Intramolecular Fluorescence Resonance Energy Transfer (FRET) Sensors of the Orexin OX1 and OX2 Receptors Identify Slow Kinetics of Agonist Activation

• Published in: The Journal of biological chemistry Vol. 287; no. 18; pp. 14937 - 14949
• Main Authors: Xu, Tian-Rui, Ward, Richard J., Pediani, John D., Milligan, Graeme
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 27.04.2012; American Society for Biochemistry and Molecular Biology
• Subjects: 7-Helix receptor; Amino Acid Substitution; Biosensing Techniques - methods; Fluorescence Resonance Energy Transfer; Fluorescence Resonance Energy Transfer (FRET); G protein-coupled receptors (GPCR); HEK293 Cells; Humans; Insomnia; Intracellular Signaling Peptides and Proteins - pharmacology; Kinetics; MAP Kinase Signaling System - drug effects; Mitogen-Activated Protein Kinase 1 - genetics; Mitogen-Activated Protein Kinase 1 - metabolism; Mitogen-Activated Protein Kinase 3 - genetics; Mitogen-Activated Protein Kinase 3 - metabolism; Mutation, Missense; Narcolepsy; Neuropeptides - pharmacology; Orexin Receptors; Orexins; Peptides - pharmacology; Phosphorylation - drug effects; Protein Structure, Secondary; Receptor, Muscarinic M3 - agonists; Receptor, Muscarinic M3 - genetics; Receptor, Muscarinic M3 - metabolism; Receptors, G-Protein-Coupled - agonists; Receptors, G-Protein-Coupled - genetics; Receptors, G-Protein-Coupled - metabolism; Receptors, Neuropeptide - agonists; Receptors, Neuropeptide - genetics; Receptors, Neuropeptide - metabolism; Signal Transduction; Signal Transduction; Insomnia; 7-Helix receptor; G protein-coupled receptors (GPCR); Fluorescence Resonance Energy Transfer (FRET); Narcolepsy; Kinetics
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M111.334300

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 OX1 and OX2 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 [Ca2+]i, promote phosphorylation of the ERK1/2 MAP kinases and become internalized effectively upon addition of the native orexin peptides. Detailed characterization of the OX1 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 OX1 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 M3 acetylcholine receptor sensor upon addition of agonist, response of the OX1 and OX2 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. Background: Orexin receptors are potential targets for the treatment of narcolepsy and insomnia. Results: Intramolecular FRET sensor forms of these receptors were functional and able to report the kinetics of agonist-mediated activation. Conclusion: The 33 amino acid peptide orexin A activates the receptors slowly. Significance: Such sensors provide a unique means to explore the kinetics of receptor activation.