Real-Time Ligand Binding of Fluorescent VEGF-A Isoforms that Discriminate between VEGFR2 and NRP1 in Living Cells

• Published in: Cell chemical biology Vol. 25; no. 10; pp. 1208 - 1218.e5
• Main Authors: Peach, Chloe J, Kilpatrick, Laura E, Friedman-Ohana, Rachel, Zimmerman, Kris, Robers, Matthew B, Wood, Keith V, Woolard, Jeanette, Hill, Stephen J
• Format: Journal Article
• Language: English
• Published: United States Cell Press 18.10.2018
• Subjects: Energy Transfer; Fluorescent Dyes - metabolism; HEK293 Cells; Human Umbilical Vein Endothelial Cells; Humans; Ligands; Luminescent Measurements; Neuropilin-1 - metabolism; Protein Binding; Protein Isoforms - metabolism; Rhodamines - metabolism; Vascular Endothelial Growth Factor A - metabolism; Vascular Endothelial Growth Factor Receptor-2 - metabolism; VEGFR2; NanoBRET; ligand binding kinetics; VEGF isoforms; neuropilin-1; receptor mechanisms
• ISSN: 2451-9456; 2451-9448; 2451-9456
• DOI: 10.1016/j.chembiol.2018.06.012

Fluorescent VEGF-A isoforms have been evaluated for their ability to discriminate between VEGFR2 and NRP1 in real-time ligand binding studies in live cells using BRET. To enable this, we synthesized single-site (N-terminal cysteine) labeled versions of VEGF a, VEGF b, and VEGF a. These were used in combination with N-terminal NanoLuc-tagged VEGFR2 or NRP1 to evaluate the selectivity of VEGF isoforms for these two membrane proteins. All fluorescent VEGF-A isoforms displayed high affinity for VEGFR2. Only VEGF a-TMR bound to NanoLuc-NRP1 with a similar high affinity (4.4 nM). Competition NRP1 binding experiments yielded a rank order of potency of VEGF a > VEGF a > VEGF a. VEGF b, VEGF-Ax, VEGF a, and VEGF a were unable to bind to NRP1. There were marked differences in the kinetic binding profiles of VEGF a-TMR for NRP1 and VEGFR2. These data emphasize the importance of the kinetic aspects of ligand binding to VEGFR2 and its co-receptors in the dynamics of VEGF signaling.