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)

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Publisher's URL: http://dx.doi.org/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 OX<sub>1</sub> 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 OX<sub>1</sub> 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 OX<sub>1</sub> receptor complexes, whereas selective OX<sub>1</sub> antagonists increased the proportion the OX<sub>1</sub> 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 OX<sub>1</sub> receptor identified cell-surface OX<sub>1</sub> 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:Milligan, Professor Graeme and Pediani, Dr John and Xu, Professor Tianrui and Ward, Dr Richard
Authors: Xu, T.‑R., Ward, R. J., Pediani, J. D., and Milligan, G.
College/School:College of Medical Veterinary and Life Sciences > Institute of Neuroscience and Psychology
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 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)G0900050Institute of Neuroscience and Psychology