Insertion of an electronegative sulfur atom in the side chain of position 5 of angiotensin II: changes in the tachyphylactic properties of the peptide.

Angiotensin II (AII) analogs bearing n-Leu, Met or S-substituted groups for cysteine at position 5 were studied regarding their agonistic and tachyphylactic properties. It was shown that these analogs lowered the relative affinity towards the AT1 receptor as determined by contractile responses, which could be due to the removal of the beta-branching residue at position 5. Insertion of a sulfur atom in a different position away from the attached backbone carbon atom presented no significant difference in EC50 values for these analogs. Interestingly, the S-bearing analogs at position 5 were full agonists but the tachyphylactic property was lost, in contrast to [n-Leu5]AII, which still induced reduction of the contractile responses. Nevertheless after replacing the Asp with Sar in position 1 (Sar1) tachyphylaxis was again established. It is concluded that the insertion of Met or an S-substituted cysteine into the side chain at position 5 of AII may promote interactions with its receptor due to the slight electronegative character of the sulfur atom and changes in the restricted conformational freedom of the Ile5 residue in the AII molecule. This was overcome by Sar1, probably through interactions due to its fully protonated N-terminal amino group and favoring the conformation responsible for the tachyphylaxis phenomenon.     

Effect of 3-5 monocyclizations of angiotensin II and 4-aminoPhe6-Ang II on AT2 receptor affinity

The endogenous angiotensin II (Ang II) and the synthetic AT(2) selective agonist 4-aminoPhe(6)-Ang II respond very differently to identical cyclizations. Cyclizations of Ang II by thioacetalization, involving the 3 and 5 amino acid residue side chains, provided ligands with almost equipotent binding affinities to Ang II at the AT(2) receptor. In contrast, the same cyclization procedures applied on the AT(2) selective 4-aminoPhe(6)-Ang II delivered significantly less potent AT(2) receptor ligands, although the AT(2)/AT(1) selectivity was still very high. The fact that different structure-activity relationships are observed after imposing conformational restrictions on Ang II and 4-aminoPhe(6)-Ang II, respectively, suggests that the peptides, despite large similarities might adopt quite different backbone conformations when binding to the AT(2) receptor.     

Characterization by high performance liquid chromatography of angiotensin peptides in the plasma and cerebrospinal fluid of the dog

A high performance liquid chromatography (HPLC) method is described for the separation of angiotensin (Ang) peptides and their subsequent quantification by radioimmunoassay in plasma and cerebrospinal fluid (CSF). The use of the ion-pair solvent heptafluorobutyric acid in gradient HPLC achieves baseline resolution of Ang I, Ang II, and the C-terminal fragments des-[Asp1]-Ang I, des-[Asp1]-Ang II, des-[Asp1,Arg2]-Ang II and des-[Asp1,Arg2,Val3]-Ang II in approximately 25 min. Recovery of synthetic Ang standards after phenylsilica extraction and HPLC separation was greater than 70% for each peptide in both plasma and CSF. Ang I and Ang II were shown to be the major immunoreactive Ang components in plasma, and Ang II, des-[Asp1,Arg2]-Ang II and des-[Asp1,Arg2,Val3]-Ang II in CSF.     

Angiotensin-(1-7) binds at the type 1 angiotensin II receptors in rat renal cortex.

Significant angiotensin (Ang) (1-7) production occurs in kidney and effects on renal function have been observed. The present study was undertaken to investigate binding characteristics of the heptapeptide to Ang II receptors present in rat renal cortex. [125I]-Ang II binding to rat glomeruli membranes was analyzed in the presence of increasing concentrations of Ang II, Ang-(1-7), DUP 753 and PD 123319. Linearity of the Scatchard plot of the [125I]-Ang II specific binding to rat glomeruli membranes indicated a single population of receptors, with a Kd value of 0.7 +/- 0.1 nM and a Bmax of 198 +/- 0.04 fmol/mg protein. DUP 753, an specific AT1 receptor antagonist, totally displaced the specific binding of [125I]-radiolabelled hormone with a Ki of 15.8 +/- 0.9 nM, while no changes were observed in the presence of the selective AT2 receptor antagonist, PD 123319. The specific [125I]-Ang II binding to rat glomerular membranes was displaced by Ang-(1-7) with high affinity (Ki = 8.0 +/- 3.2 nM). We conclude that radioligand binding assays in the presence of selective Ang II antagonists DUP 753 and PD 123319 suggest the unique presence of AT1, receptors in rat glomeruli and a possible role in the control of the biological renal effects of Ang-(1-7).     

Synthesis and AT2 receptor-binding properties of angiotensin II analogues.

The present study investigates the importance of the amino acid side chains in the octapeptide angiotensin II (Ang II) for binding to the AT2 receptor. A Gly scan was performed where each amino acid in Ang II was substituted one-by-one with glycine. The resulting set of peptides was tested for affinity to the AT2 receptor (porcine myometrial membranes). For a comparison, the peptides were also tested for affinity to the AT1 receptor (rat liver membranes). Only the substitution of Arg2 reduced affinity to the AT2 receptor considerably (92-fold when compared with Ang II). For the other Gly-substituted analogues the affinity to the AT2 receptor was only moderately affected. To further investigate the role of the Arg2 side chain for receptor binding, we synthesized some N-terminally modified Ang II analogues. According to these studies a positive charge in the N-terminal end of angiotensin III [Ang II (2-8)] is not required for high AT2 receptor affinity but seems to be more important in Ang II. With respect to the AT1 receptor, [Gly2]Ang II and [Gly8]Ang II lacked binding affinity (Ki > 10 microM). Replacement of the Val3 or Ile5 residues with Gly produced only a slight decrease in affinity. Interestingly, substitution of Tyr4 or His6, which are known to be very important for AT1 receptor binding, resulted in only 48 and 14 times reduction in affinity, respectively.     

Characterization of the AT(4) receptor in a human neuroblastoma cell line (SK-N-MC)

Angiotensin IV (Ang IV), the 3-8 fragment of angiotensin II (Ang II), binds to a distinct receptor designated the AT(4) receptor. The peptide elicits a range of vascular and central actions including facilitation of memory retention and retrieval in several learning paradigms. The aim of this study was to characterize the AT(4) receptor in a human cell line of neural origin. Receptor binding studies indicate that the human neuroblastoma cell line SK-N-MC cells express a high-affinity Ang IV binding site with a pharmacological profile similar to the AT(4) receptor: (125)I]-Ang IV and (125)I]-Nle(1)-Ang IV bind specifically to the SK-N-MC cell membranes (K(d) = 0.6 and 0.1 nM) in a saturable manner (B(max) = 1.2 pmol/mg of protein). AT(4) receptor ligands, Nle(1)-Ang IV, Ang IV and LVV-haemorphin 7 (LVV-H7), compete for the binding of [(125)I]-Ang IV or [(125)I]-Nle(1)-Ang IV to the SK-N-MC cell membranes with rank order potencies of Nle(1)-Ang IV > Ang IV > LVV-H7 with IC(50) values of 1.4, 8.7 and 59 nM ([(125)I]-Ang IV) and 1.8, 20 and 168 nM ([(125)I]-Nle(1)-Ang IV), respectively. The binding of [(125)I]-Ang IV or [(125)I]-Nle(1)-Ang IV to SK-N-MC cell membranes was not affected by the presence of GTP gamma S. Both Ang IV and LVV-H7 stimulated DNA synthesis in this cell line up to 72 and 81% above control levels, respectively. The AT(4) receptor in the SK-N-MC cells is a 180-kDa glycoprotein; under non-reducing conditions a 250-kDa band was also observed. In summary, the human neuroblastoma cell line, SK-N-MC, expresses functional AT(4) receptors that are responsive to Ang IV and LVV-H7, as indicated by an increase in DNA synthesis. This is the first human cell line of neural origin shown to express the AT(4) receptor.     

Role of the His273 located in the sixth transmembrane domain of the angiotensin II receptor subtype AT2 in ligand-receptor interaction

Angiotensin II receptor subtypes AT1 and AT2 are proteins with seven transmembrane domain (TMD) topology and share 34% homology. It was shown that His256, located in the sixth TMD of the AT1 receptor, is needed for the agonist activation by the Phe8 side chain of angiotensin II, although replacing this residue with arginine or glutamine did not significantly alter the affinity binding of the receptor. We hypothesized that the His273 located in the sixth transmembrane domain of the AT2 receptor may play a similar role in the functions of the AT2 receptor, although this residue was not identified as a conserved residue in the initial homology comparisions. Therefore, we replaced His273 of the AT2 receptor with arginine or glutamine and analyzed the ligand-binding properties of the mutant receptors using Xenopus oocytes as an expression system. Our results suggested that the AT2 receptor mutants His273Arg and His273 Glu have lost their affinity to [125I-Sar1-Ile8]Ang II, a peptidic ligand that binds both the AT1 and AT2 receptors and to 125I-CGP42112A, a peptidic ligand that binds specifically to the AT2 receptor. Thus, His273 located in the sixth TMD of the AT2 receptor seems to play an important role in determining the binding properties of this receptor. Moreover, these results along with our previous observation that the Lys215 located in the 5th TMD of the AT2 receptor is essential for its high affinity binding to [125I-Sar1-Ile8]Ang II indicate that key amino acids located in the 5th and 6th TMDs of the AT2 receptor are needed for high affinity binding of the AT2 to its ligands.     

Angiotensin AT4 ligands are potent,competitive inhibitors of insulin regulated aminopeptidase (IRAP)

Angiotensin IV (Ang IV) exerts profound effects on memory and learning, a phenomenon ascribed to its binding to a specific AT4 receptor. However the AT4 receptor has recently been identified as the insulin-regulated aminopeptidase (IRAP). In this study, we demonstrate that AT4 receptor ligands, including Ang IV, Nle1-Ang IV, divalinal-Ang IV, and the structurally unrelated LVV-hemorphin-7, are all potent inhibitors of IRAP catalytic activity, as assessed by cleavage of leu-beta-naphthylamide by recombinant human IRAP. Both Ang IV and divalinal-Ang IV display competitive kinetics, indicating that AT4 ligands mediate their effects by binding to the catalytic site of IRAP. The AT4 ligands also displaced [125I]-Nle1-Ang IV or [125I]-divalinal1-Ang IV from IRAP-HEK293T membranes with high affinity, which was up to 200-fold greater than in the catalytic assay; this difference was not consistent among the peptides, and could not be ascribed to ligand degradation. Although some AT4 ligands were subject to minor cleavage by HEK293T membranes, none were substrates for IRAP. Of a range of peptides tested, only vasopressin, oxytocin, and met-enkephalin were rapidly cleaved by IRAP. We propose that the physiological effects of AT4 ligands result, in part, from inhibition of IRAP cleavage of neuropeptides involved in memory processing.     

Identification of the region of AT2 receptor needed for inhibition of the AT1 receptor-mediated inositol 1,4,5-triphosphate generation

Increase in the intracellular inositol triphosphate (IP3) levels in Xenopus oocytes in response to expression and activation of rat angiotensin II (Ang II) receptor AT1 was inhibited by co-expression of rat AT2 receptor. To identify which region of the AT2 was involved in this inhibition, ability of three AT2 mutants to abolish this inhibition was analyzed. Deletion of the C-terminus of the AT2 did not abolish this inhibition. Replacing Ile249 in the third intracellular loop (3rd ICL) of the AT2 with proline, corresponding amino acid in the AT1, in the mutant M6, resulted in slightly reduced affinity to [125I]Ang II (K(d)=0.259 nM), however, did not abolish the inhibition. In contrast, replacing eight more amino acids in the 3rd ICL of the AT2 (at positions 241-244, 250-251 and 255-256) with that of the AT1 in the mutant M8, not only increased the affinity of the AT2 receptor to [125I]Ang II (K(d)=0.038 nM) but also abolished AT2-mediated inhibition. Interestingly, activation of the M8 by Ang II binding also resulted in increase in the intracellular IP(3) levels in oocytes. These results imply that the region of the 3rd ICL of AT2 spanning amino acids 241-256 is sufficient for the AT2-mediated inhibition of AT1-stimulated IP3 generation. Moreover, these nine mutations are also sufficient to render the AT2 with the ability to activate phospholipase C.     

Role of Asp297 of the AT2 receptor in high-affinity binding to different peptide ligands

To determine how ligand-receptor interaction is affected by the charges of the amino acids at position 2 of the ligands and position 297 of the AT2 receptor, we generated the Asp297Lys mutant of AT2 and a ligand SarAsp²Ile. Asp297Lys mutant lost affinity to Ang II and SarIle however retained partial affinity to ¹²⁵I-CGP42112A. The SarAsp²Ile had high affinity to Asp297Lys (IC₅₀3.5nM) and partial affinity to the AT2 (IC₅₀15nM). Therefore, not only the charge, but also the length of the side arms of the amino acids at position 2 of the ligand and position 297 of the receptor affect their interaction.     

Characterization of Binding Sites for the Angiotensin II Antagonist 125I-[Sarc1,Ile8]-Angiotensin II on Murine Neuroblastoma N1E-115 Cells

The murine neuroblastoma N1E-115 cell line contains binding sites for the angiotensin II (Ang II) receptor antagonist 125I-[Sarc1,Ile8]-Ang II (125I-SARILE). Binding of 125I-SARILE to N1E-115 membranes was rapid, reversible, and specific for Ang II-related peptides. The rank order potency of 125I-SARILE binding was the following: [Sarc1]-Ang II = [Sarc1,Ile8]-Ang II greater than Ang II greater than Ang III = [Sarc1,Thr8]-Ang II much greater than Ang I. Scatchard analysis of membranes prepared from confluent monolayers revealed a homogenous population of high affinity (KD = 383 +/- 60 pM) binding sites with a Bmax of 25.4 +/- 1.6 fmol/mg of protein. Moreover, the density, but not the affinity, of the binding sites increased as the cells progressed from logarithmic to stationary growth in culture. Finally, agonist, but not antagonist, binding to N1E-115 cells was regulated by guanine nucleotides. Collectively, these results suggest that the murine neuroblastoma N1E-115 cell line may provide a useful model in which to investigate the signal transduction mechanisms utilized by neuronal Ang II receptors.     

Angiotensin IV is a potent agonist for constitutive active human AT1 receptors. Distinct roles of the N-and C-terminal residues of angiotensin II during AT1 receptor activation

The octapeptide hormone, angiotensin II (Ang II), exerts its major physiological effects by activating AT(1) receptors. In vivo Ang II is degraded to bioactive peptides, including Ang III (angiotensin-(2-8)) and Ang IV (angiotensin-(3-8)). These peptides stimulate inositol phosphate generation in human AT(1) receptor expressing CHO-K1 cells, but the potency of Ang IV is very low. Substitution of Asn(111) with glycine, which is known to cause constitutive receptor activation by disrupting its interaction with the seventh transmembrane helix (TM VII), selectively increased the potency of Ang IV (900-fold) and angiotensin-(4-8), and leads to partial agonism of angiotensin-(5-8). Consistent with the need for the interaction between Arg(2) of Ang II and Ang III with Asp(281), substitution of this residue with alanine (D281A) decreased the peptide's potency without affecting that of Ang IV. All effects of the D281A mutation were superseded by the N111G mutation. The increased affinity of Ang IV to the N111G mutant was also demonstrated by binding studies. A model is proposed in which the Arg(2)-Asp(281) interaction causes a conformational change in TM VII of the receptor, which, similar to the N111G mutation, eliminates the constraining intramolecular interaction between Asn(111) and TM VII. The receptor adopts a more relaxed conformation, allowing the binding of the C-terminal five residues of Ang II that switches this "preactivated" receptor into the fully active conformation.     

Endogenous and Expressed Angiotensin II Receptors on Xenopus Oocytes

Intact Xenopus oocytes contain a homogeneous population of binding sites for the angiotensin II (Ang II) receptor antagonist 125I-[Sarc1,Ile8]-Ang II (125I-SARILE). Binding of 125I-SARILE to intact oocytes was saturable and of high affinity with an apparent KD of 0.7 nM and maximal density of 0.12 fmol/oocyte. Binding of 125I-SARILE to oocytes also was specific for Ang II-related peptides with a rank order potency of: [Sarc1]-Ang II greater than Ang II greater than Ang III much greater than Ang I. However, these endogenous binding sites were present only in follicle-enclosed oocytes and within the follicular layer itself. On the other hand, injection of poly(A)+ RNA isolated from murine N1E-115 neuroblastoma cells into oocytes resulted in the appearance of 125I-SARILE binding sites even in defolliculated oocytes. These expressed receptors exhibited pharmacological properties similar to those endogenously present in the follicular layer, although their levels were much less. Collectively, these results suggest that endogenous Ang II receptors are present on Xenopus oocyte follicle cells, whereas Ang II receptors expressed from exogenous N1E-115 RNA are found on the oocytes themselves. In addition, the high density of Ang II receptors on the follicle cells emphasizes the necessity for care in using Xenopus oocytes for the expression of receptors encoded by exogenous RNAs.     

Identification of amino acid residues of rat angiotensin II receptor for ligand binding by site directed mutagenesis.

To determine the specific mechanism of ligand binding to angiotensin (Ang II) receptor AT1, mutagenized rat receptor cDNAs were expressed transiently in COS-7 cells and the effect of the mutations on the binding to peptidic and non-peptidic ligands was analyzed by Scatchard plots. Mutation of Lys199 to Gln in the intramembrane domain strongly reduced the affinity to both [125I] Ang II and [125I]-1Sar, 8Ile-Ang II whereas mutation of two other Lys had little effect, indicating involvement of Lys199 in binding ligands. Replacement of each of four Cys in the extracellular domain markedly reduced binding affinity, indicating the importance of two putative disulfide bridges in the formation of active receptor conformation. Substitution of Asp for Asn in N-glycosylation had no effect on ligand binding or expression of the receptor. These studies indicate mutated receptors are expressed in the plasma membrane and are amenable for further detailed studies.     

Selective down-regulation of AT2 receptors in uterine arteries from pregnant ewes given 24-h intravenous infusions of angiotensin II

Previously, we showed that uterine arteries from late gestation pregnant ewes infused intravenously with angiotensin II (Ang II) for 24 h, displayed heightened responsiveness to Ang II in vitro. Furthermore, we found that a small population of ewes with a "preeclampsia-like" disorder also displayed this. Therefore, we have investigated the density and affinity of Ang II receptor subtypes in the uterine arteries from these groups. Ang II receptor binding was measured using 125I [Sar1Ile8] Ang II. Proportions of AT1 and AT2 receptors were determined by inhibiting 125I [Sar1Ile8] Ang II with losartan (AT1 antagonist) or PD 123319 (AT2 antagonist). Uterine arteries from 24-h Ang II-infused ewes had a lower proportion of AT2 receptors (56.2+/-2.3%) than control (saline-infused) ewes (84.1+/-1.0%; P<0.05). The density of AT2 receptors was reduced (P<0.05) while the density of AT1 receptors was not different. Thus, 24-h infusions of Ang II selectively down-regulated AT2 receptors in the uterine artery, resulting in heightened Ang II reactivity. By contrast, the binding properties of Ang II receptor subtypes in uterine arteries from ewes with the "preeclampsia-like" disorder were not different from control ewes.     

Autoradiographic localization and characterization of angiotensin II binding sites in the spleen of rats and mice

Specific binding sites for angiotensin II (Ang II) were localized in the red pulp of the spleen of rats and mice by quantitative autoradiography using 125I-Sar1-Ang II as a ligand. In the rat, the binding was saturable and specific, and the rank order for Ang II derivatives as competitors of 125I-Sar1-Ang II binding correlates well with their affinity for Ang II receptors in other tissues. Kinetic analysis in the rat spleen revealed a single class of binding sites with a KD of 1.11 nM and a Bmax value of 81.6 fmol/mg protein. Ang II binding sites were also localized on isolated rat spleen cells with similar affinity but with much lower Bmax, 9.75 fmol/mg protein. Ang II receptors were not detected in thymus sections from rats or mice, or on isolated rat thymocytes. The binding sites described here might represent a functional Ang II receptor with a role in the regulation of splenic volume and blood flow and in the modulation of the lymphocyte function.     

A frame shifted disulfide bridged analogue of angiotensin II

N-(2-Mercaptoethyl)glycine [NMGly] was incorporated into the 3 and 5 positions of angiotensin II and oxidized to give the corresponding cyclized disulfide c[NMGly(3,5)]Ang II. The binding affinity to the angiotensin II receptor (AT(1)) of this conformationally constrained analogue, which is related to the potent Ang II agonist c[Hcy(3,5)]Ang II, was examined. The analogue had no affinity to the AT(1) receptor. Theoretical conformational analysis was performed to compare the conformational characteristics of model compounds of c[Hcy(3,5)]Ang II and the frame shifted analogue c[NMGly(3,5)]Ang II in an attempt to explain the lack of affinity.     

Role of the third intracellular loop of the angiotensin II receptor subtype AT2 in ligand-receptor interaction

Angiotensin II (Ang II) receptor subtypes AT1 and AT2 share 34% overall homology, but the least homology is in their third intracellular loop (3rd ICL). In an attempt to elucidate the role of the 3rd ICL in determining the similarities and differences in the functions of the AT1 and the AT2 receptors, we generated a chimeric receptor in which the 3rd ICL of the AT2 receptor was replaced with that of the AT1 receptor. Ligand-binding properties and signaling properties of this receptor were assayed by expressing this receptor in Xenopus oocytes. Ligand-binding studies using [125I-Sar1-Ile8] Ang II, a peptidic ligand that binds both the AT1 and the AT2 receptor subtypes, and 125I-CGP42112A, a peptidic ligand that is specific for the AT2 receptor, showed that the chimeric receptor has lost affinity to both ligands. However, IP3 levels of the oocytes expressing the chimeric receptor were comparable to the IP3 levels of the oocytes expressing the AT1 receptor, suggesting that the chimeric receptors could couple to phospholipase C pathway in response to Ang II. We have shown previously that the nature of the amino acid present in the position 215 located in the fifth transmembrane domain (TMD) of the AT2 receptor plays an important role in determining its affinity to different ligands. Our results from the ligand-binding studies of the chimeric receptor further support the idea that the structural organization of the region spanning the 5th TMD and the 3rd ICL of the AT2 receptor has an important role in determining the ligand-binding properties of this receptor.