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Novel BRET approaches to unravel the molecular pharmacology of VEGFR2 receptors: Insights into ligand binding, allosterism and signalling bias

Reference	BB/L019418/1
Principal Investigator / Supervisor	Professor Stephen Hill
Co-Investigators / Co-Supervisors	Professor Jeanette Woolard
Institution	University of Nottingham
Department	School of Life Sciences
Funding type	Research
Value (£)	406,283
Status	Completed
Type	Research Grant
Start date	01/10/2014
End date	30/09/2017
Duration	36 months

Abstract

The novel bioluminescent protein NanoLuc will be fused to the N-terminus of VEGFR2 or its co-receptor neuropilin-1 (NRP1) to provide the basis for a unique and exquisitly sensitive live cell binding assay utilizing bioluminescence energy transfer (BRET) to monitor the direct binding of fluorescent VEGFR isoforms. Competition binding between fluorescent VEGF (VEGF-165 or VEGF-121; red fluorophores) and non-labelled VEGF isoforms will allow a detailed molecular pharmacology (in terms of binding affinity) to be deduced for the full VEGF isoform family. Fluorescent VEGF-121 will be used as a specific probe for VEGFR2 since it does not bind to NRP1. A non-VEGF binding mutant of NRP1 (Y297A) will be used to define non-specific binding to NRP1. Co-expression of wild-type NRP1 and NanoLuc-VEGFR2 will also be used to evaluate the impact of NRP1 on the ligand binding characteristics of fluorescent VEGF-121 and the consequent intracellular signalling (PI3K, ERK, Calcium, NFAT or SRE reporter genes). N- or C-terminal NanoLuc VEGFR2 constructs will also be used in combination with N- or C-terminal HaloTag (red fluorophore) fusions of VEGFR2, VEGFR1, VEGFR2 or NRP1 to evaluate the ability of specific VEGF isoforms to induce the formation of VEGFR2 homodimers, heterodimers or VEGFR2-NRP1 protein complexes. The BRET technology will also be used to monitor the impact of co-expression with wild-type VEGFR1, VEGFR3 and NRP1 on the ability of Halo-tagged signalling and adapter proteins to bind to C-terminal NLuc VEGFR2 in response to different VEGF isoforms. This will provide some insight into the propensity of different VEGFR2-protein complexes to induce signalling bias. However, NanoLuc bimolecular luminescence complementation will also be used in combination with fluorescent ligands or fluorescent signalling proteins to evaluate directing by BRET VEGF binding, efficacy and signalling bias from identified VEGFR2 protein complexes (homodimers, heterodimers, NRP1 complexes).

Summary

The way in which cells communicate with each other and mediate cellular responses is an integral part of all life and controls the inner workings of organs within the body allowing them to respond, adapt and survive. This cellular communication is largely chemically based and messenger molecules can be both small (e.g. adrenaline) and large (e.g. many growth factors including vascular endothelial growth factor, VEGF, which is the subject of this proposal). VEGF is released from cells in response to low oxygen and during wound healing and has an important role in the development and expansion of the microcirculation (growth of new blood vessels from a pre-existing vasculature; angiogenesis) in diseases such as cancer. VEGF mediates its physiological roles by interacting with a receptor on the cell surface of endothelial cells that then undergoes a conformational change to cause tyrosine residues on its intracellular surfaces to become phosphorylated. This sets in motion a chain of events that leads to the activation of a range of different intracellular signalling proteins. These mediate a variety of changes such as cell motility, protein expression and cell survival. VEGF has three different cell surface receptors that it interacts with but it is VEGFR2 which is the most important for angiogenesis. A complication of VEGF signalling is that there are multiple isoforms of VEGF that differ in size and may have markedly different abilities to bind to VEGFR2 and trigger responses. Furthermore, VEGFR2 normally functions as a homodimer (i.e. a complex of one VEGFR2 molecule bound to a second molecule of VEGFR2). However it is also known that VEGFR2 can also interact with the other two VEGF receptors (VEGFR1 and VEGFR3) to form heterodimers. In addition, co-receptors for VEGFR2 exist on the cell membrane (e.g. Neuropilin-1, NRP1) that can either enhance the binding of VEGF to VEGFR2 or can modulate subsequent functional responses. It is unknown, however, how different VEGF isoforms bind to the receptor in these complexes. Quantitative evaluation of ligand-receptor interactions has been the cornerstone of the drug discovery process, particularly in the case of other types of cell surface receptor (G protein-coupled receptors) which are the target for over 40% of all known drugs. However, binding of VEGF isoforms to VEGFR2 has not received the same degree of analysis because of the protein nature of the growth factor and the complexity of its interactions. This proposal aims to exploit the detailed and quantitative analytical skills of the PI and CI to unravel ligand-binding and signalling characteristics (molecular pharmacology) of specific VEGF isoforms utilizing new biotechnology approaches (developed by the industrial partner) to the study of ligand-receptor and receptor-protein interactions in living cells. This uses measurement of luminescence (biologically generated light output) of different colours. The industrial partner has engineered a novel bioluminescent protein (NanoLuc) that was originally isolated from a deep-sea shrimp. This is blue, bright, stable and visible to the naked eye. It can be attached to extracellular or intracellular terminals of VEGFR2 or its interacting proteins without loss of function. The light produced is of an appropriate wavelength to excite a neighbouring fluorescent molecule if it is in very close proximity to NanoLuc and thus can then generate light of a different colour (e.g. red). This strategy will be used to monitor in living cells the binding of fluorescently-labelled VEGF to VEGFR2 and to establish the detailed molecular pharmacology of each VEGF isoform in binding to specific VEGFR2 dimers, VEGFR2-NRP1 complexes and to determine whether they are capable of selectively triggering specific intracellular signalling pathways leading to signalling bias. This will provide considerable insight into VEGF function and provide new opportunities for drug discovery.

Impact Summary

There are two major impact areas of the proposed research and technologies developed during this proposed programme of research: The first is in terms of new and detailed quantitative molecular pharmacology knowledge regarding an important receptor tyrosine kinase (VEGFR2). The work has direct relevance to the academic community, the pharmaceutical industry and the biotechnology industry. Ultimately, the healthcare and animal wellfare sectors may benefit as a consequence of new therapeutic opportunities arising out of the new knowledge gained. The strategies taken for VEGFR2 may also have wide implications for receptor tyrosine kinases generally and other cell surface receptors involved in signalling in both health and disease. VEGFR2 play a major role in cell survival, proliferation and angiogenesis and this has wide implications for the cardiovascular system and the treatment of cancer and macular degeneration. The project will provide for the first time extensive quantitative information on the binding affinities and efficacies of the broad spectrum of VEGF isoforms found in nature that can underpin academic programmes in this area globally and provided essential background knowledge for new and existing drug discovery programmes. Our planned research on VEGFR2 therefore has the potential to provide significant long term impact in meeting future clinical need, delivering and improving therapies for a range of debilitating diseases and contributing to animal and human health. The second major impact will come from the technologies and methodologies developed and implemented to interrogate the molecular pharmacology of VEGFR2. The application of NanoLuc to novel BRET technologies for the evaluation ligand-binding and protein-protein interactions should revolutionize the study of cell surface receptors and have application for most drug discovery targets including intracellular proteins. Beneficiaries will include academics and drug discovery scientists in research institutes, major pharma, SMEs and the biotechnology industry. Furthermore, the application of bimolecular luminescence complementation (BiLC) in concert with BRET provides powerful and totally unique strategies to interrogate the molecular pharmacology of specific protein complexes. This will be a major advance and should foster proof of concept studies across the spectrum of biology. In addition, the work should lead to the development of new instrumentation (both plate reader and luminescence microscopes) to take advantage of the novel imaging tools developed. In addition, to the BRET and NanoLuc technologies, the programme will develop and implement new approaches to the labeling of proteins (including both site-specific labeling of protein agonists such as VEGF and general proteins using the HaloTag technology) and the optimization of NanoLuc substrates. The impact of the work will be initially through publication of our research findings and technology advances, according to the research councils' open access policy. However, in parallel, steps will be taken to showcase the power of the technology and to collaborate on Proof of Concept studies in other research areas. The project will also train research staff (both the postdoc applied for from BBSRC and the postdoc recruited by Promega) in multidisciplinary skills essential in both the industrial and academic sectors. In addition, the management of this project by both the PI and CI and the project team within the Advanced Technologies Group at Promega will provide a unique forum for developing new technologies and applications to biological research. For example, a longer term aspiration is to develop the NanoLuc BRET approaches for in vivo measurements.

Committee	Research Committee D (Molecules, cells and industrial biotechnology)
Research Topics	Pharmaceuticals
Research Priority	X – Research Priority information not available
Research Initiative	LINK: Responsive Mode [2010-2015]
Funding Scheme	X – not Funded via a specific Funding Scheme

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