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_2AR) 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_2AR-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_2AR interior in a biotinylation assay. Overall, we show that cholesterol’s impact on A_2AR-binding affinity goes beyond pure allosteric modulation and unveils a new interaction mode between cholesterol and the A_2AR that could potentially apply to other GPCRs.

Adenosine, hypoxanthine, xanthine, guanosine and inosine levels were assessed by HPLC, and the activity of related enzymes 5′‐nucleotidase (5′‐NT), adenosine deaminase (ADA) and purine nucleoside phosphorylase (PNP) measured in frontal (FC), parietal (PC) and temporal (TC) cortices at different stages of disease progression in Alzheimer's disease (AD) and in age‐matched controls. Significantly decreased levels of adenosine, guanosine, hypoxanthine and xanthine, and apparently less inosine, are found in FC from the early stages of AD; PC and TC show an opposing pattern, as adenosine, guanosine and inosine are significantly increased at least at determinate stages of AD whereas hypoxanthine and xanthine levels remain unaltered. 5′‐NT is reduced in membranes and cytosol in FC mainly at early stages but not in PC, and only at advanced stages in cytosol in TC. ADA activity is decreased in AD when considered as a whole but increased at early stages in TC. Finally, PNP activity is increased only in TC at early stages. Purine metabolism alterations occur at early stages of AD independently of neurofibrillary tangles and β‐amyloid plaques. Alterations are stage dependent and region dependent, the latter showing opposite patterns in FC compared with PC and TC. Adenosine is the most affected of the assessed purines.