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 A2A receptor (A2AR) 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 A2AR 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 A2AR, 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.

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 A2A receptor (A2AR) in reconstituted phospholipid bilayers. GTP hydrolysis assays show that cholesterol is a weak positive allosteric modulator of A2AR, as seen through enhanced basal signaling and a small decrease in agonist EC50. Fluorine nuclear magnetic resonance (19F NMR) spectroscopy suggests that this enhancement arises from an increase in the receptor's active state populations and stronger G protein coupling. 19F NMR of fluorinated cholesterol analogs reveals transient and non-specific interactions with A2AR, indicating a lack of high-affinity binding sites or direct allosteric modulation. This is confirmed by computational analysis which suggests that cholesterol contacts confer a weak and possibly negative allosteric effect. The combined results suggest that the observed cholesterol allostery in A2AR is likely a result of indirect membrane effects through cholesterol-mediated changes in membrane properties, as shown by membrane fluidity measurements and high-pressure NMR.

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