Characterization of a Bicyclic Peptide Neuropilin-1 (NP-1) Antagonist (EG3287) Reveals Importance of Vascular Endothelial Growth Factor Exon 8 for NP-1 Binding and Role of NP-1 in KDR Signaling

Jia, Hao; Bagherzadeh, Azadeh; Hartzoulakis, Basil; Jarvis, Ashley; Löhr, Marianne; Shaikh, Shaheda; Aqil, Rehan; Cheng, Lili; Tickner, Michelle; Esposito, Diego; Harris, Richard; Driscoll, Paul C.; Selwood, David L.; Zachary, Ian

doi:10.1074/jbc.m512121200

Cited by 121 publications

(148 citation statements)

References 59 publications

(44 reference statements)

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“…2). It was further shown that a peptide corresponding to the exon 8 encoded region effectively blocked VEGF165 binding to Nrp expressing cells (36). Our data strongly support that it is the C-terminal tail encoded by exon 8 that is the site of direct interaction between VEGF165 and Nrp and provides a structural basis for this interaction (Fig.…”

Section: Discussionsupporting

confidence: 68%

“…The C terminus of Tuftsin interacts with the third ligand binding loop, with specific interactions with residues S346, T349, and Y353. The importance of a C-terminal arginine for interactions with Nrp at this site explains the observation that chemical alteration of the C terminus of inhibitory peptides to a carboxyamide results in complete loss of inhibition (36). Additionally, placental growth factor-2, which binds N1b1b2, also possesses a C-terminal arginine (24,37).…”

Section: Resultsmentioning

confidence: 96%

“…The coagulation factor domains of Nrp are responsible for binding basic regions of both VEGF and semaphorin (24,25). Additionally, peptide inhibitors of Nrp are currently in development for use as antiangiogenic agents, all of which are basic in character and function by competing for ligand binding (18,(34)(35)(36). Tuftsin, an immunostimulatory tetrapeptide (TKPR), is very similar to the VEGF165 C terminus (DKPRR) and competes with VEGF165 for Nrp binding (34).…”

Section: Resultsmentioning

confidence: 99%

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Structural basis for ligand and heparin binding to neuropilin B domains

Kooi

Jusino

Perman

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

211

259

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Neuropilin (Nrp) is a cell surface receptor with essential roles in angiogenesis and axon guidance. Interactions between Nrp and the positively charged C termini of its ligands, VEGF and semaphorin, are mediated by Nrp domains b1 and b2, which share homology to coagulation factor domains. We report here the crystal structure of the tandem b1 and b2 domains of Nrp-1 (N1b1b2) and show that they form a single structural unit. Cocrystallization of N1b1b2 with Tuftsin, a peptide mimic of the VEGF C terminus, reveals the site of interaction with the basic tail of VEGF on the b1 domain. We also show that heparin promotes N1b1b2 dimerization and map the heparin binding site on N1b1b2. These results provide a detailed picture of interactions at the core of the Nrp signaling complex and establish a molecular basis for the synergistic effects of heparin on Nrp-mediated signaling.semaphorin ͉ Tuftsin ͉ VEGF N europilins (Nrps) are essential cell surface receptors with central roles in both angiogenesis and axon guidance (1-3). During angiogenesis, Nrp directly binds VEGF and functions as a coreceptor for VEGF along with VEGF receptor (VEGFR)-2, one of the three VEGFR tyrosine kinases (3, 4). During neural development, Nrp directly binds semaphorin and functions as a semaphorin coreceptor with members of the plexin family (5). Additionally, interactions with both neural adhesion protein L1 and Nrp-interacting protein (NIP), have been shown to be involved in a variety of other cellular processes (6-8).Nrp plays a stimulatory role in angiogenesis, a process critical for growth of solid tumors (reviewed in refs. 4 and 9-11). Nrp expression is observed in tumor vasculature, and overexpression promotes tumorigenesis in vivo for a variety of solid tumors including pituitary, prostate, breast, and colon cancers (12-15). In contrast, a soluble splice form containing only part of the extracellular domain of Nrp inhibits tumorigenesis (16) as do a number of peptides that block VEGF binding to Nrp (17,18). Recent evidence has also demonstrated a role for Nrp in hematological malignancies. Nrp overexpression is observed in both multiple myeloma and acute myeloid leukemia and, in the latter case, is associated with significantly reduced survival (19,20). Strategies to inhibit Nrp activity are thus being developed as potential antitumor therapies (reviewed in ref. 11).Higher eukaryotes possess two Nrp homologs, Nrp-1 and Nrp-2, which share 44% amino acid sequence identity (1). Nrp extracellular regions are composed of two complement binding CUB domains (a1 and a2) followed by two coagulation factor domains (b1 and b2), a MAM (meprin, A5, ) domain (c1), a single membrane-spanning region, and a short cytoplasmic tail (Fig. 1A) (21, 22). The a1 and a2 domains of Nrp are essential for binding to the core seven-bladed Sema domain of semaphorin as well as contributing to interactions with VEGF (23, 24). The coagulation factor domains b1 and b2 contain the high-affinity binding site for the basic heparin binding domain (HBD) of VEGF165 as well a...

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Section: Discussionsupporting

confidence: 68%

Section: Resultsmentioning

confidence: 96%

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

Structural basis for ligand and heparin binding to neuropilin B domains

Kooi

Jusino

Perman

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

211

259

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“…In fact, the R14A/R49A mutant was only minimally affected in the NRP1 binding assay, suggesting that the key residues required for NRP1 binding to VEGF164 are distinct from those at the core of heparin interactions. Based on the present results, we speculate that the NRP1 binding site is composed of multiple amino acids, including those associated with heparin binding, that function cooperatively in forming an extended binding surface and that may also include residues derived from exon 8 (55,56). A surprising discovery reported by Shintani et al (57) was that binding of VEGF to NRP1 is increased due to the presence of heparan sulfate or chondroitin sulfate chains covalently linked to the NRP1 core protein in endothelial and smooth muscle cells.…”

Section: Discussionmentioning

confidence: 99%

Molecular Mapping and Functional Characterization of the VEGF164 Heparin-binding Domain

Krilleke

DeErkenez²,

Schubert³

et al. 2007

Journal of Biological Chemistry

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The longer splice isoforms of vascular endothelial growth factor-A (VEGF-A), including mouse VEGF164, contain a highly basic heparin-binding domain (HBD), which imparts the ability of these isoforms to be deposited in the heparan sulfate-rich extracellular matrix and to interact with the prototype sulfated glycosaminoglycan, heparin. The shortest isoform, VEGF120, lacks this highly basic domain and is freely diffusible upon secretion. Although the HBD has been attributed significant relevance to VEGF-A biology, the molecular determinants of the heparin-binding site are unknown. We used site-directed mutagenesis to identify amino acid residues that are critical for heparin binding activity of the VEGF164 HBD. We focused on basic residues and found Arg-13, Arg-14, and Arg-49 to be critical for heparin binding and interaction with extracellular matrix in tissue samples. We also examined the cellular and biochemical consequences of abolishing heparin-binding function, measuring the ability of the mutants to interact with VEGF receptors, induce endothelial cell gene expression, and trigger microvessel outgrowth. Induction of tissue factor expression, vessel outgrowth, and binding to VEGFR2 were unaffected by the HBD mutations. In contrast, the HBD mutants showed slightly decreased binding to the NRP1 (neuropilin-1) receptor, and analyses suggested the heparin and NRP1 binding sites to be distinct but overlapping. Finally, mutations that affect the heparin binding activity also led to an unexpected reduction in the affinity of VEGF164 binding specifically to VEGFR1. This finding provides a potential basis for previous observations suggesting enhanced potency of VEGF164 versus VEGF120 in VEGFR1-mediated signaling in inflammatory cells.

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“…This peptide significantly inhibited VEGF-A165a binding to NRP1 while lacking most of the exon 7-encoded domain and comprising largely of the exon 8a-encoded sequence (Fig. 3 of 25 ). The peptide inhibited VEGF-A165a-induced VEGFR2, PLC-γ and ERK activation in HUVECs.…”

Section: Evidence That the Exon 8a Domain Which Vegf-a121a Has Is Rmentioning

confidence: 99%

VEGF-A121a binding to Neuropilins – A concept revisited

Sarabipour

Gabhann

2017

Cell Adhesion & Migration

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All known splice isoforms of vascular endothelial growth factor A (VEGF-A) can bind to the receptor tyrosine kinases VEGFR-1 and VEGFR-2. We focus here on VEGF-A121a and VEGF-A165a, two of the most abundant VEGF-A splice isoforms in human tissue1, and their ability to bind the Neuropilin co-receptors NRP1 and NRP2. The Neuropilins are key vascular, immune, and nervous system receptors on endothelial cells, neuronal axons, and regulatory T cells respectively. They serve as co-receptors for the Plexins in Semaphorin binding on neuronal and vascular endothelial cells, and for the VEGFRs in VEGF binding on vascular and lymphatic endothelial cells, and thus regulate the initiation and coordination of cell signaling by Semaphorins and VEGFs.2 There is conflicting evidence in the literature as to whether only heparin-binding VEGF-A isoforms – that is, isoforms with domains encoded by exons 6 and/or 7 plus 8a – bind to Neuropilins on endothelial cells. While it is clear that VEGF-A165a binds to both NRP1 and NRP2, published studies do not all agree on the ability of VEGF-A121a to bind NRPs. Here, we review and attempt to reconcile evidence for and against VEGF-A121a binding to Neuropilins. This evidence suggests that, in vitro, VEGF-A121a can bind to both NRP1 and NRP2 via domains encoded by exons 5 and 8a; in the case of NRP1, VEGF-A121a binds with lower affinity than VEGF-A165a. In in vitro cell culture experiments, both NRP1 and NRP2 can enhance VEGF-A121a-induced phosphorylation of VEGFR2 and downstream signaling including proliferation. However, unlike VEGFA-165a, experiments have shown that VEGF-A121a does not ‘bridge’ VEGFR2 and NRP1, i.e. it does not bind both receptors simultaneously at their extracellular domain. Thus, the mechanism by which Neuropilins potentiate VEGF-A121a-mediated VEGFR2 signaling may be different from that for VEGF-A165a. We suggest such an alternate mechanism: interactions between NRP1 and VEGFR2 transmembrane (TM) and intracellular (IC) domains.

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Characterization of a Bicyclic Peptide Neuropilin-1 (NP-1) Antagonist (EG3287) Reveals Importance of Vascular Endothelial Growth Factor Exon 8 for NP-1 Binding and Role of NP-1 in KDR Signaling

Abstract: Neuropilin-1 (NP-1) is a receptor for vascular endothelial growth factor-A 165 (VEGF-A

Cited by 121 publications

References 59 publications

Structural basis for ligand and heparin binding to neuropilin B domains

Structural basis for ligand and heparin binding to neuropilin B domains

Molecular Mapping and Functional Characterization of the VEGF164 Heparin-binding Domain

VEGF-A121a binding to Neuropilins – A concept revisited

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