The ligand-bound state of a G protein-coupled receptor stabilizes the interaction of functional cholesterol molecules.

Abstract Cholesterol is a major component of mammalian plasma membranes that affects the physical properties of the lipid bilayer but also the function of many membrane proteins including G protein-coupled receptors (GPCRs). The oxytocin receptor (OXTR) is involved in parturition and lactation of mammals, and in their emotional and social behaviors. Cholesterol acts on OXTR as an allosteric modulator inducing a high-affinity state for orthosteric ligands through a molecular mechanism that has yet to be determined. Using the ion channel-coupled receptor (ICCR) technology, we developed a functional assay of cholesterol modulation of GPCRs that is independent of intracellular signaling pathways and operational in living cells. Using this assay, we discovered a stable binding of cholesterol molecules to the receptor when it adopts an orthosteric ligand-bound state. This stable interaction preserves the cholesterol-dependent activity of the receptor in cholesterol-depleted membranes. This mechanism was confirmed using time-resolved FRET (TR-FRET) experiments on WT OXTR expressed in CHO cells. Consequently, a positive cross-regulation sequentially occurs in OXTR between cholesterol and orthosteric ligands. The possibility to selectively stabilize the interaction of functional cholesterol molecules, also offers new opportunities for the challenging identification of the functional cholesterol binding site(s) and for deeper understanding of the molecular mechanisms.