Regulation of angiotensin II type 1 (AT1) receptor function1

Regulatory Peptides 79 (1999) 9–23

Invited review

Regulation of angiotensin II type 1 (AT 1 ) receptor function q Walter G. Thomas* Molecular Endocrinology Laboratory, Baker Medical Research Institute, P.O. Box 6492, Melbourne 8008, Australia Received 16 September 1998

Abstract The type 1 angiotensin receptor (AT 1 ) mediates the important biological actions of the peptide hormone, angiotensin II (AngII), by activating an array of intracellular signaling pathways. The unique temporal arrangement and duration of AngII-stimulated signals suggests a hierarchy of post-AT 1 receptor binding events that permits activation of selective effector pathways. Moreover, it predicts that the coupling of AT 1 receptors is tightly regulated, allowing cells to differentiate acute responses from those requiring longer periods of stimulation. Recent studies have concentrated on delineating the molecular processes involved in modulating AT 1 receptor activity. In addition to AT 1 receptor modification (phosphorylation), trafficking (internalization and degradation) and interaction with regulatory intracellular proteins, other processes may include receptor dimerization, cross-regulation by other receptor systems, and receptor isomerization between activated and non-activated forms. This review focuses on recent advances in this area of research, highlighting directions for future investigation.  1999 Elsevier Science B.V. All rights reserved.

1. Introduction The peptide hormone angiotensin II (AngII) is the active component of the renin–angiotensin system, which maintains arterial blood pressure and fluid and electrolyte homeostasis [1]. AngII is formed within the blood and the interstitium of tissues from a precursor protein, angiotensinogen, through the sequential actions of two enzymes: renin and angiotensin-converting enzyme (Fig. 1). AngII in the extracellular space is recognized and bound by high affinity, cell surface angiotensin receptors within AngIIresponsive tissues. This interaction leads to the initiation of intracellular signaling pathways that mediate immediate responses, such as vasoconstriction (blood vessels), aldosterone release (adrenal cortex) or thirst (brain), as well as longer-term activation of gene programs that subserve the known growth and remodeling effects of AngII on cardiac, renal and vascular tissue. Signaling through AngII receptors needs to be tightly regulated because over-activity of the renin–angiotensin system, through aberrant production of angiotensin II and / or inappropriate re-

sponses to this hormone, is associated with hypertension and cardiovascular disease. Drugs that inhibit production of angiotensin II or prevent its interaction with receptors are used clinically to treat high blood pressure and reduce

*Tel.: 1 61-3-95224367; fax: 1 61-3-95211362; e-mail: [email protected] q 100 years of Renin. 0167-0115 / 99 / $ – see front matter  1999 Elsevier Science B.V. All rights reserved. PII: S0167-0115( 98 )00140-2

Fig. 1. Renin–angiotensin system.

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the morbidity and mortality associated with cardiovascular dysfunction. The actions of AngII are mediated through two types of cell surface AngII receptor (AT 1 and AT 2 ). AT 1 receptors, which exist as two closely related subtypes (AT 1A and AT 1B ) in rodents, are the principle mediators of the biological actions of AngII [2,3]. Cloning of AT 1 receptors has revealed that they are members of the guanyl nucleotide-binding protein (G protein)-coupled receptor (GPCR) superfamily [4]. GPCRs are characterized by a seven transmembrane domain topology and a capacity to associate with, and activate, heterotrimeric G-proteins, consisting of a, b and g subunits (Fig. 2). The established dogma is that ligand binds to, and activates, a single receptor complexed to a heterotrimeric G protein. The G protein complex then dissociates to release the a subunit from the bg subunit and both have been shown to promote intracellular signaling [4]. For AT 1 receptors, activation by AngII results in G protein mediated signaling, including phospholipase C-b1-dependent activation of protein kinase C (PKC) and release of calcium from intracellular stores. Interestingly, AT 1 receptors also activate intracellular signaling pathways traditionally associated with growth factor and cytokine receptors (e.g., stimulation of tyrosine kinase activity, tyrosine phosphorylation and activation of phospholipase C-g1, MAP kinase, and the JAK-STAT pathway [5–10]. The processes that couple AT 1 receptor activation to these diverse, growth factor-like, signaling pathways are not known, but may involve the ‘‘classical’’ activation of G proteins or ‘‘non-classical’’ mechanisms such as the direct interaction and activation of signaling molecules, other than heterotrimeric G proteins, with the AT 1 receptor. Investigation of the events that follow the immediate (seconds to minutes) activation of AT 1 receptors has been the focus of recent studies and forms the basis of this review. From these studies, a theme has developed which maintains that a temporal arrangement exists with respect to: the initial signals generated by activated receptors, the subsequent phosphorylation and internalization of the receptor which desensitizes these early signals, and latter signals that develop only after prolong agonist presentation to the receptor. Thus, it appears that signaling through the AT 1 receptor can be selectively desensitized and that this phenomena allows cellular responses to AngII that are appropriate for the degree of stimulation. Central to all these processes is the 54 amino acid carboxyl-terminus of the AT 1 receptor. As will be discussed in the following sections, the carboxyl-terminus is a site for G protein coupling and activation and is phosphorylated in response to AngII stimulation. It also contains motifs and regions important for receptor internalization and is a putative binding site for accessory signaling molecules that may explain the myriad, and temporal arrangement, of signal transduction pathways activated through AT 1 receptors. The four stages of receptor activation and regulation are illustrated in Fig. 2A.

2. Activation of AT 1 receptors

2.1. Initiation of AT1 signaling The 359 amino acid AT 1A receptor is an integral membrane type II protein with a molecular mass (Mr ) deduced from the cloned cDNA of 40 889 [11]. Structural predictions suggest an extracellular N-terminus followed by seven transmembrane-spanning a-helices, which are connected by three extracellular and three intracellular loops, linked to an intracellular carboxyl-terminus (see Fig. 2B). Three consensus sites for N-glycosylation can be recognized (Asn 4 in the N-terminus and Asn 176 and Asn 188 within extracellular loop two) and are probably modified because the mature, plasma membrane-localized, receptor protein migrates on sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) as a broad band (Mr , 70 000–130 000). This can be reduced to approximately 41 000 by enzymatic removal of N-linked carbohydrate groups [12,13]. The intracellular carboxylterminus (residues 305–359) contains numerous serine, threonine and tyrosine residues, some of which are phosphorylated in an agonist-dependent manner ([13] and discussed in detail below) and are predicted to play an important role in receptor activation and regulation. The high affinity interaction (Kd , | 1 nM) of AngII (Asp 1 –Arg 2 –Val 3 –Tyr 4 –Ile 5 –His 6 –Pro 7 –Phe 8 ) with the AT 1 receptor is sustained through multiple contacts between amino acid sidechains on the octapeptide, AngII, and residues on the receptor positioned by the arrangement of the seven transmembrane a-helices (TM-I–TM-VII) and the extracellular loops (EC-1, EC-2, EC-3). Docking of the agonist, AngII, presumably selects or induces a conformational change in the AT 1 receptor that initiates intracellular signaling (see Noda et al. [14], for discussion). This alteration in receptor structure is then transmitted across the membrane to the cytoplasmic regions of the receptor, which make contact with, and thereby activate, the associated heterotrimeric G proteins to cause signaling. Based on a number of biochemical and mutagenesis studies [14–20], a general consensus is that high affinity binding requires a crucial docking interaction between the positively charged sidechain of Lys 199 (TM-V) on the receptor with the a-carboxyl group of Phe 8 in AngII. This coupling, which is stabilized by additional interactions (e.g. His 183 (EC-2) with Asp 1 of AngII; Asp 281 (top of TM-VII) with Arg 2 of AngII [21]) brings AngII into a position so that interactions and switches, which predicate receptor activation and initiate signaling, can be established. For example, the interactions between His 256 (TM-VI) [17], and perhaps Val 254 and Phe 259 (TM-VI) [22], and the aromatic sidechain of Phe 8 in AngII are required for full agonist activity with respect to signaling. In addition, the interaction of Tyr 4 in AngII with Asn 111 (TM-III) may destabilize a preexisting interaction between Asn 111 (TM-III) and Asn 295 (TM-VII) [23] or Tyr 292 (TM-VII) [24], with a resultant activation and signaling of the receptor. Interest-

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Fig. 2. (A) Proposed four stage (I to IV) schematic for AT 1A receptor activation that would explain the observed hierarchy of AngII-induced signal transduction pathways. In this scheme, AngII binding to the AT 1A receptor initiates processes that result in the activation and release of the a- and bg-subunits of heterotrimeric G proteins from the receptor (Stage I). This leads to the transmission of ‘‘classical’’ signals (e.g. phospholipase C mediated production of diacylglycerol, which activates protein kinase C, and inositol phosphates, which cause release of calcium from intracellular stores). In Stage II, the AngII-activated AT 1A receptor is rapidly phosphorylated on residues within the carboxyl-terminal region, resulting in the attraction and binding of regulatory proteins (arrestins?) that uncouple the receptor from G protein and mediate the internalization (Stage III). Both processes presumably participate in the observed desensitization of ‘‘classical’’ AngII-mediated responses. With continuous exposure to AngII (stage IV), accessory signaling molecules are recruited to the receptor, either at the cell surface or during the process of receptor trafficking, to initiate the ‘‘alternate’’ signal transduction cascades (e.g. JAK-STAT pathway). (B) Proposed topology of the 359 amino acid AT 1A receptor, detailing the seven transmembrane-spanning conformation characteristic of G protein coupled receptors. Cytoplasmic serine, threonine and tyrosine residues (putative phosphorylation sites) are highlighted and particular note should be made of the presence of multiple serines and threonines within the carboxyl-terminus, a region thought to play a crucial role in receptor regulation. Indicated on the carboxyl-terminus is the position of truncations that were introduced into the AT 1A receptor to investigate the role of the carboxyl-terminus in AngII-stimulated AT 1A receptor phosphorylation (Thomas et al., 1998 32 [13]). (C) AngII-induced phosphorylation of the AT 1A receptor is abolished by truncation of the receptor carboxyl-terminus. Shown is the degree of P-labeled phosphate incorporation into the AT 1A receptor in the absence (2) or presence ( 1 ) of AngII stimulation for a series of truncated mutants. Part (C) of this figure is modified from data previously published ([13];  The Endocrine Society). See text for full discussion of AT 1 receptor phosphorylation.

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ingly, other residues within this region of TM-VII (Tyr 292 to Tyr 302 ) have been postulated to contribute to AngII binding and / or to participate in signal transmission [23– 26] suggesting a significant role for TM-VII in receptor activation. Given that the membrane-proximal region of the cytoplasmic carboxyl-terminus is an a-helical extension of TM-VII, predicts an important role for the end of TM-VII and the beginning of the carboxyl-terminus in receptor activation and G protein binding and activation. Indeed, studies using site-directed mutagenesis of, and synthesized peptides corresponding to, the various cytoplasmic regions of the AT 1 receptor have revealed that the proximal region of the AT 1 receptor carboxyl-terminus (with contributions from the second and third intracellular loops) is most important for G protein binding and activation [27–29]. Such an arrangement between the carboxyl-terminus and TM-VII may also explain the capacity of regulatory processes associated with the carboxyl-terminus (e.g. phosphorylation, internalization, and as a binding site for cytoplasmic proteins) to dynamically regulate receptor function.

is most likely that a combination of all these processes is involved. The exquisite control that the AT 1 receptor has over selective receptor coupling is most clearly demonstrated by the recent study of Ushio-Fukai et al. [33]. These researchers demonstrated that, in vascular smooth muscle cells, two phases of phospholipase C (PLC) activation could be resolved: an immediate first phase, comprising the coupling of the AT 1 receptor to PLC-b1 through Ga q / 11 and Ga 12 , and their respective bg subunits; and a second delayed phase involving a downstream tyrosine kinase activation of PLC-g1. An obvious explanation for such a temporal arrangement of AT 1 signaling is to coordinate and control various steps and responses to AngII. Such coordination may be appropriate, and required, to separate the seconds to minutes regulation of blood pressure (vasoconstriction) from signals required for the known growth effects of AngII, which require prolong exposure to agonist.

3. Regulation of AT 1 receptors

2.2. Temporal organization of signaling

3.1. Multiple mechanisms of AT1 receptor regulation

From multiple studies, focused on AT 1 receptor signal transduction pathways, it has become apparent that the temporal arrangement of signaling varies from seconds (e.g. the activation of phospholipase C and generation of inositol phosphates) to minutes (e.g. MAP kinase activation) to hours (e.g. JAK-STAT pathway), following agonist stimulation. The exact mechanism(s) by which a single AT 1 receptor is able to differentially couple to disparate signal transduction pathways is not clear, but presumably involves a complex series of steps that selectively recruits, activates and then inactivates each signaling system in a time-dependent manner. This may be achieved through an isomerization of the receptor between multiple conformations, each one capable of selecting a unique signaling target. Receptor isomerization, between inactive and multiple active forms, has been postulated as a mechanism for coupling to multiple signal transduction pathways and to explain pharmacological data, which cannot be rationalized by other models (see [30,31]). The temporal arrangement of signaling may also involve a hierarchical arrangement of receptor phosphorylation, such that early phosphorylation of the receptor in response to agonist stimulation may uncouple one pathway while promoting another. Such a hierarchy was recently observed for the b 2 -adrenergic receptor, which can switch from coupling to G s to G i as a result of phosphorylation within its third intracellular loop [32]. The recruitment and interaction of additional signaling molecules with the receptor may also provide a mechanism to selectively and temporally couple the receptor to individual signaling pathways. Given the complexity of activation and inactivation of multiple signals, it

In this section, the regulation of AT 1 receptor function will be discussed, focusing on those processes that occur immediately following AngII stimulation of the AT 1 receptor at the cell surface. In particular, the role of the AT 1A carboxyl-terminus in receptor activation, desensitization, phosphorylation, internalization and interaction with cytoplasmic proteins, which is central to receptor regulation, will be discussed in detail below. However, it should be recognized that AngII receptor regulation is multifaceted and involves additional mechanisms other than those occurring at the level of the receptor protein and, specifically, within the carboxyl-terminus. For instance, angiotensin receptors exist as several subtypes (AT 1A , AT 1B , AT 2 , AT 4 ) [2] with distinct tissue and temporal expression and function, which may permit multiple complementary or opposing actions to angiotensin peptides. Polymorphism of the AT 1 receptor has also been described that may generate additional ‘‘subtypes’’ with subtle variations in function [34–40]. Post-transcriptional alteration of the AT 1 receptor mRNA transcript (RNA editing), as described for the serotonin receptor [41], may also lead to changes in the amino acid sequence of translated AT 1 receptors. Whether RNA editing occurs for AT 1 receptors remains to be determined, but it may explain slight variations in the reported sequence of AT 1 (AT 1A and AT 1B ) receptors in the literature. Apart from receptor subtypes, the amount of AngII receptor mRNA (and hence receptor protein) can be modulated by a variety of hormonal, physiological and regulatory stimuli through activation of transcription factors that interact with upstream promoter sequences within AngII receptor genes [42–48]. In addition, alternatively

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spliced 59- and 39-untranslated regions of the transcribed AT 1 mRNA can affect the efficiency of translation and stability of the mature AT 1 receptor [49–51]. Finally, AngII stimulation may lead to the induction of other genes whose protein products regulate receptor function, such as GRK5, a member of the G-protein coupled receptor kinase family, that phosphorylates AngII receptors and thereby mediates the desensitization of receptor responses [52]. Altogether, these processes provide cells with a diverse repertoire of mechanisms for regulating AT 1 receptors.

by cytoplasmic proteins termed arrestins that prevent association of receptor and G-protein and therefore cause desensitization. The binding of arrestins to activated GPCRs may also serve to target the receptors to clathrincoated pits and internalize receptors from the cell surface. So it appears that the balance between signaling, phosphorylation, arrestin binding and internalization, and the subsequent dephosphorylation and recycling of receptors to the cell surface, determines short-term receptor responsiveness.

3.2. AT1 receptor desensitization

3.3. Evidence for AT1 receptor phosphorylation

The important cardiovascular actions of AngII are mediated through AT 1 receptor activation of multiple intracellular signaling pathways. Overactivity of the renin– angiotensin system and / or inappropriate responses to AngII is associated with hypertension and cardiovascular dysfunction. As an elegant example of the consequences of uncontrolled AT 1 signaling, Hein et al. [53] demonstrated that transgenic mice with targeted overexpression of the AT 1A receptor in cardiac myocytes displayed a newborn phenotype of myocyte hyperplasia with an associated bradycardia and heart block and premature death. Hence, to maintain cardiovascular homeostasis and tight control of AT 1A receptor function, cells have evolved, as outlined above, numerous mechanisms for regulating AT 1 receptor activity and density, both acutely and gradually. As a consequence, stimulation by AngII causes rapid termination and desensitization of many receptor responses [7,9]. For example, AngII stimulates potent vasoconstriction with an accompanying tachyphylaxis, which is considered a protective device. The mechanism behind this tachyphylaxis is presumably a desensitization of AngII-mediated intracellular signaling. Desensitization of AT 1 receptor signaling has been observed in cells naturally expressing endogenous AT 1 receptors [54–57], as well as in cell lines transfected with cloned AT 1 receptors [12,58– 63]. These desensitizing processes probably involve a complex synergy between receptor uncoupling from heterotrimeric G-proteins, receptor modifications (e.g. phosphorylation), trafficking (i.e. internalization and recycling), down-regulation [64], and the interaction of regulatory proteins with the cytoplasmic regions of the receptor protein. Many of the regulatory mechanisms for AT 1 receptors are analogous to those detailed from extensive studies into other GPCRs, in particular, the prototypical rhodospin and b-adrenergic receptors (see [65,66]). For these receptors, activation by agonist (b-adrenergic receptor) or a photon of light (rhodopsin receptor), promotes a conformation in the receptor that activates the heterotrimeric G-protein to cause cell signaling. This conformation is also a substrate for phosphorylation by specific G-protein receptor kinases (GRKs) and / or by second messenger activated kinases (PKA and PKC). Phosphorylated receptors are then bound

The standard biochemical approach for investigating agonist-stimulated receptor phosphorylation is to radioactively load intracellular ATP pools by incubating cells, expressing the receptor of interest, with inorganic 32 P. Following stimulation with agonist, phosphorylated receptors are immunoprecipitated and resolved by SDSPAGE and autoradiography or phosphoimaging. This approach is acutely dependent upon the availability of antibodies that can efficiently and specifically immunoprecipitate the receptor protein. The capacity of anti-AT 1 receptor antibodies to detect the receptor by Western blotting and to harvest the receptor by immunoprecipitation has been one the major controversies in this field of research. While many groups have attempted to raise specific antibodies against peptides sequences of the AT 1 receptor, these have generally failed to prove universally capable of detecting and immunoprecipitating the receptor (discussed in [13,67,68]). Nevertheless, several groups have published preliminary reports of AT 1 receptor phosphorylation [69–71]. Using an antibody raised against the AT 1A receptor N-terminus, Paxton et al. [69] reported phosphorylated bands at 49 000 and 68 000 immunoprecipitated from rat aortic smooth muscle cells. Surprisingly, AngII did not stimulate this phosphorylation, which was on serine and tyrosine residues. A serine- and tyrosine-directed phosphorylation of the vascular AT 1A receptor was also observed by Kai et al. [70], while Yang et al. [71] reported that the AT 1 receptor in neuronal cultures was phosphorylated by mitogen-activated protein (MAP) kinase. Unfortunately, the antibodies used in all three studies were not proven to unequivocally immunoprecipitate the receptor and this inadequate characterization makes for ambiguous conclusions. In light of this controversy, Smith et al. [68], in a series of well-controlled experiments, recently reported the successful production of a polyclonal antibody against a fusion protein containing a carboxyl-terminal 92 amino acid fragment of the AT 1B receptor. They provide compelling evidence that this antibody is capable of detecting and immunoprecipitating the endogenous AT 1 receptor expressed in bovine adrenal glomerulosa cells. Moreover, they used the antibody to clearly demonstrate a time- and agonist-dependent phosphorylation of the adrenal AT 1

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receptor. Interestingly, they were unable to duplicate their results with two commercial antibodies (sc579 and sc1173) and it appears that these widely used antibodies are incapable of immunoblotting or immunoprecipitating the receptor. The conclusions of studies using these commercial antibodies should be tempered until independently confirmed using well-characterized antibodies, such as those described by Smith et al. [68], or by using AT 1 receptors bearing epitope-tags to allow unambiguous immunoprecipitation. Epitope-tagging of receptors and other proteins, with the subsequent detection and immunoprecipitation of the tagged-protein with commercially available, and well-characterized, anti-‘‘tag’’ antibodies, is a common experimental methodology. Three recent studies have utilized this approach to unequivocally demonstrate the phosphorylation of AT 1 receptors [12,13,61]. Oppermann et al. [61] demonstrated that the AT 1A receptor, tagged with the HA (influenza hemagglutinin antigen) epitope at the N-terminus and transiently transfected into human HEK293 cells, was phosphorylated in an agonist-, time- and dosedependent manner. The AT 1A receptor was visualized as a broad band of 60 000–150 000 on SDS-PAGE and was phosphorylated by specific GRKs (GRK2 and GRK5) and by PKC. Balmforth et al. [12] used a cleavable N-terminal hexahistidine tag to purify AT 1A receptors transfected into HEK293 cells. They demonstrated an AngII stimulated phosphorylation of the AT 1A receptor which also ran as a broad band between 60 000 and 130 000. Moreover, they demonstrated co-migration of the phosphorylated band with the AT 1A receptor photolabelled with 125 I-Sar1(49N 3 )L –Phe 8 –AngII, unambiguously identifying the band as the AT 1A receptor. The AT 1A receptor was phosphorylated at low (1 nM) agonist concentrations by PKC and at higher concentrations (100 nM) by an additional unidentified kinase, presumably a GRK. In their earlier-mentioned study, Smith et al. [68] also demonstrated the immunodetection of an HA-tagged AT 1 receptor, heterologously expressed in Cos-7 cells, as a corollary to their phosphorylation data obtained using the anti-AT 1 receptor antibody on native, endogenously expressed AT 1 receptors. The AT 1A receptor resolved on SDS-PAGE showed the characteristic broad banding expected for a heavily glycosylated receptor and also co-migrated with the receptor radioactively labeled with 125 I-Sar 1 (49-N 3 )L –Phe 8 –AngII. Despite an unequivocal demonstration of AT 1A receptor phosphorylation in response to AngII stimulation, none of these three studies identified the site(s) and residues phosphorylated on the receptor. Hence, as reported in a recent study [13], we have utilized the HA-epitope-tagging approach to study the sites of AT 1A receptor phosphorylation and, in particular, the contribution of amino acids within the carboxyl-terminus to this modification. Our approach was to examine the phosphorylation of fulllength AT 1A receptors and compare this to receptors with truncations and mutations of the carboxyl-terminus. We

observed that epitope-tagged full-length AT 1A receptors, when transiently transfected in CHO-K1 cells, displayed a basal level of phosphorylation that was significantly enhanced by AngII stimulation (see Fig. 2C). Phosphorylation of AT 1A receptors was progressively reduced by serial truncation of the carboxyl-terminus, indicating multiple sites for phosphate modification in this region. Truncation to Lys 325 , which removed the last 34 amino acids, almost completely inhibited AngII-stimulated 32 P-incorporation into the AT 1A receptor. This result documents that most, if not all, AT 1A receptor phosphorylation occurs within the serine- and threonine-rich region of the cytoplasmic tail. Further site-directed mutagenesis of the carboxyl-terminus indicated that multiple residues within the carboxyl-terminus are phosphorylated, including amino acids within the region Thr 332 to Ser 338 that have previously been identified as important for AT 1A receptor internalization ([72], see Fig. 3, and discussed in detail below).

3.4. Receptor desensitization and the AT1 A carboxylterminus The serine- and threonine-rich carboxyl-terminus of the AT 1A receptor, which we have shown is phosphorylated in response to AngII stimulation, would be expected to play a key role in the desensitization of AT 1A responses. Desensitization is characterized by a reduction in magnitude of signaling in response to repeated stimulation by ligand. Surprisingly, we observed that a truncated AT 1A receptor mutant, lacking all carboxyl-terminal serine and threonine residues, when expressed in CHO-K1 cells, still displayed desensitization to AngII after an initial stimulation [60]. This result contrasts with subsequent studies, which demonstrated that removal of the AT 1A receptor carboxylterminus indeed modulates receptor desensitization. In cells expressing the wild type full-length AT 1A receptor, the capacity to mobilize inositol phosphates in response to AngII was reduced following pretreatment with a PKC activator, phorbol 12-myrisate 13-acetate (PMA) [12]. This reduction in inositol phosphate signaling was not observed in an AT 1A receptor mutant, truncated at Tyr 319 , to remove three putative PKC phosphorylation sites within the carboxyl-terminus. However, a role for direct PKC phosphorylation in AT 1A receptor desensitization remains controversial [12,61,63,73], although it is supported by a recent study by Feng et al. [74], who observed a heterologous desensitization of the AT 1A receptor following stimulation of the endothelin A (ETA ) receptor, presumably as a result of PKC activation and translocation to the plasma membrane. Conchon et al. [62], examined the activation and desensitization of six AT 1A receptor mutants truncated after Gly 303 , Phe 313 , Ser 328 , Ser 335 , Arg 340 and Ser 346 . They reported that truncation of AT 1A after Ser 328 produced a receptor that displayed an amplification of AngII-induced intracellular signaling as a consequence of reduced desensitization. A small (20–30%) desensitization

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Fig. 3. Schematic of the AT 1A receptor carboxyl-terminus, summarizing aspects pertaining to structure-function relationships. The amino acids comprising the end of the 7th transmembrane segment and the entire cytoplasmic carboxyl-terminal region (residues 305–359) are shown in detail. See text and indicated references, in parentheses, for details.

to PMA, which was observed with the full-length receptor, was also abolished in this mutant. Truncated mutants at 340 and 346 were indistinguishable from wild type receptor, suggesting the region 328 to 340 is important for AT 1A receptor desensitization. Finally, Tang et al. [63] examined the homologous (AngII induced) and heterologous (PMA induced) desensitization of full length, and truncated, AT 1A receptors. They observed that the region 328 347 between Ser and Ser within the carboxyl-terminus is critical for heterologous as well as homologous desensitization. They also proposed that homologous (AngII induced) desensitization is the result of a heparin-sensitive

kinase, indicative of a GRK, other than GRK2, GRK5 and GRK6. GRK2 and GRK5 have been previously implicated in the phosphorylation of AT 1A receptors expressed in HEK293 cells [61]. In agreement with Balmforth et al. [12], Tang et al. [63], report that the desensitization to low doses of AngII (1 nM) are mediated by PKC and that at higher concentrations (100 nM), the unidentified GRK is responsible. When comparing and interpreting these studies on AT 1A receptor desensitization, it is important to consider the sometimes substantial differences in experimental approach. For instance, some have [61,63], or have not

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[60,62], used an acid-wash step to strip cell surface receptors prior to restimulation with AngII. While acidwashing does not reflect an in vivo situation, in studies where it is omitted, ‘‘genuine’’ desensitization may be confused with receptor occupancy. Moreover, some have used intracellular calcium transients as the readout of receptor activity [60], while other studies have measured inositol phosphate generation or membrane GTPase activity [61]. Another important consideration is the role of receptor endocytosis to the desensitization of receptormediated signaling. With the exception of Oppermann et al. [61], all desensitization studies have been performed on whole cells expressing wild type and mutated receptors. Because the truncated mutants that are displaying desensitization are also severely inhibited with respect to internalization, it is difficult to separate these phenomena. In fact, the apparent desensitization of wild type (internalizing) receptors, when compared to non-internalizing truncated or mutated receptors, may be nullified if AngII stimulated responses for the wild type were corrected for reduced receptor number at the cell surface after the initial ‘‘desensitizing’’ treatment. Although desensitization is observed even when internalization is inhibited chemically (see [63] for discussion), a fully internalizing, non-desensitizing, AT 1A receptor mutant has not been reported. It is interesting to note that adrenal glomerulosa cells, which express endogenous AT 1 receptors, are resistant to shortterm desensitization and that long term AngII stimulation, with an accompanying down-regulation of receptor number, is required to dampen cell responses [64].

3.5. AT1 receptor endocytosis and trafficking Receptor endocytosis may subserve other functions besides a putative role in receptor desensitization (for review, see [75]). For example, certain tissues, particularly those (e.g. the adrenal gland) with highest concentrations of AT 1 receptor, sequester AngII from the circulation and accumulate the peptide intracellularly [76]. Because AT 1 , but not AT 2 receptors, undergo AngII stimulated endocytosis [72,77], it appears likely that AT 1 receptor internalization is the major route of entry for circulating AngII. Sequestration of AngII may represent a cellular mechanism for storing AngII for later release and to permit paracrine / autocrine actions. Sequestered AngII may stimulate cytoplasmic [78] or nuclear [79] angiotensin receptors, and AT 1 receptor internalization and trafficking may be required for the translocation of cell surface AT 1 receptors to the nucleus [80]. As discussed below, considerable effort has been directed towards elucidating the molecular mechanisms that underlie AngII-induced internalization of AT 1 receptors [75], and in particular, the contribution of the receptor carboxyl-terminus in AT 1A endocytosis to this process. Internalization motifs have been identified within the cytoplasmic regions of receptors that supposedly mediate

the recruitment of activated receptors into clathrin-coated pits and vesicles (for review see, [81,82]). These motifs are presumed to bind adaptor protein complexes, such as AP-2, which interact with the clathrin lattice to promote internalization. Truncation of the AT 1A receptor carboxylterminus severely impairs agonist-induced endocytosis, implicating the carboxyl-terminal tail in receptor internalization [72,83,84]. These truncated receptors displayed high affinity binding for AngII, coupled to G-proteins and signaled in response to AngII stimulation, demonstrating that other receptor functions were unaffected. However, serial truncation of the AT 1A receptor does lead to a concomitant decrease in functional receptor expression at the cell surface [13,85]. Severely truncated AT 1A receptors are poorly expressed and a region (Lys 307 –Lys 311 ) important for efficient receptor expression has been identified [85]. Extensive mutagenesis of the AT 1A receptor, aimed at identifying endocytotic motifs, has revealed two separate regions of the carboxyl-terminus and a region at the Nterminus of the third cytoplasmic loop that are important for internalization [15,60,62,63,75,83,84,86–88]. Hunyady et al. [72] reported that the amino acid triplet, Ser 335 – Thr 336 –Leu 337 , the so-called ‘‘STL’’ motif, within the carboxyl-terminus contributed significantly to receptor endocytosis (see Fig. 3). In a follow-up study [83], we utilized various truncations and deletions of the carboxylterminus of the AT 1A receptor, as well as point mutations, to identify the critical amino acids required for internalization. Our results suggested that the ‘‘STL’’ motif was important, but not sufficient, for endocytosis, and that two separate motifs are present: one distal to Lys 333 (i.e. the ‘‘STL’’ motif identified by Hunyady et al. [72]) and one located in the region 315–329. In particular, two of the mutants (Leu 316 Phe and Tyr 319 Ala), that fall within the 315–329 region, displayed significantly reduced internalization rates. We also have observed [Thomas, unpublished] that mutation of the a-helix-terminating, Pro 321 and Pro 322 , causes marked inhibition of AT 1A receptor endocytosis. Substitution of Leu 316 is the most effective single-point mutant yet described in inhibiting AT 1A receptor endocytosis, implying a key role for this residue and region in the internalization process. That Leu 316 is part of a dileucine pair (Leu 316 Leu 317 ) may be informative because dileucine internalization motifs have been proposed for numerous receptors and very recent evidence suggests that a carboxyl-terminal dileucine motif is crucial for the internalization of another GPCR, namely the b 2 -adrenergic receptor [89]. It remains unclear whether the proximal or distal site within the AT 1A receptor carboxyl-terminus is the actual motif engaged by the endocytotic machinery and what role the third cytoplasmic loop plays in the internalization process. G-protein coupled receptors appear to internalize through a clathrin-mediated process slightly modified from that previously described for other receptors. Thus, the

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internalization of the prototypical b-adrenergic receptor can be reduced by overexpression of a mutant form (K44A) of dynamin I [66,90], a GTPase involved in the formation and internalization of clathrin-coated vesicles. However, there is no evidence for any GPCR that AP-2 complexes interact directly with the cytoplasmic internalization motifs to target receptors to clathrin-coated pits, as is the case for the ‘‘classical’’ pathway. Instead, recent evidence indicates that arrestin proteins, which bind with high affinity to phosphorylated GPCRs and mediate their desensitization, also play a crucial role in endocytosis [91]. Arrestin has been shown to interact with clathrin [92,93], suggesting that arrestin acts as an adaptor to link activated GPCRs directly to the endocytotic machinery, independent of AP-2 complexes. Overexpression of arrestin dominant / negative mutants causes inhibition of b-adrenergic receptor internalization [90,91,94], especially when receptor internalization is stimulated by over-expression of GRKs to phosphorylate receptors and promote endogenous arrestin binding. Interestingly, the internalization of AT 1A receptors is not blocked by overexpression of the K44A dynamin I mutant [90], suggesting a dynamin-independent mechanism. Alternatively, other isoforms of dynamin may be involved that are not subject to inhibition by the dynamin I K44A mutant. Dynamin exists as two major forms: dynamin I and dynamin II, with additional subtypes of dynamin II [95]. However, we [Thomas and Liu, unpublished data] have been unable to inhibit AT 1A receptor internalization with dynamin I K44A and dynamin II K44A constructs or by overexpressing dynamin I or dynamin II Pleckstrin Homology domains, which potently inhibit endocytosis of other receptors. Hence, AT 1A receptor endocytosis appears to be independent of the major dynamin isoforms. In addition, the internalization of AT 1A receptors appears to be independent of the function of b-arrestin, in contrast to the trafficking of the b 2 -adrenergic receptor [90]. However, when b-arrestins are overexpressed some AT 1A receptors are targeted to a dynamin-dependent pathway (i.e. blocked by dynamin I K44A) [90]. These data were interpreted to mean that GPCRs could utilize distinct internalization pathways, as distinguished by dynamin and b-arrestin, and that b-arrestins are adaptor proteins that target GPCRs for dynamin-dependent endocytosis via clathrin-coated vesicles. The identity of the presumed alternate internalization pathway is not known, but may involve specialized structures within the plasma membrane called caveolae [96,97]. If arrestins are to play a role in receptor internalization, then the phosphorylation of receptors, which promotes arrestin binding, must also be important. The identification of a crucial serine / threonine rich region (Thr 332 Lys 333 Met 334 Ser 335 Thr 336 Leu 337 Ser 338 , including the ‘‘STL’’ motif) within the carboxyl-terminus of the AT 1A receptor subtype, suggests that phosphorylation may be involved in endocytosis [72]. As already discussed (see

17

also Fig. 2), we have initiated studies to examined the phosphorylation of AT 1A receptors and compared this to receptors with truncations and mutations of the carboxylterminus [13]. Serial truncation of the AT 1A carboxylterminus causes a progressive loss of AT 1A receptor phosphorylation in response to AngII stimulation, indicating multiple phosphorylation sites within the cytoplasmic tail. Truncation to Lys 325 , which removed the last 34 amino acids including all serine and threonine residues, completely inhibited AngII-stimulated 32 P-incorporation into the AT 1A receptor. When the carboxyl-terminal res332 335 336 338 idues, Thr , Ser , Thr and Ser , previously identified as important for receptor internalization, were substituted with alanine, the mutant receptor displayed a significantly reduced AngII-stimulated phosphorylation and internalization. Moreover, the reduced endocytosis of this quadruple alanine mutant could be rescued if these residues, in particular Ser 335 and Thr 336 were substituted with acidic amino acids (i.e. glutamic acid and aspartic acid) to mimic the acidic charge created by phosphorylation. Taken together, these data suggest an important role for phosphorylation in maximizing AT 1A receptor endocytosis, perhaps due to the attraction of adaptor proteins for internalization such as arrestins. However, as yet, no group has reported that arrestins can bind activated, phosphorylated AT 1A receptors, although it is expected. The use of epitope-tagged AT 1 receptors to immunoprecipitate the phosphorylated receptor (see Fig. 2 and [13,61]) provides the means to determine if arrestin proteins, or other components and adaptor proteins (such as the adaptin proteins of the AP-2 complex) directly associate with the AT 1A receptor. Once the adaptor proteins are identified, it will also possible to use receptor mutants to identify the sites of interaction and the impact of receptor phosphorylation on these processes. Fig. 3 summarizes our current understanding of the AT 1 receptor regulatory process as it relates to the structure–function aspects of the AT 1A carboxyl-terminus.

3.6. Receptor dimerization For many growth factor and cytokine receptors, the principle mechanism for initiating receptor-mediated signaling is ligand-induced receptor dimerization or ligandinduced receptor proximity [98]. Binding of growth factor (or cytokine) to its monomeric receptor results in the dimerization of receptors which can then bring about the cross-phosphorylation and / or recruitment of additional intracellular signaling molecules to the dimer. As a result, intracellular signaling cascades and growth and cytokine responses are initiated. An intriguing possibility, based on exciting preliminary data from other GPCRs, is that AT 1 receptors may undergo agonist-regulated dimerization, and that this process is involved in modulating receptor function.

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W.G. Thomas / Regulatory Peptides 79 (1999) 9 – 23

Recently, the dimerization of a number of GCPRs has been observed, including the b 2 -adrenergic [99], muscarinic [100], dopamine D2 [101], dopamine D3 [102], metabotropic glutamate receptor 5 [103], and the d-opioid receptors [104]. For b 2 -adrenergic and dopamine D2 receptors, agonist stimulation results in stabilization of receptor dimers that are dependent on sequences in transmembrane domain VI or VII. For, muscarinic receptors, dimerisation appears dependent on sequences with the third intracellular loop of the receptor. In contrast, Cvejic and Devi [104], reported that d-opioid receptors pre-exist predominantly as dimers and that binding of agonists resulted in the formation of monomeric receptors. This dimerization was dependent on residues within the last 15 amino acids of the intracellular carboxyl-terminus. Little is known about the dimerization of AT 1 receptors and its possible role in the AngII-induced signaling and receptor regulation. Indirect evidence for AT 1 receptor dimerization has come from a study by Monnot et al. [18], who demonstrated that co-expression of two binding defective AT 1 receptor mutants rescued ligand binding, suggesting that receptor dimers exist, which can interchange transmembrane regions, and thereby form functional binding sites. It will be of interest to determine directly whether AT 1 receptors dimerize in response to agonist stimulation. With the current availability of epitope-tagged AT 1 receptors, that allow unambiguous receptor immunoprecipitation and detection, such experiments are now tenable.

3.7. Receptor cross-talk and regulation Receptor cross-talk can be broadly defined as the modification of one receptor’s actions by another. This can result from direct interaction of the two receptors in the plasma membrane or as a consequence of signals generated by one receptor that impinge on, or modify, the function of the other. As an example of the latter, the phosphorylation of GPCRs (i.e. AT 1 receptors) may result from signals generated by the activated receptor itself (homologous) or may arise from activation of other receptors (heterologous). In the case of AT 1 receptors, Feng et al. [74] observed that activation of the ETA receptor was capable of provoking AT 1A receptor desensitization, as measured by an inhibited capacity of AngII to promote translocation of PKC from the cytoplasm to the plasma membrane. Because PKC phosphorylates and dampens the responsiveness of the AT 1A receptor [61], Feng et al. [74] proposed that an ETA receptor mediated activation of PKC and subsequent heterologous phosphorylation of the AT 1A receptor was the probable mechanism. Interestingly, AngII stimulation, which is known to activate PKC, had no effect on the subsequent activity of the ETA receptor. Apart from serine and threonine phosphorylation and cross-talk, GPCRs can also be phosphorylated on tyrosine residues by growth factor receptors with intrinsic tyrosine

kinase activity [105] and thereby have their activity regulated. For example, insulin causes tyrosine phosphorylation of the carboxyl-terminus of the b 2 -adrenergic receptor and influences its function [106], while insulinlike growth factor also tyrosine phosphorylates the b 2 adrenergic receptor, but at different sites [107]. Similarly, insulin stimulation in vivo has been reported to cause tyrosine phosphorylation of the cardiac AT 1 receptor [108], although this requires confirmation. Conversely, stimulation of the AT 1 receptor by AngII may affect the activity of other receptor systems and / or usurp additional signaling capabilities. Considerable evidence exists to suggest that there is important cross-talk between the signaling pathways and receptors of the angiotensin and insulin system [108,109] and AngII is a inhibitor of interleukin-6 activation of the JAK-STAT pathway [110,111]. Moreover, a series of recent papers have identified an AngII-stimulated tyrosine phosphorylation [112] and calcium-dependent transactivation [113–115] of the epidermal growth factor receptor that serves as a scaffold to recruit signaling molecules and adaptors for the activation of the MAP kinase and mitogenic pathways.

3.8. Interaction of proteins with the AT1 receptor carboxyl-terminus The activation, phosphorylation, internalization, desensitization and cross-talk of AT 1 receptors is likely to involve dynamic changes in the association of various proteins with the AT 1A receptor carboxyl-terminus. Coupling of the receptor to heterotrimeric G-proteins is dependent upon the proximal region of the AT 1A receptor carboxyl-terminus, specifically, the hydrophobic cluster of Tyr 312 , Phe 313 and Leu 314 (see Fig. 3) [27,28]. Site-directed mutations of Tyr 312 , Phe 313 , and Leu 314 yielded receptors that were insensitive to GTPgS and displayed a reduced capacity to liberate inositol phosphates following AngII stimulation, indicating uncoupling from G-protein. Also, purified peptides containing the wild type sequence (residues 306–320), but not with mutations at Tyr 312 , Phe 313 and Leu 314 , were able to activate the binding of GTPgS to purified G-proteins. This region of the AT 1A carboxyl-terminus also interacts with proteins other than G-proteins. Following the initial report of the induction of the JAK-STAT pathway by AT 1A receptors [116], Marrero et al. [117] provide evidence for a direct interaction between Jak2 kinase and the AT 1A receptor, offering a mechanism for the activation. Subsequently, the same group [118] proposed that Jak2 associates with the AT 1A 319 320 321 322 receptor through the sequence Tyr Ile Pro Pro within the proximal AT 1A receptor carboxyl-terminus. While this ‘‘YIPP’’ motif may function as a SH2 targeting sequence upon phosphorylation of Tyr 319 , Jak2 contains no SH2 domains that could putatively mediate the association. Moreover, Oppermann et al. [61] found little evidence for

W.G. Thomas / Regulatory Peptides 79 (1999) 9 – 23

tyrosine phosphorylation of the AT 1A receptor following AT 1A receptor stimulation, although Venema et al. [119] recently reported robust tyrosine phosphorylation of the AT 1A receptor in vascular smooth muscle cells following AngII treatment. The controversy of interpreting data obtained using current AT 1A receptor antibodies [119] versus that obtained using epitope-tagged receptors [13,61] (see earlier), means that further studies are required to resolve the issue of whether the AT 1A receptor becomes tyrosine phosphorylated, especially on Tyr 319 , following AngII stimulation. Nevertheless, the apparent incongruity of Jak2 binding to the AT 1A YIPP motif in the absence of Jak2 SH2 domains, predicts that an SH2 containing adaptor protein may link the YIPP motif to Jak2. Indeed, the SH2 domain-containing SHP-2 phosphotyrosine phosphatase has been identified as the possible adaptor molecule [119]. The YIPP motif has also been implicated in the recruitment of PLC-g1 to the carboxyl-terminal in an AngII- and tyrosine phosphorylation-dependent manner [119]. Binding was dependent on one of the two SH2 domains in PLC-g1 and the tyrosine phosphorylation of the receptor at Tyr 319 [119]. Thus, two effector molecules, PLC-g1 and the putative SHP-2 phosphotyrosine phosphatase-Jak2 tyrosine kinase complex, from separate signaling pathways, are able to engage the YIPP motif. This motif is very close to the Tyr 312 Phe 313 Ile 314 triplet responsible for binding and activating the G-protein, which suggests that competition may exist between these signaling molecules. Moreover, we have shown that the integrity of this region, in particular the residues encompassing and surrounding Leu 316 to Pro 322 , is critical for efficient endocytosis of the AT 1A receptor [83]. This highlights the possibility that components of the internalization machinery may also compete for binding at the proximal carboxyl-terminus. The positively-charged amphipathic a-helical region between Leu 305 and Ile 320 also shows high affinity binding for Ca 21 -calmodulin [Thomas, unpublished observations] which suggests a possible feedback loop involving AngIImediated intracellular calcium release and the formation of Ca 21 –calmodulin complexes that interact with the receptor. Finally, an in vitro association of endothelial nitric oxide synthase (eNOS) with a fusion protein containing the entire AT 1A carboxyl-terminus has been reported [120]. Although the binding site for eNOS on the AT 1A receptor carboxyl-terminus was not mapped, this observation is of interest because of the established association between AngII and eNOS activation.

4. Future directions The identification of multiple binding sites, together with the proposed competition between binding proteins, prompts speculation as to the hierarchy of interactions as this proximal region of the AT 1A receptor. Following

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receptor activation, the carboxyl-terminus is phosphorylated at multiple sites and presumably this results in arrestin (or other proteins) binding to the receptor and displacing the G-protein or perhaps other signaling molecules. At what point, if at all, during this cycle of receptor activation, phosphorylation, desensitization, internalization, dephosphorylation and recycling, do Jak2 and PLC-g1 interact with the receptor? Is it possible that PLC-g1 or the SHP-2 phosphatase–Jak2 complex only gain access to the YIPP motif after the G-protein has been activated and released from this a-helical amphipathic sequence [121]? Does the recruitment of proteins that interact on or near the YIPP motif prevent reassociation of the G-protein with the receptor and therefore contribute to desensitization? Whatever the merit of these speculations, it is obvious that the proximal carboxyl-terminus of the AT 1A receptor is a site of complex receptor activity (see Fig. 3). Finally, given the central importance of phosphorylation to both receptor coupling and regulation, it is clearly important to identify and characterize the exact sites and hierarchy of AngII induced AT 1 phosphorylation. Inherent problems with ‘‘loss-of-function’’ mutagenesis studies (i.e. the mutation may cause local or global alterations / aberrations that make interpretation equivocal), means complementary approaches are required. With the ability to immunoprecipitate AT 1 receptors, one approach will be to isolate proteolytic fragments of phosphorylated receptor and to purify these by high-performance liquid chromatography. In-line, ion-spray mass spectroscopy can then be used to unambiguously identify the exact sites of phosphorylation.

Acknowledgements A National Health and Medical Research Council of Australia Institute Block Grant to the Baker Medical Research Institute and a National Heart Foundation of Australia Grant-in-Aid to W.G.T supported this work. The assistance of Ms. Luisa Pipolo and Dr. Hongwei Qian is gratefully acknowledged and I thank Mr. Brian Jones for help with the production of Fig. 2.

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