Novel complementation biosensors to investigate G protein coupled receptors kinetics and signalling bias
Lead Research Organisation:
University of Nottingham
Department Name: School of Life Sciences
Abstract
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.
Dixon A.S., Schwinn M.K., et al. (2016). ACS Chemical Biology, 11:400-408
Motulsky, H. J., & Mahan, L. C. (1984). Molecular pharmacology, 25(1), 1-9.
Herenbrink C.K., Sykes D.A., et al. (2016). Nature communications, 7:10842.
Dixon A.S., Schwinn M.K., et al. (2016). ACS Chemical Biology, 11:400-408
Motulsky, H. J., & Mahan, L. C. (1984). Molecular pharmacology, 25(1), 1-9.
Herenbrink C.K., Sykes D.A., et al. (2016). Nature communications, 7:10842.
Organisations
People |
ORCID iD |
Steven Charlton (Primary Supervisor) |
Studentship Projects
Project Reference | Relationship | Related To | Start | End | Student Name |
---|---|---|---|---|---|
BB/M008770/1 | 30/09/2015 | 31/03/2024 | |||
1944276 | Studentship | BB/M008770/1 | 30/09/2017 | 29/09/2021 |