title: Quantifying biased signaling in GPCRs using BRET-based biosensors.
abstract: There has been a growing appreciation that G protein-coupled receptor (GPCR) functional selectivity (viz. biased signaling), in particular between G protein- and β-arrestin-dependent signaling, can be achieved with specific ligands, and that such directed signaling represents a promising avenue for improving drug efficacy and therapy. Thus, for any given GPCRs it is important to define means to pharmacologically characterize and classify drugs for their propensity to bias signaling. Here we describe an experimental protocol and step-by-step approach to assess functional selectivity between Gαq and β-arrestin-dependent responses using the prototypical angiotensin II (AngII) type 1 receptor (AT1R) expressed in HEK 293 cells. The protocol describes the expression of Bioluminescence Resonance Energy Transfer (BRET) sensors for either Gαq or β-arrestin with AT1R, and the use of the operational model of pharmacological agonism to quantify ligand bias. Such methods are equally applicable to other GPCRs and their downstream signaling effectors.
authors.affiliation: Department of Medicine, McGill University Health Center Research Institute, McGill University, Montréal, Canada.
authors.forename: Yoon
authors.initials: Y
authors.lastname: Namkung
authors.affiliation: Department of Medicine, McGill University Health Center Research Institute, McGill University, Montréal, Canada.
authors.forename: Olivier
authors.initials: O
authors.lastname: Radresa
authors.affiliation: Department of Medicine, McGill University Health Center Research Institute, McGill University, Montréal, Canada.
authors.forename: Sylvain
authors.initials: S
authors.lastname: Armando
authors.affiliation: Department of Pharmacology and Therapeutics, McGill University, Montréal, Canada.
authors.forename: Dominic
authors.initials: D
authors.lastname: Devost
authors.affiliation: Department of Biochemistry, Institute for Research in Immunology and Cancer, Université de Montréal, Montréal, Canada.
authors.forename: Alexandre
authors.initials: A
authors.lastname: Beautrait
authors.affiliation: Department of Biochemistry, Institute for Research in Immunology and Cancer, Université de Montréal, Montréal, Canada.
authors.forename: Christian
authors.initials: C
authors.lastname: Le Gouill
authors.affiliation: Department of Medicine, McGill University Health Center Research Institute, McGill University, Montréal, Canada; Department of Pharmacology and Therapeutics, McGill University, Montréal, Canada; Department of Anatomy and Cell Biology, McGill University, Montréal, Canada. Electronic address: stephane.laporte@mcgill.ca.
authors.forename: Stephane A
authors.initials: SA
authors.lastname: Laporte
mesh_terms: D000804:Angiotensin II / Q000378:metabolism; D060807:Bioluminescence Resonance Energy Transfer Techniques / Q000379:methods*; D015374:Biosensing Techniques / Q000379:methods*; D004305:Dose-Response Relationship, Drug; D057809:HEK293 Cells; D006801:Humans; D008024:Ligands; D044140:Receptor, Angiotensin, Type 1 / Q000032:analysis* / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology*
chemical_list: C544065:AGTR1 protein, human; D008024:Ligands; D044140:Receptor, Angiotensin, Type 1; D000804:Angiotensin II
keywords: Angiotensin II type 1 receptor; BRET; Biased signaling; G proteins; Operational model; β-Arrestin