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VEGF signaling plays an important role in the development and maintenance of blood vessels and it has received much attention due to its role in tumor development and other pathologies. Recent developments in therapeutics have highlighted the complex and often contradictory features of cell signaling mediated by the VEGF ligand and receptor family. The aim of this study is to constrain the effective parameters of a minimal mathematical model [1] of the first steps in VEGF signaling, namely, the molecular transformations that occur from the presentation of VEGF ligand in the extracellular space to the formation of activated ligand-receptor complexes on the cell membrane. This study addresses unique properties of VEGF due to its bivalent nature. Bivalence complicates signal initiation, which requires the formation of a complex of two receptors cross-linked by a ligand. Unlike the initial capture of ligand from the surrounding volume, the second binding step occurs between proteins bound to the cell membrane, and is therefore modulated by the lateral mobility of the receptors, which in turn reflects the limitations to free movement due to various features of the cell membrane. The kinetics of cross-linking are reflected in (i) the degree of reversibility of VEGF binding to the cell, and (ii) the phenomenon of high dose inhibition. Thus, the empirically derived kinetics that defines the well mixed model [1] help integrate binding data with high resolution imaging and single particle tracking studies of the cell membrane, two classes of experimental information that are hard to compare directly. The binding of VEGF to human umbilical vascular endothelial cells (HUVEC) has been well studied in the literature, and was the basis of the original parameterization of the model [1]. However, those experiments did not pursue the distinction between the two different binding steps, and the kinetics of cross-linking was not well determined. To address this, we use a synthetic compound, single-chain VEGF [2], conjugated to a fluorescent tag (scVEGF-Cy5.5). This ligand has two functional binding sites and its binding affinity and physiological effect on cells expressing VEGFR-2 closely reproduce those of VEGF165. We used flow cytometry to quantify the binding of scVEGF-Cy5.5 to HUVEC in vitro. We obtained dose response curves and time courses for total binding in solution, for incubation times from 30 - 120 minutes and ligand concentrations from 0.1 - 200 nM. The data show possible signs of high dose inhibition in the 100 nM range, and allow us to estimate a range for the characteristic time of cross-linking. Subsequent experiments will directly probe this time range, based on use of this versatile probe for imaging studies of receptor localization and mobility in the cell membrane. References: [1] MacGabhann F and Popel AS, Biophys.Chem. 128:125-139 (2007) [2] Backer MV et al, Nat.Med. 13:504-509 (2007) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3970. doi:1538-7445.AM2012-3970