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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.
Highlights
In mammalian cell membranes, cholesterol accounts for ~5–45% of the total lipid content across different cell types and subcellular components (Casares et al, 2019; Ingólfsson et al, 2017)
19F labeling site we find that cholesterol is a weak positive allosteric modulator of A2A receptor (A2AR)
We reconstituted A2AR in nanodiscs containing a 3:2 ratio of 1-palmitoyl-2 -o leoyl-s n-glycero-3-phosphocholine (POPC) and 1-palmit oyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-g lycerol) (POPG), supplemented with different amounts of cholesterol
Summary
Cholesterol accounts for ~5–45% of the total lipid content across different cell types and subcellular components (Casares et al, 2019; Ingólfsson et al, 2017). It is a critical metabolic precursor to steroid hormones, bile salts, and vitamin D, while numerous cardiovascular and nervous system disorders are attributed to abnormalities in cholesterol metabolism (Arsenault et al, 2009; Martín et al, 2014). The rigid planar structure of cholesterol promotes ordering of. Structural Biology and Molecular Biophysics bilayer lipids, modulating membrane fluidity and thickness. Cholesterol drives the formation of raft-like microdomains and commonly interacts with membrane proteins as a ligand or allosteric modulator (Hulce et al, 2013)
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