The orexin OX1 receptor exists predominantly as a homo-dimer in the basal state: Potential regulation of receptor organisation by both agonist and antagonist ligands

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.