Fluorescence‐ and bioluminescence‐based approaches to study GPCR ligand binding

Abstract

Ligand binding is a vital component of any pharmacologist's toolbox and allows the detailed investigation of how a molecule binds to its receptor. These studies enable the experimental determination of binding affinity of labelled and unlabelled compounds through kinetic, saturation (Kd) and competition (Ki) binding assays. Traditionally, these studies have used molecules labelled with radioisotopes; however, more recently, fluorescent ligands have been developed for this purpose. This review will briefly cover receptor ligand binding theory and then discuss the use of fluorescent ligands with some of the different technologies currently employed to examine ligand binding. Fluorescent ligands can be used for direct measurement of receptor‐associated fluorescence using confocal microscopy and flow cytometry as well as in assays such as fluorescence polarization, where ligand binding is monitored by changes in the free rotation when a fluorescent ligand is bound to a receptor. Additionally, fluorescent ligands can act as donors or acceptors for fluorescence resonance energy transfer (FRET) with the development of assays based on FRET and time‐resolved FRET (TR‐FRET). Finally, we have recently developed a novel bioluminescence resonance energy transfer (BRET) ligand binding assay utilizing a small (19 kDa), super‐bright luciferase subunit (NanoLuc) from a deep sea shrimp. In combination with fluorescent ligands, measurement of RET now provides an array of methodologies to study ligand binding. While each method has its own advantages and drawbacks, binding studies using fluorescent ligands are now a viable alternative to the use of radioligands.Linked ArticlesThis article is part of a themed section on Molecular Pharmacology of G Protein‐Coupled Receptors. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v173.20/issuetoc

Quantitative Single-Cell Analysis of Signaling Pathways Activated Immediately Downstream of Histamine Receptor Subtypes

“…FRET reporters can be developed to measure every step in the GPCR signaling cascade. FRET biosensors are available to measure ligand binding to the GPCR (Stoddart et al, 2015), GPCR activation (Vilardaga et al, 2003), GPCR and G-protein interaction (Hein et al, 2005;Stumpf and Hoffmann, 2016), G-protein activation (Janetopoulos et al, 2001;Adjobo-Hermans et al, 2011), Ca 21 release (Nagai et al, 2004), cAMP production (Klarenbeek et al, 2015), and activation of downstream effectors such as protein kinase C (Verbeek et al, 2008), RhoA (van Unen et al, 2015a), and inositol 1,4,5-trisphosphate (Gulyás et al, 2015). The preferred option is to use FRET biosensors that report on the specific activation of one of the heterotrimeric G-protein subfamilies directly stimulated by a GPCR.…”

Quantification of Membrane Protein-Detergent Complex Interactions

“…13 f The slope factor or transition steepness was calculated at the midpoint of the dissociation phase. g Free energies were determined using the standard thermodynamic relationship Δ G = R T ln K d . h The quantitative balance between the adhesive protein-detergent ( F adh ) and cohesive detergent-detergent interactions ( F coh ) of the proteomicelles. i Not determined. …”

“…Here, we show that we can overcome these challenges using rational membrane protein design, along with targeted chemical modification and steady-state fluorescence polarization (FP) spectroscopy, 12–13 to probe the detergent desolvation transitions of membrane proteins. The FP spectroscopy was previously used to inspect: (i) the interactions of mild 14 and harsh 15 detergents with water-soluble proteins, (ii) the harsh detergent-induced unfolding 16 and resistance of soluble proteins to denaturation, 17 (iii) the detergent-mediated oligomerization of hydrophobic proteins into proteomicelles, 18 and (iv) the impact of detergent on conformational changes 14 and enzymatic activity 19 of soluble proteins.…”

Cell-based in vitro models in environmental toxicology: a review

“…Some fluorescence-based binding assays performed in cells utilize the increase in polarization of light as a readout, which indicates the loss of rotational freedom after binding (fluorescence polarization assay) [172]. Both ligand and receptor labeling is required for Förster resonance energy transfer (FRET) detection, where energetic exchange between the two close fluorophores in proximity elicits a detectable intensity shift in donor and acceptor emission [173].…”