Use of Bioluminescence Resonance Energy Transfer (BRET) And Fluorescent Vascular Endothelial Growth Factor (VEGF) Isoforms To Characterize The Molecular Pharmacology Of Neuropilin‐1 Receptors In Living HEK293 Cells

Neuropilin‐1 (NRP‐1) receptors have been suggested to promote angiogenesis and tumourigenesis by binding vascular endothelial growth factor (VEGF) ligands and forming complexes with Vascular Endothelial Growth Factor receptor 2 (VEGFR2). Alternative splicing of the VEGF gene results in isoforms of VEGF, with distinct physiological roles and abilities to interact with NRP‐1 or VEGFR2 (1). However, determination of the binding affinities of these ligands at the NRP‐1 receptor expressed in the absence of endogenous VEGFR subtypes is currently lacking. Here we have used the novel luciferase NanoLuc® (NLuc; Promega) in conjunction with VEGF isoforms single site labeled with tetramethylrhodamine (TMR), to characterize the binding of different isoforms to NRP‐1 or a ligand binding mutant (NRP‐1 Y297A) stably expressed in living HEK293 cells. Methods Bioluminescence resonance energy transfer (BRET) was measured between N terminal NLuc NRP‐1 or NRP‐1 Y297A and VEGF 165 a‐TMR (1hr; 37°C in Hanks Buffered Saline Solution/0.1% bovine serum albumin (HBSS/0.1% BSA)). Competition binding experiments with unlabeled VEGF isoforms were conducted in the presence of fixed concentrations of VEGF 165 a‐TMR (0.1–5nM). Saturation binding experiments were performed (0.1–20nM VEGF 165 a‐TMR; 1hr; 37°C) with nonspecific binding determined using unlabeled VEGF 165 a (30nM). The NLuc substrate furimazine was added 5 min prior to plates being read on a Pherastar plate reader (BMG). Raw BRET ratios were calculated from acceptor to donor emission ratios. Ligand binding affinities were calculated assuming competition using K D values for VEGF 165 a‐TMR from one site saturation analysis. Imaging of fluorescently labelled VEGF isoforms: N terminal HaloTag (Promega) NRP‐1 or NRP‐1 Y297A stably expressed in HEK293 cells were labelled with the membrane impermeant substrate HaloTag AF488 (0.2μM; 30min at 37°C in HBSS). Cells were then treated with fluorescently labeled VEGF 165 a‐TMR, VEGF 121 a‐TMR or VEGF 165 b‐TMR (10nM; 60min at 37°C in HBSS/0.1% BSA) and imaged live at 63× magnification using a LSM Exciter confocal microscope (Zeiss). Results NanoBRET studies detected saturable binding of VEGF 165 a‐TMR to NLuc NRP‐1 (K D = 2.54 ± 0.92nM; n=4) that was largely prevented by co‐incubation with 30nM VEGF 165 a. In competition experiments at each fixed concentration of VEGF 165 a‐TMR, specific binding was inhibited by VEGF 165 a in a concentration dependent manner (pK i 9.86 ± 0.15; n=6). Live imaging showed that NRP‐1 did not internalize in response to VEGF 165 a‐TMR, but receptor/ligand complexes were co‐localized at the plasma membrane. Additionally NanoBRET competition experiments revealed no binding of VEGF 121 a or VEGF 165 b to the NRP‐1, which was confirmed with imaging. No binding of VEGF isoforms was observed at the NRP‐1 Y297A binding mutant using either method. This work has, determined for the first time the ligand binding affinities of VEGF isoforms for NRP‐1 in living cells in the absence of VEGFR2. Support or Funding Information Research supported by a BBSRC link grant in collaboration with Promega Corporation.