Quantifying the internalisation of the vascular endothelial growth factor receptor 2 (VEGFR2) in HEK293 cells using fluorescent VEGF165a-TMR

Introduction: The vascular endothelial growth factor receptor 2 (VEGFR2) is a crucial mediator of angiogenesis, cell proliferation and survival (1). VEGFR2 has been shown previously to internalise in human umbilical vascular endothelial cells (HUVECs) in response to its cognate ligand VEGF165a (2). Here a fluorescent form of VEGF165a, labelled at an N-terminal cysteine residue with the fluorophore tetramethylrhodamine (VEGF165a-TMR), has been used in conjunction with high content imaging to quantify the internalisation of VEGF165a-TMR/VEGFR2 complexes in HEK293 cells.

Methods: For all imaging experiments, HEK293 cells stably expressing human VEGFR2 with an N terminal NanoLuc® (Promega) tag were seeded at 25,000 cells/well in black 96 well plates (Greiner, Bio-one). To track the time course of VEGF165a-TMR internalisation, 3nM VEGF165a-TMR was added at set time points (1-120 min prior to cell fixation; at 37°C/5% CO2 in serum free Dulbecco’s Modified Eagle’s medium (DMEM/0.1% bovine serum albumin (BSA)). Cells were then fixed (3% para-formaldehyde (PFA)/phosphate buffered saline (PBS), permeabilised (0.025% Triton-X-100/PBS) and labelled with a rabbit antibody targeting pY1214 of VEGFR2, a key marker of VEGFR2 kinase activation (1:100 dilution; overnight at 4°C), prior to labelling with a secondary antibody (chicken anti rabbit AlexaFluor-488; 1:500 dilution; 1hr at room temperature). For concentration response experiments, increasing concentrations of VEGF165a-TMR were added to triplicate wells (60min; in serum free DMEM /0.1% BSA). For competition experiments, cells were simultaneously treated with 3nM VEGF165a-TMR and increasing concentrations of unlabelled VEGF165a (60min in serum free DMEM/0.1% BSA). Fixed cells (3% PFA/PBS) were then incubated with the nuclear stain H33342 (2mg/ml/PBS; 10mins) and imaged on an IX Micro widefield platereader (20x objective; 4 sites per well). An algorithm was used to quantify intracellular granules (number and intensity; 2-15μm in diameter) containing VEGF165a-TMR/VEGFR2 complexes in each cell. Competition experiments were also performed in live cells seeded at 40,000 cells/well in white 96 well plates (Greiner) and bioluminescence resonance energy transfer (BRET; BMG PheraStar) was used to monitor the proximity of NanoLuc-VEGFR2 and fluorescently labelled VEGF165a-TMR (3nM; 60min; 37°C), in the presence and absence of VEGF165a (Hanks buffered saline solution/0.1% BSA). Data are expressed as mean ± S.E.M.

Results: Stimulation of the internalisation of VEGF165a-TMR/VEGFR2 complexes by VEGF165a-TMR was concentration dependent (pEC50 = 8.68 ± 0.03; n=8). The time course of the resulting VEGFR2 phosphorylation (pY1214) showed a similar profile to that observed for VEGF165a-TMR/VEGFR2 complex internalisation, and indicated that VEGFR2 was largely intracellular after 30min. Comparable pIC50 values were derived for unlabelled VEGF165a for inhibition of the internalisation of VEGF165a-TMR/VEGFR2 complexes and for inhibition of the direct binding of VEGF165a-TMR to NanoLuc VEGFR2 as monitored by BRET (60min incubation; Table 1).

Table 1: Inhibition of 3nM VEGF 165a-TMR binding by increasing concentrations of unlabelled VEGF165a

Discussion: The use of fluorescent VEGF165a-TMR, in conjunction with high content imaging, allowed the binding characteristics and time course of the internalisation of agonist occupied VEGFR2 to be quantified for the first time in HEK293 cells.

(1) Woolard et al.(2009) Microcirculation 7: 572-92;

(2) Gourlaouen M et al.(2013) J Biol Chem 288: 7467-7480.