Intramolecular fluorescence resonance energy transfer (FRET) sensors of the orexin OX1 and OX2 receptors identify slow kinetics of agonist activation

Xu, T.-R., Ward, R. J., Pediani, J. D. and Milligan, G. (2012) Intramolecular fluorescence resonance energy transfer (FRET) sensors of the orexin OX1 and OX2 receptors identify slow kinetics of agonist activation. Journal of Biological Chemistry, 287, pp. 14937-14949. (doi: 10.1074/jbc.M111.334300)

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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.

Item Type:Articles
Glasgow Author(s) Enlighten ID:Pediani, Dr John and Xu, Professor Tianrui and Ward, Dr Richard and Milligan, Professor Graeme
Authors: Xu, T.-R., Ward, R. J., Pediani, J. D., and Milligan, G.
College/School:College of Medical Veterinary and Life Sciences > School of Molecular Biosciences
Journal Name:Journal of Biological Chemistry
Publisher:American Society for Biochemistry and Molecular Biology, Inc.
ISSN (Online):1083-351X
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Project CodeAward NoProject NamePrincipal InvestigatorFunder's NameFunder RefLead Dept
510631The organisational structure of class A GPCRs: implications for function and drug designGraeme MilliganMedical Research Council (MRC)G0900050RI NEUROSCIENCE & PSYCHOLOGY