Fluctuation Spectroscopy approaches to studying GPCR pharmacology in single cells
Dr Steve Briddon – School of Life Sciences and Centre for Membrane Proteins and Receptors, Medical School, University of Nottingham
BioLight CoB seminar, Thursday 11 May 2023, 1pm BSRC Seminar Room, in person only seminar
G protein-coupled receptors (GPCRs) are a large family of cell surface receptors which are major clinical targets for many clinically used drugs as well as those in development. Spatial organisation of GPCRs and their associated signalling molecules in the cell membrane and other cellular compartments is a major determinant of their signalling profile and interaction with ligands. This ability of GPCRs to exhibit differential pharmacology depending on where they are expressed in the cell has considerable significance to the design of more selective drugs with fewer on-target side effects.
It is therefore imperative to develop methods to study the pharmacology of GPCRs at the subcellular level, in their native cellular environment and at low levels of expression, where standard pharmacological techniques do not show enough sensitivity. We have used fluorescence fluctuation spectroscopy (FFS) approaches, including fluorescence correlation spectroscopy (FCS) and photon counting histogram (PCH) analysis, alongside other advanced imaging approaches, to develop ways of quantifying the pharmacology, diffusion, clustering and spatial organisation of a number of GPCRs in live cell membranes. Using fluorescent ligand technologies, we have been able to quantify ligand-receptor complexes and interactions in small areas (~0.1mm2) of the cell membrane. More recently, work with detergent-free purification of GPCRs has allowed us to extend this work to the use FFS based approaches to screening.
The talk will describe some examples, illustrating how combining FFS with chemical biology and fluorescent technologies provides a powerful toolbox for studying the pharmacology of GPCRs in subcellular compartments.