G Protein-selective GPCR Conformations Measured Using FRET Sensors in a Live Cell Suspension Fluorometer Assay.
Fӧrster resonance energy transfer (FRET)-based studies have become increasingly common in the investigation of GPCR signaling. Our research group developed an intra-molecular FRET sensor to detect the interaction between Gα subunits and GPCRs in live cells following agonist stimulation. Here, we detail the protocol for detecting changes in FRET between the β2-adrenergic receptor and the Gαs C-terminus peptide upon treatment with 100 µM isoproterenol hydrochloride as previously characterized(1). Our FRET sensor is a single polypeptide consisting serially of a full-length GPCR, a FRET acceptor fluorophore (mCitrine), an ER/K SPASM (systematic protein affinity strength modulation) linker, a FRET donor fluorophore (mCerulean), and a Gα C-terminal peptide. This protocol will detail cell preparation, transfection conditions, equipment setup, assay execution, and data analysis. This experimental design detects small changes in FRET indicative of protein-protein interactions, and can also be used to compare the strength of interaction across ligands and GPCR-G protein pairings. To enhance the signal-to-noise in our measurements, this protocol requires heightened precision in all steps, and is presented here to enable reproducible execution.
Genetics, Cell Biology, and Development, University of Minnesota.
Ansley
A
Semack
Department of Cell and Developmental Biology, University of Michigan.
Rabia U
RU
Malik
Genetics, Cell Biology, and Development, University of Minnesota; sivaraj@umn.edu.
Sivaraj
S
Sivaramakrishnan
D015374:Biosensing Techniques / Q000295:instrumentation / Q000379:methods*; D031541:Fluorescence Resonance Energy Transfer / Q000295:instrumentation / Q000379:methods*; D057809:HEK293 Cells; D006801:Humans; D008024:Ligands; D011487:Protein Conformation; D018343:Receptors, Adrenergic, beta-2 / Q000737:chemistry / Q000378:metabolism; D043562:Receptors, G-Protein-Coupled / Q000032:analysis / Q000737:chemistry* / Q000378:metabolism; D015398:Signal Transduction
D008024:Ligands; D018343:Receptors, Adrenergic, beta-2; D043562:Receptors, G-Protein-Coupled
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