The Effect of Lipid Oxidation on the Activity of the Membrane Protein Adenosine 2A Receptor

Abstract

The Adenosine 2A receptor (A2AR) is a prototypical G-protein coupled receptor studied as a drug target to treat inflammatory and neurodegenerative diseases involving oxidative stress. Lipids surrounding membrane proteins are important modulators of their conformation and activity; during oxidative stress these lipids can be oxidised and affect protein function. Understanding of the interaction of unoxidised and oxidised lipids with the  A2AR and its consequences is limited, so the aim of this work was to address this knowledge gap. Recombinant human C-terminal-truncated A2AR was overexpressed in Pichia pastoris SMD1163 and solubilized with the co-polymer styrene-maleic acid (SMA2000) into SMA lipid particles (SMALPs) or the detergent n-Dodecyl β-D-maltoside (DDM). The purified receptor was obtained in its native lipid environment (A2AR-SMALP) or surrounded by detergent micelles (A2AR-DDM), which were identified by LC-MS/MS. Lipids in the A2AR-SMALP were characterised, and compared to those in bulk membranes and SMA-solubilized lipids by LC-MS/MS. The effects of oxidative treatments on protein conformation, thermostability, and ligand binding were studied using Trp fluorescence, CPM fluorescence, and radioligand-binding assays. Additionally, the A2AR was overexpressed in HEK293T cells to study the effect of oxidation on receptor function by measuring the cAMP levels. A2AR-SMALP was purified from P. pastoris proteins, whereas A2AR-DDM co-eluted with alcohol dehydrogenase (mADH) contaminant. These were identified with low sequence coverage, which limited the analysis of protein lipoxidation. A2AR-SMALP was enriched in unsaturated anionic phospholipids compared to bulk membranes, which contribute to receptor activity. A2AR-SMALP was folded and able to undergo conformational changes under physiological and oxidising conditions, and its thermostability and ligand-binding capability were not affected by oxidation. cAMP levels were upregulated under oxidative conditions, and its production increased with an A2AR agonist in a dose-dependent manner, suggesting A2AR upregulation during oxidation. This study demonstrated the feasibility of characterizing membrane proteins in their native lipid environment using SMALPs under physiological and oxidative conditions, bringing insights into their roles in inflammatory conditions.