Monitoring agonist-promoted conformational changes of β-arrestin in living cells by intramolecular BRET

• Published in: EMBO reports Vol. 6; no. 4; pp. 334 - 340
• Main Authors: Charest, Pascale G, Terrillon, Sonia, Bouvier, Michel
• Format: Journal Article
• Language: English
• Published: Chichester, UK John Wiley & Sons, Ltd 01.04.2005
• Subjects: arrestin; Arrestins - chemistry; Arrestins - metabolism; Bacterial Proteins - metabolism; beta-Arrestins; Biosensing Techniques - methods; biosensor; BRET; Cell Line; Genetic Vectors - genetics; GPCR; Humans; Luciferases, Renilla - metabolism; Luminescent Proteins - metabolism; Microscopy, Fluorescence; Protein Conformation; Receptors, G-Protein-Coupled - metabolism; Scientific Report; Signal Transduction
• ISSN: 1469-221X; 1469-3178
• DOI: 10.1038/sj.embor.7400373

Recruitment of β‐arrestin (β‐arr) to agonist‐stimulated G‐protein‐coupled receptors (GPCRs) has a crucial role in controlling signalling efficacy and selectivity. When translocated to the receptor, β‐arr is believed to undergo important conformational rearrangement necessary for its downstream actions. To probe these changes in living cells, we constructed an intramolecular bioluminescence resonance energy transfer (BRET)‐based biosensor, in which β‐arr is sandwiched between the Renilla luciferase (Luc) and the yellow fluorescent protein (YFP). We show that the intramolecular BRET between Luc and YFP was significantly increased following GPCR activation, suggesting a conformational rearrangement bringing the amino terminus and carboxyl terminus of β‐arr in closer proximity. Kinetic analysis showed that this conformational change follows the initial β‐arr/receptor engagement. In addition to providing new insights into the agonist‐induced conformational rearrangements of β‐arr in living cells, the double‐brilliance β‐arr offers a universal biosensor for GPCR activation, allowing the study of native receptors in large‐scale screening analysis.