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

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Abstract

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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