Angiotensin II signal transduction pathways1

Regulatory Peptides 78 (1998) 19–29

Invited review

Angiotensin II signal transduction pathways 1 Peter P. Sayeski, M. Showkat Ali, Dan J. Semeniuk, Thanh N. Doan, Kenneth E. Bernstein* Department of Pathology and Laboratory Medicine, 1639 Pierce Drive, 7107 WMB, Emory University School of Medicine, Atlanta, GA 30322, USA Received 27 August 1998

Abstract It has been 100 years since the discovery of renin by Tigerstedt and Bergman. Since that time, numerous discoveries have advanced our understanding of the renin–angiotensin system, including the observation that angiotensin II is the effector molecule of this system. A remarkable aspect of angiotensin II is the many different physiological responses this simple peptide induces in different cell types. Here, we focus on the signal transduction pathways that are activated as a consequence of angiotensin II binding to the AT 1 receptor. Classical signaling pathways such as the activation of heterotrimeric G proteins by the AT 1 receptor are discussed. In addition, recent work examining the role of tyrosine phosphorylation in angiotensin II-mediated signal transduction is also examined. Understanding how these distinct signaling pathways transduce signals from the cell surface will advance our understanding of how such a simple molecule elicits such a wide variety of specific cellular responses.  1998 Elsevier Science B.V. All rights reserved. Keywords: Renin; Renin–angiotensin system; Effector molecule; Signal transduction pathways; Heterotrimeric G protein; Tyrosine phosphorylation

1. Historical overview of the renin–angiotensin system One hundred years ago, Tigerstedt and Bergman [88] demonstrated that intravenous injections of rabbit kidney extracts resulted in a significant and sustained increase in blood pressure 2 . They postulated that there was a vasoactive substance in the extract and named it renin. In the 1930s, Harry Goldblatt hypothesized that the disturbance of intrarenal hemodynamics associated with vascular disease could be the cause of hypertension and not the result of hypertension. He established a model of ‘experimental hypertension’ by constricting blood flow through the renal arteries and demonstrated that the increased blood pressure

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100 years of Renin. *Corresponding author. Tel.: 1 1-404-7273134; fax: 1 1-4047278540; e-mail: [email protected] 2 See Skeggs LT, Dorer FE, Kahn JR, Lentz KE, Levine M. Experimental renal hypertension: The discovery of the renin–angiotensin system. In: Soffer RL, (Ed.). Biochemical Regulation of Blood Pressure. New York, NY: Wiley Interscience 1981:3–38. This is a history of research in the renin–angiotensin system.

that followed renal artery constriction did not require the gonads, pituitary, nervous system, pancreas, or thyroid [26]. However, it did require the adrenal cortex. He also demonstrated that the hypertension could be prevented by either clamping the renal veins or by removing the offending kidney. Thus, Goldblatt showed that (1) experimental hypertension was of renal origin and (2) that the causative agent was humoral in nature. The most likely candidate was renin. Renin became the subject of intense investigation. The next major observation came from Braun-Menendez in Argentina and from Page and Helmer in the USA. Independently, they demonstrated that renin was not the direct causative agent of experimental hypertension, but that renin acted on a substance in the plasma (angiotensinogen) to yield a heat stable peptide possessing both pressor and vasoconstrictor properties [11,61]. BraunMenendez named the peptide ‘hypertensin’ while Page called it ‘angiotonin.’ In 1958, they agreed that the substance should be called ‘angiotensin.’ The first laboratory to successfully purify angiotensin was that of Leonard T. Skeggs Jr., who achieved this by incubating large quantities of hog renin with horse plasma [81]. Amino acid

0167-0115 / 98 / $ – see front matter  1998 Elsevier Science B.V. All rights reserved. PII: S0167-0115( 98 )00137-2

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analysis of angiotensin found it to be a decapeptide, but in one experiment, Skeggs accidentally purified angiotensin in the presence of 0.15 M NaCl instead of the usual distilled water. The result was an octapeptide derived from angiotensin, but missing the last two C-terminal amino acids [47,82]. Thus, Skeggs and his colleagues demonstrated that angiotensin was in two forms; the decapeptide called angiotensin I and the octapeptide termed angiotensin II. Subsequent studies by the same group characterized angiotensin-converting enzyme (ACE), the enzyme which converts angiotensin I to angiotensin II. They found that ACE activity required monovalent anions such as chloride [83]. Also, enzymatic activity was blocked by cyanide and the divalent chelator EDTA, suggesting that ACE was a metalloprotease. In addition to the plasma ACE described by Skeggs, there must be a tissue source of ACE to account for the in vivo conversion of angiotensin I to II. The quantitative measurements supporting the claim of tissue ACE came in 1967 when Ng and Vane painstakingly measured the activity of ACE from various circulatory beds. Their work suggested that the lung was the most likely source of tissue ACE as measured by the conversion of angiotensin I to angiotensin II in pulmonary blood [59]. Shortly thereafter, Bakhle [4] demonstrated that ACE activity was present in the particulate fraction of homogenized dog lung and Sander and Huggins demonstrated that the apical membrane of the lung contained ACE enzymatic activity [73]. The circle was closed several years later when ACE was localized to the luminal surface of the pulmonary endothelial cells demonstrating that the vascular bed of the lung was positioned as the major site controlling the levels of angiotensin II entering the systemic circulation [69]. Collectively, these studies defined the biochemical pathway that starts with renin and ends with angiotensin II. The conclusion that angiotensin II was the effector molecule of this important biological system set in motion an effort to clone the angiotensin II receptor. The cloned sequence of the angiotensin II type 1A (AT 1A ) receptor was independently published in 1991 by two groups: the laboratory of Kenneth E. Bernstein cloned the receptor from rat aortic smooth muscle cells while the laboratory of Tadashi Inagami cloned the same receptor from bovine adrenal [58,75]. The open reading frame of what is now termed the AT 1 receptor encodes a protein of 359 amino acids with a calculated molecular weight of 40 889 Da. Amino acid sequence analysis found that it shares structural features with the G protein-coupled receptor (GPCR) superfamily. Only a single gene encoding the AT 1 receptor has been identified in humans, rabbits, and cows. Rodents, in contrast, have two highly homologous AT 1 receptor genes that encode the receptor isozymes termed AT 1A and AT 1B . These two proteins are 95% homologous in amino acid sequence [40,43,72]. While there are differences in the tissue pattern expression of the rodent AT 1 isozymes, both receptors bind ligand and signal in an identical fashion.

Targeted disruption of each gene in mice demonstrated that the AT 1A receptor (the predominant form in vascular smooth muscle) is far more important in the control of blood pressure; mice lacking the AT 1A receptor have a marked reduction of systolic blood pressure while mice lacking the AT 1B receptor are indistinguishable from wild type control animals [15,39,85]. We now know that the renin–angiotensin system impacts nearly every major organ system in animals through its effector molecule, angiotensin II. Yet, each tissue responds to angiotensin II in different ways. Responses include vasoconstriction (vascular smooth muscle), release of aldosterone (adrenal), gluconeogenesis (liver), sodium absorption (gut and kidney), increased cardiac fibroblast matrix formation (heart), increased thirst (brain), and increased b-adrenergic activity (nervous system). This observation clearly begets the question of how a ligand binding to a single cell surface receptor generates such a diverse cellular response. In part, the answer lies in the different second messenger systems and signal transduction pathways that are activated as a consequence of angiotensin II binding to the AT 1 receptor. This review will focus on two of these pathways, namely, the role of heterotrimeric G proteins and the role of tyrosine phosphorylation signal transduction cascades in mediating angiotensin II responses. Understanding recent advances and outlining the questions that will be the focus of future work will help explain the physiology and pathophysiology of the renin–angiotensin system 100 years after Tigerstedt and Bergman.

2. The GPCR superfamily and the pharmacology of the angiotensin II receptor subtypes

2.1. The GPCR superfamily The AT 1 receptor has both the structural features (seven discreet hydrophobic domains) and signaling characteristics (linkage to heterotrimeric G proteins) common to all members of the GPCR superfamily. Recent studies estimate that over 1000 different GPCRs are encoded in the human genome. To date, roughly half this number have been cloned. GPCRs have been identified in organisms ranging from yeast to man. The ligands that bind to GPCRs form a very diverse superfamily including amines (acetylcholine, adrenaline, dopamine, and serotonin), amino acid derivatives (g-aminobutyric acid and glutamate), fatty acid derivatives (leukotrienes, prostaglandins, and thromboxanes), nucleotides (ADP and ATP), peptides (angiotensin II, bradykinin, and luteinizing hormone), phospholipids (lysophosphatidic acid and platelet-activating factor), olfactory odorants, and photons which activate the rhodopsin receptor. In addition to an impressive array of diverse ligands, the biology of GPCRs also has clinical relevance. One of the best known examples is a thyroid-

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stimulating hormone (TSH) receptor mutation. The TSH receptor is normally expressed on the thyroid gland and activation / inactivation of the receptor is tightly regulated. A specific TSH receptor mutation results in constitutive activation of the receptor. The activated receptor in turn leads to an overactive thyroid gland and clinical hyperthyroidism [64]. Similarly, an activating autosomal mutation in the luteinizing hormone (LH) receptor results in precocious puberty [92].

2.2. Pharmacology of the angiotensin II type AT1 and AT2 receptors Prior to the cloning of the G protein-coupled AT 1 receptor, many important observations pertaining to the function of the receptor were made through the use of nonpeptidic angiotensin II-receptor antagonists. The initial breakthrough came from publications describing the synthesis and actions of imidazole-5-acetic acid derivatives. While these compounds only moderately opposed the vasoconstrictive properties of angiotensin II, they were nonetheless specific for the angiotensin II receptor [25]. These patented compounds served as the starting material for drug development programs worldwide. Two separate classes of compounds were eventually found to be highaffinity angiotensin II receptor antagonists. The first was losartan developed by Du Pont Pharmaceuticals while the other was PD123177, a spinacine derivative made by Warner Lambert [22]. These compounds demonstrated the existence of two types of the angiotensin II receptor referred to as the AT 1 and AT 2 subtypes; the AT 1 receptor is blocked by losartan while the AT 2 receptor is blocked by PD123177. Selection of the PD123177 compound came through serendipity as the binding assays for this compound were done in the presence of 5 mM dithiothreitol which disrupted AT 1 receptor binding [16]. Subsequent studies demonstrated that virtually all of the known hemodynamic effects of angiotensin II are mediated by the AT 1 receptor and are therefore blocked by losartan. These include the angiotensin II-mediated (1) increase in intracellular Ca 21 , (2) contraction of rabbit aorta, and (3) drinking behavior in rats [17,93]. Furthermore, losartan was found to significantly lower blood pressure in spontaneously hypertensive rats. These and other data demonstrated that the AT 1 receptor mediates angiotensin II pressure and fluid responses in many tissues including vascular smooth muscle, adrenal, brain, gut, and kidney. Publication of the AT 1 receptor cDNA sequence preceded the cloning of the AT 2 receptor cDNA sequence [57,44]. Like the AT 1 receptor, the AT 2 receptor also possesses the characteristics of a seven transmembrane GPCR. Expression of the AT 2 receptor appears highest during fetal development, but drops precipitously after birth. The generation of mice lacking the AT 2 receptor gene found that the animals develop normally, but have an exaggerated pressure response to angiotensin II, an im-

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paired drinking response to water, and a reduction in locomotor activity [30,35]. In summary, these and other studies indicate that the AT 1 receptor falls within the diverse GPCR superfamily. However, it is capable of transducing specific effects that increase systemic blood pressure through the constriction of vascular smooth muscle, increase renal reabsorption of sodium, and increase thirst in the central nervous system.

3. The role of heterotrimeric G proteins in angiotensin II signal transduction

3.1. Overview of heterotrimeric G proteins Heterotrimeric G proteins mediate the transduction of signals from the outside to the inside of the cell by coupling ligand occupancy of GPCRs with the activation of intracellular effector molecules. Effector molecules that are activated by G proteins include adenylyl cyclase, phospholipase C, and ion channels such as K 1 and Ca 21 channels. Heterotrimeric G proteins are guanine nucleotide-binding proteins. Because of their ability to hydrolyze GTP, they belong to the GTPase superfamily of proteins. This superfamily is subdivided into two families, namely the monomeric GTPases and heterotrimeric G proteins. The monomeric GTPases are cytoplasmic proteins that become membrane associated upon activation and are involved in a diverse set of signal transduction events. Members include the Rho, Ras, and Arf proteins. In contrast, the heterotrimeric G proteins are membrane-bound molecules which appear to couple exclusively with seven transmembrane spanning receptors. Heterotrimeric G proteins are formed by three distinct protein chains designated as the a-, b-, and g-subunits. These protein subunits are distinguishable by several criteria, including size: a-range in size from 39 to 52 kDa, b- are 36 kDa, and g- are 6–8 kDa. To date, 23 different a-, 6 b-, and 11 g-subunits have been identified. The a-subunits have been further subdivided into four families designated G as , G ai , G aq , and G a12 . The expression of these different subunits in a cell could result in numerous G abg combinations assuming that all are expressed equally and all associate randomly. However, it appears that (1) each cell expresses different subunits, (2) the association of these subunits may not be random, and (3) the interaction of a specific G abg trimer with a specific receptor is unique. This last point has been observed in several instances. For example, in the GH 3 pituitary cell line, the inhibition of Ca 21 channel activity by somatostatin is mediated by the a i – o1 / b 1 / g3 complex, whereas inhibition of the same Ca 21 channel by the M4 muscarinic receptor occurs through the a i – o1 / b 3 / g4 -trimer [5]. Thus, one level of specificity for a given GPCR signal is determined by the expression of the appropriate receptor,

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the expression of the appropriate G abg subunits, and the specific interaction of these subunits with the receptor. In the inactive state, the G abg trimer is bound to GDP via the Ga-subunit. Activation of heterotrimeric G proteins is initiated by its interaction with specific cytoplasmic segments of a ligand-activated GPCR. As a result, GDP is released from the Ga-subunit. The Ga-subunit then binds GTP and Mg 21 with very high affinity and dissociates from the Gbg complex. Both the activated Ga-GTP and the Gbg subunits modulate effector proteins until hydrolysis of the GTP bound to the Ga subunit terminates signaling via the intrinsic GTPase activity contained within the a-subunit. Following GTP hydrolysis, the inactive Ga-GDP subunit dissociates from the effector molecule and re-associates with the Gbg subunit to return to the inactive G abg heterotrimer.

3.2. The role of G proteins in angiotensin II /AT1 receptor signaling The primary amino acid structure of the AT 1 receptor indicated that it fell within the GPCR superfamily. That said, efforts were made to establish whether it was in fact capable of G protein activation. The most reliable way to demonstrate this was to exploit two characteristics that are hallmarks of G protein activation: its membrane association and its ability, when activated, to bind GTP with very high affinity. Plasma membranes are prepared from angiotensin II-treated cells and incubated with a non-hydrolyzable analogue of GTP, 35 S-GTPgS. The activated Gasubunit is capable of binding GTPgS with very high affinity, but the g-sulfur makes it insensitive to the GTPase activity of the a-subunit. Heterotrimeric G protein activation is directly quantitated by the amount of radioactivity bound to the membranes. Early studies indicated that angiotensin II-stimulated membranes were in fact capable of activating heterotrimeric G proteins as measured by the amount of 35 S-GTPgS bound to the filters when compared with unstimulated membrane controls [36,20]. These results set in motion a new series of studies to identify the specific complement of G protein subunits that are coupled to AT 1 receptor activation. The addition of purified Ga subunits into these in vitro assays demonstrated that the AT 1 receptor may be coupled to G aq , G aq / 11 , and G i / 0 [42,60]. However, these studies had several limitations: the measurements were done outside the cell and the appropriate complement of Gbg subunits could not be identified. In subsequent work, the functional assay was the angiotensin II-dependent entry of extracellular Ca 21 via the L-type Ca 21 channel in cultured rat portal vein myocytes [51]. Antisense oligonucleotides directed against various a-, b-, and g-subunits were injected into cells and ligand-dependent Ca 21 entry was measured. The authors found that oligonucleotides directed against the mRNA coding for the a 13 / b 1 / g3 subunits blocked the angiotensin II-mediated entry of extracellular

Ca 21 . A corresponding reduction in a 13 / b 1 g3 protein levels was confirmed by the immunohistochemical staining of antisense injected cells. Furthermore, these studies suggested that the interaction of the heterotrimeric complex with the AT 1 receptor was mediated by the C terminal region of the G a13 subunit, as addition of a synthetic peptide corresponding to the carboxyl terminal end of G a13 inhibited, in a concentration-dependent manner, the angiotensin II-dependent Ca 21 response. Collectively, these studies demonstrated that activation of the AT 1 receptor by angiotensin II resulted in coupling to heterotrimeric G proteins. More importantly, the coupling to a specific heterotrimer G protein complex distal to AT 1 receptor activation was found to have functional significance. Another important signal transduction pathway in vascular cells that is distal to G protein activation is the protein kinase C (PKC) signaling pathway. PKCs are serine / threonine kinases and their substrates include proteins that are important in cellular proliferation such as histones and the myristolated alanine-rich C kinase substrates (MARCKS). There are currently 11 members belonging to the PKC family and they are differentiated by their mechanisms of activation (i.e. Ca 21 , phospholipids, diacylglycerol, or phorbol esters). The activation of phospholipases through both G protein-dependent and G protein-independent mechanisms causes PIP2 to be hydrolyzed to IP3 and diacylglycerol (DAG). DAG in turn activates PKC. The role of PKC in angiotensin II signaling has been studied by down-regulating PKC activity through prolonged exposure to phorbol esters. Down-regulation of PKC significantly reduced the angiotensin II-mediated induction of the early response genes and cellular proliferation of VSMCs [86,48]. Thus, it is likely that events such as smooth muscle cell proliferation seen in some vascular diseases will have a PKC-dependent component. Activation of G proteins by the AT 1 receptor initiated a search for the region of the AT 1 receptor that mediates G protein activation. Initial studies were done by generating CHO cell lines that stably expressed either the wild-type AT 1 receptor or various AT 1 receptor mutants. These studies indicated that Asp-74 was important in angiotensin II signaling as cell lines containing a point mutation at this residue were unable to produce an angiotensin II-dependent increase in inositol phosphates or an increase of intracellular Ca 21 [9]. Interestingly, it appears that the importance of Asp-74 lies in the binding of angiotensin II and not the direct coupling to heterotrimeric G proteins. Subsequent studies have demonstrated that, in response to ligand, there is a rotation in the membrane helices that results in Asp-74 forming a hydrogen bond with Tyr-292 and that this appears to be critical for receptor activation [37]. Thus, the Asp-74 mutant is unable to couple with G proteins because of altered binding affinities for ligand as opposed to disruption of the region that mediates G protein activation. Subsequent experiments using the in vitro G protein

P.P. Sayeski et al. / Regulatory Peptides 78 (1998) 19 – 29 Table 1 Structure / function mapping of the AT 1 receptor Function

AT 1 amino acid(s)

Reference

Constitutive activation G protein activation

Asn-111 Trp-219–Ala-225 Tyr-312–Leu-314 Tyr-319–Pro-322 Asp-74 Asp-278 and Asp-281 Tyr-215 Tyr-292 Arg-234–Arg-240 Ala-221–Tyr-226 Ser-329–Ser-335 Ser-335–Leu-337

[27] [91] [74] [2] [9] [31] [34] [52] [18] [32] [19] [33]

Jak2 kinase activation Ligand binding

Mitogenesis Receptor internalization

activation assay described above indicated that two regions within the AT 1 receptor were important for the activation of G a – i1 , G a – i2 , and G a – o . The first region was AT 1 receptor amino acids 216–230 found within the third intracellular loop and the second region spanned amino acids 306–320 found within the intracellular carboxyl tail of the receptor [80]. In vivo analysis quickly confirmed the importance of these two regions. Work by Escobedo and co-workers demonstrated that amino acids 219–225, contained within the third intracellular loop of the receptor, were critical for coupling to G a – q [91]. In an elegant

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experiment, they conferred AT 1 receptor signaling properties to the AT 2 receptor by substituting the third intracellular loops between the two receptors. In similar work that was published 2 years later, the region contained within the carboxyl terminal tail which mediates G a – q activation was mapped to amino acids 312–314 [74]. Finally, Kai used synthetic peptides to interfere with angiotensin II-induced membrane GTPase activity. This approach suggested that the second intracellular loop (residues 125–137), the Nterminal region of the third intracellular loop (217–227) and a portion of the C-terminal tail (304–316) were important for this G-protein-dependent process [41]. The power of structure / function analysis has not been limited to mapping G protein-mediated events. Numerous studies have mapped regions of the receptor that are critical for activation of kinases, mitogenesis, and receptor desensitization. Table 1 lists the various regions of the AT 1 receptor that have been mapped and the functional significance they confer. Also shown is a schematic representation of the AT 1 receptor indicating the relative position of each amino acid (Fig. 1). Collectively, these studies demonstrate that the AT 1 receptor couples with heterotrimeric G proteins and this coupling is transduced to effector molecules such as Ca 21 channels, phospholipase C, or adenylyl cyclase. Thus, G proteins play an integral part in angiotensin II signal transduction and it is very

Fig. 1. The rat AT 1 receptor. The receptor is thought to form seven helices which transverse the membrane seven times. At positions in which two amino acids are indicated, the first is the sequence of the rat AT 1A receptor and the second is the sequence of the AT 1B receptor. Figure courtesy of Dr Kathy K. Griendling, Division of Cardiology, Emory University School of Medicine.

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likely that they partake in the diversity of responses that follows AT 1 receptor activation.

4. The role of tyrosine phosphorylation in angiotensin II signal transduction

4.1. Overview of tyrosine kinases The existence of regulatory kinases was promoted by Fischer and Krebs when they observed both an active phosphorylated and an inactive dephosphorylated form of the enzyme glycogen phosphorylase [24]. Since then, numerous protein kinases have been characterized, and it is estimated that they may represent as much as 1% of the mammalian genome [29]. Protein kinases are classified as either serine / threonine or tyrosine kinases. The majority of target proteins are serine / threonine phosphorylated as the phosphotyrosine moiety represents less than 0.1% of the total phosphorylated proteins in a cell [66]. In spite of this, tyrosine phosphorylation is known to be a critical event in cellular biology. As quickly as new tyrosine kinases are discovered, pharmacological inhibitors that block the function of these kinases are also synthesized. These compounds are one of the first tools used in the laboratory as they allow for the rapid dissection of intracellular signaling pathways by specifically blocking a certain type of tyrosine kinase or an entire family of kinases. For example, genistein was found to be an effective inhibitor of many different types of tyrosine kinases while the compound PP1 is specific for the Src family of tyrosine kinases [1,28]. The use of these and other inhibitors, either alone or in combination, has allowed for insights to be made into many different signaling systems including angiotensin II signaling pathways.

4.2. The AT1 receptor and tyrosine kinase signal transduction In contrast to membrane-bound growth factor receptors, the AT 1 receptor harbors no intrinsic kinase activity. However, examination of cellular lysates demonstrated that numerous proteins are tyrosine phosphorylated in response to angiotensin II. This suggested that non-receptor cytoplasmic tyrosine kinases may be activated by angiotensin II and thus allow for a tyrosine phosphorylation signal transduction cascade to move through the cell. Subsequent studies have shown that this hypothesis is in fact true. We will focus on three such tyrosine kinases: pp60 c-src (c-Src), Janus kinase 2 (Jak2), and p125 FAK (FAK).

4.3. The role of c-Src in angiotensin II tyrosine phosphorylation Members belonging to the Src family of tyrosine kinases

are approximately 55–62 kDa in mass and contain several structural features that are shared by all family members. These include an amino terminal myristoylation sequence for membrane targeting, an SH2 domain, an SH3 domain, and a kinase domain. There are nine different genes encoding Src family kinases. However, alternate splicing of the mRNA by different tissues yields at least 14 different gene products [10]. While the expression of most members is restricted to hematopoietic cells, three family members (Fyn, c-Src, and Yes) are expressed in most tissues. In vascular smooth muscle cells (VSMCs), only Fyn and c-Src are expressed. Studies from several laboratories suggest that c-Src plays an important role in angiotensin II signal transduction. Stimulation of VSMCs with angiotensin II lead to a rapid activation of c-Src as measured by either autophosphorylation or the phosphorylation of a synthetic substrate [38]. Pretreatment of VSMCs with the tyrosine kinase inhibitor genistein significantly decreased the angiotensin II-dependent PLCg1 tyrosine phosphorylation and the generation of IP3 [53]. These results suggested a tyrosine kinase-dependent mechanism for the angiotensin II signaling pathway leading to the release of intracellular Ca 21 . To specifically block Src kinase activity, a method of introducing Src kinase neutralizing antibodies into cells was employed. Since the antibodies were made against the more conserved kinase domain, it showed cross-reactivity for Src, Fyn, and Yes. The inhibition of c-Src kinase activity by the electroporation of Src-neutralizing antibodies markedly decreased PLCg1 tyrosine phosphorylation and the generation of IP3 in response to angiotensin II [54]. This effect appears specific as inhibition was not observed using several different control antibodies. Similar work in cardiac myocytes has shown that angiotensin II activates both Fyn and c-Src [70]. The net effect of Fyn and c-Src activation is the subsequent activation of p21ras via the Shc-Grb2-Sos pathway, which in turn activates the MAP kinase signaling pathway. These data demonstrate that Src activation is one of the earliest events in angiotensin II signal transduction and suggest that Fyn and / or c-Src play an important role in both the PLC / IP3 / Ca 21 signaling pathway and the Shc / Grb2 / Sos pathway. One functional consequence of src family kinase activation is organization of the cytoskeleton as assessed in mice lacking various src family tyrosine kinases. The lack of both Fyn and Src results in embryonic lethality indicating a critical role in growth and development for the Src family of tyrosine kinases [84,87].

4.4. The role of Jak2 in angiotensin II tyrosine phosphorylation The Janus kinase (Jak) family of non-receptor tyrosine kinases includes Jak1, Jak2, Jak3, Tyk2, and the Drosophila homologue hopscotch [12]. Each protein is approximately 130 kDa in mass and contains seven conserved Jak

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homology domains. Initial review of the amino acid sequence indicated that this family of kinases contained two kinase domains and thus was named after Janus, the Roman god of two faces. Subsequent analysis revealed that the N-terminal kinase domain was inactive and was therefore termed the pseudokinase domain. Unlike almost all other protein tyrosine kinases, members of the Jak family bear no SH2 or SH3 domains. Nonetheless, Jaks are key mediators of mRNA expression. Jaks accomplish this by phosphorylating the signal transducers and activators of transcription (STATs). Phosphorylated STATs translocate to the nucleus where they bind to Sis-inducible promoter elements and stimulate transcription of the early growth response genes such as c-fos, c-jun, and c-myc. The increased tyrosine phosphorylation, nuclear translocation, and DNA binding activity of the STATs occurs in the presence of cycloheximide, suggesting that this signaling pathway uses a post-translational modification of existing proteins and does not require de novo protein synthesis. Thus, Jaks are capable of transducing a signal from the cell surface to the nucleus through a tyrosine phosphorylation signal transduction cascade. Studies have now shown that angiotensin II directly activates the Jak / STAT pathway via the AT 1 receptor. Initially, it was demonstrated that angiotensin II rapidly and transiently activates Jak2 and Tyk2 in VSMCs [55]. In addition, STAT1, STAT2, and STAT3 are tyrosine phosphorylated in response to angiotensin II and both STAT1 and STAT3 translocate to the nucleus. Angiotensin II also causes a transient association of Jak2 with the AT 1 receptor. Subsequent work demonstrated that this association requires amino acids 319–322 of the AT 1 receptor encoding the YIPP protein motif [2]. Similar results have been obtained in cell systems other than VSMCs. A CHO cell line that stably expressed the AT 1 receptor was found to (1) activate Stat1, (2) cause nuclear translocation of Stat1 and (3) increase DNA binding activity of the Stat1 in response to angiotensin II [6,7]. Furthermore, the angiotensin II-dependent activation of STAT1 and STAT3 was found to require Jak2 in neonatal rat cardiac myocytes [56]. In addition to angiotensin II, other GPCR ligands have been reported to activate the Jak / STAT pathway. Thrombin has been shown to increase Jak2 kinase activity and cause STAT phosphorylation [68,8]. Activation of the ETA receptor by endothelin also leads to STAT activation [65]. Collectively, these studies suggest that GPCR can activate the Jak / STAT pathway and thus transduce a signal from the cell surface to transcriptional events within the nucleus. The observation that Jak2 can physically associate with the AT 1 receptor also indicates that the activation of the Jak / STAT pathway is one of the earliest events in angiotensin II tyrosine phosphorylation signal transduction cascades. Targeted disruption of the Jak2 gene results in an embryonic lethal phenotype in which myeloid and erythroid development are absent [62]. The functional

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consequence of Jak2 activation in VSMC is less clear and is under current investigation.

4.5. The role of FAK in angiotensin II tyrosine phosphorylation Several years ago, a 125-kDa tyrosine kinase was observed to localize at focal adhesions in v-Src transformed chicken embryo fibroblasts [45]. The kinase was termed p125 FAK (focal adhesion kinase) and more recently has simply been called FAK. It lacks SH2 and SH3 domains and shares homology with other tyrosine kinases only through its kinase domain. FAK is activated by growth factor receptors, G protein-coupled receptors, and by integrin receptor activation [23]. Tyr-397 is the site of FAK autophosphorylation and a Y397F mutation results in a dominant negative FAK molecule. However, FAK kinase activity can be greatly enhanced by phosphorylation of Tyr-407, Tyr-576, and Tyr-577 [14]. These sites can be phosphorylated by c-Src and stable protein–protein complexes between a FAK phosphotyrosine and the SH2 domain of c-Src have been described [78]. Perhaps most interesting is the observation that upon phosphorylation, FAK translocates to focal adhesions where it phosphorylates substrates such as paxillin and p130 CAS , thus promoting cytoskeletal rearrangement [13]. Angiotensin II rapidly activates FAK in VSMCs [67]. Activation appears to have an autophosphorylation component and a trans-phosphorylation component by a tyrosine kinase such as c-Src. Activated FAK then translocates to focal adhesions where it presumably acts on its substrates [49]. One such substrate is paxillin. In VSMCs, this 68-kDa protein is rapidly and robustly tyrosine phosphorylated in response to angiotensin II [46]. Phosphorylation of paxillin requires the release of intracellular Ca 21 as it is blocked by the intracellular Ca 21 chelator BAPTA-AM. The tyrosine phosphorylation of paxillin correlates temporally with the formation of focal adhesion contacts in VSMCs and suggests that FAK may play a role in coordinating VSMC proliferation / remodeling with changes in cell structure [89]. Another potential FAK substrate is the adaptor protein p130 CAS (Crk associated substrate). This 130-kDa protein has several protein–protein binding domains including proline-rich domains, an SH3 domain, and numerous binding motifs for the SH2 domains of v-Crk and v-Src [71]. In VSMCs, angiotensin II rapidly induces the tyrosine phosphorylation of p130 CAS , and this is mediated through the AT 1 receptor [76]. Like paxillin, the tyrosine phosphorylation of p130 CAS requires the release of intracellular Ca 21 . The tyrosine phosphorylation of p130 CAS correlates temporally with its ability to bind at least 11 different phosphate-containing proteins in response to angiotensin II, supporting the concept that it is an adaptor molecule. Three of the 11 proteins have been identified as c-Src, PKCa, and the integrin signaling

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Table 2 Proteins that are tyrosine phosphorylated in response to angiotensin II Protein

MW (kDa)

Reference

AT 1 receptor FAK Fyn IGF-1 receptor IRS-1 Jak2 p120 Ras GAP p130 CAS p190 Rho GAP pp120 Paxillin PDGF receptor PLCg1 Pyk2 Shc SHP2 (PTP-1D) Src STAT1 STAT2 Tyk2

41 125 59 97 165 130 120 130 190 120 68 180 148 116 46 / 52 / 66 72 60 84 / 91 113 135

[63] [49] [70] [21] [21] [54] [77] [76] [77] [53] [46] [50] [53] [49] [79] [3] [50] [55] [55] [55]

molecule pp120. Thus, it appears that p130 CAS serves as a convergence point for three very different signaling pathways, namely, the c-Src tyrosine kinase pathway, the serine / threonine PKC pathway, and the cell adhesionmediated pp120 pathway. The importance of tyrosine phosphorylation in angiotensin II signal transduction goes beyond the few proteins that have been described in this review. To illustrate this point, Table 2 lists proteins that are known to be tyrosine phosphorylated as a consequence of angiotensin II binding to the AT 1 receptor; a receptor which lacks intrinsic kinase activity. As time passes, more proteins will be added to this list. Also, new insights into the pathways that mediate the tyrosine phosphorylation and the functional consequences of the phosphorylation will be discovered. This will permit a better understanding of both the physiology and pathophysiology of the angiotensin II signaling system; a system that has clinical relevance to many tissues including vascular smooth muscle and the kidney.

5. Conclusions and direction of future research The discovery of the renin–angiotensin system began with Tigerstedt and Bergman in 1898 and continues today. Numerous studies have demonstrated that both heterotrimeric G proteins and tyrosine phosphorylation signaling are vital for normal AT 1 receptor signal transduction. Fig. 2 outlines the features of G protein and tyrosine phosphorylation signaling that were discussed in this review, as well as the functional significance of each pathway. However, several key questions remain. For instance, what is the relationship, if any, between heterotrimeric G proteins and tyrosine phosphorylation signaling events? Do

Fig. 2. Summary of angiotensin II signal transduction pathways involving heterotrimeric G proteins and tyrosine phosphorylation signaling cascades. Arrowheads signify pathways that are well understood and question marks (?) indicate those pathways that are poorly understood. The pathways shown include the activation of L-type Ca 21 channels by heterotrimeric G proteins and the activation of FAK, Src, and Jak2 as well as their downstream targets.

these two diverse signaling systems operate independently of one another or does a level of crosstalk exist so that they may work in unison? Such crosstalk is suggested by a recent report that there is both a G protein component and a tyrosine phosphorylation component to the angiotensin II-mediated generation of IP3 in VSMCs [90]. These two events appear to be temporally coordinated in that the peak formation of IP3 (15 s after angiotensin II stimulation) is mediated in large part by the G protein-coupled PLC-b1 isoform of phospholipase C while the later phase of IP3 formation (30 s after angiotensin II) is mediated by the tyrosine kinase-coupled PLC-g1 isoform. As time passes, our understanding of the interplay of G protein and tyrosine phosphorylation events will certainly increase as will our knowledge of the signal transduction mechanisms that follow AT 1 receptor activation.

Acknowledgements The authors wish to thank Dr. Kathy K. Griendling for critically reviewing the manuscript and Shaun Benford for administrative assistance.

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