Abstract
Information contained in the structure of extracellular ligands is transmitted across the cell membrane through allosterically induced changes in G protein-coupled receptor (GPCR) conformation that occur upon ligand binding. These changes, in turn, are imprinted upon intracellular effectors like arrestins and help determine which of its many functions are performed. Intramolecular fluorescein arsenical hairpin (FlAsH) bioluminescence resonance energy transfer (BRET), in which both the fluorescence donor and acceptor are contained within the same protein, can be used to report on activation-induced changes in protein conformation. Here, we describe a method using a series of Rluc-arrestin3-FlAsH-BRET biosensors to measure stimulus-induced changes in arrestin conformation in live cells. Each Rluc-arrestin3-FlAsH-BRET construct contains an N-terminal Renilla luciferase fluorescence donor that excites a fluorescent arsenical targeted to a different position within the protein by mutational insertion of a tetracysteine tag motif. Changes in net BRET upon GPCR stimulation can thus be viewed from multiple vantage points within the protein and used to develop an arrestin3 “conformational signature” that is receptor- and ligand-specific. This method can be used to determine how differences in GPCR and ligand structure influence information transfer across the plasma membrane and to classify GPCRs and/or ligands based on their capacity to induce different arrestin3 activation modes.
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Acknowledgments
This work was supported by National Institutes of Health Grants R01 DK055524 (LML), R01 GM095497 (LML), Department of Veterans Affairs Merit Review Grant I01 BX003188 (LML), and the Research Service of the Charleston, SC Veterans Affairs Medical Center. The contents of this article do not represent the views of the Department of Veterans Affairs or the United States Government.
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Strungs, E.G., Luttrell, L.M., Lee, MH. (2019). Probing Arrestin Function Using Intramolecular FlAsH-BRET Biosensors. In: Scott, M., Laporte, S. (eds) Beta-Arrestins. Methods in Molecular Biology, vol 1957. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-9158-7_19
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DOI: https://doi.org/10.1007/978-1-4939-9158-7_19
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