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 VEGF 165 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 VEGF 165 a, VEGF 165 b, and VEGF 121 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.

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

“…The et al, 2019). In contrast, the profile for VEGF 165 a-TMR at the HiBiT complex had a small decrease at latter time points, albeit to a lesser extent than at NanoLuc-VEGFR2 in intact cells (Peach et al, 2019).…”

“…In contrast, the profile for VEGF 165 a‐TMR at the HiBiT complex had a small decrease at latter time points, albeit to a lesser extent than at NanoLuc‐VEGFR2 in intact cells (Peach et al, 2019). This reduction in BRET signal for NanoLuc‐VEGFR2 following 20 min has been linked to VEGF‐A/VEGFR2 endocytosis leading to a change in localisation and local pH, as this decline was absent in membrane preparations and not observed for binding to NanoLuc‐NRP1 (Peach et al, 2019). These data suggest that the presence of NRP1 in VEGFR2 heteromeric complexes may reduce the extent of VEGFR2 endocytosis normally seen when VEGFR2 is expressed alone.…”

“…This can excite a nearby fluorophore in close proximity (<10 nm), such as a compatible fluorescent ligand bound at the receptor's orthosteric site. We previously developed fluorescent VEGF‐A isoforms that were single site labelled with tetramethylrhodamine (TMR), to monitor ligand binding at full‐length VEGFR2 or NRP1 tagged with NanoLuc (Kilpatrick et al, 2017; Peach, Kilpatrick, et al, 2018; Peach et al, 2019). Despite having a similar nanomolar binding affinity, VEGF 165 a‐TMR binding kinetics were significantly faster at NRP1 than VEGFR2 (Peach, Kilpatrick, et al, 2018).…”

Differences in Activation of Intracellular Signalling in Primary Human Retinal Endothelial Cells Between Isoforms of VEGFA-165

“…We began with immunoblotting to examine the activation of MAPK and AKT in primary HRMECs. Although VEGFA 165 b does not bind the neuropilin 1 coreceptor, it does bind to VEGFR2 with similar or less affinity as VEGFA 165 a depending on the cell model and method used [ 31 , 32 ]. Regardless of relative binding affinity, VEGFA 165 b is reported to bind and activate VEGFR2 based on immunoblotting studies [ 31 , 47 , 48 ].…”

“…Similar to VEGFA 121 , VEGFA 165 b is reported to be less angiogenic than VEGFA 165 a [ 13 , 29 , 30 ]. VEGFA 165 b’s binding affinity for VEGFR2 itself is similar to that of VEGFA 165 a in human umbilical vein endothelial cells (HUVECs) and less than VEGFA 165 a in direct binding analysis in transformed human embryonic kidney cells 293 (HEK293) cell assays [ 31 , 32 ]. VEGFA 165 b also displays weaker activation of VEGFR2 and ERK1/2 (MAPK) than VEGFA 165 a in transfected porcine aortic endothelial (PAE) cells [ 31 ].…”