Allosteric modulation of the adenosine A2A receptor by cholesterol

Abstract

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.

Ribose and Non-Ribose A2A Adenosine Receptor Agonists: Do They Share the Same Receptor Recognition Mechanism?

“…A second limitation is represented by the fact that the lipid constitution of the phospholipid membrane does not include the presence of cholesterol, which could exert some form of allosteric modulation on the AR [ 22 ].…”

Allosteric Modulation of Adenosine A2A Receptors as a New Therapeutic Avenue

“…Subsequent simulations of the molecular dynamics, structureactivity relationship studies of the ligand, and nuclear magnetic resonance analyses in solution revealed the unique binding mode and antagonistic properties of Fg754, which is distinctly different from HMA [72]. In addition, cholesterol is reported to be a weak PAM of A 2A Rs [73].…”

“…Increased the dissociation rate of the antagonist ZM-241,385 at rat A2ARs [71]. Decreased the dissociation rate of the agonist NECA at A 2A Rs-embedded nanodiscs [73].…”

Mapping the conformational landscape of the stimulatory heterotrimeric G protein

“…Cloning of the wild type G s α construct has been described previously (Huang et al, 2022). The human GNAS2 gene was subcloned into a pET15b vector immediately downstream of an open reading frame for hexahistidine-tagged maltose binding protein (MBP) and tobacco etch virus (TEV) cleavage site.…”

“…Gβγ, and A 2A R were expressed and puri ed as previously described (Huang et al, 2021). Empty nanodiscs were prepared as previously described (Huang et al, 2022). Both the A 2A R and the empty nanodiscs were reconstituted using the MSPΔH5 membrane scaffold protein (Hagn et al, 2013) and a 3:2 ratio of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) to 1-palmitoyl-2-oleoyl-sn-glycero-3phospho-(1'-rac-glycerol) (POPG).…”