title: A Molecular Mechanism for Sequential Activation of a G Protein-Coupled Receptor.
abstract: Ligands targeting G protein-coupled receptors (GPCRs) are currently classified as either orthosteric, allosteric, or dualsteric/bitopic. Here, we introduce a new pharmacological concept for GPCR functional modulation: sequential receptor activation. A hallmark feature of this is a stepwise ligand binding mode with transient activation of a first receptor site followed by sustained activation of a second topographically distinct site. We identify 4-CMTB (2-(4-chlorophenyl)-3-methyl-N-(thiazol-2-yl)butanamide), previously classified as a pure allosteric agonist of the free fatty acid receptor 2, as the first sequential activator and corroborate its two-step activation in living cells by tracking integrated responses with innovative label-free biosensors that visualize multiple signaling inputs in real time. We validate this unique pharmacology with traditional cellular readouts, including mutational and pharmacological perturbations along with computational methods, and propose a kinetic model applicable to the analysis of sequential receptor activation. We envision this form of dynamic agonism as a common principle of nature to spatiotemporally encode cellular information.
authors.affiliation: Molecular-, Cellular- and Pharmacobiology Section, Institute for Pharmaceutical Biology, University of Bonn, 53115 Bonn, Germany. Electronic address: grundmann@uni-bonn.de.
authors.forename: Manuel
authors.initials: M
authors.lastname: Grundmann
authors.affiliation: Molecular Therapeutics, School of Pharmacy, Medical Biology Centre, Queen's University, Belfast BT7 1NN Northern Ireland.
authors.forename: Irina G
authors.initials: IG
authors.lastname: Tikhonova
authors.affiliation: Molecular Pharmacology Group, Institute of Molecular, Cell and Systems Biology, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow G12 8QQ Scotland.
authors.forename: Brian D
authors.initials: BD
authors.lastname: Hudson
authors.affiliation: Molecular Pharmacology Group, Institute of Molecular, Cell and Systems Biology, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow G12 8QQ Scotland; Molecular Cardiology Division, Victor Chang Cardiac Research Institute, Faculty of Medicine, University of New South Wales, Darlinghurst, NSW 2010, Australia.
authors.forename: Nicola J
authors.initials: NJ
authors.lastname: Smith
authors.affiliation: Pharmacology and Toxicology, University of Bonn, 53347 Bonn, Germany.
authors.forename: Klaus
authors.initials: K
authors.lastname: Mohr
authors.affiliation: Department of Physics, Chemistry and Pharmacy, University of Southern Denmark, 5230 Odense M, Denmark.
authors.forename: Trond
authors.initials: T
authors.lastname: Ulven
authors.affiliation: Molecular Pharmacology Group, Institute of Molecular, Cell and Systems Biology, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow G12 8QQ Scotland.
authors.forename: Graeme
authors.initials: G
authors.lastname: Milligan
authors.affiliation: Department of Pharmacology, University of North Carolina School of Medicine, Chapel Hill, NC 27599, USA.
authors.forename: Terry
authors.initials: T
authors.lastname: Kenakin
authors.affiliation: Molecular-, Cellular- and Pharmacobiology Section, Institute for Pharmaceutical Biology, University of Bonn, 53115 Bonn, Germany. Electronic address: kostenis@uni-bonn.de.
authors.forename: Evi
authors.initials: E
authors.lastname: Kostenis
mesh_terms: D000494:Allosteric Regulation / Q000187:drug effects; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D006801:Humans; D008024:Ligands; D008958:Models, Molecular; D015394:Molecular Structure; D043562:Receptors, G-Protein-Coupled / Q000819:agonists*; D054852:Small Molecule Libraries / Q000737:chemistry / Q000494:pharmacology*; D013329:Structure-Activity Relationship; D013997:Time Factors
chemical_list: D008024:Ligands; D043562:Receptors, G-Protein-Coupled; D054852:Small Molecule Libraries