Quantifying the Kinetics of Signaling and Arrestin Recruitment by Nervous System G-Protein Coupled Receptors

Abstract

Neurons integrate inputs over different time and space scales. Fast excitatory synapses at boutons (ms and μm), and slow modulation over entire dendritic arbors (seconds and mm) are all ultimately combined to produce behavior. Understanding the timing of signaling events mediated by G-protein-coupled receptors is necessary to elucidate the mechanism of action of therapeutics targeting the nervous system. Measuring signaling kinetics in live cells has been transformed by the adoption of fluorescent biosensors and dyes that convert biological signals into optical signals that are conveniently recorded by microscopic imaging or by fluorescence plate readers. Quantifying the timing of signaling has now become routine with the application of equations in familiar curve fitting software to estimate the rates of signaling from the waveform. Here we describe examples of the application of these methods, including (1) Kinetic analysis of opioid signaling dynamics and partial agonism measured using cAMP and arrestin biosensors; (2) Quantifying the signaling activity of illicit synthetic cannabinoid receptor agonists measured using a fluorescent membrane potential dye; (3) Demonstration of multiplicity of arrestin functions from analysis of biosensor waveforms and quantification of the rates of these processes. These examples show how temporal analysis provides additional dimensions to enhance the understanding of GPCR signaling and therapeutic mechanisms in the nervous system.

Cannabinoid 1 (CB1) receptor arrestin subtype‐selectivity and phosphorylation dependence

“…Raw BRET ratios were baseline-corrected by subtracting the time course of the vehicle-treated condition of each stimulation (ΔBRET ratio). These data were modelled by previously described kinetic equations (Hoare et al, 2020(Hoare et al, , 2022. Arrestin translocation data were modelled by the 'rise to steady state' equation, which quantifies translocation steady state, translocation half-time, and approximates the initial rate of signal generation.…”

Kinetic analysis of endogenous β2‐adrenoceptor‐mediated cAMP GloSensor™ responses in HEK293 cells

“…It is worth emphasising that the measurement of initial rate of signal generation does not account for ligand binding affinity for the receptor (Hoare et al, 2018(Hoare et al, , 2022. Therefore, at submaximal concentrations of ligands, the ligand association rate may distort observed initial signalling rate values, due to ligand-receptor binding becoming the rate-limiting step, rather than the agonist-occupied receptor's generation of the signal (Hoare et al, 2018(Hoare et al, , 2022.…”

“…It is worth emphasising that the measurement of initial rate of signal generation does not account for ligand binding affinity for the receptor (Hoare et al, 2018(Hoare et al, , 2022. Therefore, at submaximal concentrations of ligands, the ligand association rate may distort observed initial signalling rate values, due to ligand-receptor binding becoming the rate-limiting step, rather than the agonist-occupied receptor's generation of the signal (Hoare et al, 2018(Hoare et al, , 2022. Salmeterol has a high binding affinity for the β 2 -adrenoceptor, relative to isoprenaline, formoterol and salbutamol, due to a fast association rate with the receptor but a slow dissociation rate (Sykes et al, 2014;Sykes & Charlton, 2012).…”

“…The recent development of new and improved biosensor technologies has enabled the continuous measurement of GPCR signals, thus allowing quantification of the entire time-course of the response (Goulding et al, 2018;Greenwald et al, 2018;Lohse et al, 2008Lohse et al, , 2012Wright & Bouvier, 2021). The derivation of equations to fit these time-course data has made it possible to estimate kinetic signalling parameters such as kinetic potency (L 50 ) and maximal initial rate (IR max ), which is related to efficacy (Hoare et al, 2020(Hoare et al, , 2022. This kinetic analysis could uncover new information about the pharmacological and kinetic properties of ligands, which may ultimately allow for more accurate characterisation of ligand-receptor interactions.…”

Metadynamics simulations leveraged by statistical analyses and artificial intelligence-based tools to inform the discovery of G protein-coupled receptor ligands

“…However, it is not the only element at play. Temporal analyses of ligand binding to GPCRs, as well as GPCR-induced G protein activation/coupling and barrestin recruitment evoked by ligands with different efficacies, demonstrate the existence of another dimension of functional bias by GPCRs that directly impacts our understanding of GPCR signaling and therapeutic implications, thus suggesting the importance of incorporating quantifications of ligand binding and signaling kinetics in modern drug discovery efforts (8)(9)(10).…”