title: Conformational Profiling of the AT1 Angiotensin II Receptor Reflects Biased Agonism, G Protein Coupling, and Cellular Context.
abstract: Here, we report the design and use of G protein-coupled receptor-based biosensors to monitor ligand-mediated conformational changes in receptors in intact cells. These biosensors use bioluminescence resonance energy transfer withRenillaluciferase (RlucII) as an energy donor, placed at the distal end of the receptor C-tail, and the small fluorescent molecule FlAsH as an energy acceptor, its binding site inserted at different positions throughout the intracellular loops and C-terminal tail of the angiotensin II type I receptor. We verified that the modifications did not compromise receptor localization or function before proceeding further. Our biosensors were able to capture effects of both canonical and biased ligands, even to the extent of discriminating between different biased ligands. Using a combination of G protein inhibitors and HEK 293 cell lines that were CRISPR/Cas9-engineered to delete Gαq, Gα11, Gα12, and Gα13or β-arrestins, we showed that Gαqand Gα11are required for functional responses in conformational sensors in ICL3 but not ICL2. Loss of β-arrestin did not alter biased ligand effects on ICL2P2. We also demonstrate that such biosensors are portable between different cell types and yield context-dependent readouts of G protein-coupled receptor conformation. Our study provides mechanistic insights into signaling events that depend on either G proteins or β-arrestin.
authors.affiliation: From the Department of Pharmacology and Therapeutics, McGill University, Montréal, Québec H3G 1Y6, Canada.
authors.forename: Dominic
authors.initials: D
authors.lastname: Devost
authors.affiliation: From the Department of Pharmacology and Therapeutics, McGill University, Montréal, Québec H3G 1Y6, Canada.
authors.forename: Rory
authors.initials: R
authors.lastname: Sleno
authors.affiliation: From the Department of Pharmacology and Therapeutics, McGill University, Montréal, Québec H3G 1Y6, Canada.
authors.forename: Darlaine
authors.initials: D
authors.lastname: Pétrin
authors.affiliation: From the Department of Pharmacology and Therapeutics, McGill University, Montréal, Québec H3G 1Y6, Canada.
authors.forename: Alice
authors.initials: A
authors.lastname: Zhang
authors.affiliation: the Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai, Miyagi 980-8578, Japan.
authors.forename: Yuji
authors.initials: Y
authors.lastname: Shinjo
authors.affiliation: From the Department of Pharmacology and Therapeutics, McGill University, Montréal, Québec H3G 1Y6, Canada.
authors.forename: Rakan
authors.initials: R
authors.lastname: Okde
authors.affiliation: the Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai, Miyagi 980-8578, Japan.|the Japan Agency for Medical Research and Development-Core Research for Evolutional Science and Technology (AMED-CREST), Chiyoda-ku, Tokyo 100-0004, Japan, and.
authors.forename: Junken
authors.initials: J
authors.lastname: Aoki
authors.affiliation: the Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai, Miyagi 980-8578, Japan.|the Precursory Research for Embryonic Science and Technology (PRESTO), Japan Science and Technology Agency (JST), Kawaguchi, Saitama 332-0012, Japan.
authors.forename: Asuka
authors.initials: A
authors.lastname: Inoue
authors.affiliation: From the Department of Pharmacology and Therapeutics, McGill University, Montréal, Québec H3G 1Y6, Canada, terence.hebert@mcgill.ca.
authors.forename: Terence E
authors.initials: TE
authors.lastname: Hébert
mesh_terms: D000804:Angiotensin II / Q000378:metabolism; D060846:Cell Engineering / Q000379:methods; D002460:Cell Line; D031541:Fluorescence Resonance Energy Transfer; D019204:GTP-Binding Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D006801:Humans; D008024:Ligands; D008722:Methods; D011487:Protein Conformation; D044140:Receptor, Angiotensin, Type 1 / Q000737:chemistry*; D015398:Signal Transduction; D000071557:beta-Arrestins / Q000378:metabolism
chemical_list: D008024:Ligands; D044140:Receptor, Angiotensin, Type 1; D000071557:beta-Arrestins; D000804:Angiotensin II; D019204:GTP-Binding Proteins
keywords: G protein; G protein-coupled receptor (GPCR); angiotensin II; cell signaling; signaling