Use of NanoBiT and NanoBRET to monitor fluorescent VEGF-A binding kinetics to VEGFR2/NRP1 heteromeric complexes in living cells.

Chloe J Peach,Laura E Kilpatrick,Stephen J Hill,Jeanette Woolard

doi:10.1111/bph.15426

Chloe J Peach, Laura E Kilpatrick + Show 2 more

Open Access

https://doi.org/10.1111/bph.15426

Copy DOI

Abstract

VEGF-A is a key mediator of angiogenesis, primarily signalling via VEGF receptor 2 (VEGFR2). Endothelial cells also express the co-receptor neuropilin-1 (NRP1) that potentiates VEGF-A/VEGFR2 signalling. VEGFR2 and NRP1 had distinct real-time ligand binding kinetics when monitored using BRET. We previously characterised fluorescent VEGF-A isoforms tagged at a single site with tetramethylrhodamine (TMR). Here, we explored differences between VEGF-A isoforms in living cells that co-expressed both receptors. Receptor localisation was monitored in HEK293T cells expressing both VEGFR2 and NRP1 using membrane-impermeant HaloTag and SnapTag technologies. To isolate ligand binding pharmacology at a defined VEGFR2/NRP1 complex, we developed an assay using NanoBiT complementation technology whereby heteromerisation is required for luminescence emissions. Binding affinities and kinetics of VEGFR2-selective VEGF165 b-TMR and non-selective VEGF165 a-TMR were monitored using BRET from this defined complex. Cell surface VEGFR2 and NRP1 were co-localised and formed a constitutive heteromeric complex. Despite being selective for VEGFR2, VEGF165 b-TMR had a distinct kinetic ligand binding profile at the complex that largely remained elevated in cells over 90 min. VEGF165 a-TMR bound to the VEGFR2/NRP1 complex with kinetics comparable to those of VEGFR2 alone. Using a binding-dead mutant of NRP1 did not affect the binding kinetics or affinity of VEGF165 a-TMR. This NanoBiT approach enabled real-time ligand binding to be quantified in living cells at 37°C from a specified complex between a receptor TK and its co-receptor for the first time.

Highlights

Angiogenesis involves the growth of new blood vessels from existing vascular networks (Carmeliet, 2005)
NanoBiT technologies were used to quantify the real-time binding of two fluorescent VEGF-A isoforms at a defined receptor/co-receptor complex between VEGF receptor 2 (VEGFR2) and NRP1 in living cells at 37C
We first demonstrated that full-length VEGFR2 and NRP1 constitutively formed a heteromeric complex in living HEK293T cells

Summary

| INTRODUCTION

Angiogenesis involves the growth of new blood vessels from existing vascular networks (Carmeliet, 2005). These techniques were limited to quantifying protein–protein interactions at NanoLuc-tagged VEGFR2 or NRP1 expressed in isolation; endothelial cells and tumour cells endogenously express both VEGFR2 and NRP1 in the same cell (Fantin et al, 2013; Koch et al, 2014; Lee-Montiel et al, 2015; Prahst et al, 2008; Whitaker et al, 2001) As these receptors have distinct ligand binding dynamics and subcellular localisation, approaches are required that isolate the pharmacology of VEGF-A ligand binding to distinct complexes involving both VEGFR2 and NRP1. We have used this technology to investigate the kinetics of ligand binding of VEGF165a-TMR (Kilpatrick et al, 2017) and VEGF165b-TMR (Peach, Kilpatrick, et al, 2018) to oligomeric complexes containing both VEGFR2 and NRP1

| METHODS

| RESULTS

Findings

| DISCUSSION