Studying GPCR Pharmacology in Membrane Microdomains: Fluorescence Correlation Spectroscopy Comes of Age. (February 2018)

• Record Type: Journal Article
• Authors: Briddon, Stephen J.; Kilpatrick, Laura E.; Hill, Stephen J.
• Is Part Of: Trends in pharmacological sciences. Volume 39:Number 2(2018)
• Page Start: 158
• Page End: 174
• Publication Date: 2018-02
• Subjects: fluorescence correlation spectroscopy -- fluorescence fluctuation spectroscopy -- photon counting histogram -- G protein coupled receptor -- single cell pharmacology -- fluorescent ligands -- oligomerization; Pharmacology -- Periodicals; Pharmacology -- trends -- Periodicals; Pharmacologie -- Périodiques; Pharmacology; Electronic journals; Periodicals; 615.1
• DOI: 10.1016/j.tips.2017.11.004
• Languages: English
• ISSNs: 0165-6147

Abstract

Abstract : G protein-coupled receptors (GPCRs) are organised within the cell membrane into highly ordered macromolecular complexes along with other receptors and signalling proteins. Understanding how heterogeneity in these complexes affects the pharmacology and functional response of these receptors is crucial for developing new and more selective ligands. Fluorescence correlation spectroscopy (FCS) and related techniques such as photon counting histogram (PCH) analysis and image-based FCS can be used to interrogate the properties of GPCRs in these membrane microdomains, as well as their interaction with fluorescent ligands. FCS analyses fluorescence fluctuations within a small-defined excitation volume to yield information about their movement, concentration and molecular brightness (aggregation). These techniques can be used on live cells with single-molecule sensitivity and high spatial resolution. Once the preserve of specialist equipment, FCS techniques can now be applied using standard confocal microscopes. This review describes how FCS and related techniques have revealed novel insights into GPCR biology. Highlights: Advances in both structural biology and imaging show that GPCRs are organised in highly specialised domains in the cell membrane. Coupled with receptor oligomerisation, compartmentalisation of signalling proteins and continued signalling from intracellular compartments, this adds a significant layer of complexity to the pharmacology of GPCRs, and determining whether domain-level pharmacology can be exploited. FFS techniques analyse fluorescence fluctuations in time or space from small detection volumes (0.2 fl, 0.2 μm 2 of membrane) which can be precisely positioned on or around living cells. Analysis of these fluctuations provides quantitative information about the speed of diffusion, concentration and aggregation of fluorescent molecules, such as receptors, effectors and fluorescent ligands. This is a powerful tool for analysing receptor properties and ligand-receptor interactions at a subcellular level. FFS has been used to quantify GPCR diffusion and expression, oligomer stoichiometry, aggregation and the properties of ligand–receptor complexes, and has particularly application for endogenous receptors at low levels of expression. Recent advances in FFS methods and analysis allow more comprehensive mapping of these properties across cell surfaces, and have allowed FFS to become one of several complimentary imaging technologies to probe GPCR spatial organisation.