Characterizing GPCR Allostery by NMR Spectroscopy

Abstract

Forty percent of current pharmaceuticals target G protein-coupled receptors (GPCRs), a class of membrane proteins that regulate diverse biological processes including sensory perception, hormonal regulation, and immune response. Structurally, GPCRs exist in a dynamic ensemble of conformations spanning inactive, intermediate, and active states capable of engaging G proteins. The adenosine A2A receptor (A2AR) is a prototypical GPCR and a drug target for the treatment of inflammation, cancer, diabetes, and Parkinson's disease. While X-ray crystal structures reveal only an inactive and an active conformation, nuclear magnetic resonance (NMR) spectroscopy show that detergent-reconstituted A2AR exhibits at least four functional states spanning inactive, intermediate, and active states capable of engaging G proteins. Though mechanistically poorly understood, the lipid bilayer plays an important role in GPCR regulation. Several GPCRs are known to require membrane cholesterol for function, including A2AR. Here, we employ fluorine (19F) NMR to characterize A2AR activity and drug-response in a synthetic lipid bilayer (nanodisc). We show that cholesterol is an allosteric effector of A2AR through modulation of its functional states. This knowledge furthers our understanding of GPCR allostery and protein-membrane interactions.