title: Intramolecular fluorescence resonance energy transfer (FRET) sensors of the orexin OX1 and OX2 receptors identify slow kinetics of agonist activation.
abstract: Intramolecular fluorescence resonance energy transfer (FRET) sensors able to detect changes in distance or orientation between the 3rd intracellular loop and C-terminal tail of the human orexin OX(1) and OX(2) G protein-coupled receptors following binding of agonist ligands were produced and expressed stably. These were directed to the plasma membrane and, despite the substantial sequence alterations introduced, in each case were able to elevate [Ca(2+)](i), promote phosphorylation of the ERK1/2 MAP kinases and become internalized effectively upon addition of the native orexin peptides. Detailed characterization of the OX(1) sensor demonstrated that it was activated with rank order of potency orexin A > orexin B > orexin A 16-33, that it bound antagonist ligands with affinity similar to the wild-type receptor, and that mutation of a single residue, D203A, greatly reduced the binding and function of orexin A but not antagonist ligands. Addition of orexin A to individual cells expressing an OX(1) sensor resulted in a time- and concentration-dependent reduction in FRET signal consistent with mass-action and potency/affinity estimates for the peptide. Compared with the response kinetics of a muscarinic M(3) acetylcholine receptor sensor upon addition of agonist, response of the OX(1) and OX(2) sensors to orexin A was slow, consistent with a multistep binding and activation process. Such sensors provide means to assess the kinetics of receptor activation and how this may be altered by mutation and sequence variation of the receptors.
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, United Kingdom.
authors.forename: Tian-Rui
authors.initials: TR
authors.lastname: Xu
authors.forename: Richard J
authors.initials: RJ
authors.lastname: Ward
authors.forename: John D
authors.initials: JD
authors.lastname: Pediani
authors.forename: Graeme
authors.initials: G
authors.lastname: Milligan
mesh_terms: D019943:Amino Acid Substitution; D015374:Biosensing Techniques / Q000379:methods; D031541:Fluorescence Resonance Energy Transfer; D057809:HEK293 Cells; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000494:pharmacology*; D007700:Kinetics; D020935:MAP Kinase Signaling System / Q000187:drug effects*; D019950:Mitogen-Activated Protein Kinase 1 / Q000235:genetics / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000235:genetics / Q000378:metabolism; D020125:Mutation, Missense; D009479:Neuropeptides / Q000494:pharmacology*; D064446:Orexin Receptors; D000068797:Orexins; D010455:Peptides / Q000494:pharmacology*; D010766:Phosphorylation / Q000187:drug effects; D017433:Protein Structure, Secondary; D043587:Receptor, Muscarinic M3 / Q000819:agonists / Q000235:genetics / Q000378:metabolism; D043562:Receptors, G-Protein-Coupled / Q000819:agonists* / Q000235:genetics / Q000378:metabolism*; D018013:Receptors, Neuropeptide / Q000819:agonists* / Q000235:genetics / Q000378:metabolism*
chemical_list: C000596229:HCRT protein, human; D047908:Intracellular Signaling Peptides and Proteins; D009479:Neuropeptides; D064446:Orexin Receptors; D000068797:Orexins; D010455:Peptides; D043587:Receptor, Muscarinic M3; D043562:Receptors, G-Protein-Coupled; D018013:Receptors, Neuropeptide; C535150:MAPK1 protein, human; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3