Cholesterol-induced conformational changes in the oxytocin receptor.

Recent studies suggest that cholesterol binding is widespread among GPCRs (G-protein-coupled receptors). In the present study, we analysed putative cholesterol-induced changes in the OTR [OT (oxytocin) receptor], a prototype of cholesterol-interacting GPCRs. For this purpose, we have created recombinant OTRs that are able to bind two small-sized fluorescence-labelled ligands simultaneously. An OTR antagonist was chosen as one of the ligands. To create a second ligand-binding site, a small-sized α-BTB (bungarotoxin binding) site was inserted at the N-terminus or within the third extracellular loop of the OTR. All receptor constructs were functionally active and bound both ligands with high affinity in the nanomolar range. Measurements of the quenching behaviour, fluorescence anisotropy and energy transfer of both receptor-bound ligands were performed to monitor receptor states at various cholesterol concentrations. The quenching studies suggested no major changes in the molecular environment of the fluorophores in response to cholesterol. The fluorescence anisotropy data indicated that cholesterol affects the dynamics or orientation of the antagonist. The energy transfer efficiency between both ligands clearly increased with increasing cholesterol. Overall, cholesterol induced both a changed orientation and a decreased distance of the receptor-bound ligands, suggesting a more compact receptor state in association with cholesterol.