Interactions between NRP1 and VEGFR2 molecules in the plasma membrane

Abstract

Here we use a quantitative FRET approach, specifically developed to probe membrane protein interactions, to study the homo-association of neuropilin 1 (NRP1) in the plasma membrane, as well as its hetero-interactions with vascular endothelial growth factor receptor 2 (VEGFR2). Experiments are performed both in the absence and presence of the soluble ligand vascular endothelial growth factor A (VEGFA), which binds to both VEGFR2 and NRP1. We demonstrate the presence of homo-interactions between NRP1 molecules, as well as hetero-interactions between NRP1 and VEGFR2 molecules, in the plasma membrane. Our results underscore the complex nature of the interactions between self-associating receptors, co-receptors, and their ligands in the plasma membrane. They also highlight the need for new methodologies that capture this complexity, and the need for precise physiological measurements of local receptor surface densities in the membrane of cells. This article is part of a Special Issue entitled: Emergence of Complex Behavior in Biomembranes edited by Marjorie Longo.

Direct measurements of VEGF–VEGFR2 binding affinities reveal the coupling between ligand binding and receptor dimerization

“…The AF594-scVEGF conjugate is singly labeled in a site-specific manner at the Cys tag and exhibits 95-100% VEGF activity (28). We used a VEGFR2 ECTM construct, labeled with YFP, that has been described previously (22,23,33).…”

“…6 or 7). The discrepancy is well-documented in the literature and may be due to the interactions of VEGF with the 3D extracellular matrix as opposed to 2D cell culture or possibly due to the effects of accessory proteins like NRP1 that also bind VEGF on orthogonal binding sites (33,43,44). These additional VEGF interactions in vivo may prolong signaling, or they may simply serve to enhance the local effective concentration of VEGF or VEGFR2.…”

“…We seek to measure the two-dimensional VEGFR2 surface density in the plasma membranes of HEK293T cells as well as the concentration (surface density) of the bound scVEGF. To achieve this goal, we subject the cells to reversible osmotic swelling as described previously (22,32,33). This treatment is required for quantitative determination of the concentration of receptors and bound ligands on the surface of the cells as the complex topology of the resting plasma membrane prevents the conversion of effective 3D concentrations into 2D membrane protein surface densities (22).…”

Optical Imaging of the Nanoscale Structure and Dynamics of Biological Membranes

“…2,3 The fluorescent probe's lifetime is used to generate the image, which has the benefit of being independent of probe concentration. FRET is also used to elucidate membrane protein interactions and conformational kinetics, [4][5][6][7][8][9] and is exquisitely sensitive to a separation distance of less than 10 nanometers. FRAP, [10][11][12][13][14] SPT 15,16 and FCS 17,18 are well suited to measure membrane dynamics.…”

Tumor and endothelial cells collaborate via transcellular receptor complexes

“…VEGFA binding to both VEGFR2 and NRP1 on the same endothelial cell forms a cis NRP1‐VEGFA‐VEGFR2 complex; the stoichiometry is likely two VEGFR2s, two NRP1s, and one VEGFA. This cis complex and the resulting signaling pathways have been studied extensively (reviewed in ). Now, NRP1‐VEGFA‐VEGFR2 interactions in trans (i.e.…”