title: cAMP biosensors applied in molecular pharmacological studies of G protein-coupled receptors.
abstract: Cyclic adenosine monophosphate (cAMP) is a common second messenger that mediates numerous biological responses. Intracellular cAMP levels are increased by activation of G(s)-coupled G protein-coupled receptors (GPCRs) and decreased by activation of G(i)-coupled GPCRs via the adenylyl cyclase. Many end-point assays for quantifying GPCR-mediated changes in intracellular cAMP levels exist. More recently, fluorescence resonance energy transfer (FRET)-based cAMP biosensors that can quantify intracellular cAMP levels in real time have been developed. These FRET-based cAMP biosensors have been used primarily in single cell FRET microscopy to monitor and visualize changes in cAMP upon GPCR activation. Here, a similar cAMP biosensor with a more efficient mCerulean/mCitrine FRET pair is described for use in the 384-well plate format. After cloning and expression in HEK293 cells, the biosensor is characterized in the 384-well plate format and used for measuring the signaling of the G(s)-coupled β(2)-adrenergic receptor. The procedures described may be applied for other FRET-based biosensors in terms of characterization and conversion to the 384-well plate format.
authors: Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark. jmm@sund.ku.dk
authors: Jesper Mosolff
authors: JM
authors: Mathiesen
authors: Line
authors: L
authors: Vedel
authors: Hans
authors: H
authors: Bräuner-Osborne
mesh_terms: D015374:Biosensing Techniques; D000242:Cyclic AMP / Q000032:analysis*; D018076:DNA, Complementary / Q000235:genetics / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer; D015870:Gene Expression / Q000187:drug effects; D005822:Genetic Vectors; D020662:Guanine Nucleotide Exchange Factors / Q000235:genetics / Q000378:metabolism*; D057809:HEK293 Cells; D057166:High-Throughput Screening Assays; D006801:Humans; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology; D018343:Receptors, Adrenergic, beta-2 / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects
chemical_list: D018076:DNA, Complementary; D020662:Guanine Nucleotide Exchange Factors; D010726:Phosphodiesterase Inhibitors; C116122:RAPGEF3 protein, human; D018343:Receptors, Adrenergic, beta-2; D000242:Cyclic AMP