Quantifying binding of VEGF‐A isoforms at VEGFR2 in membranes

Abstract

Vascular endothelial growth factor A (VEGF‐A) is a key mediator of angiogenesis, primarily signalling via VEGF Receptor 2 (VEGFR2) [1]. There are numerous endogenous isoforms of VEGF‐A resulting from alternative mRNA splicing. We previously used bioluminescence resonance energy transfer (BRET) to quantify binding of VEGF‐A isoforms single‐site labelled with tetramethylrhodamine (TMR) at NanoLuciferase (NanoLuc)‐tagged VEGFR2 in living cells [2,3]. However, VEGFR2 undergoes ligand‐dependent and ‐independent endocytosis, therefore we aimed to compare the pharmacology of VEGF‐A isoforms at VEGFR2 in membrane preparations. Membranes were homogenised from HEK293T cells stably expressing NanoLuc‐VEGFR2 as in [4], stored in Phosphate Buffered Saline and plated on the day of experimentation as 5μg/well in 40μl vehicle (Hanks Buffered Saline Solution/0.1% Bovine Serum Albumin; pH 7.4). For kinetic experiments, membranes were incubated with the NanoLuc substrate furimazine (10μM) for 5min, then 1–20nM fluorescent VEGF‐A was added. BRET ratios were measured every 30sec for 90min at 37°C using a BMG Pherastar. To quantify binding affinities, membranes were incubated with increasing concentrations of VEGF165a‐TMR, VEGF165b‐TMR or VEGF121a‐TMR (0.1–20nM) in the presence and absence of 100nM unlabelled VEGF. Alternatively, membranes were co‐stimulated with increasing concentrations of unlabelled VEGF‐Ax and a fixed concentration of fluorescent VEGF‐A (0.5–5nM). Following 60min stimulation at 37°C, furimazine was added and BRET ratios were measured. The kinetic binding profiles of each fluorescent VEGF‐A isoform in membranes reached a plateau at NanoLuc‐VEGFR2 and binding was maintained for 90min. This contrasted with the characteristic decrease in BRET ratio observed following 20min in intact cells [2]. Each fluorescent VEGF‐A isoform had saturable binding at NanoLuc‐VEGFR2, with a similarly low level of non‐specific binding compared to cells [3]. Derived equilibrium dissociation constants in membranes showed VEGF165a‐TMR, VEGF165b‐TMR and VEGF121a‐TMR bound NanoLuc‐VEGFR2 with nanomolar affinity. Each fluorescent VEGF‐A isoform was displaced by unlabelled VEGF‐Ax, from which nanomolar affinities were derived for VEGF‐Ax. Using NanoBRET to monitor protein‐protein interactions therefore showed that while the kinetic binding profile was strikingly different in membrane preparations compared to intact cells, derived affinity constants were identical. This quantitative approach retains the ability for receptor oligomerisation in an intact membrane bilayer, while isolating molecular ligand/receptor interactions at 37°C without the complex spatio‐temporal dynamics governing full‐length VEGFR2 in living cells.Support or Funding InformationThis project is supported by the British Pharmacological Society's AJ Clark Scholarship.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.