Monitoring agonist-promoted conformational changes of beta-arrestin in living cells by intramolecular BRET.
Recruitment of beta-arrestin (beta-arr) to agonist-stimulated G-protein-coupled receptors (GPCRs) has a crucial role in controlling signalling efficacy and selectivity. When translocated to the receptor, beta-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 beta-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 beta-arr in closer proximity. Kinetic analysis showed that this conformational change follows the initial beta-arr/receptor engagement. In addition to providing new insights into the agonist-induced conformational rearrangements of beta-arr in living cells, the double-brilliance beta-arr offers a universal biosensor for GPCR activation, allowing the study of native receptors in large-scale screening analysis.
Charest
Pascale G
PG
Department of Biochemistry and Groupe de Recherche sur le Système Nerveux Autonome, Université de Montréal, Montréal, Québec H3C 3J7, Canada.
Terrillon
Sonia
S
Bouvier
Michel
M
D019390:Arrestins / Q000737:chemistry* / Q000378:metabolism; D001426:Bacterial Proteins / Q000378:metabolism; D015374:Biosensing Techniques / Q000379:methods*; D002460:Cell Line; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D049410:Luciferases, Renilla / Q000378:metabolism; D008164:Luminescent Proteins / Q000378:metabolism; D008856:Microscopy, Fluorescence; D011487:Protein Conformation; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism*; D015398:Signal Transduction; D000071557:beta-Arrestins
D019390:Arrestins; D001426:Bacterial Proteins; D008164:Luminescent Proteins; D043562:Receptors, G-Protein-Coupled; D000071557:beta-Arrestins; C054562:yellow fluorescent protein, Bacteria; D049410:Luciferases, Renilla