title: A multiplexed fluorescent assay for independent second-messenger systems: decoding GPCR activation in living cells.
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 (Ca(2+)). 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 Ca(2+) 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.
authors.affiliation: Montana Molecular, Bozeman, MT 59717, USA.
authors.forename: Paul H
authors.initials: PH
authors.lastname: Tewson
authors.forename: Anne Marie
authors.initials: AM
authors.lastname: Quinn
authors.forename: Thomas E
authors.initials: TE
authors.lastname: Hughes
mesh_terms: D000255:Adenosine Triphosphate / Q000502:physiology; D001681:Biological Assay; D015374:Biosensing Techniques; D020013:Calcium Signaling; D004075:Diglycerides / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000096:biosynthesis; D057809:HEK293 Cells; D006801:Humans; D008164:Luminescent Proteins / Q000096:biosynthesis; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000378:metabolism; D051745:Protein Kinase C-delta / Q000096:biosynthesis; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D015290:Second Messenger Systems; D013050:Spectrometry, Fluorescence; D010738:Type C Phospholipases / Q000378:metabolism; D000097573:Red Fluorescent Protein
chemical_list: D000255:Adenosine Triphosphate; D004075:Diglycerides; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D019269:Phosphatidylinositol 4,5-Diphosphate; D051745:Protein Kinase C-delta; D043562:Receptors, G-Protein-Coupled; D011993:Recombinant Fusion Proteins; D010738:Type C Phospholipases; D000097573:Red Fluorescent Protein
keywords: G-protein–coupled receptors (GPCR); cell-based assays; fluorescence methods; multiplex assays and technology