A membrane-associated, fluorogenic reporter for mammalian phospholipase C isozymes

A diverse group of cell-surface receptors, including many G protein-coupled receptors and receptor tyrosine kinases, activate phospholipase C (PLC) isozymes to hydrolyze phosphatidylinositol 4,5-bisphosphate into the second messengers diacylglycerol and 1,4,5-inositol trisphosphate. Consequently, PL...

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Bibliographic Details
Published in:	The Journal of biological chemistry Vol. 293; no. 5; pp. 1728 - 1735
Main Authors:	Huang, Weigang, Wang, Xiaoyang, Endo-Streeter, Stuart, Barrett, Matthew, Waybright, Jarod, Wohlfeld, Christian, Hajicek, Nicole, Harden, T. Kendall, Sondek, John, Zhang, Qisheng
Format:	Journal Article
Language:	English
Published:	United States Elsevier Inc 02-02-2018 American Society for Biochemistry and Molecular Biology
Subjects:	Cell Membrane - enzymology Cell Membrane - genetics Fluorescence fluorogenic reporter G protein-coupled receptor (GPCR) Genes, Reporter GTP-Binding Protein alpha Subunits - genetics GTP-Binding Protein alpha Subunits - metabolism GTP-Binding Protein beta Subunits - genetics GTP-Binding Protein beta Subunits - metabolism HEK293 Cells Humans inositol phospholipid Isoenzymes - genetics Isoenzymes - metabolism lipid metabolism Lipids Phosphatidylinositol Phosphates - genetics Phosphatidylinositol Phosphates - metabolism Phospholipase C Phospholipase C beta - genetics Phospholipase C beta - metabolism receptor tyrosine kinase G protein-coupled receptor (GPCR) receptor tyrosine kinase inositol phospholipid fluorogenic reporter lipid metabolism Phospholipase C
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Summary:	A diverse group of cell-surface receptors, including many G protein-coupled receptors and receptor tyrosine kinases, activate phospholipase C (PLC) isozymes to hydrolyze phosphatidylinositol 4,5-bisphosphate into the second messengers diacylglycerol and 1,4,5-inositol trisphosphate. Consequently, PLCs control various cellular processes, and their aberrant regulation contributes to many diseases, including cancer, atherosclerosis, and rheumatoid arthritis. Despite the widespread importance of PLCs in human biology and disease, it has been impossible to directly monitor the real-time activation of these enzymes at membranes. To overcome this limitation, here we describe XY-69, a fluorogenic reporter that preferentially partitions into membranes and provides a selective tool for measuring the real-time activity of PLCs as either purified enzymes or in cellular lysates. Indeed, XY-69 faithfully reported the membrane-dependent activation of PLC-β3 by Gαq. Therefore, XY-69 can replace radioactive phosphatidylinositol 4,5-bisphosphate used in conventional PLC assays and will enable high-throughput screens to identify both orthosteric and allosteric PLC inhibitors. In the future, cell-permeable variants of XY-69 represent promising candidates for reporting the activation of PLCs in live cells with high spatiotemporal resolution.
Bibliography:	ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23 Edited by George M. Carman Both authors contributed equally to the results of this work.
ISSN:	0021-9258 1083-351X
DOI:	10.1074/jbc.RA117.000926