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• W3128779600 abstract "Abstract Heterotrimeric G proteins constitute the primary transducers of G protein-coupled receptor (GPCR) signaling. Besides mediating ligand-induced GPCR activation, G proteins transduce basal levels of activity in various physiological and pathophysiological settings evoked by constitutively active, native GPCRs or disease-related receptor mutants. Several generations of optical biosensors were developed and optimized to monitor GPCR ligand-induced G protein activation, however, quantitative approaches to detect constitutively active GPCRs are not available. Here, we designed and validated a set of eight bioluminescence-resonance-energy-transfer (BRET)-based G protein sensors, covering all four major families of G proteins, and established a protocol to identify constitutive GPCR/G protein signaling in living cells. These sensors rely on the encoding of all three G protein subunits on a single plasmid, enabling their cellular expression at desired relative levels and resulting in reduced signal variability in mammalian cells. Based on this sensor platform, we further present here an experimental protocol to quantify constitutive signaling of native and mutated GPCRs through these heterotrimeric transducers. This approach will aid in the characterization of constitutively active GPCRs and the exploration of their role in health and disease. One Sentence Summary This Resource article describes the validation of a biophysical approach to directly assess the constitutive signaling activity of G protein-coupled receptors through heterotrimeric G proteins in living cells using optical biosensors." @default.
• W3128779600 created "2021-02-15" @default.
• W3128779600 creator A5000142376 @default.
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• W3128779600 date "2021-02-06" @default.
• W3128779600 modified "2023-10-11" @default.
• W3128779600 title "Quantitative assessment of constitutive G protein-coupled receptor activity with BRET-based G protein biosensors" @default.
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• W3128779600 doi "https://doi.org/10.1101/2021.02.05.429900" @default.
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