Structural Insights into Activation of a Human G Protein‐Coupled Receptor by Membrane Phospholipids

The 826 human G protein‐coupled receptors (GPCRs) are involved in nearly every physiological process and comprise the largest class of “druggable” proteins. For over the past decade, crystal and cryo‐EM structures have focused on revealing molecular details of GPCR interactions with small molecules and intracellular partner proteins, which are well‐established regulators of GPCR function. More recently, literature data from biological and biophysical studies have demonstrated that phospholipids found in the endogenous membrane environment also control GPCR function and act as allosteric modulators. In particular, phospholipids with charged headgroups have been reported to be especially important regulators of GPCR function and facilitate the formation of signaling complexes. Using nuclear magnetic resonance (NMR) spectroscopy in aqueous solutions, we investigated the structural basis for the regulation of GPCR activity by phospholipids in nanodiscs containing the human A 2A adenosine receptor (A 2A AR), a representative class A GPCR. Lipid nanodiscs containing mixtures of uncharged and charged phospholipids were produced, yielding homogeneous preparations over a wide range of lipid compositions. NMR data of uniformly stable‐isotope labeled A 2A AR showed the receptor was properly folded in lipid nanodiscs and shared a similar global conformation to detergent micelle preparations of A 2A AR. Using 19 F NMR with conformationally‐sensitive probes located at the A 2A AR intracellular surface, we systematically investigated the influence of lipid composition over a wide range of mixtures of charged and uncharged phospholipids. A 2A AR complexes with antagonists showed almost no response to variation in lipid composition. In striking contrast to this, A 2A AR complexes with agonists showed a clear response to changes in the relative proportion of charged phospholipids. NMR data of agonist complexes in nanodiscs containing lower proportions of charged lipids were highly similar to data of antagonist complexes, showing a higher proportion of neutral lipids negated the efficacy of bound agonists. As the proportion of charged phospholipids increased, the relative population of an A 2A AR active conformation also increased. The dependence of the receptor response to bound agonists on lipid composition suggested specific interactions between lipid headgroups and charged amino acids at the A 2A AR intracellular surface underlie the regulation of activity. Results from this study indicate that lipid composition must be carefully selected in studies of GPCRs in nanodiscs. Our data also provide a potential molecular mechanism for how changes in membrane composition, due to disease for example, can directly impact the efficacy of drugs.