INTRODUCTION

Angiogenesis involves the growth of new blood vessels from existing vascular networks (Carmeliet, 2005). This important physiological process can also be dysregulated in numerous pathologies, such as in tumour development (Chung and Ferrara, 2011). Vascular endothelial growth factor A (VEGF-A) is a key mediator of angiogenesis that primarily signals via its cognate receptor tyrosine kinase (RTK), the VEGF Receptor 2 (VEGFR2) (Simons et al., 2016; Peach et al., 2018b). VEGF-A binds across immunoglobulin-like domains 2 and 3 of VEGFR2 (Ruch et al., 2007; Leppanen et al., 2010). Agonist binding results in conformational changes throughout the VEGFR2 dimer that lead to auto- and trans-phosphorylation of key intracellular tyrosine residues. This triggers numerous signalling cascades that ultimately initiate endothelial cell proliferation, migration and survival, as well as increased vascular permeability (Koch et al., 2011).
VEGFR2 is subject to complex trafficking via clathrin-dependent and clathrin-independent endocytosis (Ewan et al., 2006; Basagiannis and Christoforidis, 2016; Basagiannis et al., 2016). It internalises in both the presence and absence of VEGF-A (Ewan et al., 2006; Jopling et al., 2009; Jopling et al., 2011). VEGF-A can also bind to the VEGFR2 co-receptor Neuropilin-1 (NRP1), a type 1 transmembrane glycoprotein (Soker et al., 1998, 2002). VEGFR2 signalling is upregulated by NRP1 (Fantin et al., 2011; Djordjevic and Driscoll, 2013; Gelfand et al., 2014). Endothelial cells express both VEGFR2 and NRP1 (Soker et al., 1998; Witmer et al., 2002). NRP1 is also overexpressed in numerous tumour subtypes (Jubb et al., 2012; Goel and Mercurio, 2013; Lee et al., 2014) and immune cells in the tumour microenvironment (Roy et al., 2017). VEGF-A interacts with VEGFR2 via residues encoded at the N-terminus of VEGF-A (Leppanen et al., 2010; Brozzo et al., 2011), while the C-terminus can interact with NRP1 (Mamluk et al., 2002; Vander Kooi et al., 2007; Parker et al., 2012).
VEGF-A is an anti-parallel, disulphide-linked homodimer. Alternative splicing of VEGF-A mRNA leads to a number of distinct VEGF-A isoforms (Woolard et al., 2009; Peach et al., 2018b). VEGF-A isoforms have different signalling properties in physiological systems with distinct expression profiles in health and disease (Vempati et al., 2014). VEGF-A isoforms differ in length, such as pro-angiogenic VEGF165a or the shorter VEGF121a isoform. A major site of splicing occurs at exon 8, where proximal splicing results in VEGFxxxa isoforms that contain exon 8a-encoded residues (CDKPRR) and VEGFxxxb isoforms that instead contain exon 8b-encoded residues (SLTKDD). While VEGF165a stimulates angiogenesis as a full agonist, VEGF165b is a partial agonist with reported anti-angiogenic activity in vivo (Woolard et al., 2004; Cébe Suarez et al., 2006; Eswarappa et al., 2014). The b1 domain of NRP1 can interact with VEGF165a via an arginine residue encoded by exon 8a (Mamluk et al., 2002; Vander Kooi et al., 2007; Parker et al., 2012). In contrast, ‘anti-angiogenic’ VEGF165b isoforms are unable to interact with NRP1 (Cébe Suarez et al., 2006; Kawamura et al., 2008; Delcombel et al., 2013).
Fluorescence-based technologies have been used to advance our pharmacological understanding of G protein-coupled receptors (GPCRs), RTKs and other classes of membrane protein (Stoddart et al., 2017). For example, Bioluminescence Resonance Energy Transfer (BRET) is a proximity-based assay that can quantify real-time binding at 37°C in living cells (Stoddart et al., 2015). A receptor is tagged at the N-terminus with a 19 kDa NanoLuciferase (NanoLuc) such that NanoLuc emits luminescence upon oxidation of the furimazine substrate. This can excite a nearby fluorophore in close proximity (<10 nm), such as a compatible fluorescent ligand bound at the receptor’s orthosteric site. We previously developed fluorescent VEGF-A isoforms that were single-site labelled with tetramethylrhodamine (TMR), to monitor ligand binding at full-length VEGFR2 or NRP1 tagged with NanoLuc (Kilpatrick et al., 2017; Peach et al., 2018a, 2019). Despite having a similar nanomolar binding affinity, VEGF165a-TMR binding kinetics were significantly faster at NRP1 than VEGFR2 (Peach et al., 2018a). VEGFR2 and NRP1 were also subject to distinct subcellular trafficking in the absence or presence of ligand when expressed alone. These techniques were limited to quantifying protein-protein interactions at NanoLuc-tagged VEGFR2 or NRP1 expressed in isolation, however endothelial cells and tumour cells endogenously express both VEGFR2 and NRP1 in the same cell (Whitaker et al., 2001; Prahst et al., 2008; Fantin et al., 2013; Koch et al., 2014; Lee-Montiel et al., 2015). Since these receptors have distinct ligand binding dynamics and subcellular localisation, approaches are required that isolate the pharmacology of VEGF-A ligand binding to distinct complexes involving both VEGFR2 and NRP1.
NanoLuc Binary Technology (NanoBiT) uses a modified NanoLuc split into a large fragment (LgBiT; 156 amino acids) and a small 11 amino acid tag (HiBiT or SmBiT; Dixon et al., 2016). Complementation of fragments is required for luminescence emission. Numerous variants were developed of the small tag with different intrinsic affinities for complementation with the LgBiT fragment, including the ‘higher affinity’ HiBiT fragment (Kd ~0.7 nM) and the lower affinity SmBiT fragment (Kd ~190 µM). Used in combination with a fluorescent ligand, interactions between the ligand and a particular protein pairing can be monitored using NanoBiT and BRET. Here we have used this technology to investigate the kinetics of ligand binding of VEGF165a-TMR (Kilpatrick et al., 2017) and VEGF165b-TMR (Peach et al., 2018a) to oligomeric complexes containing both VEGFR2 and NRP1.