Applications of BRET to study dynamic G-protein coupled receptor interactions in living cells

2001 ◽  
Vol 8 (3-5) ◽  
pp. 155-162 ◽  
Author(s):  
Karen M. Kroeger ◽  
Aylin C. Hanyaloglu ◽  
Karin A. Eidne
Keyword(s):  
G Protein ◽  
Living Cells ◽  
G Protein Coupled Receptor ◽  
Receptor Interactions ◽  
G Protein Coupled

A FlAsH-based FRET approach to determine G protein–coupled receptor activation in living cells

2005 ◽  
Vol 2 (3) ◽  
pp. 171-176 ◽  
Author(s):  
Carsten Hoffmann ◽  
Guido Gaietta ◽  
Moritz Bünemann ◽  
Stephen R Adams ◽  
Silke Oberdorff-Maass ◽  
...  
Keyword(s):  
G Protein ◽  
Receptor Activation ◽  
Living Cells ◽  
G Protein Coupled Receptor ◽  
G Protein Coupled

scholarly journals Determination of Stoichiometry and Geometry of G Protein Coupled Receptor Homo-Oligomers in Living Cells Using Spectrally Resolved FRET

2010 ◽  
Vol 98 (3) ◽  
pp. 395a
Author(s):  
Michael R. Stoneman ◽  
Deo R. Singh ◽  
Arron Sullivan ◽  
Valerica Raicu
Keyword(s):  
G Protein ◽  
Living Cells ◽  
G Protein Coupled Receptor ◽  
Spectrally Resolved ◽  
G Protein Coupled

scholarly journals Measurement of Ligand-G Protein-Coupled Receptor Interactions

2010 ◽  
pp. 1-29 ◽  
Author(s):  
Katie Leach ◽  
Celine Valant ◽  
Patrick M. Sexton ◽  
Arthur Christopoulos
Keyword(s):  
G Protein ◽  
G Protein Coupled Receptor ◽  
Receptor Interactions ◽  
G Protein Coupled

scholarly journals Quantitative assessment of constitutive G protein-coupled receptor activity with BRET-based G protein biosensors

2021 ◽  
Author(s):  
Hannes Schihada ◽  
Rawan Shekhani ◽  
Gunnar Schulte
Keyword(s):  
G Protein ◽  
G Proteins ◽  
Mammalian Cells ◽  
Living Cells ◽  
Optical Biosensors ◽  
Heterotrimeric G Proteins ◽  
G Protein Coupled Receptor ◽  
Cellular Expression ◽  
G Protein Coupled ◽  
Constitutively Active

AbstractHeterotrimeric 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 SummaryThis 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.

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