VEGFR2 dimerization kinetics on the cell surface determined by microscopy and mathematical modeling

Inter-receptor interactions play a key role in cell signaling in response to external stimuli. For quantitative understanding and modeling of transmembrane signal transduction, it is necessary to quantify receptor interaction kinetics (i.e., association and dissociation rate constants) on the cell surface. Live-cell single-molecule imaging (SMI) has the powerful ability to capture transient receptor interaction events in their native cellular environment with high spatiotemporal resolution. However, it reveals these interactions for the labeled subset only, which is a small fraction of the full population of receptors. We have previously shown that mathematical modeling, combined with SMI data, offers a route to compensate for this lost information and infer the population-level receptor interaction kinetics from SMI data. Here, we applied this approach to vascular endothelial growth factor receptor 2 (VEGFR2), both wild-type full-length VEGFR2 (FLR2)-the dimerization of which is necessary for activation-and a truncated mutant consisting of the extracellular and transmembrane domains (ECTM), which has been shown to exhibit reduced homotypic interactions in the absence of ligand. We developed a stochastic mathematical model mimicking VEGFR2 diffusion and homotypic interactions and determined the unknown model parameters (most importantly, association and dissociation rate constants) through a combination of direct experimental measurements and stochastic model calibration. As expected, we found that ECTM was primarily monomeric, while FLR2 exhibited substantial reversible dimerization. Importantly, our inference revealed that the difference between FLR2 and ECTM was primarily in their dimer association rate constant, which was about an order of magnitude higher for FLR2 than for ECTM. To our knowledge, this is the first time that the dimerization kinetics of VEGFR2, i.e., its association and dissociation rate constants, have been measured in intact live cells.