Allosteric modulation of the adenosine A(2A) receptor by cholesterol

Cholesterol is a major component of the cell membrane and commonly regulates membrane protein function. Here, we investigate how cholesterol modulates the conformational equilibria and signaling of the adenosine A(2A) receptor (A(2A)R) in reconstituted phospholipid nanodiscs. This model system conveniently excludes possible effects arising from cholesterol-induced phase separation or receptor oligomerization and focuses on the question of allostery. GTP hydrolysis assays show that cholesterol weakly enhances the basal signaling of A(2A)R while decreasing the agonist EC(50). Fluorine nuclear magnetic resonance ((19)F NMR) spectroscopy shows that this enhancement arises from an increase in the receptor’s active state population and a G-protein-bound precoupled state. (19)F NMR of fluorinated cholesterol analogs reveals transient interactions with A(2A)R, indicating a lack of high-affinity binding or direct allosteric modulation. The combined results suggest that the observed allosteric effects are largely indirect and originate from cholesterol-mediated changes in membrane properties, as shown by membrane fluidity measurements and high-pressure NMR.