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A luminescent assay for real-time measurements of receptor endocytosis in living cells.

Research paper by Matthew B MB Robers, Brock F BF Binkowski, Mei M Cong, Chad C Zimprich, Cesear C Corona, Mark M McDougall, George G Otto, Christopher T CT Eggers, Jim J Hartnett, Thomas T Machleidt, Frank F Fan, Keith V KV Wood

Indexed on: 19 Aug '15Published on: 19 Aug '15Published in: Analytical Biochemistry: Methods in the Biological Sciences



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.