G protein-coupled receptors (GPCRs) are embedded in phospholipids that strongly influence drug-stimulated signaling. Anionic lipids are particularly important for GPCR signaling complex formation, but a mechanism for this role is not understood. Using NMR spectroscopy, we explore the impact of anionic lipids on the function-related conformational equilibria of the human A2A adenosine receptor (A2AAR) in bilayers containing defined mixtures of zwitterionic and anionic phospholipids. Anionic lipids prime the receptor to form complexes with G proteins through a conformational selection process. Without anionic lipids, signaling complex formation proceeds through a less favorable induced fit mechanism. In computational models, anionic lipids mimic interactions between a G protein and positively charged residues in A2AAR at the receptor intracellular surface, stabilizing a pre-activated receptor conformation. Replacing these residues strikingly alters the receptor response to anionic lipids in experiments. High sequence conservation of the same residues among all GPCRs supports a general role for lipid-receptor charge complementarity in signaling.
Highlights d Single-molecule fluorescence observes slow dynamic behavior of A 2A AR complexes d Three different fluorescence emission states observed for A 2A AR complexes with agonists d Reversible transitions among different states occurred in a sequential order d A functionally critical transition state not represented among available structures
G protein-coupled receptors (GPCRs) bind a broad array of extracellular molecules and transmit intracellular signals that initiate physiological responses. The signal transduction functions of GPCRs are inherently related to their structural plasticity, which can be experimentally observed by spectroscopic techniques. Nuclear magnetic resonance (NMR) spectroscopy in particular is an especially advantageous method to study the dynamic behavior of GPCRs. The success of NMR studies critically relies on the production of functional GPCRs containing stable-isotope labeled probes, which remains a challenging endeavor for most human GPCRs. We report a protocol for the production of the human histamine H1 receptor (H1R) in the methylotrophic yeast Pichia pastoris for NMR experiments. Systematic evaluation of multiple expression parameters resulted in a ten-fold increase in the yield of expressed H1R over initial efforts in defined media. The expressed receptor could be purified to homogeneity and was found to respond to the addition of known H1R ligands. Two-dimensional transverse relaxation-optimized spectroscopy (TROSY) NMR spectra of stable-isotope labeled H1R show well-dispersed and resolved signals consistent with a properly folded protein, and 19F-NMR data register a response of the protein to differences in efficacies of bound ligands.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.