The orexin OX₁ receptor exists predominantly as a homodimer in the basal state: potential regulation of receptor organization by both agonist and antagonist ligands

Xu, T.‑R., Ward, R. J., Pediani, J. D. and Milligan, G. (2011) The orexin OX₁ receptor exists predominantly as a homodimer in the basal state: potential regulation of receptor organization by both agonist and antagonist ligands. Biochemical Journal, 439(1), pp. 171-183. (doi: 10.1042/BJ20110230)

Abstract

It is unclear what proportion of a G-protein-coupled receptor is present in cells as dimers or oligomers. Saturation bioluminescence resonance energy transfer studies demonstrated the orexin OX₁ receptor to be present in such complexes. Forms of this receptor containing a minimal epitope tag, with the C-terminus linked to yellow fluorescent protein or modified at the N-terminus to incorporate a SNAP tag, migrated in SDS/PAGE gels as monomers, indicating a lack of covalent interactions. Solubilization with dodecylmaltoside, followed by Blue native-PAGE, indicated that the receptor constructs migrated predominantly as anticipated for dimeric species with evidence for further, higher-order, complexes, and this was true over a wide range of expression levels. Addition of SDS prior to separation by Blue native-PAGE resulted in much of the previously dimeric, and all of the higher-order, complexes being dissociated and now migrating at the size predicted for monomeric species. Expression of forms of the OX₁ receptor capable of generating enzyme complementation confirmed that solubilization itself did not result in interaction artefacts. Addition of the endogenous agonist orexin A enhanced the proportion of higher-order OX₁ receptor complexes, whereas selective OX₁ antagonists increased the proportion the OX₁ receptor migrating in Blue native-PAGE as a monomer. The antagonist effects were produced in a concentration-dependent manner, consistent with the affinity of the ligands for the receptor. Homogeneous time-resolved fluorescence resonance energy transfer studies using Tag-Lite™ reagents on cells expressing the SNAP-tagged OX₁ receptor identified cell-surface OX₁ homomers. Predominantly at low receptor expression levels, orexin A increased such fluorescence resonance energy transfer signals, also consistent with ligand-induced reorganization of the homomeric complex.

• Item Type: Articles
• Status: Published
• Refereed: Yes
• 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 Psychology & Neuroscience
• Journal Name: Biochemical Journal
• Publisher: Portland Press Ltd.
• ISSN: 0264-6021
• ISSN (Online): 1470-8728
• Published Online: 14 September 2011

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Project Code	Award No	Project Name	Principal Investigator	Funder's Name	Funder Ref	Lead Dept
51063	1	The organisational structure of class A GPCRs: implications for function and drug design	Graeme Milligan	Medical Research Council (MRC)	G0900050	Institute of Neuroscience and Psychology