The human adenosine A_2A receptor co-isolates with anionic phospholipids in SMALPs from Pichia pastoris membranes

The adenosine A_2A receptor (A_2AR) is a GPCR that binds adenosine, leading to modulation of inflammation, platelet aggregation and vasodilation of coronary arteries. It is known that phospholipids surrounding GPCRs affect their activity but these effects have largely been determined by studies in reconstituted liposomes or by molecular dynamics simulations. Therefore, the aim was to investigate the phospholipid composition of the A_2AR in a native-like environment to determine its preference for specific phospholipids. Recombinant human truncated A_2AR (A316) was overexpressed in Pichia pastoris and solubilized by SMA2000 into SMA lipid particles (SMALPs). Phospholipids in the A_2AR-SMALP were characterised and compared to those in bulk membranes and SMA-solubilized lipids by LC-MS/MS. In all samples, species of phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylglycerol (PG), phosphatidylinositol (PI), lysophosphatidylcholine (LPC), and lysophosphatidylethanolamine (LPE) were detected. Principal component analysis showed clear differences in the lipid profiles of P. pastoris bulk membranes, SMA-solubilized fraction, and purified A2AR-SMALP. Bulk membranes were enriched in PC and LPE, whereas the SMA-solubilized fraction was enriched in PE, PS, and PG. In contrast, the A_2AR-SMALP was enriched in the anionic lipids, especially PG and PA but contained lower intensities of PE. The data provides the first practical evidence suggesting that the A_2AR co-isolates with anionic lipids that are positive regulators for GPCRs and has lower levels of the negative regulator PE than non-specific SMA-solubilized membrane. The study also provides evidence that SMA2000 does preferentially solubilize membrane regions containing certain phospholipids.