al³⁶ identified a highly conserved tyrosine (Y326) in the motif NPXXY, which is involved in the sequestration of the ß₂-adrenergic receptor, although these investigators did not demonstrate tyrosine phosphorylation at this site. This motif is present in the AT₁ receptor at Tyr³⁰² (NPLFY). However, AT₁ receptors in which Tyr³⁰² was mutated to Ala or in which triple alanine replacements of Phe³⁰¹, Tyr³⁰², and Phe³⁰⁴ were performed showed no substantial changes in their internalization kinetics.⁴⁰ More recently, an AT₁ receptor truncated at Leu³¹⁴, which still contained the NPXXY motif, failed to show normal receptor sequestration.³³ These findings demonstrate that the NPLFY sequence of the AT₁ receptor is not an important determinant of agonist-induced sequestration, unlike the ß₂-adrenergic receptor.

Tyrosine phosphorylation of the AT₁ receptor has been shown to occur,^{41 42} suggesting a role in other receptor-mediated events such as signal transduction. There is controversy regarding the extent to which tyrosine phosphorylation is regulated by exposure to angiotensin II. Immunoprecipitation of the AT₁ receptor and Western blot analysis using anti-phosphotyrosine antibody showed a small increase in agonist-dependent tyrosine phosphorylation up to 6 minutes after angiotensin II treatment.⁴⁴ In contrast, when VSMCs were metabolically labeled with ³²P-orthophosphate and stimulated with angiotensin II and the AT₁ receptor was immunoprecipitated, a slow increase in phosphotyrosine content was observed.⁴² It should be noted that both of these techniques measure total phosphotyrosine content; increases and decreases in phosphorylation of individual tyrosine residues and the actual site(s) of AT₁ receptor phosphorylation have not been determined after angiotensin II binding. Tyr³⁰² appears to be important in signal transduction, because two groups^{40 45} have shown that mutating this residue to Ala or Phe caused alterations in G-protein activation and IP₃ production. Tyr³¹⁹ is of interest because it is part of the motif Tyr-Ile-Pro-Pro, which is analogous to SH2 binding motifs found within the PDGF receptor (Tyr-Ile-Ile-Pro) and within the EGF receptor (Tyr-Leu-Pro-Pro).^{46 47} In the PDGF and EGF receptors, these motifs have been shown to be target sequences for signaling mediators when the tyrosine is phosphorylated. SH2 binding domains contain a critical tyrosine residue that, when phosphorylated, promotes interactions with signaling proteins that contain the SH2 domain. For example, PLC- contains an SH2 domain that interacts with the Tyr-Ile-Ile-Pro and Tyr-Leu-Pro-Pro sequences present in the PDGF and EGF receptors.⁴⁷ Thus, tyrosine phosphorylation of the AT₁ receptor may be important in signal transduction events mediated by binding of SH2 domain–containing proteins.

	Overview of Tyrosine Kinase Signal Transduction by the AT1 Receptor

Top Abstract Introduction The AT1 Receptor:... The AT1 Receptor:... Overview of Tyrosine Kinase... Interactions With Tyrosine... Tyrosine Phosphorylation of... Conclusion References

 
Recent findings demonstrate that the AT₁ receptor couples to many intracellular signal transduction events. Five major tyrosine kinase–regulated pathways will be discussed in this review on the basis of activation of specific kinases shown to be regulated by angiotensin II in VSMCs (Figs 2 and 3): Janus kinases (JAK and TYK), c-Src, FAK, receptor tyrosine kinases (Axl, EGF, and PDGF), and calcium-dependent tyrosine kinases (eg, Pyk2) that phosphorylate signal substrates such as Shc, Raf, and PLC-. The purpose of these multiple receptor-activated kinases is to provide an integrated series of regulated cellular events. It is unlikely that all these effects occur simultaneously in vivo. Thus, an important area of future research is to determine the physiological variables and tissue-specific factors that determine the relative extent to which these signal transduction pathways are activated.

View larger version (39K): [in this window] [in a new window]   Figure 2. Summary of tyrosine kinase–mediated signal transduction pathways stimulated by angiotensin II in VSMCs. Depicted are several cytosolic and transmembrane protein kinases potentially activated by angiotensin II in VSMCs. Phosphorylation of the linker protein Shc is hypothesized to be mediated by Src, FAK, and other unidentified tyrosine kinases (TyrKs). Of note, it has recently been demonstrated that Pyk2 and Src may form a complex that leads to Src activation.80 These kinases are also candidates to phosphorylate Raf. Shc may also be phosphorylated in a calcium-dependent manner by Pyk2 (or Pyk2-related kinases). Transactivation of Axl, EGF, PDGF, and receptor tyrosine kinases is postulated by unknown means. Note that JAK2 and TYK2, which are directly activated at the receptor, are excluded from this figure.

View larger version (52K): [in this window] [in a new window]   Figure 3. Role of tyrosine kinases in induction of c-fos expression by angiotensin II. Angiotensin II binding stimulates tyrosine phosphorylation of the linker protein Shc, which then interacts with the GRB2-Sos complex, causing activation of Ras. Ras/Raf then stimulates ERK1/2, which phosphorylates substrates such as the transcription factor p62TCF. p62TCF translocates to the nucleus, where it forms a complex at the SRE of the c-fos gene. The c-fos promoter also contains a sis-inducing factor element (SIE), which interacts with members of the STAT family of transcription factors. Angiotensin II stimulates the phosphorylation and activity of STAT91 and STAT113 through the action of JAK2, thus providing a direct link between the AT1 receptor and the nucleus. In VSMCs, angiotensin II increases production of prostaglandins, thereby activating adenyl cyclase, increasing cAMP, and stimulating protein kinase A (PKA). In the adrenal gland, the AT1 receptor may be coupled to Gi, thereby inhibiting adenyl cyclase. Finally, angiotensin II stimulates increases in diacylglycerol and intracellular calcium, thereby activating PKC.

JAK and TYK
Recent studies indicate that the AT₁ receptor shares properties with cytokine receptors, such as the interleukin-2, IFN-, and IFN- receptors. Similar to these classical cytokine receptors, the AT₁ receptor stimulates tyrosine phosphorylation, activates the MAP kinase pathway, and induces c-fos mRNA expression (Fig 3).^{7 16 17 18 19 20} In the case of the cytokine receptors, tyrosine phosphorylation is mediated by protein tyrosine kinases that associate with the receptor, including the Src-related kinases Lck and Fyn⁴⁸ and the Janus family kinases (JAK and TYK).⁴⁹ The Janus family kinases are key mediators of mRNA expression characterized as "early growth response genes." Transcription of these genes, exemplified by c-fos and c-jun, does not require prior synthesis of any other new gene products. Recent studies have established that JAK and TYK associate with membrane receptors and, when activated, stimulate tyrosine phosphorylation of a family of transcription factors termed STAT.⁵⁰ The STAT proteins translocate to the nucleus, where they bind to SIEs and stimulate transcription of early growth response genes. We recently found that angiotensin II rapidly activates JAK2 and TYK2 and stimulates tyrosine phosphorylation of STAT113 in VSMCs,⁶ and Baker's group showed that angiotensin II activates STAT91 in cultured neonatal cardiac fibroblasts.⁵¹ These data support the role of angiotensin II as a cytokine similar to IFN- and IFN- (Table).^{49 52} In addition, JAK2 was found to immunoprecipitate with the AT₁ receptor, suggesting a role for this kinase in mediating the earliest events activated by angiotensin II.⁶ These observations and the recent demonstration that thrombin also activates JAK2⁵³ indicate the great extent to which G protein–coupled receptors share signal mechanisms activated by cytokine and growth factor receptors.

View this table: [in this window] [in a new window]   Table 1. JAK and TYK Activation by Ang II, Cytokines, and Growth Factors

Src Family Kinases
The Src family kinases are likely candidates to mediate AT₁ receptor signal events, on the basis of the recent demonstration that angiotensin II stimulates tyrosine phosphorylation of PLC-,⁶ a Src substrate.⁵⁴ The 60-kD c-Src is the best characterized member of a family of nine cytoplasmic protein tyrosine kinases that participate in growth factor signal transduction. Tissue-specific expression of alternatively spliced gene products yields at least 14 different Src-related kinases.⁵⁵ Three family members (c-Src, Fyn, and Yes) are expressed ubiquitously and appear to have partially overlapping functions, on the basis of studies with transgenic mice.⁴⁶ Functional domains shared by Src family kinases include an amino-terminal myristoylation sequence for membrane targeting, SH2 and SH3 domains, a kinase domain, and a carboxy-terminal noncatalytic domain. These regions participate in a complex tonic inhibition of Src family kinases that can be overcome in cells exposed to mitogens. One of the residues that appears to be critical for regulation of c-Src is Tyr⁵³⁰, which is not present in v-Src. Phosphorylation of Tyr⁵³⁰ by Csk inhibits c-Src activity,⁵⁶ whereas dephosphorylation of this residue appears to be an activating mechanism. Autophosphorylation of Tyr⁴¹⁹ in the catalytic domain may be an activating signal. c-Src activity is also inhibited via intramolecular interactions of the carboxy-terminal catalytic domains with both the SH2 and SH3 domains.⁵⁷ These SH2 and SH3 domains probably also stimulate c-Src activity through interactions with regulators and downstream/kinase substrates.

Recent studies from several laboratories,^{58 59} including our laboratory,⁶⁰ suggest that c-Src plays an important role in angiotensin II signal transduction. We demonstrated that angiotensin II stimulation of VSMCs was associated with a rapid activation of c-Src.⁶⁰ Specifically, angiotensin II stimulated a 2- to 3-fold increase in activity of c-Src within 2 minutes, measured by either autophosphorylation or kinase activity toward enolase.⁶⁰ Bernstein's laboratory⁵⁹ studied the functional consequences of inhibiting c-Src activity by electroporating a monoclonal anti–c-Src antibody into cultured VSMCs. They demonstrated significantly greater inhibition of PLC- phosphorylation and IP₃ formation by the anti–c-Src antibody compared with purified murine IgG. Data from Izumo's laboratory⁶¹ support the importance of Src family kinases as mediators of angiotensin II function. They showed in cardiac fibroblasts that angiotensin II activated both c-Src and Fyn.⁵³ These findings demonstrate that activation of Src family kinases is one of the earliest signal events stimulated by angiotensin II and strongly suggest that c-Src is involved in angiotensin II–stimulated tyrosine phosphorylation of PLC-.

FAK
Because angiotensin II stimulates changes in smooth muscle cell shape and volume and promotes migration, alterations in cell-matrix interaction are likely to be critical to angiotensin II signal transduction. It has become clear that focal adhesion complexes, specialized sites of cell adhesion, act as supramolecular structures for the assembly of signal transduction mediators. The best characterized tyrosine kinase localized to focal adhesion complexes is a 125-kD protein termed FAK. This protein was originally isolated from v-Src–transformed chicken embryo fibroblasts by Kanner et al.⁶² FAK exhibits protein tyrosine kinase activity toward other proteins with which it colocalizes at these sites, such as paxillin.⁶³ FAK lacks modular domains such as SH2 or SH3 and resembles known protein tyrosine kinases only in its catalytic domain. FAK is autophosphorylated at Tyr³⁹⁷ in resting substrate-attached cells, and it possesses sites favored for phosphorylation by Src, such as tyrosines 407, 576, and 577. Phosphorylation of these tyrosines markedly increases the protein tyrosine kinase activity of FAK.⁶⁴ FAK appears to play an important role in events leading to cell proliferation, as suggested by the finding in v-Src–transformed fibroblasts that hyperphosphorylation of FAK occurs in concert with the loss of a requirement for cell attachment for growth.⁶⁵

Angiotensin II rapidly stimulates tyrosine phosphorylation of FAK in cultured VSMCs.^{66 67} Tyrosine phosphorylation of FAK appears to be due to autophosphorylation and activation by unclear mechanisms, although major roles for Src and Csk have been proposed, with an initial event, possibly attachment-dependent activation of Csk, enhancing FAK autophosphorylation.⁶⁸ The upstream activators of Csk are unknown, but it has been proposed that activation by G protein–coupled receptors may be mediated by the Rho family of GTPases, as suggested by studies using the Rho inhibitor C3 botulinum exoenzyme.⁶⁹ These events have in common the formation of a multimeric complex at focal adhesion contacts, including adaptors such as paxillin and SH2-rich p130^Cas,⁷⁰ GTPases such as Sos and dynamin,^{71 72} and effectors, such as PLC-.⁷³ Autophosphorylation of FAK on tyrosine generates a site for binding of SH2 domain–containing proteins and leads to recruitment and activation of Src-like protein kinases including c-Src itself, Fyn, and Csk.^{74 75} The signal transduction particle consisting of FAK and a Src-like kinase then phosphorylates paxillin.⁶³ It has become clear that phosphorylation of paxillin in response to angiotensin II is one of the earliest and most prominent tyrosine phosphorylation events in VSMCs.^{66 67 76 77} The functional significance of this activation and the alterations in integrin signal transduction remain unknown.

Two laboratories have independently reported the cloning of a second FAK family member, denoted Pyk2⁷⁸ or cell adhesion kinase-ß.⁷⁹ In cells of neural origin, Pyk2 was found to be activated by G protein–coupled receptor agonists, PKC stimulation, or increased cytoplasmic calcium levels (eg, potassium depolarization or calcium ionophore). Pyk2 has been postulated as a potential link between calcium-dependent signaling pathways and protein tyrosine kinase pathways (Fig 2). Recently, Dikic et al⁸⁰ reported that activated Pyk2, like FAK, complexes with c-Src and that this interaction is required for Pyk2-mediated ERK activation. As such, Pyk2 is a candidate both to regulate c-Src and to link G protein–coupled vasoconstrictor receptors with protein tyrosine kinase–mediated contractile, migratory, and growth responses.

	Interactions With Tyrosine Kinase–Coupled Receptors

Top Abstract Introduction The AT1 Receptor:... The AT1 Receptor:... Overview of Tyrosine Kinase... Interactions With Tyrosine... Tyrosine Phosphorylation of... Conclusion References

 
Cross talk between G protein–coupled receptors and tyrosine kinase receptors has been shown recently by two groups^{81 82} to include rapid activation of tyrosine kinase receptors by agonist binding to G protein–coupled receptors. In one study, stimulation of Rat-1 cells with endothelin-1, lysophosphatidic acid, or thrombin stimulated a rapid increase in tyrosine phosphorylation of the EGF receptor and the neu oncoprotein.⁸¹ Specific inhibition of EGF receptor function by either the selective tyrphostin, AG1478, or a dominant-negative EGF receptor mutant suppressed ERK1/2 activation and strongly inhibited induction of fos gene expression and DNA synthesis by endothelin-1 or thrombin. A study by Rao et al⁸³ reported that thrombin stimulated tyrosine phosphorylation of the IGF-1 receptor as well as IRS-1 and PLC-. In a similar series of experiments,⁸² stimulation of smooth muscle cells with angiotensin II resulted in tyrosine phosphorylation of Shc proteins, complex formation between Shc and GRB2, and increased tyrosine phosphorylation of PDGF ß-receptors that coprecipitated with Shc-GRB2 complexes. Autocrine release of PDGF failed to account for Shc complex formation at the PDGF receptor after angiotensin II treatment, and a specific angiotensin II type I receptor antagonist, losartan, abolished the response. These studies suggest that angiotensin II signal transduction is mediated in part by PDGF (EGF?) receptor transactivation. However, the number of distinct cytoplasmic and membrane tyrosine kinases that become activated after AT₁ receptor stimulation suggest that a general "autocrine" mechanism is unlikely and that specific intracellular events mediate the transactivation that has been reported.

In addition to signal events mediated by rapid activation of tyrosine kinase receptors, angiotensin II may exert long-term autocrine growth effects through interactions with receptor tyrosine kinases, such as Axl (also called UFO or Ark⁸⁴ ). Axl was originally cloned as a transforming gene from human myeloid leukemia cells.⁸⁵ Axl is a member of a family of cell adhesion molecule–related tyrosine kinase receptors that includes the Rse/Tyro3 proteins (also called Sky, Brt, or Tif⁸⁶ ) and the Nyk/Mer proteins.⁸⁷ The ligand for Axl was recently shown to be Gas6,⁸⁸ a 70-kD -carboxyglutamic acid–containing protein related to protein S. Analysis of the structure of Gas6 showed that only the tandem globular domains (related to domains used by laminin and agrin for binding to the dystroglycan complex) were required for receptor binding and autophosphorylation.⁸⁹ Gas6 protein was originally isolated as a growth-potentiating factor from the conditioned medium of VSMCs.⁸⁸ Concomitant stimulation of VSMCs with Gas6 (activation of Axl) and thrombin or endothelin (activation of their respective G protein–coupled receptors) caused a synergistic increase in DNA synthesis. More recently, signal transduction events stimulated by Axl⁹⁰ and the related Nyk/Mer⁸⁷ were shown to include PLC-, p70 S6 kinase, Shc, GRB2, Raf-1, and ERK1/2, demonstrating important similarities to angiotensin II signal transduction.

	Tyrosine Phosphorylation of Signal Mediators

Top Abstract Introduction The AT1 Receptor:... The AT1 Receptor:... Overview of Tyrosine Kinase... Interactions With Tyrosine... Tyrosine Phosphorylation of... Conclusion References

 
PLC
Angiotensin II binding to the AT₁ receptor stimulates the phosphoinositide-specific PLC to hydrolyze phosphatidylinositol 4,5-bisphosphate, thereby generating the second messengers IP₃ and diacylglycerol. PLC is a family of at least three related genes: PLC-ß, PLC-, and PLC-.⁹¹ Isoforms of PLC-ß are differentially regulated by G-protein and ß subunits.^{92 93} In contrast, the PLC- isoforms are regulated by tyrosine phosphorylation.⁹⁴ Regulation of PLC- isoforms is unclear but may involve changes in intracellular calcium. For most G protein–coupled receptors, it appears that PLC-ß is critical for production of IP₃. Two different, but not mutually exclusive, mechanisms may account for IP₃ formation by angiotensin II. Similar to G protein–coupled receptors, such as the ₁-adrenergic receptor, G_q activated by angiotensin II may stimulate PLC-ß. Alternatively, angiotensin II may activate PLC-, via tyrosine phosphorylation. This distinction is important because activation of calcium-dependent tyrosine kinases, such as Pyk2/CAK-ß, would be "downstream" from PLC-, if tyrosine phosphorylation of PLC- is the primary mechanism by which angiotensin II increases intracellular calcium. Alternatively, if angiotensin II increases calcium by stimulating PLC-ß (via G_q), then activation of Pyk2/CAK-ß would be an important "upstream" tyrosine kinase event.

Our group has made several observations suggesting that in VSMCs (especially in culture or in neointima) activation of PLC by angiotensin II is mediated by tyrosine phosphorylation of PLC-. First, we observed in cultured rat aortic VSMCs that PLC-, but not PLC-ß or PLC-, was expressed constitutively. Second, angiotensin II activated the PLC- isoform, as shown by rapid tyrosine phosphorylation.⁹⁵ Third, the time course for tyrosine phosphorylation of PLC- was very similar to the time course for IP₃ formation, suggesting that PLC- activation by tyrosine phosphorylation is responsible for IP₃ generation. Fourth, pretreating VSMCs with Na₂VO₄, a potent inhibitor of tyrosine phosphatases, increased subsequent angiotensin II–induced IP₃ formation.⁴⁴ Fifth, when c-Src activity was inhibited by electroporation of anti–c-Src antibodies, there was a corresponding inhibition of PLC- phosphorylation and IP₃ formation, suggesting that PLC- is the predominant pathway for IP₃ formation in response to angiotensin II in cultured VSMCs.⁵⁹ All these responses were completely inhibited by losartan, indicating that the AT₁ receptor is the receptor by which angiotensin II stimulates PLC-. In light of the anti–c-Src antibody electroporation studies discussed above, these data support the concept that c-Src is likely to be the angiotensin II–stimulated PLC- tyrosine kinase in VSMCs. However, we have been unable to coprecipitate c-Src and PLC- (U. Schmitz, unpublished data, 1996), suggesting that another kinase and/or linker protein may mediate c-Src–dependent activation of PLC-.

c-Raf-1
Historically, the first molecules to be identified as being tyrosine-phosphorylated in response to angiotensin II were the 44- and 42-kD MAP kinases, also termed ERK1/2.^{19 20} Activation of ERK1/2 requires dual phosphorylation on threonine and tyrosine residues found within the motif Thr-Glu-Tyr, which is mediated by the MAP kinase kinase or MEK. MEK is, in turn, regulated by serine (and probably tyrosine) phosphorylation by the kinase c-Raf-1, although Raf-independent pathways for ERK1/2 activation have also been proposed.⁹⁶ Angiotensin II has been shown to stimulate serine/threonine phosphorylation of Raf-1,^{18 97 98} although the significance of serine/threonine phosphorylation for Raf-1 activity is uncertain. We have recently found that PKC downregulation inhibits angiotensin II–stimulated Raf-1 association with Ras but does not inhibit ERK1/2 activation, further suggesting that Raf-1 may not be required for MEK activation in VSMCs.⁹⁹ More recently, we have observed that PKC may interact with Ras and appears to be required for activation of ERK1/2 (Fig 2).¹⁰⁰ It is clear that recruitment of Raf-1 to the plasma membrane and interaction with the low-molecular-weight guanine nucleotide–binding protein Ras^{101 102} is required for Raf activity. In addition, tyrosine phosphorylation of Raf is an early signal event associated with translocation and likely activation.^{103 104 105} We have recently shown that angiotensin II stimulates tyrosine phosphorylation of Raf (T. Ishida, unpublished data, 1996), although the function of Raf tyrosine phosphorylation and the identity of the putative Raf tyrosine kinase remain undetermined.

Shc-GRB2-Sos-Ras
The role of Shc-GRB2-Sos and Ras in signal transduction by G_q-coupled receptors, such as the AT₁ receptor, is unclear. Importantly, tyrosine phosphorylation of Shc^{61 82 106} and activation of Ras by angiotensin II were recently demonstrated.¹⁰⁷ Tyrosine phosphorylation of a linker protein called Shc appears to be an important mechanism used by all G protein–coupled receptors on the basis of studies to date.¹⁰⁸ For G_i-coupled receptors, a pathway for activation of Ras and ERK1/2 has been proposed (Fig 2)^{106 107 108} in which release of G-protein ß subunits from G protein–coupled receptors stimulates downstream events leading to tyrosine phosphorylation of Shc.¹⁰⁹ c-Src has been proposed as a likely candidate.^{108 110 111} Additional signal mediators that may be required include PI 3-K and a protein tyrosine phosphatase.¹⁰⁸ Once Shc is tyrosine-phosphorylated, it now binds GRB2 via SH2 domain interactions (Fig 2). GRB2 also binds to the guanine nucleotide exchange factor Sos via SH3 domains.¹¹² Sos stimulates the release of GDP for GTP on Ras, which then recruits Raf to the plasma membrane, where it is then activated (probably via tyrosine and serine phosphorylation). Active Raf phosphorylates and activates MEK, which can then phosphorylate and activate ERK1/2. The evidence for this pathway is strong and includes the fact that transient expression of G-protein ß subunits in COS-7 and other cells stimulates ERK1/2 activity.^{113 114 115} Inhibiting G-protein ß subunit activity by expression of the ß subunit–binding site of ßARK blocks ERK1/2 activation by G_i-coupled receptors.¹¹⁰ The stimulatory effects of G-protein ß subunits were shown to be due to Ras activation, as demonstrated by increased Ras-GTP formation and the ability of dominant negative Ras to block ERK1/2 activation.¹¹³

In contrast to the well-defined role of G-protein ß subunits in signal transduction by G_i-coupled receptors, their role in signal events stimulated by G_q-coupled receptors, such as the AT₁ receptor, is much less clear. Crespo et al¹¹⁴ showed that overexpression of the subunit of transducin, to inhibit G-protein ß subunit function, significantly decreased ERK1/2 activation by the G_q-coupled m₁ muscarinic receptor. However, Faure et al¹¹⁵ showed that the subunit of transducin failed to block ERK1/2 activation by the G_q-coupled bombesin receptor. Further evidence that G-protein ß subunits do not mediate ERK1/2 activation by G_q-coupled receptors was demonstrated by failure of a truncated form of ßARK to inhibit ERK1/2 activation by the _1B-adrenergic and m₁ muscarinic receptors.^{110 113} Thus, it appears unlikely that G-protein ß subunits are necessary for angiotensin II–mediated activation of ERK1/2 in VSMCs.

There may be convergence of G_i- and G_q-coupled receptor signal transduction with regard to the tyrosine kinase(s) that phosphorylates Shc. Recently, van Biesen et al¹⁰⁹ demonstrated that the G-protein ß subunit–dependent pathway by which G_i-coupled receptors stimulated Shc phosphorylation and ERK1/2 activation was inhibited by genistein, implying that a tyrosine kinase was required. Because the G_i-coupled thrombin receptor was shown to increase c-Src autophosphorylation and activity,^{83 116} c-Src is a likely candidate to mediate these effects. The results with thrombin are very relevant for angiotensin II–mediated signal transduction, because both thrombin and angiotensin II stimulate genistein-dependent tyrosine phosphorylation of phospholipase C-1 in VSMCs.^{59 83} Angiotensin II was recently shown to stimulate tyrosine phosphorylation of Shc in cardiac fibroblasts,¹⁰⁶ cardiac myocytes,⁶¹ and smooth muscle cells.⁸² More recently, Touhara et al¹⁰⁸ showed that overexpression of G-protein ß subunits promoted Shc tyrosine phosphorylation, which was inhibited by wortmannin, implying a role for PI 3-K. We have shown similar results for angiotensin II signal transduction in VSMCs, including activation of c-Src,⁶⁰ c-Src–dependent stimulation of ERK1/2 (M. Ishida, unpublished data, 1996), and wortmannin-sensitive activation of ERK1/2 (D.-F. Liao, unpublished data, 1996). It has been suggested that Fyn (a Src family member) is the Shc tyrosine kinase in cardiac myocytes.⁶¹ Thus, a putative signal transduction cascade for angiotensin II may be proposed in which activation of c-Src (or Fyn) causes tyrosine phosphorylation of Shc, activation of Ras, and stimulation of ERK1/2 (Fig 2).

However, it is possible that angiotensin II–stimulated signal events may be mediated by tyrosine kinases other than c-Src. A candidate kinase is Pyk2, a calcium-dependent tyrosine kinase that phosphorylates Shc independent of G-protein ß subunit activation and thereby activates ERK1/2 in brain⁷⁸ (Fig 2). We have studied this pathway in VSMCs and can find no evidence for a Pyk2-mediated pathway. Specifically, inhibiting calcium mobilization in VSMCs failed to block angiotensin II–mediated ERK1/2 activation,¹¹⁷ and PCR screening failed to identify Pyk2 or a closely related intracellular tyrosine kinase (D. Wuthrich, unpublished data, 1996). Thus, the most likely tyrosine kinase to mediate angiotensin II–dependent activation of Ras is c-Src. Current studies with VSMCs from c-Src knockout mice and with cells expressing dominant-negative c-Src constructs should characterize the role of c-Src (and its dependence on G-protein ß subunits) in angiotensin II–mediated activation of Ras. However, the present findings suggest that multiple, possibly cell type–and receptor-specific, mechanisms may exist for Ras activation by G_q protein–coupled receptors, such as the AT₁ receptor. Questions that remain to be answered regarding activation of Ras by angiotensin II include the role of G-protein ß subunits (characterizing other calcium-activated tyrosine kinases, such as Pyk2, which may be important in angiotensin II–responsive tissues, such as brain, adrenal gland, and liver²¹ ) and the identity of tyrosine kinases in addition to c-Src that may phosphorylate Shc as well as Raf and PLC-1.

	Conclusion

Top Abstract Introduction The AT1 Receptor:... The AT1 Receptor:... Overview of Tyrosine Kinase... Interactions With Tyrosine... Tyrosine Phosphorylation of... Conclusion References

 
The concept that the AT₁ receptor acts as a multifunctional receptor by activating multiple tyrosine kinases is well illustrated by regulation of c-fos expression by angiotensin II (Fig 3). Angiotensin II induces c-fos mRNA expression rapidly in a PKC- and calcium-dependent fashion,⁷ requiring activation of PLC- by tyrosine phosphorylation. The c-fos promoter contains the SRE. Induction of the SRE occurs upon formation of a ternary complex with serum response factor, p62^TCF, and the SRE.¹¹⁸ ERK1/2 was shown to phosphorylate p62^TCF (also known as elk-1 or SAP-1), resulting in enhanced ternary complex formation.¹¹⁹ Thus, the SRE present in the c-fos promoter may be regulated by angiotensin II–stimulated ERK1/2 phosphorylation of p62^TCF, as shown for EGF.¹¹⁹ Activation of ERK1/2 requires activation of Ras and Raf, events that are regulated by tyrosine phosphorylation. Finally, an SIE is present in the c-fos promoter and interacts with members of the STAT family of transcription factors (including STAT91 and STAT113).⁴⁹ The diversity of signal transduction pathways stimulated by angiotensin II, which leads to the expression of c-fos and other early growth response genes, demonstrates the ability of angiotensin II to act as a cytokine and regulate more general cellular functions.

Angiotensin II was initially identified by its physiological role in regulation of salt and water metabolism, blood pressure, and vascular tone. However, as demonstrated in this review, angiotensin II may participate in a diversity of cellular functions via its effects mediated by the AT₁ receptor. Exciting new findings point to functions of the AT₁ receptor related to growth and vasoconstriction (mediated by c-Src or Fyn), transactivation by PDGF and Axl, and functions related to inflammation and migration (mediated by JAK, TYK, c-Src, and FAK). It is clear that future work will be required to determine the nature of the interactions among these different kinase pathways and their role in the pathogenesis of hypertension and atherosclerosis.

	Selected Abbreviations and Acronyms

 

ßARK = ß-adrenergic receptor kinase ACE = angiotensin-converting enzyme c-Src, v-Src = pp60c-src kinase, pp60v-src kinase Csk = c-Src kinase EGF, FGF, PDGF = epidermal, fibroblast, and platelet-derived growth factors ERK = extracellular signal–regulated kinase FAK = focal adhesion kinase GRK = G protein–coupled receptor kinase IFN = interferon IP3 = inositol 1,4,5-trisphosphate IRS-1 = insulin receptor substrate-1 JAK, TYK = Janus kinases JNK = c-Jun N-terminal kinase MAP = mitogen-activated protein MEK = MAP kinase kinase or ERK kinase p62TCF = ternary complex factor PI 3-K = phosphatidylinositol 3-kinase PKC = protein kinase C PLC = phospholipase C SH2 = src-homology 2 SIE = sis-inducing factor element SRE = serum response element STAT = signal transducers and activators of transcription VSMC = vascular smooth muscle cell

	Acknowledgments

 
This work was supported by grants from the National Institutes of Health (Heart, Lung, and Blood Institute) to Drs Berk and Corson. Dr Berk is an Established Investigator of the American Heart Association. We gratefully acknowledge the scientific contributions of our colleagues and collaborators, including K. Bernstein, M. Marrero, W. Paxton, J. Abe, J. Duff, M. Ishida, T. Ishida, M. Kusuhara, D.-F. Liao, P. Lucchesi, D. Wuthrich, and U. Schmitz.

	Footnotes

 
This manuscript was sent to Robert J. Lefkowitz, Consulting Editor, for review by expert referees, editorial decision, and final disposition.

Received July 24, 1996; accepted January 3, 1997.

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