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_2AR) 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_2AR while decreasing the agonist EC₅₀. Fluorine nuclear magnetic resonance (¹⁹F NMR) spectroscopy shows that this enhancement arises from an increase in the receptor's active state population and a G-protein-bound precoupled state. ¹⁹F NMR of fluorinated cholesterol analogs reveals transient interactions with A_2AR, 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.