title: Designing BRET-based conformational biosensors for G protein-coupled receptors.
abstract: Ligand-biased signaling is starting to have significant impact on drug discovery programs in the pharmaceutical industry and has reinvigorated our understanding of pharmacological efficacy. As such, many investigators and screening campaigns are now being directed at a larger section of the signaling responses downstream of an individual G protein-coupled receptor. Many biosensor-based platforms have been developed to capture signaling signatures. Despite our growing ability to use such signaling signatures, we remain hampered by the fact that signaling signatures may be particular to an individual cell type and thus our platforms may not be portable from cell to cell, necessitating further cell-specific biosensor development. Here, we provide a complementary strategy based on capturing receptor-proximal conformational profiles using intra-molecular BRET-based sensors composed of a Renilla luciferase donor engineered into the carboxy-terminus and CCPGCC motifs which bind fluorescent hairpin arsenical dyes engineered into different positions in intracellular loop 3 of FP, the receptor for PGF2α. We discuss the design and optimization of such sensors for orthosteric and allosteric ligands.
authors.affiliation: Department of Pharmacology and Therapeutics, McGill University, Canada.
authors.forename: Rory
authors.initials: R
authors.lastname: Sleno
authors.affiliation: Department of Pharmacology and Therapeutics, McGill University, Canada.
authors.forename: Darlaine
authors.initials: D
authors.lastname: Pétrin
authors.affiliation: Department of Pharmacology and Therapeutics, McGill University, Canada.
authors.forename: Dominic
authors.initials: D
authors.lastname: Devost
authors.affiliation: Department of Pharmacology and Therapeutics, McGill University, Canada.
authors.forename: Eugénie
authors.initials: E
authors.lastname: Goupil
authors.affiliation: Department of Pharmacology and Therapeutics, McGill University, Canada.
authors.forename: Alice
authors.initials: A
authors.lastname: Zhang
authors.affiliation: Department of Pharmacology and Therapeutics, McGill University, Canada. Electronic address: terence.hebert@mcgill.ca.
authors.forename: Terence E
authors.initials: TE
authors.lastname: Hébert
mesh_terms: D058917:Adenosine A2 Receptor Antagonists / Q000138:chemical synthesis / Q000378:metabolism; D000595:Amino Acid Sequence; D060807:Bioluminescence Resonance Energy Transfer Techniques / Q000379:methods*; D015374:Biosensing Techniques / Q000379:methods*; D015195:Drug Design; D005456:Fluorescent Dyes / Q000138:chemical synthesis* / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D049410:Luciferases, Renilla / Q000138:chemical synthesis / Q000378:metabolism; D008969:Molecular Sequence Data; D017433:Protein Structure, Secondary; D043705:Receptor, Adenosine A2A / Q000032:analysis / Q000378:metabolism; D043562:Receptors, G-Protein-Coupled / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D058917:Adenosine A2 Receptor Antagonists; D005456:Fluorescent Dyes; D043705:Receptor, Adenosine A2A; D043562:Receptors, G-Protein-Coupled; D049410:Luciferases, Renilla
keywords: Biased signaling; Biosensor; G protein-coupled receptor; Resonance energy transfer; Screening