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

© 2018 The Author(s) 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 VEGF165a, VEGF165b, and VEGF121a. 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 VEGF165a-TMR bound to NanoLuc-NRP1 with a similar high affinity (4.4 nM). Competition NRP1 binding experiments yielded a rank order of potency of VEGF165a > VEGF189a > VEGF145a. VEGF165b, VEGF-Ax, VEGF121a, and VEGF111a were unable to bind to NRP1. There were marked differences in the kinetic binding profiles of VEGF165a-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. Peach et al. have used fluorescent VEGF-A isoforms to demonstrate that they can discriminate between VEGFR2 and its co-receptor NRP1 in real-time ligand binding studies in live cells. This precision chemical biology approach showed that fluorescent VEGF165a binds more rapidly to NRP1 than VEGFR2

Real-time ligand binding of fluorescent VEGF-A isoforms that discriminate between VEGFR2 and NRP1 in living cells

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, single-site (N-terminal cysteine) labelled versions of VEGF165a, VEGF165b and VEGF121a were synthesised. 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 VEGF165a-TMR bound to NanoLuc- NRP1 with a similar high affinity (4.4nM). Competition NRP1 binding experiments yielded a rank order of potency of VEGF165a > VEGF189a > VEGF145a. VEGF165b, VEGF-Ax, VEGF121a and VEGF111a were unable to bind to NRP1. There were marked differences in the kinetic binding profiles of VEGF165a-TMR for NRP1 and VEGFR2. These data emphasise the importance of the kinetic aspects of ligand binding to VEGFR2 and its co-receptors in the dynamics of VEGF signalling

Real-time analysis of the binding of fluorescent VEGF₁₆₅a to VEGFR2 in living cells: Effect of receptor tyrosine kinase inhibitors and fate of internalized agonist-receptor complexes

Vascular endothelial growth factor (VEGF) is an important mediator of angiogenesis. Here we have used a novel stoichiometric protein-labeling method to generate a fluorescent variant of VEGF (VEGF₁₆₅a-TMR) labeled on a single cysteine within each protomer of the antiparallel VEGF homodimer. VEGF₁₆₅a-TMR has then been used in conjunction with full length VEGFR2, tagged with the bioluminescent protein NanoLuc, to undertake a real time quantitative evaluation of VEGFR2 binding characteristics in living cells using bioluminescence resonance energy transfer (BRET). This provided quantitative information on VEGF-VEGFR2 interactions. At longer incubation times, VEGFR2 is internalized by VEGF₁₆₅a-TMR into intracellular endosomes. This internalization can be prevented by the receptor tyrosine kinase inhibitors (RTKIs) cediranib, sorafenib, pazopanib or vandetanib. In the absence of RTKIs, the BRET signal is decreased over time as a consequence of the dissociation of agonist from the receptor in intracellular endosomes and recycling of VEGFR2 back to the plasma membrane

Dynamics of VEGF-A binding at VEGFR2 and NRP1

Vascular Endothelial Growth Factor A (VEGF-A) is a key mediator of angiogenesis, a process dysregulated in tumour development. Alternative splicing of the Vegfa gene leads to distinct endogenous VEGF-A isoforms with differential signalling and expression. These include pro-angiogenic VEGF165a, ‘anti-angiogenic’ VEGF165b and freely diffusible VEGF121a. VEGF-A primarily signals via its cognate receptor tyrosine kinase (RTK), VEGF Receptor 2 (VEGFR2). Signalling can be potentiated by its co-receptor Neuropilin-1 (NRP1), a transmembrane protein also found at high levels in malignant tumours. Despite numerous approved anti-cancer therapeutics targeting VEGF-A/VEGFR2 signalling, there is limited quantitative pharmacological understanding of VEGF-A isoforms at full-length VEGFR2 or NRP1 in living cells. This thesis explored the spatial and temporal dynamics of ligand binding to VEGFR2 and its co-receptor NRP1. First, VEGF165a, VEGF165b and VEGF121a were stoichiometrically labelled with tetramethylrhodamine (TMR) in collaboration with Promega Corporation. VEGFxxxx-TMR ligand binding was quantified in real-time at 37°C using bioluminescence resonance energy transfer (BRET) with VEGFR2 or NRP1 tagged with NanoLuciferase (NanoLuc). This technique was used to discriminate between VEGF-A binding to two distinct classes of membrane protein expressed in isolation in HEK293T cells. While all VEGF-A isoforms had similar nanomolar affinities at NanoLuc-VEGFR2, not all isoforms were able to interact with NanoLuc-NRP1. This also revealed marked differences in the kinetic binding profiles of VEGF165a-TMR for NRP1 and VEGFR2, despite similar binding affinities. Using live cell imaging, we identified differences between the localisation of HaloTag-VEGFR2 and HaloTag-NRP1. Whereas NRP1 remained at the plasma membrane, VEGFR2 was subject to constitutive and ligand-driven endocytosis in HEK293T cells. Second, we investigated the relationship between receptor localisation and ligand binding given the complex trafficking of VEGFR2 observed in Chapter 3. Each fluorescent VEGF-A isoform was internalised with VEGFR2 within 30 minutes. At NanoLuc-VEGFR2, there was a decline in BRET signal for each fluorescent VEGF-A isoform following a peak at 20 minutes in living cells. This was absent for ligand binding at NanoLuc-NRP1. We further exploited these techniques to gain insight into how inhibition of VEGFR2 phosphorylation influenced ligand binding and endocytosis using a tyrosine phosphorylation deficient receptor mutant. In the absence of phosphorylation, there was an elevation in the BRET signal upon stimulation with fluorescent VEGF-A. VEGFR2 phosphorylation at Y951, Y1054, Y1059, Y1175 or Y1214 was not required for endocytosis. Membrane preparations were then used to probe VEGF-A/VEGFR2 binding in the absence of this endocytic component. Here, ligand binding profiles were maintained for 90 minutes and reached equilibrium. This assay was exploited to directly probe how ligand/receptor interactions were influenced by the acidic pH in the endosomal microenvironment. Interestingly, VEGF-TMR had a shorter residence time at NanoLuc-VEGFR2 at a pH similar to that in endosomes. Third, we investigated the effect of co-expressing VEGFR2 and NRP1 in the same living cell. Colocalisation was monitored between HaloTag-VEGFR2 and SnapTag-NRP1 in live cells upon stimulation with VEGF165b or VEGF165a. Using receptor-receptor BRET, we confirmed that VEGFR2 and NRP1 were in close proximity in the absence of ligand. We isolated the real-time pharmacology of VEGFxxxx-TMR at a defined VEGFR2/NRP1 complex using split NanoLuc Binary Technology (NanoBiT). As NanoBiTs require complementation to emit luminescence, BRET can only occur from a heteromeric complex of LgBiT-VEGFR2 and HiBiT-NRP1. Despite having faster kinetics at NRP1 in isolation, VEGF165a-TMR bound to the VEGFR2/NRP1 complex with dynamics comparable to those of NanoLuc-VEGFR2. VEGF165b-TMR had a ligand binding profile that largely remained elevated in cells over 90 minutes, despite being selective for VEGFR2. This thesis applied quantitative technologies to monitor real-time ligand binding at receptors that contribute to physiological and patho-physiological angiogenesis. These findings have implications for how NRP1 modulates VEGFR2 as a potential target in drug discovery

Dynamics of VEGF-A binding at VEGFR2 and NRP1

Vascular Endothelial Growth Factor A (VEGF-A) is a key mediator of angiogenesis, a process dysregulated in tumour development. Alternative splicing of the Vegfa gene leads to distinct endogenous VEGF-A isoforms with differential signalling and expression. These include pro-angiogenic VEGF165a, ‘anti-angiogenic’ VEGF165b and freely diffusible VEGF121a. VEGF-A primarily signals via its cognate receptor tyrosine kinase (RTK), VEGF Receptor 2 (VEGFR2). Signalling can be potentiated by its co-receptor Neuropilin-1 (NRP1), a transmembrane protein also found at high levels in malignant tumours. Despite numerous approved anti-cancer therapeutics targeting VEGF-A/VEGFR2 signalling, there is limited quantitative pharmacological understanding of VEGF-A isoforms at full-length VEGFR2 or NRP1 in living cells. This thesis explored the spatial and temporal dynamics of ligand binding to VEGFR2 and its co-receptor NRP1. First, VEGF165a, VEGF165b and VEGF121a were stoichiometrically labelled with tetramethylrhodamine (TMR) in collaboration with Promega Corporation. VEGFxxxx-TMR ligand binding was quantified in real-time at 37°C using bioluminescence resonance energy transfer (BRET) with VEGFR2 or NRP1 tagged with NanoLuciferase (NanoLuc). This technique was used to discriminate between VEGF-A binding to two distinct classes of membrane protein expressed in isolation in HEK293T cells. While all VEGF-A isoforms had similar nanomolar affinities at NanoLuc-VEGFR2, not all isoforms were able to interact with NanoLuc-NRP1. This also revealed marked differences in the kinetic binding profiles of VEGF165a-TMR for NRP1 and VEGFR2, despite similar binding affinities. Using live cell imaging, we identified differences between the localisation of HaloTag-VEGFR2 and HaloTag-NRP1. Whereas NRP1 remained at the plasma membrane, VEGFR2 was subject to constitutive and ligand-driven endocytosis in HEK293T cells. Second, we investigated the relationship between receptor localisation and ligand binding given the complex trafficking of VEGFR2 observed in Chapter 3. Each fluorescent VEGF-A isoform was internalised with VEGFR2 within 30 minutes. At NanoLuc-VEGFR2, there was a decline in BRET signal for each fluorescent VEGF-A isoform following a peak at 20 minutes in living cells. This was absent for ligand binding at NanoLuc-NRP1. We further exploited these techniques to gain insight into how inhibition of VEGFR2 phosphorylation influenced ligand binding and endocytosis using a tyrosine phosphorylation deficient receptor mutant. In the absence of phosphorylation, there was an elevation in the BRET signal upon stimulation with fluorescent VEGF-A. VEGFR2 phosphorylation at Y951, Y1054, Y1059, Y1175 or Y1214 was not required for endocytosis. Membrane preparations were then used to probe VEGF-A/VEGFR2 binding in the absence of this endocytic component. Here, ligand binding profiles were maintained for 90 minutes and reached equilibrium. This assay was exploited to directly probe how ligand/receptor interactions were influenced by the acidic pH in the endosomal microenvironment. Interestingly, VEGF-TMR had a shorter residence time at NanoLuc-VEGFR2 at a pH similar to that in endosomes. Third, we investigated the effect of co-expressing VEGFR2 and NRP1 in the same living cell. Colocalisation was monitored between HaloTag-VEGFR2 and SnapTag-NRP1 in live cells upon stimulation with VEGF165b or VEGF165a. Using receptor-receptor BRET, we confirmed that VEGFR2 and NRP1 were in close proximity in the absence of ligand. We isolated the real-time pharmacology of VEGFxxxx-TMR at a defined VEGFR2/NRP1 complex using split NanoLuc Binary Technology (NanoBiT). As NanoBiTs require complementation to emit luminescence, BRET can only occur from a heteromeric complex of LgBiT-VEGFR2 and HiBiT-NRP1. Despite having faster kinetics at NRP1 in isolation, VEGF165a-TMR bound to the VEGFR2/NRP1 complex with dynamics comparable to those of NanoLuc-VEGFR2. VEGF165b-TMR had a ligand binding profile that largely remained elevated in cells over 90 minutes, despite being selective for VEGFR2. This thesis applied quantitative technologies to monitor real-time ligand binding at receptors that contribute to physiological and patho-physiological angiogenesis. These findings have implications for how NRP1 modulates VEGFR2 as a potential target in drug discovery

Use of NanoBiT and NanoBRET to monitor fluorescent VEGF-A binding kinetics to VEGFR2/NRP1 heteromeric complexes in living cells

Background and Purpose: VEGF‐A is a key mediator of angiogenesis, primarily signalling via VEGF receptor 2 (VEGFR2). Endothelial cells also express the co‐receptor neuropilin‐1 (NRP1) that potentiates VEGF‐A/VEGFR2 signalling. VEGFR2 and NRP1 had distinct real‐time ligand binding kinetics when monitored using BRET. We previously characterised fluorescent VEGF‐A isoforms tagged at a single site with tetramethylrhodamine (TMR). Here, we explored differences between VEGF‐A isoforms in living cells that co‐expressed both receptors.Experimental Approach: Receptor localisation was monitored in HEK293T cells expressing both VEGFR2 and NRP1 using membrane‐impermeant HaloTag and SnapTag technologies. To isolate ligand binding pharmacology at a defined VEGFR2/NRP1 complex, we developed an assay using NanoBiT complementation technology whereby heteromerisation is required for luminescence emissions. Binding affinities and kinetics of VEGFR2‐selective VEGF165b‐TMR and non‐selective VEGF165a‐TMR were monitored using BRET from this defined complex. Key Results: Cell surface VEGFR2 and NRP1 were co‐localised and formed a constitutive heteromeric complex. Despite being selective for VEGFR2, VEGF165b‐TMR had a distinct kinetic ligand binding profile at the complex that largely remained elevated in cells over 90 min. VEGF165a‐TMR bound to the VEGFR2/NRP1 complex with kinetics comparable to those of VEGFR2 alone. Using a binding‐dead mutant of NRP1 did not affect the binding kinetics or affinity of VEGF165a‐TMR. Conclusion and Implications: This NanoBiT approach enabled real‐time ligand binding to be quantified in living cells at 37°C from a specified complex between a receptor TK and its co‐receptor for the first time

Real-time ligand binding of fluorescent VEGF-A isoforms that discriminate between VEGFR2 and NRP1 in living cells

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, single-site (N-terminal cysteine) labelled versions of VEGF165a, VEGF165b and VEGF121a were synthesised. 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 VEGF165a-TMR bound to NanoLuc- NRP1 with a similar high affinity (4.4nM). Competition NRP1 binding experiments yielded a rank order of potency of VEGF165a > VEGF189a > VEGF145a. VEGF165b, VEGF-Ax, VEGF121a and VEGF111a were unable to bind to NRP1. There were marked differences in the kinetic binding profiles of VEGF165a-TMR for NRP1 and VEGFR2. These data emphasise the importance of the kinetic aspects of ligand binding to VEGFR2 and its co-receptors in the dynamics of VEGF signalling

Complex Formation between VEGFR2 and the β₂-Adrenoceptor

Vascular endothelial growth factor (VEGF) is an important mediator of endothelial cell proliferation and angiogenesis via its receptor VEGFR2. A common tumor associated with elevated VEGFR2 signaling is infantile hemangioma that is caused by a rapid proliferation of vascular endothelial cells. The current first-line treatment for infantile hemangioma is the β-adrenoceptor antagonist, propranolol, although its mechanism of action is not understood. Here we have used bioluminescence resonance energy transfer and VEGFR2 genetically tagged with NanoLuc luciferase to demonstrate that oligomeric complexes involving VEGFR2 and the β2-adrenoceptor can be generated in both cell membranes and intracellular endosomes. These complexes are induced by agonist treatment and retain their ability to couple to intracellular signaling proteins. Furthermore, coupling of β2-adrenoceptor to β-arrestin2 is prolonged by VEGFR2 activation. These data suggest that protein-protein interactions between VEGFR2, the β2-adrenoceptor, and β-arrestin2 may provide insight into their roles in health and disease

Mice expressing fluorescent PAR2 reveal that endocytosis mediates colonic inflammation and pain.

G protein-coupled receptors (GPCRs) regulate many pathophysiological processes and are major therapeutic targets. The impact of disease on the subcellular distribution and function of GPCRs is poorly understood. We investigated trafficking and signaling of protease-activated receptor 2 (PAR2) in colitis. To localize PAR2 and assess redistribution during disease, we generated knockin mice expressing PAR2 fused to monomeric ultrastable green fluorescent protein (muGFP). PAR2-muGFP signaled and trafficked normally. PAR2 messenger RNA was detected at similar levels in Par2-mugfp and wild-type mice. Immunostaining with a GFP antibody and RNAScope in situ hybridization using F2rl1 (PAR2) and Gfp probes revealed that PAR2-muGFP was expressed in epithelial cells of the small and large intestine and in subsets of enteric and dorsal root ganglia neurons. In healthy mice, PAR2-muGFP was prominently localized to the basolateral membrane of colonocytes. In mice with colitis, PAR2-muGFP was depleted from the plasma membrane of colonocytes and redistributed to early endosomes, consistent with generation of proinflammatory proteases that activate PAR2 PAR2 agonists stimulated endocytosis of PAR2 and recruitment of Gαq, Gαi, and β-arrestin to early endosomes of T84 colon carcinoma cells. PAR2 agonists increased paracellular permeability of colonic epithelial cells, induced colonic inflammation and hyperalgesia in mice, and stimulated proinflammatory cytokine release from segments of human colon. Knockdown of dynamin-2 (Dnm2), the major colonocyte isoform, and Dnm inhibition attenuated PAR2 endocytosis, signaling complex assembly and colonic inflammation and hyperalgesia. Thus, PAR2 endocytosis sustains protease-evoked inflammation and nociception and PAR2 in endosomes is a potential therapeutic target for colitis