Abstract
Background and PurposeVascular endothelial growth factor A (VEGF‐A) is a key mediator of angiogenesis. A striking feature of the binding of a fluorescent analogue of VEGF165a 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 ApproachLigand‐binding kinetics were monitored in both intact HEK293T cells and membranes (expressing nanoluciferase‐tagged VEGFR2) using BRET between tagged receptor and fluorescent analogues of VEGF165a, VEGF165b, and VEGF121a. 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 ResultsQuantitative 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 ImplicationsThese 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.
Highlights
Angiogenesis, the growth of new blood vessels from existing vascular networks, is an important physiological process that can be dysregulated in numerous pathologies including cancer and age‐ related macular degeneration (Chung & Ferrara, 2011; Peach, Mignone, et al, 2018)
Due to the complex spatiotemporal dynamics of VEGF receptor 2 (VEGFR2), kinetic profiles of ligand binding to VEGFR2 in intact living cells are likely to contain components that represent the initial ligand‐binding interaction and components that reflect receptor endocytosis, whereby the endosomal environment can impact upon the stability of these ligand–receptor complexes
In order to isolate the ligand‐binding profiles of fluorescent VEGF‐A isoforms to VEGFR2 from the potential influences of agonist‐induced receptor endocytosis, the present study was undertaken with VEGF165a‐TMR, VEGF165b‐TMR, and VEGF121a‐TMR to: (a) investigate the concentration‐dependence and temporal profile of ligand‐induced VEGFR2 endocytosis, (b) the influence of VEGFR2 phosphorylation on endocytosis and ligand binding, and (c) the kinetics of the ligand–receptor interactions in isolated membrane preparations, where the potential for parallel receptor endocytosis is not present
Summary
Angiogenesis, the growth of new blood vessels from existing vascular networks, is an important physiological process that can be dysregulated in numerous pathologies including cancer and age‐ related macular degeneration (Chung & Ferrara, 2011; Peach, Mignone, et al, 2018). The residues present within each VEGF‐A isoform determine whether they can interact with other membrane proteins (e.g., neuropilin 1; Cébe Suarez et al, 2006; Parker, Xu, Li, & Vander Kooi, 2012; Guo & Vander Kooi, 2015; Peach, Kilpatrick, et al, 2018) and extracellular matrix components (Krilleke et al, 2007; Vempati, Popel, & Mac Gabhann, 2014). Due to the complex spatiotemporal dynamics of VEGFR2, kinetic profiles of ligand binding to VEGFR2 in intact living cells are likely to contain components that represent the initial ligand‐binding interaction and components that reflect receptor endocytosis, whereby the endosomal environment can impact upon the stability of these ligand–receptor complexes. In order to isolate the ligand‐binding profiles of fluorescent VEGF‐A isoforms to VEGFR2 from the potential influences of agonist‐induced receptor endocytosis, the present study was undertaken with VEGF165a‐TMR, VEGF165b‐TMR, and VEGF121a‐TMR to: (a) investigate the concentration‐dependence and temporal profile of ligand‐induced VEGFR2 endocytosis, (b) the influence of VEGFR2 phosphorylation on endocytosis and ligand binding, and (c) the kinetics of the ligand–receptor interactions in isolated membrane preparations, where the potential for parallel receptor endocytosis is not present
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