A luminescent assay for real-time measurements of receptor endocytosis in living cells |
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Authors: | Matthew B Robers Brock F Binkowski Mei Cong Chad Zimprich Cesear Corona Mark McDougall George Otto Christopher T Eggers Jim Hartnett Thomas Machleidt Frank Fan Keith V Wood |
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Institution: | 1. Promega Corporation, Fitchburg, WI 53711, USA;2. Promega Biosciences, San Luis Obispo, CA 93401, USA |
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Abstract: | Ligand-mediated endocytosis is a key autoregulatory mechanism governing the duration and intensity of signals emanating from cell surface receptors. Due to the mechanistic complexity of endocytosis and its emerging relevance in disease, simple methods capable of tracking this dynamic process in cells have become increasingly desirable. We have developed a bioluminescent reporter technology for real-time analysis of ligand-mediated receptor endocytosis using genetic fusions of NanoLuc luciferase with various G-protein-coupled receptors (GPCRs). This method is compatible with standard microplate formats, which should decrease work flows for high-throughput screens. This article also describes the application of this technology to endocytosis of epidermal growth factor receptor (EGFR), demonstrating potential applicability of the method beyond GPCRs. |
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Keywords: | G-protein-coupled receptor Receptor tyrosine kinase Endocytosis Internalization NanoLuc |
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