TY - JOUR
T1 - cAMP biosensors applied in molecular pharmacological studies of G protein-coupled receptors
AU - Mathiesen, Jesper Mosolff
AU - Vedel, Line
AU - Bräuner-Osborne, Hans
N1 - Copyright © 2013 Elsevier Inc. All rights reserved.
PY - 2013
Y1 - 2013
N2 - Cyclic adenosine monophosphate (cAMP) is a common second messenger that mediates numerous biological responses. Intracellular cAMP levels are increased by activation of Gs-coupled G protein-coupled receptors (GPCRs) and decreased by activation of Gi-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 β2-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.
AB - Cyclic adenosine monophosphate (cAMP) is a common second messenger that mediates numerous biological responses. Intracellular cAMP levels are increased by activation of Gs-coupled G protein-coupled receptors (GPCRs) and decreased by activation of Gi-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 β2-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.
U2 - 10.1016/b978-0-12-407865-9.00011-x
DO - 10.1016/b978-0-12-407865-9.00011-x
M3 - Journal article
C2 - 23374187
SN - 0076-6879
VL - 522
SP - 191
EP - 207
JO - Methods in Enzymology
JF - Methods in Enzymology
ER -