Investigating Real-Time Internalization of Membrane Proteins using a Novel Technology Based on Bioluminescence

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2021-04-01

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The trafficking of membrane proteins including G-protein coupled receptors (GPCRs) is an exciting area in pharmacology. Proteins, acting in a diverse array of physiological systems, can have differential signaling consequences depending on their subcellular localization. Critical gene transcription and protein translation occur continuously, as well as trafficking and localization of proteins to specific functional zones of the cell membrane. As heart failure becomes an increasingly important clinical entity, growing numbers of investigative teams are examining the cell biology of healthy and diseased cardiomyocytes. However, membrane proteins are challenging to study under physiological conditions given their low endogenous expression levels. Current technologies that monitor internalization are costly and limited to a specific internalization mechanism. Thus, we hypothesized that, due to the high sensitivity of bioluminescence, we could devise a universal and powerful technology using bioluminescence to monitor a wide variety of internalization processes by targeting the early endosomes. Here we show a novel technology based on bioluminescence produced by the fragment complementation of Nano Luciferase in living cells. This novel assay allows for quantifying real-time membrane proteins internalization and recycling at endogenous expression levels in cardiomyocytes and kidney cells. Moreover, this technology can universally be applied towards a wide range of membrane proteins and used in the elucidation of novel molecular mechanisms as well as the development of therapeutic agents for several diseases such as cancer, mood disorders, cardiomyopathies and infectious diseases including COVID-19.

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