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

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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.