Membrane cholesterol access into a G-protein-coupled receptor

Cholesterol is a key component of cell membranes with a proven modulatory role on the function and ligand-binding properties of G-protein-coupled receptors (GPCRs). Crystal structures of prototypical GPCRs such as the adenosine A(2A) receptor (A(2A)R) have confirmed that cholesterol finds stable binding sites at the receptor surface suggesting an allosteric role of this lipid. Here we combine experimental and computational approaches to show that cholesterol can spontaneously enter the A(2A)R-binding pocket from the membrane milieu using the same portal gate previously suggested for opsin ligands. We confirm the presence of cholesterol inside the receptor by chemical modification of the A(2A)R interior in a biotinylation assay. Overall, we show that cholesterol's impact on A(2A)R-binding affinity goes beyond pure allosteric modulation and unveils a new interaction mode between cholesterol and the A(2A)R that could potentially apply to other GPCRs.