title: Time-resolved fluorescence ligand binding for G protein-coupled receptors.
abstract: G protein-coupled receptors (GPCRs) and their ligands are traditionally characterized by radioligand-binding experiments. These experiments yield excellent quantitative data, but have low temporal and spatial resolution. In addition, the use of radioligands presents safety concerns. Here we provide a general procedure for an alternative approach with high temporal and spatial resolution, based on Tb(+)-labeled fluorescent receptor ligands and time-resolved fluorescence resonance energy transfer (TR-FRET). This protocol and its design are detailed here for the parathyroid hormone receptor, a class B GPCR, and its fluorescently labeled 34-amino acid peptide ligand, but it can be easily modified for other receptors and their appropriately labeled ligands. We discuss three protocol options that use Tb(+)-labeled fluorescent ligands: a time-resolved fluorescence separation option that works on native receptors but requires separation of bound and unbound ligand; a TR-FRET option using SNAP-tag-labeled receptors for high-throughput screening; and a TR-FRET option that uses fluorescently labeled antibodies directed against an epitope engineered into the Flag-labeled receptors' N terminus. These protocol options can be used as standard procedures with very high signal-to-background ratios in order to characterize ligands and their receptors in living cells and in cell membranes via straightforward plate-reader measurements.
authors.affiliation: Institute of Pharmacology and Toxicology, University of Würzburg, Würzburg, Germany.
authors.forename: Alexander
authors.initials: A
authors.lastname: Emami-Nemini
authors.forename: Thomas
authors.initials: T
authors.lastname: Roux
authors.forename: Marion
authors.initials: M
authors.lastname: Leblay
authors.forename: Emmanuel
authors.initials: E
authors.lastname: Bourrier
authors.forename: Laurent
authors.initials: L
authors.lastname: Lamarque
authors.forename: Eric
authors.initials: E
authors.lastname: Trinquet
authors.forename: Martin J
authors.initials: MJ
authors.lastname: Lohse
mesh_terms: D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry; D008024:Ligands; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D043562:Receptors, G-Protein-Coupled / Q000032:analysis / Q000378:metabolism*; D013725:Terbium / Q000737:chemistry; D019379:Teriparatide / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D013997:Time Factors
chemical_list: D005456:Fluorescent Dyes; D008024:Ligands; D010446:Peptide Fragments; D043562:Receptors, G-Protein-Coupled; D013725:Terbium; D019379:Teriparatide; C035748:parathyroid hormone (1-34)amide