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Department of Biophysics, Escola Paulista de Medicina, Federal University of São Paulo; Department of Biochemistry and Immunology, Ribeirão Preto Medical School, University of São Paulo, Ribeirão Preto; and Department of Biochemistry, Institute of Chemistry, University of São Paulo, São Paulo, Brazil
ABSTRACT I. INTRODUCTION A. The Renin-Angiotensin System B. Scope of This Review II. ANGIOTENSIN II STRUCTURE-ACTIVITY RELATIONSHIPS A. Receptor Binding and Activation B. Antagonism by ANG II Analogs C. Desensitization and Tachyphylaxis III. AT1 RECEPTOR STRUCTURE-ACTIVITY CORRELATIONS A. Receptor Structure 1. Sequences of AGPCRs 2. Rhodopsin structure 3. Patterns of residue conservation 4. The AT1 receptor A) BINDING-RELATED RESIDUES. B) RESIDUES PARTICIPATING IN RECEPTOR ACTIVATION. B. Receptor Activation 1. Activation of rhodopsin 2. Physiological activation of AGPCRs and AT1 receptors A) AGONIST BINDING AND ACTIVATION. B) EVENTS AT THE 7TM BUNDLE CYTOSOLIC ENDS. 3. Constitutive activation A) AGPCR IN GENERAL. B) AT1 RECEPTORS. 4. AT1 receptor inhibition C. Mechanisms Following Receptor Activation 1. Signal transduction 2. G protein activation 3. Arrestin binding D. Overview of the AT1 Receptor 1. Activation in the 7TM bundle 2. The role of the cluster of polar residues: the sodium site 3. Events at the cytosolic domains 4. Conclusions IV. FINAL REMARKS A. Active Rhodopsin (AGPCR) Structures as a Result of Probabilistic Analyses B. The Extracellular Locus as a Site for Cross-Talking Mechanisms C. The AT2 Receptor Is a Natural Constitutively Activated Form of AGPCR D. The Cluster of Polar Residues: The Sodium Site V. SUMMARY GRANTS ACKNOWLEDGMENTS REFERENCES
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I. INTRODUCTION |
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The renin-angiotensin system (Fig. 1) regulates blood pressure and fluid homeostasis (227) by renin-mediated hydrolysis of angiotensinogen to release ANG I. This inactive decapeptide is further hydrolyzed by the angiotensin converting enzyme (ACE) yielding the vasoconstrictor octapeptide ANG II (Asp-Arg-Val-Tyr-Ile-His-Pro-Phe), the classical effector of the renin-angiotensin cascade. ANG I may also be hydrolyzed by prolyl-endopeptidase and carboxypeptidases to yield the vasodilator heptapeptide ANG-(1–7). Alternatively, ACE-2, a carboxypeptidase homolog of ACE (46, 285), cleaves a single residue from ANG I to generate ANG-(1–9), which can be hydrolyzed by ACE to yield ANG-(1–7) (Fig. 1). Degradation products of ANG II lacking the NH2-terminal Asp (ANG III) and the Asp-Arg (ANG IV) residues are particularly involved in brain and pituitary physiology (10, 300).
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Two ANG II receptor types (AT1 and AT2) were cloned and sequenced (125, 186, 187, 251), both belonging to the rhodopsin-like family (family A) of GPCRs (AGPCR). ANG-(1–7) was reported to interact with a different GPCR, the mas protooncogene product (250), exerting vasodilator and antiproliferative effects (58, 59).
AT1 and AT2 receptor types can be distinguished by their binding profiles to different ligands. Thus, whereas the AT1 receptor has a high affinity for the nonpeptide antagonist losartan (284), AT2 binds preferentially to the peptide antagonist CGP42112 (283). The two receptor types trigger distinct (sometimes opposing) signal transduction pathways (113) such as effects leading to growth and antigrowth responses (91, 92). The idea that the AT2 receptor is an AT1 receptor antagonist is also suggested by a finding not yet confirmed by others, that the AT1/AT2 receptor heterodimer inhibits AT1 signals (2).
AT1 and AT2 receptors also differ in that the former undergoes rapid internalization and desensitization upon agonist stimulation (97, 281), whereas the AT2 receptor does not internalize and is not desensitized upon interaction with the agonist (87, 108, 186, 288). However, contrasting with these findings, AT2 receptor was shown to be internalized in response to ANG II and driven to play a novel signaling pathway. This consists of a receptor-mediated translocation of a cytosolic transcription factor, the promyelocytic zinc finger protein (PLZF), to the nucleus and activation of p85alfaPI3K and p70S6 kinase leading to an increase of protein synthesis (256).
AT2 receptors have also been shown to play a role in a number of biological processes, such as vascular relaxation, antiproliferation, cellular differentiation, and apoptosis (32, 68, 98, 271), but it is the AT1 receptor that plays a critical role in signaling pathways that mediate most of the classical physiological and pathological effects of ANG II on target cells, such as vascular smooth muscle cell contraction, aldosterone release, stimulation of sympathetic transmission and cellular growth, salt appetite, thirst, sympathetic outflow, and inhibition of renin biosynthesis and secretion (9, 43, 63, 267, 282). Interestingly, each of these ANG II-induced effects is triggered at different time intervals: seconds in cell signaling, activation of phospholipase C, generation of inositol phosphate (IP) and Ca2+ release; minutes in mitogen-activated protein kinase activation; hours in activation of Janus kinase (JNK), signal transducers, and activators of transcription pathway (79).
The AT1 receptor acts by stimulating protein Gq/G11 and subsequently phospholipase C and IP production or by regulating the level of cAMP by activation of protein Gi (12, 173, 260). In addition to vasoconstriction, regulation of renal tubular electrolyte handling, aldosterone release, and facilitation of adrenergic release, the AT1 receptor is one of the most potent stimulators of hypertrophic remodeling of the vascular walls, through ANG II-mediated growth-promoting signals. It has been suggested that the intracellular signaling mechanisms by which the AT1 receptor exerts hypertrophic and/or hyperplastic effects on targets such as vascular smooth muscle cells are closely associated with receptor and nonreceptor tyrosine kinases (49). Furthermore, it has been demonstrated that G12/G13 can induce vascular smooth muscle cell contraction through a Rho/Rho-kinase (ROCK)-mediated pathway (71). These authors provided evidence that receptor AT1 couples with Gq and G12 chains to efficiently induce cell contraction via dual regulation of the myosin light chain (MLC) phosphorylation: the Ca2+-dependent stimulation of MLC kinase and the Ca2+-independent Rho/ROCK-mediated inhibition of myosin phosphatase.
ANG II-stimulated activation of some mitogen-activated protein kinases (MAPKs) has been reported (74, 76), with calcium channels being essential in this mechanism. In fact, activation of the extracellular signal-regulated kinase (ERK1/ERK2), tyrosine kinase 2, and Janus-activated kinase 2 (Jak2), as well as phosphorylation of signal transducer and activator of transcription (STAT1 and STAT3), were shown to be inhibited by the calcium channel blocker azelnidipine (152).
Superoxide generation has been found to be mediated by ANG II and to play an important role in vascular smooth muscle cell growth, contraction/relaxation, and inflammation. It was shown that ANG II-induced oxidant activity is increased and generation of reactive oxygen species is enhanced in conditions associated with vascular damage such as in hypertension, ischemia-reperfusion injury, atherosclerosis, and diabetes (30, 49).
AT1 receptors desensitize following agonist stimulation due to sequestration and endocytosis (281). Differently from this desensitization, acute loss of response upon repeated treatments with ANG II is also observed in smooth muscle cells, characterizing the event of tachyphylaxis (130, 216, 270).
Homodimerization has been described for AT1 receptors linked to the action of factor XIIIA transglutaminase (3) or as a natural phenomenon occurring in wild-type receptors (233). However, experiments demonstrating oligomerization (or at least cross-talking) in AT1 receptors consisted of coexpression of a wild-type and a nonfunctional mutant (85, 183). Heterodimerization of AT1 receptor with AT2 (2) and bradykinin B2 (1) receptors, and other AGPCRs such 
2-adrenergic receptors (19), has been claimed to occur. Interestingly, it appears that the dimerization of AT2 receptors might be regulated by disulfide bond exchange (179).
The ANG II AT1 receptor is responsible for virtually all of the known peripheral actions of ANG II. It has been most extensively studied by site-directed mutagenesis aiming at the elucidation of ligand binding, signal transduction, phosphorylation, binding to arrestins, internalization, desensitization, tachyphylaxis, and other properties. The main scope of this review is to correlate the AT1 receptor basic functions (ANG II binding, receptor activation, signal transduction across the transmembrane structure, and some events at the cytosolic ends of the receptor) with the data available about AGPCR sequences, rhodopsin structure, and mechanism of receptor action. Presentation and discussion of related events such as G protein coupling, arrestin binding, desensitization, and tachyphylaxis shall not be fully comprehensive but limited to features considered necessary to enrich the main subject. Other events such as oligo(di)merization of AGPCRs, for which no relationship with receptor activation has been completely confirmed, shall not be discussed.
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II. ANGIOTENSIN II STRUCTURE-ACTIVITY RELATIONSHIPS |
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Long before cloning of the receptors was achieved, numerous studies of the cardiovascular effects of synthetic peptide analogs allowed a detailed mapping of ANG II's structural requirements for receptor binding and activation (130, 131, 212, 213, 215). These structural requirements can be assumed to apply to the AT1 receptor, since the biological responses that were analyzed in those early reports were later found to be mediated by that receptor type.
Analyses of the effects of different ANG II analogs bearing single or multiple modifications indicated that an electrostatic interaction between the ANG II COOH-terminal carboxylate group and the receptor is an important factor for high-affinity binding (100) and that the peptide's Arg2, Tyr4, and His6 side chains are also important for high-affinity binding (31, 178, 239, 240). On the other hand, replacements of Tyr4 and mainly Phe8 by aliphatic residues were shown to generate competitive antagonists, thus attesting to the importance of these aromatic residues for receptor activation (70, 247). The Val3, Ile5, and Pro7 residues were found to be neither crucial for binding nor for activation. Asp1 was not found to be relevant, since analogs with replacements in this position, as well as des-Asp1-ANG II (ANG III), still retain significant activity. On the other hand, the introduction of a sarcosine (Sar) residue in position 1 increased the potencies of agonist and antagonist analogs (26, 81, 211, 238, 239). Pharmacological (158) and mutagenesis (249) studies on the AT1 receptor indicated that [Sar1]-ANG II has a binding mode different from that of the natural agonist, as discussed in section III, B4 and D1. Also, the presence of Sar in position 1 seems to induce a reaccommodation of other residues involved in binding, such as Arg2 (249).
ANG II was shown to be in a random conformation in aqueous solution, various conformers coexisting in equilibrium, which would be displaced towards the "active one" upon binding to the receptor (217). One of these conformers could be that of a horseshoe-shaped structure of ANG II described in a complex of the peptide with antibody (67). Other studies were published in which different conformations were proposed for ANG II or synthetic peptide analogs in aqueous or other media (57, 163, 194, 236), but no rational information was obtained about the "active conformation" that binds to the receptor. Other approaches were based on constraints introduced in the ANG II structure so as to freeze certain conformations of the peptide, thus allowing their biological activities to be checked (122, 192, 306). However, despite the great deal of effort, no conclusive results leading to knowledge about the active conformation of ANG II could be obtained using these approaches.
B. Antagonism by ANG II Analogs
As expected from the structural determinants for signaling described above, one of the first reported antagonists for ANG II was [Phe4,Tyr8]-ANG II (164), and in the following years, other antagonists were reported, mostly bearing modifications in the Phe8 position and usually also with sarcosine in place of Asp1. A milestone in such studies was the discovery of the analog saralasin ([Sar1,Ala8]-ANG II) as the first high-affinity antagonist (222).
During the first years of investigations on this subject, development of antagonists with modifications at the Tyr4 position was less explored, possibly due to the lower potency observed in the first analogs with modifications in this position (246). Later on, sarmesin, a potent antagonist with hydroxymethylated Tyr4 ([Sar1,Tyr(Me)4]-ANG II), was described (252). Combined modifications on both Tyr4 and Phe8 were shown to be nonadditive, generating antagonists of lower potencies (70, 247).
C. Desensitization and Tachyphylaxis
It has been demonstrated in whole animals, isolated tissues, and cultured cells that the AT1 receptor may be inactivated when left in prolonged contact with the agonist (96, 130, 184, 216, 218, 226, 270, 281). This loss of response has been linked to two mechanisms, tachyphylaxis and desensitization, which were first recognized by the experimental protocols used to elicit them. Tachyphylaxis is triggered by cycles of stimulation (agonist addition and washing) that have to be repeated twice, thrice, and more times to abolish the response (216, 270). Desensitization is promoted by prolonged contact of receptor with a same dose of agonist.
Differently from desensitization, tachyphylaxis seems to act at the level of agonist-receptor interaction and is dependent on the NH2-terminal ammonium group and the Arg2 guanidinium group of ANG II. [Succinyl1]-ANG II does not induce and [Sar1]-ANG II induces an enhanced tachyphylactic phenomenon (211), with this effect being explained by the increased protonation of the sarcosine secondary amino group at physiological pH (214, 216). The importance of the Arg2 guanidinium group for tachyphylaxis triggering has also been supported by the fact that [Lys2]-ANG II and even [Sar1,Lys2]-ANG II cannot induce tachyphylaxis (169, 211). Also differing from desensitization, tachyphylaxis induction depends on the aromatic ring at position 4 of ANG II, but some apparently conflicting aspects need to be analyzed. The phenolic hydroxyl of Tyr4 is vital to elicit in vitro tachyphylaxis, a property that is lost with [Phe4]-ANG II (185, 262). However, paradoxically, this ANG II analog is able to trigger tachyphylaxis in vivo (286), a condition that was not described for wild-type ANG II.
Thus tachyphylaxis is possibly due to conformational changes on the ANG II-AT1 receptor complex (126, 216, 263, 264, 270), a supposition compatible with findings showing that this phenomenon can be prevented or reversed by the AT1 receptor antagonist losartan (241).
Desensitization has been shown to be correlated with phosphorylation of Ser and Thr residues in the cytosolic domains of AT1 receptor (104, 281). Therefore, rapid desensitization involves primarily phosphorylation (27) of the receptor through two distinct types of Ser/Thr protein kinases: the second messenger-activated kinases, protein kinase A or C (75, 127, 245, 275), and the second messenger-independent G protein-coupled receptor kinases (110).
The installation of both tachyphylaxis and desensitization in AT1 receptors leads to decays of IP formation and calcium influx (4, 126, 264), but the most flagrant difference between these two states is that only tachyphylaxis is accompanied by reduction of sodium influx (4, 264).
AT1 receptor sequestration and internalization have been involved in ANG II desensitization in smooth muscle cells (6, 75, 76, 289). However, no results could be obtained from a series of studies supporting this correlation (23, 97, 102, 202, 280). Hence, sequestration does not appear to play a major role in desensitization but may instead be involved in the resensitization process (79).
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III. AT1 RECEPTOR STRUCTURE-ACTIVITY CORRELATIONS |
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Using the rhodopsin structure (151, 221) as a template, one may define the topography of the AGPCR sequence segments relative to the plasma membrane (Fig. 2) starting at the extracellular (Nt domain) and ending at the cytosolic side (Ct domain). Between these two extremes there is a seven-transmembrane (7TM) bundle (helices I–VII) intercalated by three intracellular loops (IC-1, IC-2, and IC-3) and three extracellular loops (EC-1, EC-2, and EC-3).
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2,800 AGPCRs according to the rhodopsin structure and oriented by very conserved residues in the 7TM helices and adjoining domains which were found without deletion or insertion in many receptors of the family (see Table A in the supplemental material1 and the consensual segments of sequences in Figs. 2 and 3) (13, 207, 209). Despite these conserved residues, true fingerprints which discriminate family A from other GPCR families, AGPCRs are heterogeneous because they belong to different classes of receptors, containing specific motifs inserted in the loops and terminal domains. However, within receptor types and subtypes, there is a remarkable similarity regarding both residues and lengths of the various segments (see GPCRDB).
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To allow a comparison of the different GPCR sequences, residue positions shall be identified using the general GPCRDB numbering system (99). In this system, the numbers for positions in the 7TM bundle have three digits, the first being the number of the helix (1–7) and the remaining two being the number of the position relative to the reference positions (Fig. 2).
The same method was applied to number positions of extramembrane domains that are conserved for some classes of receptors, as indicated in Figure 2 for the EC-1 and EC-2 loops of a large majority of nonolfactory AGPCRs and for the EC-3 loop of ANG II, bradykinin, endothelin, and other receptors (41; see GPCRDB). In receptor domains devoid of conserved motifs (for instance, the IC-1 and IC-3 loops), the respective sequences were divided into two halves (N and C) that were numbered according to the references used in the adjoining helices. As an example, Table 1 shows the segments of an AT1 receptor sequence (AG1R_RAT: Refs. 115, 187) with the respective natural and general GPCRDB numbers. Residue positions used as references for numbering are indicated. In the text, for every position of the AT1 receptor (or of other classes of AGPCRs), the numbering system is indicated as shown in Table 1: first the natural number (1–3 digits) followed by superscript A (AT1 receptor), superscript R (rhodopsin), and superscript G (other AGPCRs), and then the respective general GPCRDB numbers (3 digits) in parentheses.
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The ground or inactive structure of bovine rhodopsin (151, 221) consists of a 7TM bundle surrounding an ellipsoidal central cavity whose major axis is aligned with helices I-III and V-VII (Fig. 4). The broader side of this cavity is flanked by helices II-III and VI-VII, and the narrower sides are closed by helices I and V. The cavity is accessible from the intracellular side despite the existence of interactions between the cytosolic ends of helices II, III, and V-VII. The extracellular side is closed by the EC-2 loop hairpin that lies inside the central cavity parallel to the membrane surface, making interactions with side chains of helices. A remarkable interaction in rhodopsin is a disulfide bridge [Cys110R(315)-Cys187R(470)] between EC-2 and the top of helix III (Figs. 2 and 3), which is found in a majority of AGPCRs. Following the end of helix VII, the main chain forms a 90° bend and then a cytosolic helix (helix VIII) running parallel to the membrane. This helix, whose relative position was claimed to be due to crystal contacts, is tied to helix VII, to the VII-VIII bend, and to helices I and II by side chain interactions such as that involving Tyr306R(734) and Phe313R(810) (151) (Figs. 2 and 3).
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Patterns of residue conservation have previously been determined (209) on residue positions of AGPCR aligned sequences (see supplemental material Table A). For each position of the alignment, values of entropy (E) and variability (V) were determined and distributed along an E-V plot (Fig. 5; see legend of this figure for definitions of E and V). Over a broad range of values, it was possible to separate the positions of the sequence alignment according to levels of residue conservation by grouping them into boxes 11, 12, 22, 23, and 33, according to different ranges of E and V values.
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This structure-function map obtained from known proteins may be extrapolated to AGPCRs in general, what allowed us to assume that the conserved positions of these receptors (EV boxes 11 and 12 in Fig. 5) form a common main site and the variable positions (boxes 23 and 33 in Fig. 5) form modulator sites. The main site is at the cytosolic side of the 7TM bundle central cavity and may be related to a common cluster of conserved polar residues consisting of Asn(130), Asp(224), Asp(339), Arg(340), Asn(729), and Tyr(734) (see Fig. 3) which includes a part of a sodium allosteric site for some AGPCR classes (191) (see sect. IIID2). The most important modulator site (the agonist site) is at the extracellular side of the 7TM bundle central cavity (29, 205) (Fig. 6) and corresponds to the retinal site in rhodopsin structure (221). Positions with intermediate E and V values in the alignment of all AGPCR sequences are in the core of the 7TM structure, between the two sites. Thus, in the course of receptor activation, a signal may be transmitted from one site to the other through positions with intermediate EV values (209).
Additional experimental evidence suggests the existence of other modulator sites (variable positions) in other regions of the AGPCR structure, such as the COOH-terminal end of helix III [including the Tyr(339) residue of the DRY motif], the cytosolic ends of helices V and VI and adjacent sequences of the loop between these helices, helix VIII, and the VII-VIII bend (see references in supplemental material Table D).
Despite the low level of residue identity (20% in relation to rhodopsin), AT1 receptors can be modeled (203) in homology to rhodopsin structure (151, 221), at least at the level of sequence segments containing conserved residues (Fig. 7). Details and the rationale supporting this procedure are described in the supplemental material.
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A) BINDING-RELATED RESIDUES. Binding assays using radiolabeled ANG II or peptide analogs, and cloned AT1 receptors heterologously expressed in cultured cells, allowed identification of the following binding-related residues (Fig. 7, B and C): 1) the ANG II COOH-terminal carboxylate group is neutralized by the positive charge of the receptor's helix V Lys199A(512) (94, 111, 183, 195, 302, 303); 2) the peptide's Tyr4 side chain binds at the receptor's Asn111A(325) (55); 3) the receptor's Ser105A(319) is required for ANG II binding (78); 4) ANG II's His6 and Phe8 side chains are in proximity to the receptor's His256A(621) and Phe259A(624) side chains (82, 195, 196); 5) the peptide's Arg2 guanidinium group interacts either with the receptor's Asp281A(712) (53) or Asp278A(709) side chain (94) {this discrepancy seems to be due to the fact that different ligands (ANG II or [Sar1,Leu8]-ANG II) were used in the two studies, as discussed in sect. IIID1}; 6) the NH2-terminal half of the EC-2 loop, including residues Val179A(469) (94) and His183A(473) (53, 129, 303), is involved in ANG II binding; 7) Lys101A(316), located at the extracellular end of helix III, is important for binding (94, 183); 8) other residues involved in agonist binding are the Nt domain Arg23A(105) (249), His24A(106), Tyr26A(108), and Ile27A(109) (94) as well as the EC-1 loop Thr88A(238) (42) and Tyr92A(268) (42, 94).
Besides site-mutation studies, structures of AGPCRs have been analyzed by two indirect procedures: the substituted-cysteine accessibility method (16, 120, 176, 181) and the methionine proximity assay (36, 154). In these methods, residues are replaced by Cys and Met, and their relative positions in the three-dimensional structure of the receptors are defined as a function of kinetic parameters determined with Cys-specific reactants and by means of photoaffinity labeling, using (in the case of the AT1 receptor) ANG II analogs with benzophenone residues in position 8 (36).
The relative location (inside or outside the structure) of residues along the sequences of helices was estimated in many AGPCRs from cysteine accessibility results (16). Also, movements of helices upon AT1 receptor activation, as denoted by change in accessibility of strategically placed Cys residues, was studied by Miura and Karnik (176) and Miura et al. (181). A drawback linked to the Cys accessibility method is that this residue is revealed by bulky reagents. Because rhodopsin structures (151, 221) have an EC-2 loop interacting with the extracellular halves of helices and closing the extracellular mouth of the 7TM bundle central cavity, even small conformational changes, such as those produced by mildly bulky reagents, might artificially change the relative position of that loop and thus the accessibility of helices.
The Met-proximity assay also has limitations. To apply this method to AT1 receptors, the ANG II's Phe8 residue is replaced by a bulky moiety that is likely to interfere with the role of this agonist's aromatic residue to induce receptor activation (178) and with the formation of the salt bridge between the agonist's COOH-terminal carboxylate group and the receptor's helix V Lys199A(512) side chain (53, 94, 111, 303). As a consequence, the obtained results were contradictory. Against results from other laboratories (53, 94, 111, 302, 303), a different geometry for the ANG II binding site was described in which very conserved AGPCR residues [Phe249A(614), Trp253A(618), Asn294A(725), and Asn295A(726)], but not the specific Lys199A(512) and Asp281A(712) residues, are claimed to be involved in peptide binding (36).
B) RESIDUES PARTICIPATING IN RECEPTOR ACTIVATION. ANG II structure-activity studies showed that the peptide's Phe8 aromatic side chain is essential for triggering the response by interacting with the receptor's His256A(621) (178) (Fig. 7). Aromaticity and residue size in the ANG II position 4 side chain also appear to be important for activating the receptor, possibly by interaction with the receptor's Asn111A(325). In addition, experiments with Lys199A(512) receptor mutants show that this residue can modulate the effect of the His256A(621) side chain on receptor binding. Whereas the H256A(621)A mutation does not affect binding, the loss of affinity of the [K199A(512)A;H256A(621)A] double mutant is significantly larger than that of the K199A(512)A mutant (196), suggesting that, in the AT1 structure, the Lys199A (512) ammonium group is able to keep the His256A(621) imidazole ring at a specific position, perhaps by forming an intramolecular hydrogen bond. This interaction might not contribute to ligand-receptor affinity (303), but could be important to form a more productive receptor-agonist binding mode. This might involve a relay-like structure consisting of ANG II's COOH-terminal carboxylate, the receptor's Lys199A(512) ammonium group, and the His256A(621) imidazole ring (82, 195).
AGPCR activation has been associated with a transition of the receptor's structure from a constrained inactive to an expanded active state (146, 149), in contrast to inactivation by binding of nonpeptide antagonists which drive a receptor to a more constrained inactive form (253, 294).
Physiological receptor activation is triggered by agonist binding (297), whereas constitutive receptor activation occurs in the absence of agonistic stimulus by side chain mutations and other modifications of the AGPCR structure (225).
Activation of rhodopsin is seen as an expansion of the structure. Figure 8A shows that the more likely site of the 7TM bundle structure which may allow this expansion, without the breakage of covalent bonds, is that between two blocks of structure: a (helices I–V) and b (helices VI and VII). Inside the block a helices, helix V also moves in relation to helix III during activation (21, 224). Functionally, ground state rhodopsin has a compressed 7TM bundle structure with an 11-cis-retinal moiety linked to helix VII's Lys296R(723) by a Schiff bond (151, 221, 254) (Fig. 8B, 1). Light absorption causes retinal isomerization to an all-trans-form (296), followed by slight modifications of the rhodopsin structure, probably limited to the retinal pocket (150, 254). In a late stage, this effect can spread over the structure giving rise to an equilibrium between metarhodopsins I (MI) and II (MII), the latter being the fully active form (112) that triggers transducin coupling and other cytosolic events (168, 244). The activation of rhodopsin can be approached by following the structural features relative to three states.
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2) The second state is the MI form, in which the bridge between the Schiff base and Glu113R(318) (Fig. 8B, 1) is ruptured and the counterion function exerted by this residue is transferred to Glu181R(464) (243) (Fig. 8C, 5). These modifications lead to the release of the Tyr268R(621) residue and consequently of the extracellular third of helix VI.
3) The third state is the MII form, produced by all-trans-retinal contacts with aromatic side chains in the middle of the 7TM bundle: helix VI's Trp265R(618) and Tyr268R(621), and helix III's Trp126R(331) (137, 157) (Fig. 8C, 6). As a consequence of change in these residues’ positions, bonds between cytosolic ends of rhodopsin are ruptured (Fig. 8C, 7 and 8) leading to expansion (170) and activation of the receptor.
2. Physiological activation of AGPCRs and AT1 receptors
The high variability of residues in the AGPCR agonist binding sites (Fig. 6) reflects the heterogeneity of ligand specificities. In general, the locations and dimensions of the sites may differ according to size of the ligands. For small molecules with retinal-like dimensions, such as bioamines, prostanoids, phospholipids, purines, and small peptides, the binding sites overlap entirely the rhodopsin retinal pocket (29, 205). For larger ligands, e.g., peptides (43, 69, 94, 116), melanocortin (159, 210), and glycoprotein hormones (52, 153, 293), the receptor-agonist interaction also involves positions in the extracellular segments of the receptor structure.
A) AGONIST BINDING AND ACTIVATION. ANG II binding to the AT1 receptor, leading to its activation, is likely to be a two-step process as suggested by studies of Le et al. (145) and Feng et al. (56). The first step involves binding of the peptide's COOH-terminal segment (Tyr4-Ile5-His6-Pro7-Phe8) at the retinal-like locus inside the 7TM bundle central cavity, oriented by a salt bridge between the peptide's COOH-terminal carboxylate and the receptor's Lys199A(512) side chain (Fig. 7C, left). This binding, responsible for the basal agonistic activity, would lead to expansion of the 7TM bundle structure thus enlarging the space between the EC-2 loop's hairpin turn and helices I, II, VI, and VII (Fig. 7C, right). For the interaction of its NH2 terminus (Asp1-Arg2) with AT1 receptor (second step of binding), the ANG II molecule should bend at the level of Val3-Tyr4 so that the Arg2's guanidinium group would be at binding distance from the receptor's Asp278A(709) and Asp281A(712) side chains (Fig. 7B, top). By completing its interaction at the extracellular space between the EC-1, EC-3 loops, and the Nt domain, the peptide would open this space, favoring the separation of blocks a and b initiated at the 7TM bundle.
AGPCR activation due to agonist binding is to be compared with the mechanism triggered by retinal isomerization in rhodopsin, leading to MI formation (Fig. 8). Agonist binding could exert pressure on the receptor's retinal-like locus, pushing aside the 7TM bundle helices. The possibility of a role of aromatic residues on triggering AGPCR activation was raised by the higher density of these residues in the extracellular half of the receptor's 7TM bundle, flanking the limits of the agonist site (118, 209). As this site comprises variable positions (Fig. 6), activation in different AGPCRs may not be like that described above for rhodopsin, namely, breakage of the bond between EC-2 loop's Glu181R(464) and helix VI's Tyr268R(621) (Fig. 8), being specific for receptor classes and performed by different residues at different locations of that site.
In AT1 receptor activation, a role of aromatic side chains at the receptor and at the agonist in pushing the structure towards an expanded form is observed. For instance, ANG II's Phe8 and Tyr4 (and His6) aromatic side chains are involved in receptor activation (70, 178, 247) through interactions with helix V's Phe204A(517), helix VI's Phe249A(614), Trp253A(618), His256A(621) and Phe259A(624), and helix VII's Tyr292A(723) side chains (82, 175, 178, 195, 196) (Fig. 7B).
Interestingly, the AT1 receptor, like rhodopsin, has an EC-2 loop hairpin's residue, Glu173A(464), which in the inactive receptor model (Fig. 7A) is supposed to make a salt bridge with helix VI's His256A(621) side chain. This could be broken upon receptor activation by agonist binding, thus mimicking the mechanism for formation of the rhodopsin MI state. Also the disulfide bridge [Cys101A(315)-Cys180A(470)] between EC-2 and the top of helix III (Figs. 2, 3, and 8) could be broken in the course of this mechanism, thus supporting the hypothesis of a potential role which has been attributed to this bond in AT1 receptors. Previously proposed by Pederson and Ross (228), Ohyama et al. (200), and Karnik et al. (128) to regulate the activation of these receptors, but mainly to be vital to keep the 7TM bundle structure, the Cys101A(315)-Cys180A(470) disulfide bridge has now been recognized, based on solid mutagenesis experiments, as an important factor to stabilize the inactive state of C5a receptors (136).
The signal started at the agonist site level (extracellular half of the structure) is transmitted along the AGPCR 7TM bundle by changes in receptor structure which in rhodopsin are related to the formation of the fully active MII state. In AGPCRs, this mechanism might follow a pathway along the central cavity of the 7TM bundle structure (agonist-site-interface-main-site) involving positions displaying a gradient of residue conservation (Fig. 6). Signaling would start at the agonist site by different receptor-class-specific (agonist-specific) mechanisms but should converge to a single receptor-family-specific pathway at the cytosolic side of the 7TM bundle.
Translation and rotation of 7TM helices have been proposed to occur during AGPCR activation. In AT1 receptors, movements of helix II (176), helix VII (25), and helices II and VII (180, 181) have been observed. Nikiforovich et al. (193) have proposed a mechanism of extensive mobilization of the whole helix IV as a result of constitutive activation of AT1 receptors elicited by mutation in the helix III residue Asn111A(325) and adjacent residues in the three-dimensional structure. Applied to the physiological activation of AGPCRs, this mechanism would be an additional step in the scheme discussed in this and in the following sections.
B) EVENTS AT THE 7TM BUNDLE CYTOSOLIC ENDS. The activation of rhodopsin initiated at the 7TM bundle retinal site leads ultimately to the dissociation of bonds between the cytosolic ends of transmembrane helices, such as the Arg135R(340), Glu247R(600), and Tyr306R(734)-Phe313R(810) bonds shown in Figure 8B (7 and 8, respectively), which stabilize the receptors’ inactive forms (151).
Many studies on the activation and inactivation of AGPCRs due to mutations on residue positions of 7TM cytosolic ends (see Table D in the supplemental material) reveal that these mechanisms are mainly controlled by interactions between conserved positions (340), (343) and (344) of helix III, the conserved position (810) of helix VIII, the conserved position (734) of the VII-VIII bend, and the variable positions (600), (603), (604), and (606) of helix VI (see Figs. 2 and 3 for identification of positions).
In rhodopsin, the variable cytosolic half of helix VI seems to work like a stalk holding the inactive form of opsin. Thus Glu247R(600), Val250R(603), and Thr251R(604) interact with helix III's Arg135R(340), Val138R(343), and Val139R(344) (Fig. 9, green spheres), keeping together blocks a and b of the 7TM structure. In many AGPCRs, mutations at these positions lead to activation (see references in the supplemental material Table D). On the other hand, helix VI residues of Lys248R(601), Glu249R(602), and Ile253R(606), that in rhodopsin's structure are seen pointing outside or to helices VII and VIII (Fig. 9, blue spheres), can make interactions with these helices and thus favor receptor activation. Mutations at position (606) of AGPCRs lead to inactivation of receptors (see references in the supplemental material Table D).
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25% of the AGPCRs) for which position (600) contains Glu, the inactive form is stabilized by a salt bridge (15) (Fig. 9). An electrostatic bond between helix III's Arg(340) and Glu(Asp) residues in other internal positions of helix VI, such as (603) and (604), might produce a similar effect. In fact, when Glu or Asp is introduced in positions (600) and (604) of serotonin 5-HT6 (138) and mu-opioid (101) receptors, these proteins are driven from wild-type constitutively activated to inactive forms. Some positions placed externally such as those of the EC-2 loop (red spheres in Fig. 9) are involved in AGPCR phosphorylation and G protein coupling mechanisms. The same role can be recognized for external positions of helix VI cytosolic end and helix VIII (not indicated in Fig. 9) (see references in the supplemental material Table D for the respective cytosolic ends).
In AT1 receptors, position 235A(600) is occupied by Asn, whereas positions 238A(603) and 239A(604) bear Ile and Phe, respectively (Fig. 7, A and B). N235A(600)A mutation resulted in a slight increase of IP formation. Mutations carried out at positions 238A(603) and, mainly, 239A(604) revealed that replacement of Phe239A(604) with polar residues had relatively minor effects on receptor responses, but its replacement by Asp or by positively charged residues reduced or significantly increased these responses, respectively (305). As receptor activation is linked to the breakage of bonds between helices III and VI, one may suppose that positive and negative charges at helix VI's position 239A(604) are repelled and attracted, respectively, by the positively charged Arg126A(340) side chain (Fig. 7, A and B). As helix VI Asn235A(600) is able to form only a weaker hydrogen bond with helix III Arg126(340), the dissociation of helices III and VI upon N235A(600)A mutation is less sensitive to produce constitutive activation than the breakage of a homologous strong Glu(600)[Asp(600)]-Arg(340) salt bond of amine receptors and other AGPCRs. Similar mutations of Ile238A(603) led to inconclusive results (305) due perhaps to the fact that this position is not so frontally located in relation to helix III as position 239A(604). In consonance with this interpretation, mutation on positions of Asp236A(601) and Asp237A(602) led to AT1 receptor inactivation and G protein uncoupling (305) or reduction of phosphorylation (202), findings that are consistent with the accessibility of those two positions revealed in the modeled structure of Figures 7A and 9.
Besides the interactions between the cytosolic ends of helices III and VI, the inactive form of rhodopsin seems to be held by a bond between the conserved Tyr306R(734) and Phe313R(810) side chains (Fig. 9, right and bottom) (64), indicating that upon activation this bond is broken, releasing the cytosolic helix VIII from interactions with the 7TM bundle structure. A similar condition is found in other AGPCRs, such as the 5-HT(2C) receptor (242).
In short, two very conserved residues [Arg(340) and Tyr(734)] in the cytosolic ends of AGPCR 7TM bundle structure can be used as referential positions to orient the interpretation of postactivation cytosolic events (Fig. 9). It is possible to speculate that activation may be uniform or not depending on whether the structure expansion attains these two spots simultaneously or separately, respectively. In the former event, the two bonds in the cytosolic ends of the rhodopsin [the bond between Tyr306R(734) and Phe313R(810) or the bond between Arg135R(340) and Glu247R(600) at the right or left of Fig. 9, respectively] are broken at the same time, and the structure is expanded uniformly. In the latter event, one of the two bonds of the rhodopsin structure could be broken first, thus contributing to change the relative orientation of the centrally positioned helix VI, and destabilize the interactions of this helix with the other side of the structure.
The ambivalence of this mechanism of signal transduction can have important consequences. The orientation of the signal towards one cytosolic end of the 7TM bundle could be dictated by different factors such as, for instance, specific effects due to the binding of agonist side chains to the agonist site (see Refs. 105, 198, 281 for AT1 receptors and Ref. 20 for rhodopsin). This would control a mechanism of selective receptor activation to elicit preferentially either G protein coupling or another postreceptor activation event such as phosphorylation, arrestin binding, and internalization. Examples illustrating this point are shown and discussed in section III, C1 and D3.
A) AGPCR IN GENERAL. We shall limit our analysis on constitutive activation of AGPCRs (225) to the mechanisms linked to residue mutations in the receptor's 7TM bundle and adjacent extramembrane segments. In these regions of the receptor structure, one can observe (Fig. 6) 1) a high residue variation in the agonist site (residue positions of Fig. 5's EV boxes 23 and 33) and in the central part of the bundle (residue positions of Fig. 5's EV box 22), and 2) a high residue conservation in the internal cytosolic half of the bundle central cavity (residue positions of Fig. 5's EV boxes 11, 12, and 22) except the helix VI (residue positions of Fig. 5's EV boxes 23 and 33). Thus the constitutive activation due to mutations in the extracellular and central part of the AGPCR structure is specific for types of receptors (or groups of a few different types), whereas in the 7TM bundle cytosolic ends, the same process has a common component site on positions of all helices but helix VI and a type-specific component sited in helix VI positions.
As examples to illustrate the problem, some constitutive activation events in AGPCRs, due to residue mutations in the extracellular side and middle of helices, are described below.
1) Rhodopsin is activated by mutation of the EC-2 loop hairpin Glu113R(318) residue which stabilizes Schiff base positive charge, or of positions in helix II Gly90R(231), helix V Phe212R(517), helix VI Met258R(610), Phe262R(614), Trp265R(618), and Tyr269R(621) which make bonds at other parts of the opsin-retinal interface (85, 137, 157, 261, 268) (Fig. 8). Due to these facts, the apoprotein form of rhodopsin is also constitutively activated (259).
2) AGPCRs with mutations of bulky and mainly aromatic residues are constitutively activated, for instance, Trp256G(618)Phe mutation in bradykinin B2 receptors (160).
3) Besides AT1 receptors, mutation of Asn residue in helix III position (325) can give rise to constitutive activation of bradykinin B2 (160) and cysteinyl-leukotriene (48) receptors.
4) Glycoprotein hormone (follicle stimulating hormone, thyroid stimulating hormone, and lutropin-choriogonadotropic hormone) receptors are constitutively activated by Asp(614) mutation and other mutations at neighbor residues in helix VI and helices II and VII [polar cluster positions (224), (725) and (726)] (7, 8, 171, 276, 290).
5) Mutations on helix V Ser204G(513), Ser207G(516), and probably Ser203G(512) to Ala, all of them placed in the agonist pocket of 
2-adrenergic receptors, also lead to constitutive activation (119).
Constitutive activation due to mutational events in cytosolic ends of AGPCRs are discussed in detail in section III, B2a and D3.
B) AT1 RECEPTORS. Constitutive activation of AT1 receptors has been elicited by single mutations on residues located along the receptor central cavity such as helix III's Asn111A(325) (55, 77, 106, 165, 174, 195) and Asn111A(325) surroundings in three-dimensional structure (193), helix VI's Ile245A(610) (174), helix VII's Asn295A(726) (17, 55, 181), and cytosolic helix VIII's Leu305A(806) (55, 174, 223).
The N111A(325)G mutation drives the AT1 receptor to a constitutive activated state by overcoming a barrier that is normally imposed by the Asn111A(325) side chain (55, 77, 165, 195). Substitution of this Asn residue appears to release the 7TM bundle structure from a steric effect, rather than from a polar interaction with receptor helix VII residues of Tyr292A(723) (77) or Asn295A(726) (17), which keeps the 7TM helices compacted and the receptor inactivated. This interpretation comes from the fact that Asn111A(325) mutation to Gly was the most effective to elicit activation (55).
Based on modeling and mutagenesis procedures, Nikiforovich et al. (193) proposed a mechanism to elicit constitutive activation on AT1 receptor by mutation in the position of Asn111A(325) and surroundings in both helices III and IV. This mechanism would consist of a chain of conformational changes along the transmembrane helix III from Leu112A(326) to Tyr113A(327) to Phe117A(331), which can be propagated to helix IV residues Ile152A(419) and Met155A(422) leading to an expressive movement of this helix as a whole and modifications in the structure of the IC-2 loop.
A screening of a randomly mutated cDNA library of AT1 receptors (223) allowed the identification of the following 7TM mutations linked to constitutive activation of these receptors: F77A(227)Y, N111A(325)S, L112A(326)H, L112A(326)F, L118A(332)H, I193A(506)K, L195A(508)P, and L305A(806)Q. Except for positions (506), (508), and (806), the other positions are in or close to the interface between blocks a and b of the structure (Fig. 8A) and thus might contribute to the association of these receptor subdomains on the inactivated form of receptors.
Constitutive activation of AT1 receptors has also been associated with mutations on residues Leu262A(627) and Leu265A(630) at the extracellular third of helix VI which increased the receptor-mediated adenylate cyclase activity (40, 41). Residue mutations in the cytosolic ends of AT1 receptors have led to constitutive activation as described, for instance, by Gaborik et al. (66) for the DRY motif Asp125A(339) (see the supplemental material Table D for many other occurrences).
Specific mutagenesis studies (41, 53, 94) showed that competitive antagonistic peptides, such as [Sar1,Leu8]-ANG II, bind AT1 receptors in the same two-step fashion observed with the natural ligand. A minor difference is that [Sar1]-ANG II requires both the EC-3 loop's Asp278A(709) and Asp281A(712) side chains for binding, whereas ANG II is more dependent on binding to Asp281A(712) and to the Nt domain's Arg23A(105). The implication of this difference is further discussed with more details in section IIID1.
AT1 receptor inhibition by losartan is surmountable since it can be reversed by ANG II and follows a typical competitive mechanism in which only EC50 values are increased. In contrast, inhibition by losartan's carboxylated metabolite EXP3174 is insurmountable and characterized by increased EC50 values and decrease of maximum effect (291). Other carboxylate-containing derivatives, such as irbesartan (34) and candesartan (182), are also insurmountable inhibitors (274, 291). It has been proposed that this type of inhibition is dictated by a free carboxylate connected to the nonpeptide compound's imidazole ring that would interact with the receptor's Lys199A(512) (Fig. 8) leading to insurmountable inhibition (61, 188, 197, 292, 294, 295). However, the insurmountable inhibition is also regulated by the receptor's Gln257A(622) residue at the extracellular third of helix VI (274), which allows for a different binding mode of inhibitors to the receptor agonist site.
Whereas AT1 receptor residues such as Asp281A(712), at the EC-3 loop, are not required for losartan binding (129), mutagenesis screening showed that binding involves many residues along the 7TM bundle structure (17, 78, 107, 121, 129, 183, 195, 197,
(i = 1–20)fi(lnfi), where fi is the fraction of each amino acid found at each position. In the numbers designating boxes 11, 12, 22, 23, and 33, the first and second digits express increasing ranges of V and E values, respectively. Thus 11 and 33 are boxes containing highly conserved and highly variable residue positions, respectively.
