Investigating membrane lipid composition on A2A adenosine receptor state-dependent dynamics by single-molecule FRET

Sreyashi Das1, Alyssa Gonneville2, ShuShu Wei2, Sriram Tiruvadi-Krishnan2, Rajan Lamichhane2, Matthew T Eddy1

1. Department of Chemistry, College of Liberal Arts and Sciences, University of Florida, Gainesville, FL, USA.
2. Department of Biochemistry & Cellular and Molecular Biology, College of Arts and Sciences, University of Tennessee, Knoxville, TN, USA.

Abstract:

G protein-coupled receptors (GPCRs) are transmembrane sensory proteins crucial for most biological processes and major targets of many pharmaceutics.  Most studies of GPCR structure and conformational dynamics have focused on receptor-ligand interactions, ignoring the important role that membrane lipids play in regulating GPCR structure-function relationships.  Growing evidence indicates that plasma membranes in which GPCRs function also regulate GPCRs via receptor-lipid interactions.  The human A2A receptor (A2AAR) is a class A GPCR endogenously activated by adenosine and an important target in treating cancer due to the very high concentration of adenosine in tumor microenvironments.  We leveraged single-molecule fluorescence (SMF) to visualize how lipids can alter the structural plasticity of  A2AAR in lipid nanodiscs of varying lipid composition.  Samples containing two complementary fluorescence probes allowed us to record single-molecule Förster resonance energy transfer (smFRET) measurements to monitor changes in A2AAR helical positions as a function of drug efficacy and lipid composition.  These data are evaluated in the context of a growing body of literature from cryo-EM and NMR spectroscopy that highlight the crucial but understudied role of lipids in GPCR signaling.