Comparison of the ligand-binding properties of fluorescent VEGF-A isoforms to VEGF receptor 2 in living cells and membrane preparations using NanoBRET

Abstract

Background and purpose: Vascular endothelial growth factor A (VEGF-A) is a key mediator of angiogenesis. A striking feature of the binding of a fluorescent analogue of VEGF₁₆₅ a to nanoluciferase-tagged VEGF receptor 2 (VEGFR2) in living cells is that the BRET signal is not sustained and declines over time. This may be secondary to receptor internalisation. Here, we have compared the binding of three fluorescent VEGF-A isoforms to VEGFR2 in cells and isolated membrane preparations.

Experimental approach: Ligand-binding kinetics were monitored in both intact HEK293T cells and membranes (expressing nanoluciferase-tagged VEGFR2) using BRET between tagged receptor and fluorescent analogues of VEGF₁₆₅ a, VEGF₁₆₅ b, and VEGF₁₂₁ a. VEGFR2 endocytosis in intact cells expressing VEGFR2 was monitored by following the appearance of fluorescent ligand-associated receptors in intracellular endosomes using automated quantitative imaging.

Key results: Quantitative analysis of the effect of fluorescent VEGF-A isoforms on VEGFR2 endocytosis in cells demonstrated that they produce a rapid and potent translocation of ligand-bound VEGFR2 into intracellular endosomes. NanoBRET can be used to monitor the kinetics of the binding of fluorescent VEGF-A isoforms to VEGFR2. In isolated membrane preparations, ligand-binding association curves were maintained for the duration of the 90-min experiment. Measurement of the k_off at pH 6.0 in membrane preparations indicated shorter ligand residence times than those obtained at pH 7.4.

Conclusions and implications: These studies suggest that rapid VEGF-A isoform-induced receptor endocytosis shortens agonist residence times on the receptor (1/k_off ) as VEGFR2 moves from the plasma membrane to the intracellular endosomes.