Elsevier

SLAS Discovery

Volume 18, Issue 7, August 2013, Pages 797-806
SLAS Discovery

Original Research
A Multiplexed Fluorescent Assay for Independent Second-Messenger Systems: Decoding GPCR Activation in Living Cells

https://doi.org/10.1177/1087057113485427Get rights and content
Under a Creative Commons license
open access

Abstract

There is a growing need in drug discovery and basic research to measure multiple second-messenger components of cell signaling pathways in real time and in relevant tissues and cell types. Many G-protein–coupled receptors activate the heterotrimeric protein, Gq, which in turn activates phospholipase C (PLC). PLC cleaves phosphatidylinositol 4,5-bisphosphate (PIP2) to produce two second messengers: diacylglycerol (DAG), which remains in the plasma membrane, and inositol triphosphate (IP3), which diffuses through the cytosol to release stores of intracellular calcium ions (Ca2+). Our goal was to create a series of multiplex sensors that would make it possible to simultaneously measure two different components of the Gq pathway in living cells. Here we describe new fluorescent sensors for DAG and PIP2 that produce robust changes in green or red fluorescence and can be combined with one another, or with existing Ca2+ sensors, in a live-cell assay. These assays can detect multiple components of Gq signaling, simultaneously in real time, on standard fluorescent plate readers or live-cell imaging systems.

Keywords

cell-based assays
G-protein–coupled receptors (GPCR)
fluorescence methods
multiplex assays and technology

Cited by (0)