Short Chain Lipids Maintain Adenosine A2AR Ligand Binding in the Absence of Cholesterol

Abstract

Evidence of preferential interactions between sphingolipids and cholesterol, and the identification of cholesterol binding pockets in some membrane proteins highlights the importance of lipids in the regulation of membrane protein function. However, the mechanism through which these lipid-protein interactions modify the conformation and activity of membrane proteins is not yet understood. In vitro, cholesterol is known to affect micelle physical properties and has been found to interact directly with some membrane proteins. Previous studies of a G protein-coupled receptor (GPCR), human adenosine A2A receptor (hA2AR), purified using the detergent dodecylmaltoside (DDM) showed that the presence of a cholesterol analog was critical for ligand binding and the structural stability of the receptor. Here, we present biophysical characterization and ligand binding studies of the hA2AR purified using a short chain lipid (1,2-dihexanoyl-sn-glycero-3-phosphocholine, DHPC), which indicate that cholesterol is not needed to maintain the functionality and stability of the hA2AR in this lipid system. Differences in stability and ligand binding affinity are observed when the receptor is purified using these detergent and lipid systems, emphasizing the importance of the solubilization environment on receptor functionality. We will highlight the use of fluorescence polarization in characterizing this stability, for example, following protein storage or lipid addition.