Allosteric modulation of the adenosine A2A 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 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.