Development of a Novel SNAP-Epitope Tag/Near-Infrared Imaging Assay to Quantify G Protein-Coupled Receptor Degradation in Human Cells

Lee, Kyung-Soon; Navaluna, Edelmar; Marsh, Nicole M; Janezic, Eric M.; Hague, Chris

doi:10.1177/2472555220979793

Cited by 2 publications

(1 citation statement)

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“…Degradation for the V 2 receptor was markedly slow (27 h half-time) whereas that for the MOR was faster (2.8 h). The value for the MOR receptor is within the range of receptor degradation half-times reported for a broad panel of GPCRs in HEK293 cells [0.7–2.8 h (Lee et al, 2021 )]. The long half-time for the V 2 receptor might be a manifestation of the tight arrestin binding impairing degradation.…”

Section: Quantitative Mechanistic Analysis Of Arrestin Recruitment Waveformssupporting

confidence: 73%

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

Hoare

Tewson

Sachdev

et al. 2022

Front. Cell. Neurosci.

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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.

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Section: Quantitative Mechanistic Analysis Of Arrestin Recruitment Waveformssupporting

confidence: 73%