Adapting a TR-FRET platform to decipher G protein-coupled receptor internalization and recycling kinetics

G protein-coupled receptors (GPCRs) represent one of the largest family of membrane receptors, mediating an extensive array of physiological responses through the activation of diverse intracellular signaling pathways. Due to their prominence as therapeutic targets, regulation of GPCR trafficking including internalization and recycling is a critical determinant of drug efficacy and signaling bias. Traditional biochemical and imaging approaches, while informative, often lack the throughput and temporal resolution required to systematically characterize receptor kinetics across large compound libraries. Herein, we describe the adaptation of a time-resolved Förster resonance energy transfer (TR-FRET)-based platform to quantitatively monitor GPCR internalization and recycling dynamics in living cells. By integrating donor-acceptor fluorophore pairs with receptor constructs labeled at extracellular epitopes, the assay enables real-time tracking of ligand-induced receptor trafficking with high sensitivity and minimal perturbation of cellular physiology. The TR-FRET readout allows precise kinetic profiling of receptor endocytosis and recycling across different ligand classes, facilitating the discrimination of biased agonists and the elucidation of pathway-specific trafficking signatures. As an example, an energy-donor tag-modified GLP-1R cell line was stimulated by energy-acceptor-modified agonists for comparison against activation by modified GLP-1. Before internalization, a high TR-FRET signal can be observed. As the receptor gets internalized, the increased distance between the membrane-bound donor with the acceptor agonists quenches the TR-FRET signal before resurfacing as the receptor gets recycled. Altogether, this mechanism helps researchers elucidate the signal duration, intensity, de/re-sensitization of the agonist on its receptor, impacting efficacy, potency and tolerance. Altogether, this optimized TR-FRET approach provides a scalable, high-throughput framework for assessing GPCR trafficking kinetics, bridging the gap between molecular pharmacology and system-level drug screening. By enabling rapid, quantitative evaluation of receptor dynamics, this platform holds significant potential to accelerate the discovery and optimization of therapeutics that leverage GPCR signaling for improved efficacy and safety profiles.