Dataset Information

Real-Time Ligand Binding of Fluorescent VEGF-A Isoforms that Discriminate between VEGFR2 and NRP1 in Living Cells.

ABSTRACT: Fluorescent VEGF-A isoforms have been evaluated for their ability to discriminate between VEGFR2 and NRP1 in real-time ligand binding studies in live cells using BRET. To enable this, we synthesized single-site (N-terminal cysteine) labeled versions of VEGF165a, VEGF165b, and VEGF121a. These were used in combination with N-terminal NanoLuc-tagged VEGFR2 or NRP1 to evaluate the selectivity of VEGF isoforms for these two membrane proteins. All fluorescent VEGF-A isoforms displayed high affinity for VEGFR2. Only VEGF165a-TMR bound to NanoLuc-NRP1 with a similar high affinity (4.4 nM). Competition NRP1 binding experiments yielded a rank order of potency of VEGF165a > VEGF189a > VEGF145a. VEGF165b, VEGF-Ax, VEGF121a, and VEGF111a were unable to bind to NRP1. There were marked differences in the kinetic binding profiles of VEGF165a-TMR for NRP1 and VEGFR2. These data emphasize the importance of the kinetic aspects of ligand binding to VEGFR2 and its co-receptors in the dynamics of VEGF signaling.

SUBMITTER: Peach CJ

PROVIDER: S-EPMC6200776 | biostudies-literature | 2018 Oct

REPOSITORIES: biostudies-literature

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Real-Time Ligand Binding of Fluorescent VEGF-A Isoforms that Discriminate between VEGFR2 and NRP1 in Living Cells.

Peach Chloe J CJ Kilpatrick Laura E LE Friedman-Ohana Rachel R Zimmerman Kris K Robers Matthew B MB Wood Keith V KV Woolard Jeanette J Hill Stephen J SJ

Cell chemical biology 20180726 10

Fluorescent VEGF-A isoforms have been evaluated for their ability to discriminate between VEGFR2 and NRP1 in real-time ligand binding studies in live cells using BRET. To enable this, we synthesized single-site (N-terminal cysteine) labeled versions of VEGF165a, VEGF165b, and VEGF121a. These were used in combination with N-terminal NanoLuc-tagged VEGFR2 or NRP1 to evaluate the selectivity of VEGF isoforms for these two membrane proteins. All fluorescent VEGF-A is ...[more]

PMID: 30057299

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Project description:BackgroundDifferent isoforms of VEGF-A (mainly VEGF₁₂₁, VEGF₁₆₅ and VEGF189) have been shown to display particular angiogenic properties in the generation of a functional tumor vasculature. Recently, a novel class of VEGF-A isoforms, designated as VEGF(xxx)b, generated through alternative splicing, have been described. Previous studies have suggested that these isoforms may inhibit angiogenesis. In the present work we have produced recombinant VEGF₁₂₁/₁₆₅b proteins in the yeast Pichia pastoris and constructed vectors to overexpress these isoforms and assess their angiogenic potential.ResultsRecombinant VEGF₁₂₁/₁₆₅b proteins generated either in yeasts or mammalian cells activated VEGFR2 and its downstream effector ERK1/2, although to a lesser extent than VEGF₁₆₅. Furthermore, treatment of endothelial cells with VEGF₁₂₁/₁₆₅b increased cell proliferation compared to untreated cells, although such stimulation was lower than that induced by VEGF₁₆₅. Moreover, in vivo angiogenesis assays confirmed angiogenesis stimulation by VEGF₁₂₁/₁₆₅b isoforms. A549 and PC-3 cells overexpressing VEGF₁₂₁b or VEGF₁₆₅b (or carrying the PCDNA3.1 empty vector, as control) and xenotransplanted into nude mice showed increased tumor volume and angiogenesis compared to controls. To assess whether the VEGF(xxx)b isoforms are differentially expressed in tumors compared to healthy tissues, immunohistochemical analysis was conducted on a breast cancer tissue microarray. A significant increase (p < 0.05) in both VEGF(xxx)b and total VEGF-A protein expression in infiltrating ductal carcinomas compared to normal breasts was observed. A positive significant correlation (r = 0.404, p = 0.033) between VEGF(xxx)b and total VEGF-A was found.ConclusionsOur results demonstrate that VEGF₁₂₁/₁₆₅b are not anti-angiogenic, but weakly angiogenic isoforms of VEGF-A. In addition, VEGF(xxx)b isoforms are up-regulated in breast cancer in comparison with non malignant breast tissues. These results are to be taken into account when considering a possible use of VEGF₁₂₁/₁₆₅b-based therapies in patients.

Dataset Information

Real-Time Ligand Binding of Fluorescent VEGF-A Isoforms that Discriminate between VEGFR2 and NRP1 in Living Cells.

Publications

Real-Time Ligand Binding of Fluorescent VEGF-A Isoforms that Discriminate between VEGFR2 and NRP1 in Living Cells.

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