A membrane-associated, fluorogenic reporter for mammalian phospholipase C isozymes
| Autor: | Jarod Waybright, John Sondek, Stuart Endo-Streeter, T. Kendall Harden, Qisheng Zhang, Nicole Hajicek, Xiaoyang Wang, Christian Wohlfeld, Weigang Huang, Matthew O. Barrett |
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| Rok vydání: | 2017 |
| Předmět: |
0301 basic medicine
Phospholipase C beta Biochemistry Isozyme Receptor tyrosine kinase Fluorescence 03 medical and health sciences chemistry.chemical_compound Phosphatidylinositol Phosphates Genes Reporter Humans Phosphatidylinositol Receptor Molecular Biology Diacylglycerol kinase 030102 biochemistry & molecular biology biology Phospholipase C Chemistry Cell Membrane GTP-Binding Protein beta Subunits Cell Biology Lipids GTP-Binding Protein alpha Subunits Isoenzymes 030104 developmental biology HEK293 Cells Gq alpha subunit Second messenger system biology.protein |
| Zdroj: | The Journal of biological chemistry. 293(5) |
| ISSN: | 1083-351X |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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