Promega Corporation

Multiplexed Analysis of Second Messenger Signaling in Live Cells Using Aequorin and...

Multiplexed Analysis of Second Messenger Signaling in Live Cells Using Aequorin and GloSensor cAMP Scientific Poster




Matthew Robers, Pete Stecha, Natasha Karassina, Brock Binkowski, Frank Fan, and Mei Cong
Promega Corporation; 2800 Wood Hollow Road, Madison WI 53711

Detection of intracellular second messenger signaling is an established method for measuring G-protein-dependent GPCR activation. Although there are several technologies available for measurement of second messengers via endpoint analysis, technologies for monitoring second messengers in living cells include Promega’s GloSensor™ cAMP for quantifying intracellular [cAMP] and technologies such as the photoprotein Aequorin or various fluorescence-based indicators for [Ca2+]. These technologies serve to quantify second messengers in live cells and in real-time following GPCR activation, providing several advantages over lytic endpoint assays. However, it may be challenging when screening for GPCR activity modulators when G-protein-dependent signaling is uncharacterized or when the desired second messenger detection format cannot be predicted (for example, in the case of orphan receptors). Furthermore, for GPCRs capable of modulating both [cAMP] and [Ca2+] pathways concurrently, it would be desirable to measure G protein coupling simultaneously. Few technologies exist that allow for simultaneous measurement of Ca2+ and cAMP in live cells, while maintaining assay robustness and high signal-to-background for use in HTS. To address this limitation, Promega has developed a live cell method for the kinetic measurement of Ca2+ and cAMP by multiplexing of Aequorin and GloSensor™ cAMP bioluminescent sensor technologies. Using the Hamamatsu FDSS/μCell, we report simultaneously analysis of Ca2+ and cAMP mobilization following agonism of Parathyroid Hormone Receptor (PTH1R) using a promiscuous compound directing both Gαq + Gαs signaling, as well as a biased compound specifically directing Gαs coupling alone. The combination of these bioluminescence-based sensor technologies with the Hamamatsu FDSS/μCell serves as an ideal platform for the analysis of these divergent second messenger
signaling events in live cells and in real time.

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  • Part# PS124
  • Printed in USA.

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