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Automated imaging-based technique for monitoring of Ca2+ and DAG biosensors enables detailed characterization of GPCR kineticsTélécharger
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December 04, 2017
Authors: Joe Clayton and Peter Banks, BioTek Instruments, Inc.
G protein coupled receptor (GPCR)-mediated pathways are critical for cells to respond to intercellular and environmental cues, and are a major focus of drug discovery efforts. The molecules that activate GPCRs, and the resulting signaling cascades triggered by associated G proteins, are diverse. Activation of the Gq sub-family of G proteins increases phospholipase C (PLC) activity, converting phosphatidylinositol 4,5-bisphosphate (PIP2) into inositol 1,4,5-triphosphate (IP3) and diacylglycerol (DAG), both of which function as second messengers. IP3 triggers release of intracellular Ca2+ stores which regulate calcium-dependent proteins, while membrane-bound DAG activates the signal cascade via protein kinase C.
Here we describe an automated imaging-based approach to characterize the IP3/DAG signaling pathway using multiplexed R-GECO Ca2+ and DAG fluorescent biosensors from Montana Molecular. This method produces detailed kinetic profiles of Ca2+ flux and DAG levels following activation of Gq-coupled receptors within the same population of cells. Background subtraction and image analysis tools enable detection of changes in Ca2+ and DAG levels that are more sensitive than techniques relying on bulk fluorescence measurements. Additionally, this method enables single-cell analysis for detailed characterization of response dynamics and subcellular effects.