Novel complementation biosensors to investigate G protein coupled receptors kinetics and signalling bias

Acting as targets for 30% of marketed drugs, G protein coupled receptors (GPCRs) have demonstrated their importance within the clinical field. However, there is still a clinically significant issue of the incidence of side effects. One reason for this is a lack of understanding of ligand-receptor kinetics and the mechanism of GPCR activation of intracellular effectors, such as G proteins and arrestins. This project will use a luciferase complementation technique (NanoBiT; Dixon et al, 2016) to investigate ligand pharmacology and the dynamic nature of recruitment of intracellular effectors to a model receptor, the beta 2 adrenoceptor. The NanoBiT methodology will be further used to stabilise effector-bound receptor states and measure the binding kinetics using time-resolved fluorescence resonance energy transfer techniques and competition association assays (Motulsky & Mahan, 1984; Klein Herenbrink et al, 2016). The final aim of the project will be to use the intracellular bioluminescence produced by the luciferase complementation as a donor in NanoLuc based bioluminescent resonance energy transfer (NanoBRET) techniques. Here, NanoBRET assays will be developed to directly measure ligand binding kinetics at the complemented receptor-effector complexes and establish whether the nature of the effector affects the kinetic parameters of beta 2 adrenoceptor ligands.

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Herenbrink C.K., Sykes D.A., et al. (2016). Nature communications, 7:10842.