Structure of the Angiotensin receptor revealed by serial femtosecond crystallography.

Angiotensin II type 1 receptor (AT(1)R) is a G protein-coupled receptor that serves as a primary regulator for blood pressure maintenance. Although several anti-hypertensive drugs have been developed as AT(1)R blockers (ARBs), the structural basis for AT(1)R ligand-binding and regulation has remained elusive, mostly due to the difficulties of growing high-quality crystals for structure determination using synchrotron radiation. By applying the recently developed method of serial femtosecond crystallography at an X-ray free-electron laser, we successfully determined the room-temperature crystal structure of the human AT(1)R in complex with its selective antagonist ZD7155 at 2.9-Å resolution. The AT(1)R-ZD7155 complex structure revealed key structural features of AT(1)R and critical interactions for ZD7155 binding. Docking simulations of the clinically used ARBs into the AT(1)R structure further elucidated both the common and distinct binding modes for these anti-hypertensive drugs. Our results thereby provide fundamental insights into AT(1)R structure-function relationship and structure-based drug design.

Mechanism of Hormone Peptide Activation of a GPCR: Angiotensin II Activated State of AT1R Initiated by van der Waals Attraction.

We present a succession of structural changes involved in hormone peptide activation of a prototypical GPCR. Microsecond molecular dynamics simulation generated conformational ensembles reveal propagation of structural changes through key "microswitches" within human AT1R bound to native hormone. The endocrine octa-peptide angiotensin II (AngII) activates AT1R signaling in our bodies which maintains physiological blood pressure, electrolyte balance, and cardiovascular homeostasis. Excessive AT1R activation is associated with pathogenesis of hypertension and cardiovascular diseases which are treated by sartan drugs. The mechanism of AT1R inhibition by sartans has been elucidated by 2.8 Å X-ray structures, mutagenesis, and computational analyses. Yet, the mechanism of AT1R activation by AngII is unclear. The current study delineates an activation scheme initiated by AngII binding. A van der Waals "grasp" interaction between Phe8AngII with Ile2887.39 in AT1R induced mechanical strain pulling Tyr2927.43 and breakage of critical interhelical H-bonds, first between Tyr2927.43 and Val1083.32 and second between Asn1113.35 and Asn2957.46. Subsequently changes are observed in conserved microswitches DRYTM3, Yx7K(R)TM5, CWxPTM6, and NPxxYTM7 in AT1R. Activating the microswitches in the intracellular region of AT1R may trigger formation of the G-protein binding pocket as well as exposure of helix-8 to cytoplasm. Thus, the active-like conformation of AT1R is initiated by the van der Waals interaction of Phe8AngII with Ile2887.39, followed by systematic reorganization of critical interhelical H-bonds and activation of microswitches.

Divergent Spatiotemporal Interaction of Angiotensin Receptor Blocking Drugs with Angiotensin Type 1 Receptor.

Crystal structures of the human angiotensin II type 1 receptor (AT1R) complex with the antihypertensive agent ZD7155 (PDB id: 4YAY ) and the blood pressure medication Benicar (PDB id: 4ZUD ) showed that binding poses of both antagonists are similar. This finding implies that clinically used angiotensin receptor blocking (ARB) drugs may interact in a similar fashion. However, clinically observed differences in pharmacological and therapeutic efficacies of ARBs lead to the question of whether the dynamic interactions of AT1R with ARBs vary. To address this, we performed induced-fit docking (IFD) of eight clinically used ARBs to AT1R followed by 200 ns molecular dynamic (MD) simulation. The experimental Ki values for ARBs correlated remarkably well with calculated free energy with R2 = 0.95 and 0.70 for AT1R-ARB models generated respectively by IFD and MD simulation. The eight ARB-AT1R complexes share a common set of binding residues. In addition, MD simulation results validated by mutagenesis data discovered distinctive spatiotemporal interactions that display unique bonding between an individual ARB and AT1R. These findings provide a reasonably broader picture reconciling the structure-based observations with clinical studies reporting efficacy variations for ARBs. The unique differences unraveled for ARBs in this study will be useful for structure-based design of the next generation of more potent and selective ARBs.

International Union of Basic and Clinical Pharmacology. XCIX. Angiotensin Receptors: Interpreters of Pathophysiological Angiotensinergic Stimuli [corrected].

The renin angiotensin system (RAS) produced hormone peptides regulate many vital body functions. Dysfunctional signaling by receptors for RAS peptides leads to pathologic states. Nearly half of humanity today would likely benefit from modern drugs targeting these receptors. The receptors for RAS peptides consist of three G-protein-coupled receptors­the angiotensin II type 1 receptor (AT1 receptor), the angiotensin II type 2 receptor (AT2 receptor), the MAS receptor­and a type II trans-membrane zinc protein­the candidate angiotensin IV receptor (AngIV binding site). The prorenin receptor is a relatively new contender for consideration, but is not included here because the role of prorenin receptor as an independent endocrine mediator is presently unclear. The full spectrum of biologic characteristics of these receptors is still evolving, but there is evidence establishing unique roles of each receptor in cardiovascular, hemodynamic, neurologic, renal, and endothelial functions, as well as in cell proliferation, survival, matrix-cell interaction, and inflammation. Therapeutic agents targeted to these receptors are either in active use in clinical intervention of major common diseases or under evaluation for repurposing in many other disorders. Broad-spectrum influence these receptors produce in complex pathophysiological context in our body highlights their role as precise interpreters of distinctive angiotensinergic peptide cues. This review article summarizes findings published in the last 15 years on the structure, pharmacology, signaling, physiology, and disease states related to angiotensin receptors. We also discuss the challenges the pharmacologist presently faces in formally accepting newer members as established angiotensin receptors and emphasize necessary future developments.

Current topics in angiotensin II type 1 receptor research: Focus on inverse agonism, receptor dimerization and biased agonism.

Although the octapeptide hormone angiotensin II (Ang II) regulates cardiovascular and renal homeostasis through the Ang II type 1 receptor (AT1R), overstimulation of AT1R causes various human diseases, such as hypertension and cardiac hypertrophy. Therefore, AT1R blockers (ARBs) have been widely used as therapeutic drugs for these diseases. Recent basic research and clinical studies have resulted in the discovery of interesting phenomena associated with AT1R function. For example, ligand-independent activation of AT1R by mechanical stress and agonistic autoantibodies, as well as via receptor mutations, has been shown to decrease the inverse agonistic efficacy of ARBs, though the molecular mechanisms of such phenomena had remained elusive until recently. Furthermore, although AT1R is believed to exist as a monomer, recent studies have demonstrated that AT1R can homodimerize and heterodimerize with other G-protein coupled receptors (GPCR), altering the receptor signaling properties. Therefore, formation of both AT1R homodimers and AT1R-GPCR heterodimer may be involved in the pathogenesis of human disease states, such as atherosclerosis and preeclampsia. Finally, biased AT1R ligands that can preferentially activate the ß-arrestin-mediated signaling pathway have been discovered. Such ß-arrestin-biased AT1R ligands may be better therapeutic drugs for cardiovascular diseases. New findings on AT1R described herein could provide a conceptual framework for application of ARBs in the treatment of diseases, as well as for novel drug development. Since AT1R is an extensively studied member of the GPCR superfamily encoded in the human genome, this review is relevant for understanding the functions of other members of this superfamily.

Structural Basis for Ligand Recognition and Functional Selectivity at Angiotensin Receptor.

Angiotensin II type 1 receptor (AT1R) is the primary blood pressure regulator. AT1R blockers (ARBs) have been widely used in clinical settings as anti-hypertensive drugs and share a similar chemical scaffold, although even minor variations can lead to distinct therapeutic efficacies toward cardiovascular etiologies. The structural basis for AT1R modulation by different peptide and non-peptide ligands has remained elusive. Here, we report the crystal structure of the human AT1R in complex with an inverse agonist olmesartan (Benicar(TM)), a highly potent anti-hypertensive drug. Olmesartan is anchored to the receptor primarily by the residues Tyr-35(1.39), Trp-84(2.60), and Arg-167(ECL2), similar to the antagonist ZD7155, corroborating a common binding mode of different ARBs. Using docking simulations and site-directed mutagenesis, we identified specific interactions between AT1R and different ARBs, including olmesartan derivatives with inverse agonist, neutral antagonist, or agonist activities. We further observed that the mutation N111(3.35)A in the putative sodium-binding site affects binding of the endogenous peptide agonist angiotensin II but not the ß-arrestin-biased peptide TRV120027.

Structure-Function Basis of Attenuated Inverse Agonism of Angiotensin II Type 1 Receptor Blockers for Active-State Angiotensin II Type 1 Receptor.

Ligand-independent signaling by the angiotensin II type 1 receptor (AT1R) can be activated in clinical settings by mechanical stretch and autoantibodies as well as receptor mutations. Transition of the AT1R to the activated state is known to lower inverse agonistic efficacy of clinically used AT1R blockers (ARBs). The structure-function basis for reduced efficacy of inverse agonists is a fundamental aspect that has been understudied not only in relation to the AT1R but also regarding other homologous receptors. Here, we demonstrate that the active-state transition in the AT1R indeed attenuates an inverse agonistic effect of four biphenyl-tetrazole ARBs through changes in specific ligand-receptor interactions. In the ground state, tight interactions of four ARBs with a set of residues (Ser109(TM3), Phe182(ECL2), Gln257(TM6), Tyr292(TM7), and Asn295(TM7)) results in potent inverse agonism. In the activated state, the ARB-AT1R interactions shift to a different set of residues (Val108(TM3), Ser109(TM3), Ala163(TM4), Phe182(ECL2), Lys199(TM5), Tyr292(TM7), and Asn295(TM7)), resulting in attenuated inverse agonism. Interestingly, V108I, A163T, N295A, and F182A mutations in the activated state of the AT1R shift the functional response to the ARB binding toward agonism, but in the ground state the same mutations cause inverse agonism. Our data show that the second extracellular loop is an important regulator of the functional states of the AT1R. Our findings suggest that the quest for discovering novel ARBs, and improving current ARBs, fundamentally depends on the knowledge of the unique sets of residues that mediate inverse agonistic potency in the two states of the AT1R.

Critical role for lysine 685 in gene expression mediated by transcription factor unphosphorylated STAT3.

STAT3 is a pleiotropic transcription factor that is activated by the phosphorylation of tyrosine 705 in response to many cytokines and growth factors. STAT3 without Tyr-705 phosphorylation (U-STAT3) is also a potent transcription factor, and its concentration in cells increases greatly in response to STAT3 activation because the STAT3 gene can be driven by phosphorylated STAT3 dimers. We have now searched for post-translational modifications of U-STAT3 that might have a critical role in its function. An analysis by mass spectroscopy indicated that U-STAT3 is acetylated on Lys-685, and the integrity of Lys-685 is required for the expression of most U-STAT3-dependent genes. In contrast, we found only a very minor role for Lys-685 in gene expression induced in response to tyrosine-phosphorylated STAT3. U-STAT3 plays an important role in angiotensin II-induced gene expression and in the consequent development of cardiac hypertrophy and dysfunction. Mutation of Lys-685 inhibits this function of STAT3, providing new information on the role of U-STAT3 in augmenting the development of heart failure.

Angiotensin II-regulated microRNA 483-3p directly targets multiple components of the renin-angiotensin system.

Improper regulation of signaling in vascular smooth muscle cells (VSMCs) by angiotensin II (AngII) can lead to hypertension, vascular hypertrophy and atherosclerosis. The extent to which the homeostatic levels of the components of signaling networks are regulated through microRNAs (miRNA) modulated by AngII type 1 receptor (AT1R) in VSMCs is not fully understood. Whether AT1R blockers used to treat vascular disorders modulate expression of miRNAs is also not known. To report differential miRNA expression following AT1R activation by AngII, we performed microarray analysis in 23 biological and technical replicates derived from humans, rats and mice. Profiling data revealed a robust regulation of miRNA expression by AngII through AT1R, but not the AngII type 2 receptor (AT2R). The AT1R-specific blockers, losartan and candesartan antagonized >90% of AT1R-regulated miRNAs and AngII-activated AT2R did not modulate their expression. We discovered VSMC-specific modulation of 22 miRNAs by AngII, and validated AT1R-mediated regulation of 17 of those miRNAs by real-time polymerase chain reaction analysis. We selected miR-483-3p as a novel representative candidate for further study because mRNAs of multiple components of the renin-angiotensin system (RAS) were predicted to contain the target sequence for this miRNA. MiR-483-3p inhibited the expression of luciferase reporters bearing 3'-UTRs of four different RAS genes and the inhibition was reversed by antagomir-483-3p. The AT1R-regulated expression levels of angiotensinogen and angiotensin converting enzyme 1 (ACE-1) proteins in VSMCs are modulated specifically by miR-483-3p. Our study demonstrates that the AT1R-regulated miRNA expression fingerprint is conserved in VSMCs of humans and rodents. Furthermore, we identify the AT1R-regulated miR-483-3p as a potential negative regulator of steady-state levels of RAS components in VSMCs. Thus, miRNA-regulation by AngII to affect cellular signaling is a novel aspect of RAS biology, which may lead to discovery of potential candidate prognostic markers and therapeutic targets.

Long range effect of mutations on specific conformational changes in the extracellular loop 2 of angiotensin II type 1 receptor.

The topology of the second extracellular loop (ECL2) and its interaction with ligands is unique in each G protein-coupled receptor. When the orthosteric ligand pocket located in the transmembrane (TM) domain is occupied, ligand-specific conformational changes occur in the ECL2. In more than 90% of G protein-coupled receptors, ECL2 is tethered to the third TM helix via a disulfide bond. Therefore, understanding the extent to which the TM domain and ECL2 conformations are coupled is useful. To investigate this, we examined conformational changes in ECL2 of the angiotensin II type 1 receptor (AT1R) by introducing mutations in distant sites that alter the activation state equilibrium of the AT1R. Differential accessibility of reporter cysteines introduced at four conformation-sensitive sites in ECL2 of these mutants was measured. Binding of the agonist angiotensin II (AngII) and inverse agonist losartan in wild-type AT1R changed the accessibility of reporter cysteines, and the pattern was consistent with ligand-specific "lid" conformations of ECL2. Without agonist stimulation, the ECL2 in the gain of function mutant N111G assumed a lid conformation similar to AngII-bound wild-type AT1R. In the presence of inverse agonists, the conformation of ECL2 in the N111G mutant was similar to the inactive state of wild-type AT1R. In contrast, AngII did not induce a lid conformation in ECL2 in the loss of function D281A mutant, which is consistent with the reduced AngII binding affinity in this mutant. However, a lid conformation was induced by [Sar(1),Gln(2),Ile(8)] AngII, a specific analog that binds to the D281A mutant with better affinity than AngII. These results provide evidence for the emerging paradigm of domain coupling facilitated by long range interactions at distant sites on the same receptor.

Ligand-specific conformation of extracellular loop-2 in the angiotensin II type 1 receptor.

The orientation of the second extracellular loop (ECL2) is divergent in G-protein coupled receptor (GPCR) structures determined. This discovery provoked the question, is the ECL2 conformation differentially regulated in the GPCRs that respond to diffusible ligands? We have determined the conformation of the ECL2 of the angiotensin II type 1 receptor by reporter-cysteine accessibility mapping in different receptor states (i.e. empty, agonist-bound and antagonist-bound). We introduced cysteines at each position of ECL2 of an N-terminal epitope-tagged receptor surrogate lacking all non-essential cysteines and then measured reaction of these with a cysteine-reactive biotin probe. The ability of biotinylated mutant receptors to react with a steptavidin-HRP-conjugated antibody was used as the basis for examining differences in accessibility. Two segments of ECL2 were accessible in the empty receptor, indicating an open conformation of ECL2. These segments were inaccessible in the ligand-bound states of the receptor. Using the accessibility constraint, we performed molecular dynamics simulation to predict ECL2 conformation in different states of the receptor. Analysis suggested that a lid conformation similar to that of ECL2 in rhodopsin was induced upon binding both agonist and antagonist, but exposing different accessible segments delimited by the highly conserved disulfide bond. Our study reveals the ability of ECL2 to interact with diffusing ligands and to adopt a ligand-specific lid conformation, thus, slowing down dissociation of ligands when bound. Distinct conformations induced by the bound agonist and the antagonist around the conserved disulfide bond suggest an important role for this disulfide bond in producing different functional states of the receptor.

Luciferase reporter assay for unlocking ligand-mediated signaling of GPCRs.

G protein-coupled receptors (GPCRs) play an active role in numerous cellular processes, from cell proliferation to differentiation, by modulating gene transcription through various signal transduction pathways. Transcriptional regulation coupled to reporter gene expression may be used to study both G protein-dependent and G protein-independent responses activated by GPCR ligands. Reporter genes are typically used to monitor changes in receptor-mediated cellular responses at the transcription/translation level. Genetic reporter assays are based on reporter gene expression in response to activation of specific signaling cascade, followed by monitoring the presence of the reporter protein by directly measuring its enzymatic activity. These optimized genes are expressed under the control of a response element to assess its transcriptional activity that can be readily detected by a luminescent signal. Firefly luciferase gene has been widely used as a genetic reporter that responds rapidly to modulation of a GPCR by agonists or antagonists. Luciferase assays have been successfully developed for deorphanization of GPCRs, high-throughput screening (HTS) applications for drug discovery and deciphering both canonical and non-canonical signaling of numerous GPCRs. The protocol outlined for STAT3-driven luciferase assay could be adapted with appropriate changes to any aspect of GPCR signaling.

Effect of novel GPCR ligands on blood pressure and vascular homeostasis.

Maintenance of normal blood pressure under conditions of drug treatment is a measure of system-wide neuro-hormonal controls and electrolyte/fluid volume homeostasis in the body. With increased interest in designing and evaluating novel drugs that may functionally select or allosterically modulate specific GPCR signaling pathways, techniques that allow us to measure acute and long-term effects on blood pressure are very important. Therefore, this chapter describes techniques to measure acute and long-term impact of novel GPCR ligands on blood pressure regulation. We will use the angiotensin type 1 receptor, a powerful blood pressure regulating GPCR, in detailing the methodology. Normal blood pressure maintenance depends upon dynamic modulation of angiotensin type 1 receptor activity by the hormone peptide angiotensin II. Chronic activation of angiotensin type 1 receptor creates hypertension and related cardiovascular disease states which are treated with angiotensin type 1 receptor blockers (ARBs). Thus, a prototype for evaluation of blood pressure control under experimental evaluation of novel drugs.

The non-biphenyl-tetrazole angiotensin AT1 receptor antagonist eprosartan is a unique and robust inverse agonist of the active state of the AT1 receptor.

BACKGROUND AND PURPOSE: Conditions such as hypertension and renal allograft rejection are accompanied by chronic, agonist-independent, signalling by angiotensin II AT1 receptors. The current treatment paradigm for these diseases entails the preferred use of inverse agonist AT1 receptor blockers (ARBs). However, variability in the inverse agonist activities of common biphenyl-tetrazole ARBs for the active state of AT1 receptors often leads to treatment failure. Therefore, characterization of robust inverse agonist ARBs for the active state of AT1 receptors is necessary. EXPERIMENTAL APPROACH: To identify the robust inverse agonist for active state of AT1 receptors and its molecular mechanism, we performed site-directed mutagenesis, competition binding assay, inositol phosphate production assay and molecular modelling for both ground-state wild-type AT1 receptors and active-state N111G mutant AT1 receptors. KEY RESULTS: Although candesartan and telmisartan exhibited weaker inverse agonist activity for N111G- compared with WT-AT1 receptors, only eprosartan exhibited robust inverse agonist activity for both N111G- and WT- AT1 receptors. Specific ligand-receptor contacts for candesartan and telmisartan are altered in the active-state N111G- AT1 receptors compared with the ground-state WT-AT1 receptors, suggesting an explanation of their attenuated inverse agonist activity for the active state of AT1 receptors. In contrast, interactions between eprosartan and N111G-AT1 receptors were not significantly altered, and the inverse agonist activity of eprosartan was robust. CONCLUSIONS AND IMPLICATIONS: Eprosartan may be a better therapeutic option than other ARBs. Comparative studies investigating eprosartan and other ARBs for the treatment of diseases caused by chronic, agonist-independent, AT1 receptor activation are warranted.

Significance of angiotensin 1-7 coupling with MAS1 receptor and other GPCRs to the renin-angiotensin system: IUPHAR Review 22.

Angiotensins are a group of hormonal peptides and include angiotensin II and angiotensin 1-7 produced by the renin angiotensin system. The biology, pharmacology and biochemistry of the receptors for angiotensins were extensively reviewed recently. In the review, the receptor nomenclature committee was not emphatic on designating MAS1 as the angiotensin 1-7 receptor on the basis of lack of classical G protein signalling and desensitization in response to angiotensin 1-7, as well as a lack of consensus on confirmatory ligand pharmacological analyses. A review of recent publications (2013-2016) on the rapidly progressing research on angiotensin 1-7 revealed that MAS1 and two additional receptors can function as 'angiotensin 1-7 receptors', and this deserves further consideration. In this review we have summarized the information on angiotensin 1-7 receptors and their crosstalk with classical angiotensin II receptors in the context of the functions of the renin angiotensin system. It was concluded that the receptors for angiotensin II and angiotensin 1-7 make up a sophisticated cross-regulated signalling network that modulates the endogenous protective and pathogenic facets of the renin angiotensin system.

Correction: Interaction of G-Protein ßγ Complex with Chromatin Modulates GPCR-Dependent Gene Regulation.

[This corrects the article DOI: 10.1371/journal.pone.0052689.].

Constitutive activity in the angiotensin II type 1 receptor: discovery and applications.

The pathophysiological actions of the renin-angiotensin system hormone, angiotensin II (AngII), are mainly mediated by the AngII type 1 (AT1) receptor, a GPCR. The intrinsic spontaneous activity of the AT1 receptor in native tissues is difficult to detect due to its low expression levels. However, factors such as the membrane environment, interaction with autoantibodies, and mechanical stretch are known to increase G protein signaling in the absence of AngII. Naturally occurring and disease-causing activating mutations have not been identified in AT1 receptor. Constitutively active mutants (CAMs) of AT1 receptor have been engineered using molecular modeling and site-directed mutagenesis approaches among which substitution of Asn(111) in the transmembrane helix III with glycine or serine results in the highest basal activity of the receptor. Transgenic animal models expressing the CAM AT1 receptors that mimic various in vivo disease conditions have been useful research tools for discovering the pathophysiological role of AT1 receptor and evaluating the therapeutic potential of inverse agonists. This chapter summarizes the studies on the constitutive activity of AT1 receptor in recombinant as well as physiological systems. The impact of the availability of CAM AT1 receptors on our understanding of the molecular mechanisms underlying receptor activation and inverse agonism is described.

Interaction of G-protein ßγ complex with chromatin modulates GPCR-dependent gene regulation.

Heterotrimeric G-protein signal transduction initiated by G-protein-coupled receptors (GPCRs) in the plasma membrane is thought to propagate through protein-protein interactions of subunits, Gα and Gßγ in the cytosol. In this study, we show novel nuclear functions of Gßγ through demonstrating interaction of Gß(2) with integral components of chromatin and effects of Gß(2) depletion on global gene expression. Agonist activation of several GPCRs including the angiotensin II type 1 receptor specifically augmented Gß(2) levels in the nucleus and Gß(2) interacted with specific nucleosome core histones and transcriptional modulators. Depletion of Gß(2) repressed the basal and angiotensin II-dependent transcriptional activities of myocyte enhancer factor 2. Gß(2) interacted with a sequence motif that was present in several transcription factors, whose genome-wide binding accounted for the Gß(2)-dependent regulation of approximately 2% genes. These findings suggest a wide-ranging mechanism by which direct interaction of Gßγ with specific chromatin bound transcription factors regulates functional gene networks in response to GPCR activation in cells.

Domain coupling in GPCRs: the engine for induced conformational changes.

Recent solved structures of G protein-coupled receptors (GPCRs) provide insights into variation of the structure and molecular mechanisms of GPCR activation. In this review, we provide evidence for the emerging paradigm of domain coupling facilitated by intrinsic disorder of the ligand-free state in GPCRs. The structure-function and dynamic studies suggest that ligand-bound GPCRs exhibit multiple active conformations in initiating cellular signals. Long-range intramolecular and intermolecular interactions at distant sites on the same receptor are crucial factors that modulate signaling function of GPCRs. Positive or negative coupling between the extracellular, the transmembrane and the intracellular domains facilitates cooperativity of activating 'switches' as requirements for the functional plasticity of GPCRs. Awareness that allosteric ligands robustly affect domain coupling provides a novel mechanistic basis for rational drug development, small molecule antagonism and GPCR regulation by classical as well as nonclassical modes.