Hydrocarbon stapled B chain analogues of relaxin-3 retain biological activity

Relaxin-3 or insulin-like peptide 7 (INSL7) is the most recently discovered relaxin/insulin-like family peptide. Mature relaxin-3 consists of an A chain and a B chain held by disulphide bonds. According to structure activity relationship studies, the relaxin-3 B chain is more important in binding and activating the receptor. RXFP3 (also known as Relaxin-3 receptor 1, GPCR 135, somatostatin- and angiotensin- like peptide receptor or SALPR) was identified as the cognate receptor for relaxin-3 by expression profiles and binding studies. Recent studies imply roles of this system in mediating stress and anxiety, feeding, metabolism and cognition. Stapling of peptides is a technique used to develop peptide drugs for otherwise undruggable targets. The main advantages of stapling include, increased activity due to reduced proteolysis, increased affinity to receptors and increased cell permeability. Stable agonists and antagonists of RXFP3 are crucial for understanding the physiological significance of this system. So far, agonists and antagonists of RXFP3 are peptides. In this study, for the first time, we have introduced stapling of the relaxin-3 B chain at 14th and 18th positions (14s18) and 18th and 22nd position (18s22). These stapled peptides showed greater helicity than the unstapled relaxin-3 B chain in circular dichroism analysis. Both stapled peptides bound RXFP3 and activated RXFP3 as observed in an inhibition of forskolin-induced cAMP assay and a ERK1/2 activation assay, although with different potencies. Therefore, we conclude that stapling of the relaxin3 B chain does not compromise its ability to activate RXFP3 and is a promising method for developing stable peptide agonists and antagonists of RXFP3 to aid relaxin-3/RXFP3 research.     

Insulin-like growth factor binding protein-3 (IGFBP-3): Novel ligands mediate unexpected functions

In addition to its important role in the regulation of somatic growth by acting as the major circulating transport protein for the insulin-like growth factors (IGFs), IGF binding protein-3 (IGFBP-3) has a variety of intracellular ligands that point to its function within major signaling pathways. The discovery of its interaction with the retinoid X receptor has led to the elucidation of roles in regulating the function of several nuclear hormone receptors including retinoic acid receptor-α, Nur77 and vitamin D receptor. Its interaction with the nuclear hormone receptor peroxisome proliferator-activated receptor-γ is believed to be involved in regulating adipocyte differentiation, which is also modulated by IGFBP-3 through an interaction with TGFβ/Smad signaling. IGFBP-3 can induce apoptosis alone or in conjunction with other agents, and in different systems can activate caspases −8 and −9. At least two unrelated proteins (LRP1 and TMEM219) have been designated as receptors for IGFBP-3, the latter with a demonstrated role in inducing caspase-8-dependent apoptosis. In contrast, IGFBP-3 also has demonstrated roles in survival-related functions, including the repair of DNA double-strand breaks through interaction with the epidermal growth factor receptor and DNA-dependent protein kinase, and the induction of autophagy through interaction with GRP78. The ability of IGFBP-3 to modulate the balance between pro-apoptotic and pro-survival sphingolipids by regulating sphingosine kinase 1 and sphingomyelinases may be integral to its role at the crossroads between cell death and survival in response to a variety of stimuli. The pleiotropic nature of IGFBP-3 activity supports the idea that IGFBP-3 itself, or pathways with which it interacts, should be investigated as targets of therapy for a variety of diseases.     

Peptide fragment of the m3 muscarinic acetylcholine receptor activates G(q) but not G(i2).

G(q), a heterotrimeric guanine nucleotide-binding protein, plays important roles such as the regulation of calcium mobilization and cell proliferation. This protein is considered as a promising drug target for the treatment of cardiac hypertrophy. Selective activation of G(q) would be quite useful for analyzing the role of G(q) in signaling pathways. We synthesized m3i3c-a peptide with 16 amino acid residues that corresponds to the junction between the C-terminus of the third intracellular loop and the sixth transmembrane helix (TM-VI) of human m3 muscarinic acetylcholine receptor, which couples to G(q) but not G(i2). At micromolar concentrations, this peptide was found to activate G(q) but not G(i2). This peptide is the first small compound that selectively activates G(q) but not G(i2). Copyright (c) 2008 European Peptide Society and John Wiley & Sons, Ltd.     

Relaxin-3: improved synthesis strategy and demonstration of its high-affinity interaction with the relaxin receptor LGR7 both in vitro and in vivo

Relaxin-3 is a member of the human relaxin peptide family, the gene for which, RLN3, is predominantly expressed in the brain. Mapping studies in the rodent indicate a highly developed network of RLN3, RLN1, and relaxin receptor-expressing cells in the brain, suggesting that relaxin peptides have important functional roles in the central nervous system. A regioselective disulfide-bond synthesis protocol was developed and used for the chemical synthesis of human (H3) relaxin-3. The selectively S-protected A and B chains were combined by stepwise formation of each of the three insulin-like disulfides via aeration, thioloysis, and iodolysis. Judicious positioning of the three sets of S-protecting groups was crucial for acquisition of synthetic H3 relaxin in a good overall yield. The activity of the peptide was tested against relaxin family peptide receptors. Although the highest activity was demonstrated on the human relaxin-3 receptor (GPCR135), the peptide also showed high activity on relaxin receptors (LGR7) from various species and variable activity on the INSL3 receptor (LGR8). Recombinant mouse prorelaxin-3 demonstrated similar activity to H3 relaxin, suggesting that the presence of the C peptide did not influence the conformation of the active site. H3 relaxin was also able to activate native LGR7 receptors. It stimulated increased MMP-2 expression in LGR7-expressing rat ventricular fibroblasts in a dose-dependent manner and, following infusion into the lateral ventricle of the brain, stimulated water drinking in rats, activating LGR7 receptors located in the subfornical organ. Thus, H3 relaxin is able to interact with the relaxin receptor LGR7 both in vitro and in vivo.     

Specific interaction between the hop1 intracellular loop 3 domain of the human PAC(1) receptor and ARF

The PAC(1), VPAC(1) and VPAC(2) receptors are members of the secretin (Group II) family of G protein-coupled receptors. All members of this family activate adenylate cyclase and several have also been shown to activate phospholipase C. We have recently reported that the rat VPAC(1), VPAC(2) and PAC(1) receptors activate phospholipase D and that distinct pathways are utilised by two intracellular loop 3 splice variants of PAC(1), one of which is ARF-dependent. Phospholipase D activation by the hop1, but not the null (short), form of the PAC(1) receptor is sensitive to brefeldin A, an inhibitor of GTP exchange at ARF. We have expressed the null and hop1 intracellular loop 3 domains of the human PAC(1) receptor in bacteria as GST-fusion proteins and used them as peptide affinity matrices to determine whether a functional interaction exists between these domains and ARF. Using this GST pull-down assay, we have shown binding of the small G protein ARF6 to the hop1 but not the null domain of this receptor.     

Characterization of indolinones which preferentially inhibit VEGF-C- and VEGF-D-induced activation of VEGFR-3 rather than VEGFR-2.

VEGF-C and VEGF-D are lymphangiogenic factors that bind to and activate VEGFR-3, a fms-like tyrosine kinase receptor whose expression is limited almost exclusively to lymphatic endothelium in the adult. Processed forms of VEGF-C and VEGF-D can also activate VEGFR-2, a key player in the regulation of angiogenesis. There is increasing evidence to show that these receptor-ligand interactions play a pivotal role in a number of pathological situations. Inhibition of receptor activation by VEGF-C and VEGF-D could therefore be pharmaceutically useful. Furthermore, to understand the different roles of VEGF-C, VEGF-D, VEGFR-2 and VEGFR-3 in pathological situations it will be necessary to dissect the complex interactions of these ligands and their receptors. To facilitate such studies we cloned, sequenced and characterized the expression of rat VEGF-C and VEGF-D. We showed that Cys152-->Ser mutants of processed rat VEGF-C can activate VEGFR-3 but not VEGFR-2, while the corresponding mutation in rat VEGF-D inhibits its ability to activate both VEGFR-2 and VEGFR-3. We also synthesized and characterized indolinones that differentially block VEGF-C- and VEGF-D-induced VEGFR-3 kinase activity compared to that of VEGFR-2. These tools should be useful in analysing the different activities and roles of VEGF-C, VEGF-D and their ligands, and in blocking VEGFR-3-mediated lymphangiogenesis.     

Pharmacological characterization of a selective agonist for bombesin receptor subtype-3

Bombesin receptor subtype-3 (BRS-3) is an orphan G protein-coupled receptor in the bombesin receptor family that still awaits identification of its natural ligand. BRS-3 deficient mice develop a mild late-onset obesity with metabolic defects, implicating BRS-3 plays a role in feeding and metabolism. We describe here the pharmacological characterization of a synthetic compound, 16a, which serves as a potent agonist for BRS-3. This compound is selective for BRS-3 as it does not activate neuromedin B or gastrin-releasing peptide receptors, two most closely related bombesin receptors, as well as a series of other GPCRs. We assessed the receptor trafficking of BRS-3 and found that compound 16a promoted β-arrestin translocation to the cell membrane. Neither central nor peripheral administration of compound 16a affects locomotor activity in mice. Therefore compound 16a is a potential tool to study the function of the BRS-3 system in vitro and possibly in vivo.     

Melanocortin-1 receptor-mediated signalling pathways activated by NDP-MSH and HBD3 ligands

Binding of melanocortin peptide agonists to the melanocortin-1 receptor of melanocytes results in eumelanin production, whereas binding of the agouti signalling protein inverse agonist results in pheomelanin synthesis. Recently, a novel melanocortin-1 receptor ligand was reported. A β-defensin gene mutation was found to be responsible for black coat colour in domestic dogs. Notably, the human equivalent, β-defensin 3, was found to bind with high affinity to the melanocortin-1 receptor; however, the action of β-defensin as an agonist or antagonist was unknown. Here, we use in vitro assays to show that β-defensin 3 is able to act as a weak partial agonist for cAMP signalling in human embryonic kidney (HEK) cells expressing human melanocortin-1 receptor. β-defensin 3 is also able to activate MAPK signalling in HEK cells stably expressing either wild type or variant melanocortin-1 receptors. We suggest that β-defensin 3 may be a novel melanocortin-1 receptor agonist involved in regulating melanocyte responses in humans.     

NMR structure of an intracellular loop peptide derived from prostaglandin EP3alpha receptor

We found that a peptide (EP3a: TIKALVSRCRAKAAV) corresponding to the N-terminal site of the intracellular third loop of human prostaglandin EP3α receptor could activate G protein α-subunit directly. The activity was almost same as Mastoparan-X, a G protein activating peptide from wasp venom. The three-dimensional molecular structure of the peptide in SDS-d₂₅ micelles was determined by 2D ¹H NMR spectroscopy. The structure of EP3a consists of a positive charge cluster on the C-terminal helical site. The cluster was also found in several corresponding receptor peptides. Therefore, the positive charge cluster on the helical structure might play a crucial role in activation of G protein.     

Activation of the PDGF β Receptor by a Persistent Artificial Signal Peptide

Most eukaryotic transmembrane and secreted proteins contain N-terminal signal peptides that mediate insertion of the nascent translation products into the membrane of the endoplasmic reticulum. After membrane insertion, signal peptides typically are cleaved from the mature protein and degraded. Here, we tested whether a small hydrophobic protein selected for growth promoting activity in mammalian cells retained transforming activity while also acting as a signal peptide. We replaced the signal peptide of the PDGF β receptor (PDGFβR) with a previously described 29-residue artificial transmembrane protein named 9C3 that can activate the PDGFβR in trans. We showed that a modified version of 9C3 at the N-terminus of the PDGFβR can function as a signal peptide, as assessed by its ability to support high level expression, glycosylation, and cell surface localization of the PDGFβR. The 9C3 signal peptide retains its ability to interact with the transmembrane domain of the PDGFβR and cause receptor activation and cell proliferation. Cleavage of the 9C3 signal peptide from the mature receptor is not required for these activities. However, signal peptide cleavage does occur in some molecules, and the cleaved signal peptide can persist in cells and activate a co-expressed PDGFβR in trans. Our finding that a hydrophobic sequence can display signal peptide and transforming activity suggest that some naturally occurring signal peptides may also display additional biological activities by interacting with the transmembrane domains of target proteins.     

Proteinase-activated receptors (PARs)--the PAR3 Neo-N-terminal peptide TFRGAP interacts with PAR1

Thrombin activates proteinase-activated receptor (PAR)1, PAR3 and PAR4 by a unique mechanism that involves cleavage of the receptor and exposure of a new N-terminal domain acting as a tethered ligand. Synthetic peptides based on the proteolytically revealed receptor sequence can selectively activate PAR1 or PAR4 independently of receptor cleavage. However, corresponding peptides for PAR3 have not been identified thus far. Here, we demonstrate that the synthetic peptide TFRGAP representing the 1st six residues of the new amino terminus of PAR3 induced ERK activation in human A-498 carcinoma cells endogeneously expressing PAR1 and PAR3. This effect was completely abolished by single alanine substitution at positions 3, 4 and 6 in the peptide. Since the specific PAR1 antagonist RWJ 56110 completely abolished TFRGAP-induced ERK activation in A-498 cells we speculate that TFRGAP does signal MAPK via interaction with PAR1. This was underlined by experiments on PAR1-/- mouse lung fibroblasts (KOLF cells) that stably overexpress human PAR1 and PAR3, respectively. While TFRGAP was without effect on ERK activation in PAR3+ KOLF cells, it induced MAPK activation in KOLF cells transfected with PAR1. These studies provide evidence that analogues of the PAR3 tethered ligand can mediate cell signaling by interaction with PAR1-type thrombin receptors.     

4-Bromohomoibotenic Acid Selectively Activates a 1-Aminocyclopentane-1S,3R-Dicarboxylic Acid-Insensitive Metabotropic Glutamate Receptor Coupled to Phosphoinositide Hydrolysis in Rat Cortical Slices

Abstract: Glutamate activates a family of receptors, known as metabotropic glutamate receptors (mGluRs), that are coupled to various second messenger systems through G proteins. All mGluR subtypes characterized to date in rat brain slices are activated by the glutamate analogue 1-aminocyclopentane-1 S ,3 R -dicarboxylic acid (1 S ,3 R -ACPD). However, few agonists are available that selectively activate specific mGluR subtypes. We report that the glutamate analogue ( R,S )-4-bromohomoibotenate (BrHI) stimulates phosphoinositide hydrolysis in rat cerebral cortical slices in a concentration-dependent manner (EC₅₀ = 190 µ M ). The response to BrHI is stereoselective and is not blocked by ionotropic glutamate receptor antagonists. It is interesting that the responses to BrHI and 1 S ,3 R -ACPD are completely additive, suggesting that these responses are mediated by different receptor subtypes. Consistent with this, the response to BrHI is insensitive to l -2-amino-3-phosphonopropionic acid ( l -AP3), whereas the response to 1 S ,3 R -ACPD is partially blocked by l -AP3. BrHI does not activate metabotropic receptors coupled to changes in cyclic AMP accumulation or activation of phospholipase D. Thus, BrHI seems to activate specifically a phosphoinositide hydrolysis-linked mGluR that is insensitive to 1 S ,3 R -ACPD. This compound may prove useful as a tool for elucidating the roles of different mGluR subtypes in mammalian brain.     

Dual assay for MCLV3 activity reveals structure-activity relationship of CLE peptides

The dodecapeptide MCLV3 is a functional peptide, derived from the CLV3 precursor protein, which is a candidate ligand of the CLV1/CLV2 receptor complex that restricts the stem cell population in the shoot apical meristem (SAM). MCLV3 can induce shoot and root meristem consumption, the typical phenotype of transgenic plants overexpressing CLV3. We investigated the bioactivities of a series of alanine-substituted MCLV3 and related peptides on the root growth of Arabidopsis. The structure-activity relationship (SAR) of MCLV3 had high similarity with that of tracheary element differentiation inhibitory factor (TDIF). We also evaluated the binding activities of the peptides by a competitive receptor binding assay using tritiated MCLV3 and the membrane fraction of a tobacco BY-2 cell line overexpressing the MCLV3 ectodomain. This dual assay, combining a biological and receptor binding assay for evaluating the activities of MCLV3-related peptides, uncovered the SAR of MCLV3, and indicated that the terminal residues play critical roles in exerting its activity and are important for specific binding to the receptor, CLV1.     

Independent activation of endogenous p21-activated protein kinase-3 (PAK3) and JNK by thrombin in CCL39 fibroblasts

Thrombin, a potent mitogen for CCL39 hamster lung fibroblasts, activates the seven membrane-spanning receptor PAR1. To better understand the signaling pathways controlled by this receptor we analyzed a potential downstream effector, p21-activated protein kinase (PAK). Thrombin and PAR1 agonist peptide, as well as serum and lysophosphatidic acid, were found to stimulate HA-mPAK3 activity in CCL39 cells transfected with a plasmid encoding the epitope-tagged kinase. Similar results were obtained using antibodies developed against the endogenous kinase. PAK3 activation is sensitive to pertussis toxin, but insensitive to LY 294002, an inhibitor of phosphatidylinositol 3'-kinase. Thrombin and serum also activate c-jun amino terminal kinase (JNK). Similar to PAK3 activation, thrombin-stimulated JNK activity is inhibited by pertussis toxin, but not by LY 294002. In a CCL39-derived cell line expressing constitutively active mPAK3 in a tetracyline-dependent manner, induction of PAK activity does not lead to corresponding increases in JNK activity. Our findings indicate that PAK3 is responsive to thrombin and other G protein-coupled receptor systems. Furthermore, our data suggest that in CCL39 cells, JNK activation by thrombin occurs independently of PAK3.     

A monocyte-specific peptide from herpes simplex virus type 2 glycoprotein G activates the NADPH-oxidase but not chemotaxis through a G-protein-coupled receptor distinct from the members of the formyl peptide receptor family

We have recently identified a peptide derived from the secreted portion of the HSV-2 glycoprotein G, gG-2p20, to be proinflammatory. Based on its ability to activate neutrophils and monocytes via the formyl peptide receptor (FPR) to produce reactive oxygen species (ROS) that down-regulate NK cell function, we suggested it to be of importance in HSV-2 pathogenesis. We now describe the effects of an overlapping peptide, gG-2p19, derived from the same HSV-2 protein. Also, this peptide activated the ROS-generating NADPH-oxidase, however, only in monocytes and not in neutrophils. Surprisingly, gG-2p19 did not induce a chemotactic response in the affected monocytes despite using a pertussis toxin-sensitive, supposedly G-protein-coupled receptor. The specificity for monocytes suggested that FPR and its homologue FPR like-1 (FPRL1) did not function as receptors for gG-2p19, and this was also experimentally confirmed. Surprisingly, the monocyte-specific FPR homologue FPRL2 was not involved either, and the responsible receptor thus remains unknown so far. However, the receptor shares some basic signaling properties with FPRL1 in that the gG-2p19-induced response was inhibited by PBP10, a peptide that has earlier been shown to selectively inhibit FPRL1-triggered responses. We conclude that secretion and subsequent degradation of the HSV-2 glycoprotein G can generate several peptides that activate phagocytes through different receptors, and with different cellular specificities, to generate ROS with immunomodulatory properties.     

Tyrosine sulfation influences the chemokine binding selectivity of peptides derived from chemokine receptor CCR3

The interactions of chemokines with their G protein-coupled receptors play critical roles in the control of leukocyte trafficking in normal homeostasis and in inflammatory responses. Tyrosine sulfation is a common post-translational modification in the amino-terminal regions of chemokine receptors. However, tyrosine sulfation of chemokine receptors is commonly incomplete or heterogeneous. To investigate the possibility that differential sulfation of two adjacent tyrosine residues could bias the responses of chemokine receptor CCR3 to different chemokines, we have studied the binding of three chemokines (eotaxin-1/CCL11, eotaxin-2/CCL24, and eotaxin-3/CCL26) to an N-terminal CCR3-derived peptide in each of its four possible sulfation states. Whereas the nonsulfated peptide binds to the three chemokines with approximately equal affinity, sulfation of Tyr-16 gives rise to 9-16-fold selectivity for eotaxin-1 over the other two chemokines. Subsequent sulfation of Tyr-17 contributes additively to the affinity for eotaxin-1 and eotaxin-2 but cooperatively to the affinity for eotaxin-3. The doubly sulfated peptide selectively binds to both eotaxin-1 and eotaxin-3 approximately 10-fold more tightly than to eotaxin-2. Nuclear magnetic resonance chemical shift mapping indicates that these variations in affinity probably result from only subtle differences in the chemokine surfaces interacting with these receptor peptides. These data support the proposal that variations in sulfation states or levels may regulate the responsiveness of chemokine receptors to their cognate chemokines.     

Affinity modulation of the alpha IIb beta 3 integrin (platelet GPIIb-IIIa) is an intrinsic property of the receptor.

To analyze the basis of affinity modulation of integrin function, we studied cloned stable Chinese hamster ovary cell lines expressing recombinant integrins of the beta 3 family (alpha IIb beta 3 and alpha v beta 3). Antigenic and peptide recognition specificities of the recombinant receptors resembled those of the native receptors found in platelets or endothelial cells. The alpha IIb beta 3-expressing cell line (A5) bound RGD peptides and immobilized fibrinogen (Fg) but not soluble fibrinogen or the activation-specific monoclonal anti-alpha IIb beta 3 (PAC1), indicating that it was in the affinity state found on resting platelets. Several platelet agonists failed to alter the affinity state of ("activate") recombinant alpha IIb beta 3. The binding of soluble Fg and PAC1, however, was stimulated in both platelets and A5 cells by addition of IgG papain-digestion products (Fab) fragments of certain beta 3-specific monoclonal antibodies. These antibodies stimulated PAC1 binding to platelets fixed under conditions rendering them unresponsive to other agonists. Addition of these antibodies to detergent-solubilized alpha IIb beta 3 also stimulated specific Fg binding. These data demonstrate that certain anti-beta 3 antibodies activate alpha IIb beta 3 by acting directly on the receptor, possibly by altering its conformation. Furthermore, they indicate that the activation state of alpha IIb beta 3 is a property of the receptor itself rather than of the surrounding cell membrane microenvironment.