Program

PO1-8-13

Using bioluminescence resonance energy transfer (BRET) and super-resolution microscopy to investigate complex formation between VEGFR2, its co-receptor Neuropilin-1 and G protein coupled receptors

[Speaker] Laura Kilpatrick:1
[Co-author] Diana C Alcobia:1, Jackie R Glenn:1, Kris Zimmerman:2, Rachel Friedman Ohana:2, Matthew B Robers:2, Keith V Wood:2, Jeanette Woolard:1, Stephen J Hill:1
1:Centre of Membrane Proteins and Receptors (COMPARE), University of Nottingham, UK, 2:Promega Corporation, Madison, Wisconsin, USA

Background:
The vascular endothelial growth factor 2 (VEGFR2) is a receptor tyrosine kinase (RTK) that, with its co-receptor neuropilin-1 (NRP-1), is a crucial mediator of angiogenesis(1). Interestingly, two G protein coupled receptors (GPCRs)- the β2-adrenoceptor (β2AR) and the adenosine A2A receptor (A2AR)- have been implicated in enhancing VEGF driven pathological angiogenesis (2,3). One way this may occur is by forming oligomeric complexes with VEGFR2. Here, bioluminescence resonance energy transfer (BRET) using the luciferase variant NanoLuc was used to investigate this possibility.

Methods:
BRET: HEK293 cells transiently transfected with a fixed concentration of NLuc VEGFR2 (25ng/well) or NLuc NRP-1(10ng/well) alongside increasing concentrations of SnapTag labelled GPCR (β2AR, A2AR or A3AR; 0.2-40ng/well) were incubated with HBSS/0.1% BSA containing 0.2μM SnapTag AF488 membrane impermeant substrate (30min at 37°C). The NLuc substrate furimazine was added (10μM) and luminescence and fluorescence emissions recorded using a Pherastar FS (BMG). BRET ratios were calculated and data fit to a one site fit. Structured Illumination super-resolution microscopy: HEK293 cells were seeded onto high-refractive index coverglasses and transiently transfected with HaloTag receptor (VEGFR2 or NRP-1) alongside SnapTag β2AR, A2AR or A3AR in a 1:1 cDNA ratio. Cells were labelled using HaloTag AF488 or SnapTag AF647 substrates (1μM; 30min at 37°C) and then stimulated with 10nM VEGF165a or 10μM Isoprenaline (60min at 37°C). Cells were fixed mounted and imaged using a Zeiss ELYRA.

Results:
BRET experiments between NLuc VEGFR2 or NRP-1 and SnapTag β2AR or A2AR resulted in a saturable BRET signal indicative of close proximity of these receptor pairings (<10nm). No specific interaction was observed between NLuc VEGFR2 or NRP-1 and A3AR. Super resolution imaging showed cell surface co-localisation of VEGFR2 or NRP-1 with β2AR or A2AR. Stimulation with VEGF165a or Isoprenaline resulted in VEGFR2/β2AR complexes being found within the same intracellular compartments.

Conclusions:
This work provides evidence for the formation of larger oligomeric complexes of VEGFR2 or NRP-1 with β2AR and A2AR. These receptors are all endogenously expressed on endothelial cells, therefore RTK/GPCR complexes may represent novel anti cancer therapeutic targets.

(1)Woolard et al(2009)Microcirculation 16 (2)Ozeki M. et al(2016)Pediatrics International 58 (3)Acurio J. et al(2017)Purinergic Signalling 13.
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