G protein-coupled receptors (GPCRs) are integral membrane proteins crucial for sensing extracellular signals, including hormones, neurotransmitters, and environmental cues. These receptors initiate intracellular signaling cascades upon activation, ultimately regulating a myriad of physiological processes. Central to GPCR function are G proteins, comprising subfamilies such as Gs, Gi/o, Gq/11, and G12/13, which orchestrate downstream signaling events, including the modulation of cyclic adenosine monophosphate (cAMP), calcium mobilization, and extracellular signal-regulated kinase (ERK) activation [1].
Traditionally, second messenger assays measuring cAMP accumulation, calcium mobilization, and ERK phosphorylation have been pivotal in deciphering GPCR activity, particularly in drug discovery endeavors [2]. However, these conventional assays often provide limited information on intermediate signaling events due to pathway crosstalk and signal amplification [3]. Also, most second messenger assays are primarily endpoint measurements and do not allow practical measurement of the kinetics of signaling. The emergence of resonance energy transfer (RET) techniques, notably Fluorescence Resonance Energy Transfer (FRET) and Bioluminescence Resonance Energy Transfer (BRET), has revolutionized the study of GPCRs by enabling real-time monitoring of protein-protein interactions and intracellular signaling dynamics[4].
FRET and BRET sensors operate on the principle of energy transfer between a fluorescent or luminescent donor and acceptor molecules within close proximity, typically within 100Å. These biosensors have facilitated the investigation of various aspects of GPCR signaling, including ligand binding (e.g NanoBRET ligand binding [5]), effector protein recruitment assays (e.g G protein recruitment assay [6], mini-G recruitment assay [7], GRK and β-arrestin recruitment assays [8]), G protein activation (e.g TRUPATH assay [9]), receptor trafficking ( e.g FYVE assay [10]), β-arrestin dynamics ( e.g FLAsH biosensor [11]), cAMP production (e.g CAMYEL assay [12]) , and ERK activity (e.g YEN assay [13]), among others.
One of the significant advantages of FRET and BRET-based sensors is their ability to provide real-time readouts of pharmacological activity, allowing for the determination of drug kinetics—a critical aspect in drug development [14]. Moreover, these experiments can be conducted in live cells, preserving the physiological context and minimizing artifacts associated with sample preparation for endpoint measurements.
Despite their immense potential, utilizing FRET and BRET sensors in GPCR research comes with challenges, including expensive cost of reagents, optimizing sensor expression levels and adapting these systems to disease-relevant models. Addressing these hurdles is essential for translating findings from cellular models to clinically relevant scenarios. Some innovative solutions have been the development of the BERKY and the ONE-GO biosensors, designed to facilitate their application in disease models[3, 15]. BERKY consists of a membrane linker, a BRET donor, an ER/K α-helix linker, a BRET acceptor, and an active G protein detector. The ONE-GO biosensors, designed in a single vector, incorporate a G protein tagged with a YFP acceptor and a G protein detector tagged with an Nluc donor. Both BERKY and ONE-GO biosensors are engineered to detect the GTP-bound Gα subunit, serving as a proxy for G protein activation and have been optimized for use in detecting GPCR activity in primary cells.
In conclusion, FRET and BRET-based sensors have transformed the landscape of GPCR research, offering unprecedented insights into the intricacies of GPCR signaling. These techniques not only enhance our understanding of fundamental cellular processes, but also hold immense promise in accelerating drug discovery efforts by enabling the precise characterization of pharmacological interventions in real time. As technology continues to advance, leveraging RET-based sensors will undoubtedly continue to propel our quest to unravel the complexities of GPCR signaling and pave the way for novel therapeutic strategies.
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