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.

This dataset contains source data files for the article "Allosteric modulation of the adenosine A2A receptor by cholesterol", published in eLife. Included here are Excel and Prism files for Figure 2 (GTPase assays, cholesterol-NECA titration), Figure 5 (rigidity transmission allostery), Figure 7b and c (Laurdan fluorescence), Figure 2—figure supplement 1 (DLS), and Figure 6—figure supplement 1 (GTPase assays, effects of 19F-cholesterol and ligands on A2AR activity). Methods details are given in the article.

GraphPad Prism and Microsoft Excel