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 bilayers. GTP hydrolysis assays show that cholesterol is a weak positive allosteric modulator of A_2AR, 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 A_2AR, 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 A_2AR 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.