Cyclic insulin-regulated aminopeptidase (IRAP)/AT4 receptor ligands.

The angiotensin IV receptor (AT4 receptor) is the insulin-regulated aminopeptidase enzyme (IRAP, EC 3.4.11.3). This membrane-spanning enzyme belongs to the M1 family of zinc-dependent metallo-peptidases. It has been proposed that AT4 receptor ligands exert their physiological effects by binding to the active site of IRAP and thereby inhibiting the catalytic activity of the enzyme. The biological activity of a large series of linear angiotensin IV analogs was previously disclosed. Herein, the synthesis and biological evaluation of a series of angiotensin IV analogs, encompassing macrocyclic ring systems of different sizes, are presented. It is demonstrated that disulfide cyclizations of angiotensin IV can deliver ligands with high IRAP/AT4 receptor affinity. One ligand, with an 11-membered ring system (4), inhibited human IRAP and aminopeptidase N (AP-N) activity with similar potency as angiotensin IV but was considerably more stable than angiotensin IV toward enzymatic degradation. The compound provides a promising starting point for further optimization toward more drug-like derivatives. The cyclic constrained analogs allowed us to propose a tentative bioactive conformation of angiotensin IV and it seems that the peptide adopts an inverse gamma-turn at the C-terminal.     

Small potent ligands to the insulin-regulated aminopeptidase (IRAP)/AT(4) receptor.

Angiotensin IV analogs encompassing aromatic scaffolds replacing parts of the backbone of angiotensin IV have been synthesized and evaluated in biological assays. Several of the ligands displayed high affinities to the insulin-regulated aminopeptidase (IRAP)/AT(4) receptor. Displacement of the C-terminal of angiotensin IV with an o-substituted aryl acetic acid derivative delivered the ligand 4, which exhibited the highest binding affinity (K(i) = 1.9 nM). The high affinity of this ligand provides support to the hypothesis that angiotensin IV adopts a gamma-turn in the C-terminal of its bioactive conformation.Ligand (4) inhibits both human IRAP and aminopeptidase N-activity and induces proliferation of adult neural stem cells at low concentrations. Furthermore, ligand 4 is degraded considerably more slowly in membrane preparations than angiotensin IV. Hence, it might constitute a suitable research tool for biological studies of the (IRAP)/AT(4) receptor.     

Angiotensin IV interacts with a juxtamembrane site on AT(4)/IRAP suggesting an allosteric mechanism of enzyme modulation

Angiotensin IV (Ang IV), the 3-8 fragment of angiotensin II, binds to a specific receptor (AT(4)) that has recently been identified as the transmembrane aminopeptidase insulin-regulated aminopeptidase (IRAP) based on the fact that the two proteins share several pharmacological and biochemical properties. Our binding studies indicated that bovine heart expresses relatively large amounts (1.2 pmol/mg protein) of high-affinity binding sites for Ang IV (K(d)=1.8 nM). A photoaffinity-labeling approach combined with mild trypsin digestion revealed that the AT(4) receptor of bovine heart is a single transmembrane domain protein (153 kDa) with a large extracellular fragment (143 kDa). After alkaline denaturation of the AT(4) receptor, trypsin digestion produced two small membrane-associated fragments (16.9 and 6.6 kDa). These results suggest that Ang IV interacts with a juxtamembrane domain of AT(4) receptor. The location of the juxtamembrane site of contact was different from that of the active site of IRAP, suggesting that Ang IV uses an allosteric mechanism to modulate the activity of the AT(4)/IRAP.     

(Tyr(0),Bpa(4))bombesin is a GRP receptor agonist

(Tyr(0),Bpa(4))bombesin, (YB)BB was synthesized and its biologic activity evaluated using T47D breast cancer cells. ((125)I-Tyr(0), Bpa(4))BB bound with high affinity (K(d) = 5 nM) to T47D cells. Specific ((125)I-Tyr(0),Bpa(4))BB binding was inhibited with high affinity by BB, BW2258U89, GRP, GRP(14-27) and NMB (IC(50) values of 10, 2, 15, 20, and 150 nM)but not GRP(1-16) (IC(50) value of > 1000 nM). ((125)I-Tyr(0),Bpa(4))BB bound to the surface of T47D cells at 4 degrees C but was internalized at 37 degrees C. After binding at 4 degrees C followed by irradiation using ultraviolet light, ((125)I-Tyr(0),Bpa(4))BB labeled a 75 kDa protein using T47D cells. (Tyr(0),Bpa(4))BB, 10 nM, elevated cytosolic calcium using T47D cells within 10 s. Also (Tyr(0),Bpa(4))BB, 10 nM, elevated c-fos mRNA after 45 min. These results indicate that (Tyr(0),Bpa(4))BB is an agonist for GRP receptors.     

Evolution of the tRNA(Tyr)/TyrRS aminoacylation systems

The tRNA identity rules ensuring fidelity of translation are globally conserved throughout evolution except for tyrosyl-tRNA synthetases (TyrRSs) that display species-specific tRNA recognition. This discrimination originates from the presence of a conserved identity pair, G1-C72, located at the top of the acceptor stem of tRNA(Tyr) from eubacteria that is invariably replaced by an unusual C1-G72 pair in archaeal and eubacterial tRNA(Tyr). In addition to the key role of pair 1-72 in tyrosylation, discriminator base A73, the anticodon triplet and the large variable region (present in eubacterial tRNA(Tyr) but not found in eukaryal tRNA(Tyr)) contribute to tyrosylation with variable strengths. Crystallographic structures of two tRNA(Tyr)/TyrRS complexes revealed different interaction modes in accordance with the phylum-specificity. Recent functional studies on the human mitochondrial tRNA(Tyr)/TyrRS system indicates strong deviations from the canonical tyrosylation rules. These differences are discussed in the light of the present knowledge on TyrRSs.     

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.     

125I-d(CH2)5[Tyr(Me)2, Tyr(NH2)9]AVP: iodination and binding characteristics of a vasopressin receptor ligand

A radioiodinated vasopressin antagonist, d(CH2)5[Tyr(NH2)9]AVP has been prepared. Iodination was carried out at the phenyl moiety of the tyrosylamide residue at position 9, followed by HPLC purification. Non-radiolabelled monoiodinated antagonist was used as a reference for identification. 125I-d(CH2)5[Tyr(Me)2, Tyr(NH2)9]AVP binding appeared to take place with a dissociation constant of 0.28 +/- 0.09 nM (Kd +/- SD) to V1 vasopressin receptors on rat liver membranes.     

Ligands of the neuropeptide Y Y2 receptor

Neuropeptide Y (NPY) is one of the most abundant neuropeptides in the mammalian brain and exerts a variety of physiological processes in humans via four different receptor subtypes Y1, Y2, Y4 and Y5. Y2 receptor is the most abundant Y subtype receptor in the central nervous system and implicated with food intake, bone formation, affective disorders, alcohol and drugs of abuse, epilepsy, pain, and cancer. The lack of small molecule non-peptidic Y2 receptor modulators suitable as in vivo pharmacological tools hampered the progress to uncover the precise pharmacological role of Y2. Only in recent years, several potent, selective and non-peptidic Y2 antagonists have been discovered providing the tools to validate Y2 receptor as a therapeutic target. This Letter reviews Y2 receptor modulators mainly non-peptidic antagonists and their structure–activity relationships.     

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.     

Protein kinase A regulates AKAP250 (gravin) scaffold binding to the beta2-adrenergic receptor

A-kinase-anchoring protein 250 (AKAP250; gravin) acts as a scaffold that binds protein kinase A (PKA), protein kinase C and protein phosphatases, associating reversibly with the beta(2)-adrenergic receptor. The receptor-binding domain of the scaffold and the regulation of the receptor-scaffold association was revealed through mutagenesis and biochemical analyses. The AKAP domain found in other members of this superfamily is essential for the scaffold-receptor interactions. Gravin constructs lacking the AKAP domain displayed no binding to the receptor. Metabolic labeling studies in vivo demonstrate agonist-stimulated phosphorylation of gravin and enhanced gravin-receptor association. Analysis of the AKAP domain revealed two canonical PKA sites phosphorylated in response to elevated cAMP, blocked by PKA inhibitor, and essential for scaffold-receptor association and for resensitization of the receptor. The AKAP appears to provide the catalytic PKA activity responsible for phosphorylation of the scaffold in response to agonist activation of the receptor as well as for the association of the scaffold with the receptor, a step critical to receptor resensitization.     

Functional mimetic of the G-protein coupled receptor CXCR4 on a soluble antibody scaffold

G-protein coupled receptors (GPCRs) constitute major drug targets due to their involvement in critical biological functions and pathophysiological disorders. The leading challenge in their structural and functional characterization has been the need for a lipid environment to accommodate their hydrophobic cores. Here, we report an antibody scaffold mimetic (ASM) platform where we have recapitulated the extracellular functional domains of the GPCR, C-X-C chemokine receptor 4 (CXCR4) on a soluble antibody framework. The engineered ASM molecule can accommodate the N-terminal loop and all three extracellular loops of CXCR4. These extracellular features are important players in ligand recruitment and interaction for allostery and signal transduction. Our study shows that ASM^CXCR4 can be recognized by the anti-CXCR4 antibodies, MEDI3185, 2B11, and 12G5, and that ASM^CXCR4 can bind the HIV-1 glycoprotein ligand gp120, and the natural chemokine ligand SDF-1α. Further, we show that ASM^CXCR4 can competitively inhibit the SDF-1α signaling pathway, and be used as an immunogen to generate CXCR4-specific antibodies. This platform will be useful in the study of GPCR biology in a soluble receptor context for evaluating its extracellular ligand interactions.     

The scaffold protein gravin (cAMP-dependent protein kinase-anchoring protein 250) binds the beta 2-adrenergic receptor via the receptor cytoplasmic Arg-329 to Leu-413 domain and provides a mobile scaffold during desensitization

The cyclic AMP-dependent kinase-anchoring proteins (AKAPs) function as scaffolds for a wide-range of protein-protein interactions. The 250-kDa AKAP known as gravin plays a central role in organizing G-protein-coupled receptors to the protein kinases and phosphatases that regulate receptor function in desensitization, resensitization, and sequestration. Although gravin is critical for G-protein-linked receptor biology, the molecular features of the receptor necessary for interaction with this scaffold are not known. Herein, we map the regions of the beta(2)-adrenergic receptor that are required for binding to gravin. Intracellular loops 1, 2, and 3 appear not to participate in the binding of the receptor to the scaffold. In contrast, the C-terminal cytoplasmic region of the receptor (Arg-329 to Leu-413) competes readily for the binding of the beta(2)-adrenergic receptor by gravin, both using in vitro and in vivo assays. C-terminally truncated peptides with sequences ranging from Arg-329 to Leu-342 (13 aminoacyl residues), to Asn-352 (23 residues), to Tyr-366 (37 residues), to Asp-380 (51 residues), or to His-390 (61 residues), as well as N-terminally truncated peptides from Gln-391 to Leu-413 (23 residues) or Leu-381 to Leu-413 (33 residues) displayed no ability to block binding of receptor to gravin. The combination of Arg-329 to His-390 peptide and Gln-391 to Leu-413 peptide, however, reconstitutes a fragmented but full-length C-terminal region and also potently blocks the ability of gravin to bind the beta(2)-adrenergic receptor. The gravin-receptor interaction was examined in response to agonist by confocal microscopy. Remarkably, the association of the receptor with gravin was not disrupted during agonist-induced sequestration. The receptor-scaffold complex was maintained during agonist-induced sequestration. These data, in agreement with the biochemical data, reveal that gravin binds the receptor through the beta(2)-adrenergic receptor C-terminal cytoplasmic domain and that this interaction is maintained as the receptor is internalized. This is the first report of an AKAP scaffold protein translocating with its receptor, in this case a G-protein-coupled receptor.     

AT2 receptor interacting protein 1 (ATIP1) mediates COX-2 induction by an AT2 receptor agonist in endothelial cells

Angiotensin II (Ang II) type 2 receptor (AT2R) is one of the major components of the renin-angiotensin-aldosterone system. Nevertheless, the physiological role is not well defined compared to the understanding of the Ang II type 1 receptor (AT1R), which is a well characterized G-protein coupled receptor in the cardiovascular system. While the AT2R signaling pathway remains unclear, AT2 receptor interacting protein 1 (ATIP1) has been identified as a candidate molecule for interacting with the C-terminal region of AT2R. In this study, we investigated the ATIP1 dependent AT2R inducible genes in human umbilical vein endothelial cells (HUVECs). CGP42112A, an AT2R specific agonist, resulted in an upregulation of inflammatory genes in HUVECs, which were inhibited by knocking down ATIP1 with siRNA (siATIP1). Among them, we confirmed by quantitative PCR that the induction of COX-2 mRNA expression was significantly downregulated by siATIP1. COX-2 was also upregulated by Ang II stimulation. This upregulation was suppressed by treatment with the AT2R specific antagonist PD123319, which was not replicated by the AT1R antagonist telmisartan.These findings suggest that ATIP1 plays an important role in AT2R dependent inflammatory responses. This may provide a new approach to the development of cardio-protective drugs.     

G protein-coupled receptor kinase 2 (GRK2) is a Rho-activated scaffold protein for the ERK MAP kinase cascade

The G protein-coupled receptor kinases (GRKs) are best known for their role in phosphorylating and desensitising G protein-coupled receptors (GPCRs). The GRKs also regulate signalling downstream of other families of receptors and have a number of non-receptor substrates and binding partners. Here we identify RhoA_GTP and Raf1 as novel binding partners of GRK2 and report a previously unsuspected function for this kinase. GRK2 is a RhoA effector that serves as a RhoA-activated scaffold protein for the ERK MAP kinase cascade. The ability of GRK2 to bind to Raf1, MEK1 and ERK2 is dependent on RhoA_GTP binding to the catalytic domain of the kinase. Exogenous GRK2 has previously been shown to increase ERK activation downstream of the epidermal growth factor receptor (EGFR). Here we find that GRK2-mediated ERK activation downstream of the EGFR is Rho-dependent and that treatment with EGF promotes RhoA_GTP binding and ERK scaffolding by GRK2. Depletion of GRK2 expression by RNAi reveals that GRK2 is required for EGF-induced, Rho- and ERK-dependent thymidine incorporation in vascular smooth muscle cells (VSMCs). We therefore hypothesise that Rho-dependent ERK MAPK scaffolding by GRK2 downstream of the EGFR may have an important role in the vasculature, where increased levels of both GRK2 and RhoA have been associated with hypertension.     

Potent and selective MC-4 receptor agonists based on a novel disulfide scaffold

Extensive structure-activity relationship studies utilizing a beta-MSH-derived cyclic nonapeptide, Ac-Tyr-Arg-[Cys-Glu-His-D-Phe-Arg-Trp-Cys]-NH(2) (3), led to identification of a series of novel MC-4R selective disulfide-constrained hexapeptide analogs including Ac-[hCys-His-D-Phe-Arg-Trp-Cys]-NH(2) (12). The structural modifications associated with profound influence on MC-4R potency and selectivity were ring size, ring conformation, and the aromatic substitution of the D-Phe7. These cyclic peptide analogs provide novel and enhanced reagents for use in the elucidation of melanocortin-4 receptor-related physiology, and may additionally find application in the treatment of obesity and related metabolic disorders.     

Angiotensin II and III suppress food intake via angiotensin AT(2) receptor and prostaglandin EP(4) receptor in mice

Intracerebroventricularly administered angiotensin (Ang) II and III dose-dependently suppressed food intake in mice and their anorexigenic activities were inhibited by AT(2) receptor-selective antagonist. Ang II did not suppress food intake in AT(2) receptor-knockout mice, while it did significantly in wild-type and AT(1) receptor-knockout mice. The suppression of food intake in AT(1) receptor-knockout mice was smaller than that in wild-type. The anorexigenic activities of Ang II and III were also blocked by a selective antagonist for prostaglandin EP(4) receptor. Taken together, centrally administered Ang II and III may decrease food intake through AT(2) receptor with partial involvement of AT(1) receptor, followed by EP(4) receptor activation, which is a novel pathway regulating food intake.     

AT2 receptor mediates the cardioprotective effects of AT1 receptor antagonist in post-myocardial infarction remodeling

There are two subtypes of angiotensin (Ang) II receptors, AT1R and AT2R. It is established that clinical use of specific AT1R blocker (ARB) improves the long-term prognosis of heart failure. However, scientific basis for such effects of ARB is incompletely understood. The present study was designed to determine whether ARB inhibits the left ventricular (LV) remodeling that occurs early after myocardial infarction (MI) and whether the benefit of ARB is mediated by blockade of AT1R itself or by stimulation of AT2R resulting from AT1R blockade. MI was induced in AT2R-knockout mice and wild-type mice. Administration of valsartan, an ARB, or vehicle was started soon after the surgery and continued for two weeks. Infarction caused significant increase in end diastolic and end systolic LV dimensions, LV/body weight ratio, and myocyte cross-sectional area (MCSA) in both strains to a similar extent. Lung/body weight ratio, an index of pulmonary congestion, was also significantly increased in both strains, but the magnitude of increase was significantly larger in knockout mice. Valsartan significantly reduced LV dimensions, LV/body weight ratio, MCSA, and lung/body weight ratio in wild-type mice. In knockout mice, however, valsartan failed to inhibit the increases in LV dimensions and LV/body weight ratio. After the treatment, lung/body weight ratio in the mutant strain was significantly larger than that in the wild-type mice. Valsartan attenuates acute phase post-infarction remodeling and ameliorates heart failure, and a large part of its cardioprotective effect was mediated by AT2R.     

Cellular recognition of tri-/di-palmitoylated peptides is independent from a domain encompassing the N-terminal seven leucine-rich repeat (LRR)/LRR-like motifs of TLR2

Toll-like receptors (TLRs) mediate microbial pattern recognition in vertebrates. A broad variety of agonists has been attributed to TLR2 and three TLRs, TLR4, TLR2, and TLR5, have been demonstrated to bind microbial products. Distinct agonists might interact with different subdomains of the TLR2 extracellular domain. The TLR2 extracellular domain sequence includes 10 canonical leucine-rich repeat (LRR) motifs and 8-10 additional and potentially functionally relevant LRR-like motifs. Thus, the transfection of TLR2 LRR/LRR-like motif deletion constructs in human embryonic kidney 293 cells and primary TLR2-deficient mouse fibroblasts was performed for analysis of the role of the regarding domains in specific pattern recognition. Preparations applied as agonists were highly purified soluble peptidoglycan, lipoteichoic acid, outer surface protein A from Borrelia burgdorferi, synthetic mycoplasmal macrophage-activating lipoprotein-2, tripalmitoyl-cysteinyl-seryl-(lysyl)3-lysine (P3CSK4), dipalmitoyl-CSK4 (P2-CSK4), and monopalmitoyl-CSK4 (PCSK4) as well as lipopolysaccharide and inactivated bacteria. We found that a block of the N-terminal seven LRR/LRR-like motifs was not involved in TLR2-mediated cell activation by P3CSK4 and P2CSK4 ligands mimicking triacylated and diacylated bacterial polypeptides, respectively. In contrast, the integrity of the TLR2 holoprotein was compulsory for effective cellular recognition of other TLR2 agonists applied, including PCSK4. The formation of a functionally relevant subdomain by a region including the N-terminal seven LRR/LRR-like motifs rather than by single LRRs is suggested by our results. They further imply that TLR2 contains multiple binding domains for ligands that may contribute to the characterization of its promiscuous molecular pattern recognition.     

Agonist-dependent AT(4) receptor internalization in bovine aortic endothelial cells

Recent studies have characterized a specific binding site for the C-terminal 3-8 fragment of angiotensin II (Ang IV). In the present study we looked at the internalization process of this receptor on bovine aortic endothelial cells (BAEC). Under normal culture conditions, BAEC efficiently internalized (125)I-Ang IV as assessed by acid-resistant binding. Internalization of (125)I-Ang IV was considerably decreased after pretreatment of cells with hyperosmolar sucrose or after pretreatment of BAEC with inhibitors of endosomal acidification such as monensin or NH(4)Cl. About 50% of internalized (125)I-Ang IV recycled back to the extracellular medium during a 2 h incubation at 37 degrees C. (125)I-Ang IV remained mostly intact during the whole process of internalization and recycling as assessed by thin layer chromatography. As expected, internalization of (125)I-Ang IV was completely abolished by divalinal-Ang IV, a known AT(4) receptor antagonist. Interestingly, (125)I-divalinal-Ang IV did not internalize into BAEC. These results suggest that AT(4) receptor undergoes an agonist-dependent internalization and recycling process commonly observed upon activation of functional receptors.