Identification of Distinct Conformations of the Angiotensin-II Type 1 Receptor Associated with the Gq/11 Protein Pathway and the ß-Arrestin Pathway Using Molecular Dynamics Simulations.

Biased signaling represents the ability of G protein-coupled receptors to engage distinct pathways with various efficacies depending on the ligand used or on mutations in the receptor. The angiotensin-II type 1 (AT1) receptor, a prototypical class A G protein-coupled receptor, can activate various effectors upon stimulation with the endogenous ligand angiotensin-II (AngII), including the Gq/11 protein and ß-arrestins. It is believed that the activation of those two pathways can be associated with distinct conformations of the AT1 receptor. To verify this hypothesis, microseconds of molecular dynamics simulations were computed to explore the conformational landscape sampled by the WT-AT1 receptor, the N111G-AT1 receptor (constitutively active and biased for the Gq/11 pathway), and the D74N-AT1 receptor (biased for the ß-arrestin1 and -2 pathways) in their apo-forms and in complex with AngII. The molecular dynamics simulations of the AngII-WT-AT1, N111G-AT1, and AngII-N111G-AT1 receptors revealed specific structural rearrangements compared with the initial and ground state of the receptor. Simulations of the D74N-AT1 receptor revealed that the mutation stabilizes the receptor in the initial ground state. The presence of AngII further stabilized the ground state of the D74N-AT1 receptor. The biased agonist [Sar(1),Ile(8)]AngII also showed a preference for the ground state of the WT-AT1 receptor compared with AngII. These results suggest that activation of the Gq/11 pathway is associated with a specific conformational transition stabilized by the agonist, whereas the activation of the ß-arrestin pathway is linked to the stabilization of the ground state of the receptor.

Design, synthesis, and biological evaluation of CXCR4 ligands.

A combination of the CXCR4 inverse agonist T140 with N-terminal CXCL12 oligopeptides has produced the first nanomolar synthetic CXCR4 agonists. In these agonists, the inverse agonistic portion provides affinity whereas the N-terminal CXCL12 sequence induces receptor activation. Several CXCR4 crystal structures exist with either CVX15, an inverse agonist closely related to T140 and IT1t, a small molecule; we therefore attempted to produce another CXCL12 oligopeptide combination with IT1t. For this purpose, a primary amino group was introduced by total synthesis into one of the methyl groups of IT1t, serving as an anchoring point for the oligopeptide graft. The introduction of the oligopeptides on this analog however yielded antagonists, one compound displaying high affinity. On the other hand, the amino-substituted analogue itself proved to be an inverse agonist with a binding affinity of 2.6 nM compared to 11.5 nM for IT1t. This IT1t-like analog is hitherto one of the most potent non-peptidic CXCR4 inverse agonists reported.

Characterization of Angiotensin II Molecular Determinants Involved in AT1 Receptor Functional Selectivity.

The octapeptide angiotensin II (AngII) exerts a variety of cardiovascular effects through the activation of the AngII type 1 receptor (AT1), a G protein-coupled receptor. The AT1 receptor engages and activates several signaling pathways, including heterotrimeric G proteins Gq and G12, as well as the extracellular signal-regulated kinases (ERK) 1/2 pathway. Additionally, following stimulation, ßarrestin is recruited to the AT1 receptor, leading to receptor desensitization. It is increasingly recognized that specific ligands selectively bind and favor the activation of some signaling pathways over others, a concept termed ligand bias or functional selectivity. A better understanding of the molecular basis of functional selectivity may lead to the development of better therapeutics with fewer adverse effects. In the present study, we developed assays allowing the measurement of six different signaling modalities of the AT1 receptor. Using a series of AngII peptide analogs that were modified in positions 1, 4, and 8, we sought to better characterize the molecular determinants of AngII that underlie functional selectivity of the AT1 receptor in human embryonic kidney 293 cells. The results reveal that position 1 of AngII does not confer functional selectivity, whereas position 4 confers a bias toward ERK signaling over Gq signaling, and position 8 confers a bias toward ßarrestin recruitment over ERK activation and Gq signaling. Interestingly, the analogs modified in position 8 were also partial agonists of the protein kinase C (PKC)-dependent ERK pathway via atypical PKC isoforms PKCζ and PKCι.

Structure of the human angiotensin II type 1 (AT1) receptor bound to angiotensin II from multiple chemoselective photoprobe contacts reveals a unique peptide binding mode.

Breakthroughs in G protein-coupled receptor structure determination based on crystallography have been mainly obtained from receptors occupied in their transmembrane domain core by low molecular weight ligands, and we have only recently begun to elucidate how the extracellular surface of G protein-coupled receptors (GPCRs) allows for the binding of larger peptide molecules. In the present study, we used a unique chemoselective photoaffinity labeling strategy, the methionine proximity assay, to directly identify at physiological conditions a total of 38 discrete ligand/receptor contact residues that form the extracellular peptide-binding site of an activated GPCR, the angiotensin II type 1 receptor. This experimental data set was used in homology modeling to guide the positioning of the angiotensin II (AngII) peptide within several GPCR crystal structure templates. We found that the CXC chemokine receptor type 4 accommodated the results better than the other templates evaluated; ligand/receptor contact residues were spatially grouped into defined interaction clusters with AngII. In the resulting receptor structure, a ß-hairpin fold in extracellular loop 2 in conjunction with two extracellular disulfide bridges appeared to open and shape the entrance of the ligand-binding site. The bound AngII adopted a somewhat vertical binding mode, allowing concomitant contacts across the extracellular surface and deep within the transmembrane domain core of the receptor. We propose that such a dualistic nature of GPCR interaction could be well suited for diffusible linear peptide ligands and a common feature of other peptidergic class A GPCRs.

Critical hydrogen bond formation for activation of the angiotensin II type 1 receptor.

G protein-coupled receptors contain selectively important residues that play central roles in the conformational changes that occur during receptor activation. Asparagine 111 (N111(3.35)) is such a residue within the angiotensin II type 1 (AT(1)) receptor. Substitution of N111(3.35) for glycine leads to a constitutively active receptor, whereas substitution for tryptophan leads to an inactivable receptor. Here, we analyzed the AT(1) receptor and two mutants (N111G and N111W) by molecular dynamics simulations, which revealed a novel molecular switch involving the strictly conserved residue D74(2.50). Indeed, D74(2.50) forms a stable hydrogen bond (H-bond) with the residue in position 111(3.35) in the wild-type and the inactivable receptor. However, in the constitutively active mutant N111G-AT(1) receptor, residue D74 is reoriented to form a new H-bond with another strictly conserved residue, N46(1.50). When expressed in HEK293 cells, the mutant N46G-AT(1) receptor was poorly activable, although it retained a high binding affinity. Interestingly, the mutant N46G/N111G-AT(1) receptor was also inactivable. Molecular dynamics simulations also revealed the presence of a cluster of hydrophobic residues from transmembrane domains 2, 3, and 7 that appears to stabilize the inactive form of the receptor. Whereas this hydrophobic cluster and the H-bond between D74(2.50) and W111(3.35) are more stable in the inactivable N111W-AT(1) receptor, the mutant N111W/F77A-AT(1) receptor, designed to weaken the hydrophobic core, showed significant agonist-induced signaling. These results support the potential for the formation of an H-bond between residues D74(2.50) and N46(1.50) in the activation of the AT(1) receptor.

Identification of membrane-bound variant of metalloendopeptidase neurolysin (EC 3.4.24.16) as the non-angiotensin type 1 (non-AT1), non-AT2 angiotensin binding site.

Recently, we discovered a novel non-angiotensin type 1 (non-AT1), non-AT2 angiotensin binding site in rodent and human brain membranes, which is distinctly different from angiotensin receptors and key proteases processing angiotensins. It is hypothesized to be a new member of the renin-angiotensin system. This study was designed to isolate and identify this novel angiotensin binding site. An angiotensin analog, photoaffinity probe 125I-SBpa-Ang II, was used to specifically label the non-AT1, non-AT2 angiotensin binding site in mouse forebrain membranes, followed by a two-step purification procedure based on the molecular size and isoelectric point of the photoradiolabeled binding protein. Purified samples were subjected to two-dimensional gel electrophoresis followed by mass spectrometry identification of proteins in the two-dimensional gel sections containing radioactivity. LC-MS/MS analysis revealed eight protein candidates, of which the four most abundant were immunoprecipitated after photoradiolabeling. Immunoprecipitation studies indicated that the angiotensin binding site might be the membrane-bound variant of metalloendopeptidase neurolysin (EC 3.4.24.16). To verify these observations, radioligand binding and photoradiolabeling experiments were conducted in membrane preparations of HEK293 cells overexpressing mouse neurolysin or thimet oligopeptidase (EC 3.4.24.15), a closely related metalloendopeptidase of the same family. These experiments also identified neurolysin as the non-AT1, non-AT2 angiotensin binding site. Finally, brain membranes of mice lacking neurolysin were nearly devoid of the non-AT1, non-AT2 angiotensin binding site, further establishing membrane-bound neurolysin as the binding site. Future studies will focus on the functional significance of this highly specific, high affinity interaction between neurolysin and angiotensins.

Analysis of transmembrane domains 1 and 4 of the human angiotensin II AT1 receptor by cysteine-scanning mutagenesis.

The octapeptide hormone angiotensin II (AngII) exerts a wide variety of cardiovascular effects through the activation of the AT(1) receptor, which belongs to the G protein-coupled receptor superfamily. Like other G protein-coupled receptors, the AT(1) receptor possesses seven transmembrane domains that provide structural support for the formation of the ligand-binding pocket. Here, we investigated the role of the first and fourth transmembrane domains (TMDs) in the formation of the binding pocket of the human AT(1) receptor using the substituted-cysteine accessibility method. Each residue within the Phe-28((1.32))-Ile-53((1.57)) fragment of TMD1 and Leu-143((4.40))-Phe-170((4.67)) fragment of TMD4 was mutated, one at a time, to a cysteine. The resulting mutant receptors were expressed in COS-7 cells, which were subsequently treated with the charged sulfhydryl-specific alkylating agent methanethiosulfonate ethylammonium (MTSEA). This treatment led to a significant reduction in the binding affinity of TMD1 mutants M30C((1.34))-AT(1) and T33C((1.37))-AT(1) and TMD4 mutant V169C((4.66))-AT(1). Although this reduction in binding of the TMD1 mutants was maintained when examined in a constitutively active receptor (N111G-AT(1)) background, we found that V169C((4.66))-AT(1) remained unaffected when treated with MTSEA compared with untreated in this context. Moreover, the complete loss of binding observed for R167C((4.64))-AT(1) was restored upon treatment with MTSEA. Our results suggest that the extracellular portion of TMD1, particularly residues Met-30((1.34)) and Thr-33((1.37)), as well as residues Arg-167((4.64)) and Val-169((4.66)) at the junction of TMD4 and the second extracellular loop, are important binding determinants within the AT(1) receptor binding pocket but that these TMDs undergo very little movement, if at all, during the activation process.

THE CONCISE GUIDE TO PHARMACOLOGY 2021/22: G protein-coupled receptors.

The Concise Guide to PHARMACOLOGY 2021/22 is the fifth in this series of biennial publications. The Concise Guide provides concise overviews, mostly in tabular format, of the key properties of nearly 1900 human drug targets with an emphasis on selective pharmacology (where available), plus links to the open access knowledgebase source of drug targets and their ligands (www.guidetopharmacology.org), which provides more detailed views of target and ligand properties. Although the Concise Guide constitutes over 500 pages, the material presented is substantially reduced compared to information and links presented on the website. It provides a permanent, citable, point-in-time record that will survive database updates. The full contents of this section can be found at http://onlinelibrary.wiley.com/doi/bph.15538. G protein-coupled receptors are one of the six major pharmacological targets into which the Guide is divided, with the others being: ion channels, nuclear hormone receptors, catalytic receptors, enzymes and transporters. These are presented with nomenclature guidance and summary information on the best available pharmacological tools, alongside key references and suggestions for further reading. The landscape format of the Concise Guide is designed to facilitate comparison of related targets from material contemporary to mid-2021, and supersedes data presented in the 2019/20, 2017/18, 2015/16 and 2013/14 Concise Guides and previous Guides to Receptors and Channels. It is produced in close conjunction with the Nomenclature and Standards Committee of the International Union of Basic and Clinical Pharmacology (NC-IUPHAR), therefore, providing official IUPHAR classification and nomenclature for human drug targets, where appropriate.

Activation induces structural changes in the liganded angiotensin II type 1 receptor.

The octapeptide hormone angiotensin II (AngII) binds to and activates the human angiotensin II type 1 receptor (hAT(1)) of the G protein-coupled receptor class A family. Several activation mechanisms have been proposed for this family, but they have not yet been experimentally validated. We previously used the methionine proximity assay to show that 11 residues in transmembrane domain (TMD) III, VI, and VII of the hAT(1) receptor reside in close proximity to the C-terminal residue of AngII. With the exception of a single change in TMD VI, the same contacts are present on N111G-hAT(1), a constitutively active mutant; this N111G-hAT(1) is a model for the active form of the receptor. In this study, two series of 53 individual methionine mutations were constructed in TMD I, II, IV, and V on both receptor forms. The mutants were photolabeled with a neutral antagonist, (125)I-[Sar(1),p-benzoyl-L-Phe(8)]AngII, and the resulting complexes were digested with cyanogen bromide. Although no new contacts were found for the hAT(1) mutants, two were found in the constitutively active mutants, Phe-77 in TMD II and Asn-200 in TMD V. To our knowledge, this is the first time that a direct ligand contact with TMD II and TMD V has been reported. These contact point differences were used to identify the structural changes between the WT-hAT(1) and N111G-hAT(1) complexes through homology-based modeling and restrained molecular dynamics. The model generated revealed an important structural rearrangement of several TMDs from the basal to the activated form in the WT-hAT(1) receptor.

The fifth transmembrane domain of angiotensin II Type 1 receptor participates in the formation of the ligand-binding pocket and undergoes a counterclockwise rotation upon receptor activation.

The octapeptide hormone angiotensin II exerts a wide variety of cardiovascular effects through the activation of the angiotensin II Type 1 (AT(1)) receptor, which belongs to the G protein-coupled receptor superfamily. Like other G protein- coupled receptors, the AT(1) receptor possesses seven transmembrane domains that provide structural support for the formation of the ligand-binding pocket. The role of the fifth transmembrane domain (TMD5) was investigated using the substituted cysteine accessibility method. All of the residues within Thr-190 to Leu-217 region were mutated one at a time to cysteine, and after expression in COS-7 cells, the mutant receptors were treated with the sulfhydryl-specific alkylating agent methanethiosulfonate-ethylammonium (MTSEA). MTSEA reacts selectively with water-accessible, free sulfhydryl groups of endogenous or introduced point mutation cysteines. If a cysteine is found in the binding pocket, the covalent modification will affect the binding kinetics of the ligand. MTSEA substantially decreased the binding affinity of L197C-AT(1), N200C-AT(1), I201C-AT(1), G203C-AT(1), and F204C-AT(1) mutant receptors, which suggests that these residues orient themselves within the water-accessible binding pocket of the AT(1) receptor. Interestingly, this pattern of acquired MTSEA sensitivity was altered for TMD5 reporter cysteines engineered in a constitutively active N111G-AT(1) receptor background. Indeed, mutant I201C-N111G-AT(1) became more sensitive to MTSEA, whereas mutant G203C-N111G-AT(1) lost some sensitivity. Our results suggest that constitutive activation of AT(1) receptor causes an apparent counterclockwise rotation of TMD5 as viewed from the extracellular side.

A single-nucleotide polymorphism of alanine to threonine at position 163 of the human angiotensin II type 1 receptor impairs Losartan affinity.

BACKGROUND AND OBJECTIVE: AT1 is the principal receptor for angiotensin II (AngII), which regulates blood pressure and osmotic homeostasis. Earlier studies have shown that position 163 interacts with the antihypertensive nonpeptide antagonist, Losartan. A recently discovered polymorphism found in humans (rs12721226) coding for residue 163 led us to determine whether this polymorphism would affect Losartan antihypertensive therapies. The pharmacological properties of the A163T hAT1 variant are described. METHOD AND RESULTS: The A163T hAT1 mutation was evaluated by testing its affinity by dose displacement of AngII analogs in COS-7 cells expressing either wild-type hAT1 or the A163T hAT1. The expressions of the receptors were evaluated by saturation binding and the efficacies were assessed by measuring the 3H-inositol phosphate production. The results showed that the A163T hAT1 receptor is comparable with the affinity, expression, and efficacy of native hAT1 towards peptide ligands. The affinities were also tested with nonpeptide antagonists Losartan, L-158 809, valsartan, telmisartan, irbesartan, candesartan, and EXP3174. Losartan and EXP3174 displayed a 7-fold loss in affinity towards A163T hAT1. The ability of Losartan to inhibit AngII-induced inositol triphosphate production also confirmed a loss in efficacy. Molecular modeling showed a higher steric and hydrophilic hindrance of the A163T hAT1-Losartan complex. CONCLUSION: The polymorphism that codes for the A163T hAT1 variant results in a receptor with normal physiological properties toward the endogenous hormone. However, the significant reduction in affinity to Losartan and its active metabolite, EXP3174, could significantly impair the clinical effectiveness of an antihypertensive therapy using Losartan with patients bearing the A163T polymorphism.

Mutational analysis of the conserved Asp2.50 and ERY motif reveals signaling bias of the urotensin II receptor.

Class A (rhodopsin-like) G protein-coupled receptors possess conserved residues and motifs that are important for their specific activity. In the present study, we examined the role of residue Asp97(2.50) as well as residues Glu147(3.49), Arg148(3.50), and Tyr149(3.51) of the ERY motif on the functionality of the urotensin II receptor (UT). Mutations D97(2.50)A, R148(3.50)A, and R148(3.50)H abolished the ability of UT to activate phospholipase C, whereas mutations E147(3.49)A and Y149(3.51)A reduced the ability to activate PLC by 50%. None of the mutants exhibited constitutive activity. However, R148(3.50)A and R148(3.50)H promoted ERK1/2 activation, which was abolished by 4-(3-chloroanilino)-6,7-dimethoxyquinazoline (AG1478), an inhibitor of epidermal growth factor receptor (EGFR) tyrosine kinase activity. Both these mutants were capable of directly activating EGFR, which confirmed that they activated the mitogen-activated protein kinase (MAPK) pathway by a Galpha(q/11)-independent transactivation of EGFR. The D97(2.50)A, R148(3.50)A, and R148(3.50)H mutants did not readily internalize and did not promote translocation or colocalize with beta-arrestin2-GFP. Finally, the agonist-induced internalization of the E147(3.49)A mutant receptor was significantly increased compared with wild-type receptor. This study highlights the major contribution of the conserved Asp(2.50) residue to the functionality of the UT receptor. The Arg residue in the ERY motif of UT is an important structural element in signaling crossroads that determine whether Galpha(q/11)-dependent and -independent events can occur.

Novel solubility-switchable MRI agent allows the noninvasive detection of matrix metalloproteinase-2 activity in vivo in a mouse model.

A novel MRI proteinase-modulated contrast agent (PCA) was developed to detect the activity of the proinvasive enzyme matrix metalloproteinase-2 (MMP-2) in vivo. The PCA2-switch agent incorporates a solubility switch, where cleavage of a peptide substrate by MMP-2 decreases the water solubility of the agent. Evidence suggests that this leads to an accumulation of cleaved PCA2-switch in an MMP-2-positive, wild-type, MC7-L1 mammary carcinoma tumor in a Balb/c mouse model compared to a MC7-L1 MMP-2-knockdown tumor. When a scrambled peptide sequence is inserted into the agent (PCA2-scrambled), the in vitro cleavage efficiency of MMP-2 is markedly reduced. In vivo, PCA2-scrambled does not accumulate in the wild-type tumor and the pharmacokinetics is similar in both tumors. In conclusion, in vivo cleavage of PCA2-switch by MMP-2 results in a significant accumulation of the cleaved PCA2-switch in an MMP-2-positive tumor.

Ascorbic acid decreases the binding affinity of the AT1 receptor for angiotensin II.

BACKGROUND: Ascorbic acid is an essential vitamin and a powerful antioxidant. Many studies have highlighted the benefits of ascorbic acid for chronic cardiovascular diseases such as hypertension in which angiotensin II (Ang II) plays an significant role. We therefore hypothesized that ascorbic acid could modify the pharmacological properties of the AT(1) receptor for Ang II. METHODS: Binding studies and Ca(2+) mobilization studies were performed with HEK293 cells stably expressing the AT(1) receptor for Ang II. Smooth muscle contraction studies were performed with rabbit aorta strips that endogenously express the AT(1) receptor. RESULTS: Scatchard analysis revealed that ascorbic acid decreased the binding affinity of the AT(1) receptor without modifying its maximal binding capacity. Ascorbic acid did not modify the binding affinity of the AT(2) receptor for Ang II or of the UT receptor for urotensin II. In single-cell Ca(2+) imaging assays, ascorbic acid reduced the frequency of intracellular Ca(2+) oscillations induced by a low dose of Ang II. In functional assays, ascorbic acid significantly diminished the contraction of rabbit aorta pre-contracted with Ang II but not those pre-contracted with urotensin II. CONCLUSIONS: Ascorbic acid decreases the binding affinity of the AT(1) receptor. These results offer a mechanistic explanation for the reported blood pressure lowering effect of ascorbic acid.

Biological properties and functional determinants of the urotensin II receptor.

The urotensin II receptor (UT) is a member of the G protein-coupled receptor (GPCR) family and binds the cyclic undecapeptide urotensin II (U-II) as well as the octapeptide urotensin II-related peptide (URP). The active UT mediates pleiotropic effects through various signal transduction pathways, including coupling to G proteins and activating the mitogen-activated protein kinase pathway. Several highly conserved residues and motifs of class A GPCRs that are important for activity are found in UT. This review highlights some of the putative roles of these motifs in the binding, activation and desensitization of UT.

Photolabelling the urotensin II receptor reveals distinct agonist- and partial-agonist-binding sites.

The mechanism by which GPCRs (G-protein-coupled receptors) undergo activation is believed to involve conformational changes following agonist binding. We have used photoaffinity labelling to identify domains within GPCRs that make contact with various photoreactive ligands in order to better understand the activation mechanism. Here, a series of four agonist {[Bpa1]U-II (Bpa is p-benzoyl-L-phenylalanine), [Bpa2]U-II, [Bpa3]U-II and [Bpa4]U-II} and three partial agonist {[Bpa1Pen5D-Trp7Orn8]U-II (Pen is penicillamine), [Bpa2Pen5D-Trp7Orn8]U-II and [Pen5Bpa6D-Trp7Orn8]U-II} photoreactive urotensin II (U-II) analogues were used to identify ligand-binding sites on the UT receptor (U-II receptor). All peptides bound the UT receptor expressed in COS-7 cells with high affinity (Kd of 0.3-17.7 nM). Proteolytic mapping and mutational analysis led to the identification of Met288 of the third extracellular loop of the UT receptor as a binding site for all four agonist peptides. Both partial agonists containing the photoreactive group in positions 1 and 2 also cross-linked to Met288. We found that photolabelling with the partial agonist containing the photoreactive group in position 6 led to the detection of transmembrane domain 5 as a binding site for that ligand. Interestingly, this differs from Met184/Met185 of the fourth transmembrane domain that had been identified previously as a contact site for the full agonist [Bpa6]U-II. These results enable us to better map the binding pocket of the UT receptor. Moreover, the data also suggest that, although structurally related agonists or partial agonists may dock in the same general binding pocket, conformational changes induced by various states of activation may result in slight differences in spatial proximity within the cyclic portion of U-II analogues.

Angiotensin II cyclic analogs as tools to investigate AT1R biased signaling mechanisms.

G protein coupled receptors (GPCRs) produce pleiotropic effects by their capacity to engage numerous signaling pathways once activated. Functional selectivity (also called biased signaling), where specific compounds can bring GPCRs to adopt conformations that enable selective receptor coupling to distinct signaling pathways, continues to be significantly investigated. However, an important but often overlooked aspect of functional selectivity is the capability of ligands such as angiotensin II (AngII) to adopt specific conformations that may preferentially bind to selective GPCRs structures. Understanding both receptor and ligand conformation is of the utmost importance for the design of new drugs targeting GPCRs. In this study, we examined the properties of AngII cyclic analogs to impart biased agonism on the angiotensin type 1 receptor (AT1R). Positions 3 and 5 of AngII were substituted for cysteine and homocysteine residues ([Sar1Hcy3,5]AngII, [Sar1Cys3Hcy5]AngII and [Sar1Cys3,5]AngII) and the resulting analogs were evaluated for their capacity to activate the Gq/11, G12, Gi2, Gi3, Gz, ERK and ß-arrestin (ßarr) signaling pathways via AT1R. Interestingly, [Sar1Hcy3,5]AngII exhibited potency and full efficacy on all pathways tested with the exception of the Gq pathway. Molecular dynamic simulations showed that the energy barrier associated with the insertion of residue Phe8 of AngII within the hydrophobic core of AT1R, associated with Gq/11 activation, is increased with [Sar1Hcy3,5]AngII. These results suggest that constraining the movements of molecular determinants within a given ligand by introducing cyclic structures may lead to the generation of novel ligands providing more efficient biased agonism.

Stereospecific synthesis of a carbene-generating angiotensin II analogue for comparative photoaffinity labeling: improved incorporation and absence of methionine selectivity.

A stereospecific convergent synthesis of N-[(9-fluorenyl)methoxycarbonyl]-p-[3-(trifluoromethyl)-3H-diazirin-3-yl]-l-phenylalanine (Fmoc-12, Fmoc-Tdf) and its incorporation into the C-terminal position of the angiotensin II (AngII) peptide to form (125)I[Sar(1),Tdf(8)]AngII ((125)I-13) is presented. This amino acid photoprobe is a highly reactive carbene-generating diazirine phenylalanine derivative that can be used for photoaffinity labeling. Using model receptors, we compared the reactivity and the Met selectivity of 12 to that of the widely used and reputedly Met-selective p-benzoyl-l-phenylalanine (Bpa) photoprobe. Wild-type and mutant AngII type 2 receptors, a G protein-coupled receptors, were photolabeled with (125)I-13 as well as with (125)I[Sar(1),Bpa(8)]AngII ((125)I-14), and the respective incorporation yields were assessed. The carbene-generating 12 was more reactive toward inert residues and was not Met-selective compared to the biradical ketone-generating Bpa, allowing for more precise determination of ligand contact points in peptidergic receptors.

S-nitrosylation of cysteine 289 of the AT1 receptor decreases its binding affinity for angiotensin II.

1. Nitric oxide (NO) is known to affect the properties of various proteins via the S-nitrosylation of cysteine residues. This study evaluated the direct effects of the NO donor sodium nitroprusside (SNP) on the pharmacological properties of the AT1 receptor for angiotensin II expressed in HEK-293 cells. 2. SNP dose-dependently decreased the binding affinity of the AT1 receptor without affecting its total binding capacity. This modulatory effect was reversed within 5 min of removing SNP. 3. The effect of SNP was not modified in the presence of the G protein uncoupling agent GTPgammaS or the soluble guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one. 4. The binding properties of a mutant AT1 receptor in which all five cysteine residues within the transmembrane domains had been replaced by serine was not affected by SNP. Systematic analysis of mutant AT1 receptors revealed that cysteine 289 conferred the sensitivity to SNP. 5. These results suggest that NO decreased the binding affinity of the AT1 receptor by S-nitrosylation of cysteine 289. This modulatory mechanism may be particularly relevant in pathophysiological situations where the beneficial effects of NO oppose the deleterious effects of angiotensin II.

Importance of N-glycosylation positioning for cell-surface expression, targeting, affinity and quality control of the human AT1 receptor.

GPCRs (G-protein-coupled receptors) are preferentially N-glycosylated on ECL2 (extracellular loop 2). We previously showed that N-glycosylation of ECL2 was crucial for cell-surface expression of the hAT1 receptor (human angiotensin II receptor subtype 1). Here, we ask whether positioning of the N-glycosylation sites within the various ECLs of the receptor is a vital determinant in the functional expression of hAT(1) receptor at the cell surface. Artificial N-glycosylation sequons (Asn-Xaa-Ser/Thr) were engineered into ECL1, ECL2 and ECL3. N-glycosylation of ECL1 caused a very significant decrease in affinity and cell surface expression of the resulting receptor. Shifting the position of the ECL2 glycosylation site by two residues led to the synthesis of a misfolded receptor which, nevertheless, was trafficked to the cell surface. The misfolded nature of this receptor is supported by an increased interaction with the chaperone HSP70 (heat-shock protein 70). Introduction of N-glycosylation motifs into ECL3 yielded mutant receptors with normal affinity, but low levels of cell surface expression caused by proteasomal degradation. This behaviour differed from that observed for the aglycosylated receptor, which accumulated in the endoplasmic reticulum. These results show how positioning of the N-glycosylation sites altered many properties of the AT1 receptor, such as targeting, folding, affinity, cell surface expression and quality control.