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

Chloe J Peach,Laura E Kilpatrick,Rachel Friedman-Ohana,Kris Zimmerman,Matthew B Robers,Keith V Wood,Jeanette Woolard,Stephen J Hill

doi:10.1016/j.chembiol.2018.06.012

Chloe J Peach, Laura E Kilpatrick + Show 6 more

Open Access

https://doi.org/10.1016/j.chembiol.2018.06.012

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Abstract

SummaryFluorescent 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.

Highlights

Angiogenesis, the growth of new blood vessels from pre-existing vasculature, is critical in both physiology and pathology for maintaining an adequate supply of oxygen and nutrients (Chung and Ferrara, 2011)
Vascular endothelial growth factor A (VEGF-A) is an essential mediator of both angiogenesis and vascular permeability that signals via its cognate receptor VEGF receptor 2 (VEGFR2) (Koch et al, 2011; Shibuya, 2011)
Fluorescence SDS-PAGE analysis of the purified VEGF165b-TMR and VEGF121a-TMR isoforms in the presence or absence of 100 mM DTT confirmed that, in non-reducing conditions, both VEGF isoforms were largely present as homodimers (Figures S1 and S2)

Summary

Introduction

Angiogenesis, the growth of new blood vessels from pre-existing vasculature, is critical in both physiology and pathology for maintaining an adequate supply of oxygen and nutrients (Chung and Ferrara, 2011). Vascular endothelial growth factor A (VEGF-A) is an essential mediator of both angiogenesis and vascular permeability that signals via its cognate receptor VEGF receptor 2 (VEGFR2) (Koch et al, 2011; Shibuya, 2011). VEGF binds to VEGFR2 at the extracellular immunoglobulin (Ig)-like domains 2 and 3 (D2/D3) of the receptor (Ruch et al, 2007). VEGF binding stimulates receptor dimerization and initiates conformational changes across the VEGFR2 dimer interface that result in auto- and transphosphorylation of intracellular tyrosine residues (Cunningham et al, 1997). VEGFR2 is overexpressed in many solid tumors and leads to activation of pro-angiogenic signaling, which promotes tumorigenesis. A number of anti-angiogenic therapeutics have been targeted at the VEGF/VEGFR2 axis (Ferrara and Adamis, 2016)

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