Probing Allostery in Oligomeric M2 Receptors using smFRET

Abstract

G Protein-Coupled Receptors (GPCRs) possess two types of ligand binding sites: orthosteric and allosteric sites. The highly conserved structure of the orthosteric sites results in low levels of ligand selectivity across members of a single GPCR subfamily, posing challenges for drug discovery. An alternative approach is the development of receptor-specific allosteric ligands, which modulate responsiveness of GPCRs to orthosteric ligands. There is still an open question whether some GPCRs function as monomers, dimers or higher oligomers, in particular regarding cooperative interactions between orthosteric and allosteric sites in different units. Here we used the M2 muscarinic receptor (M2R) to quantify these inter-monomeric interactions using single-molecule Förster Resonance energy Transfer (smFRET). Oligomeric preparations of M2R have been designed to contain receptors with vacant orthosteric sites and intact allosteric sites. The smFRET construct added a small peptide sequence at the N-terminal (ybbR-tag) and a tetra-cysteine sequence the extracellular loop 2 (ECL2) (tetra-cysteine), which specifically bind to Co-enzyme a (CoA) conjugated fluorophores (AlexaFluoro®647, AF647), and the Fluorescein Arsenical Hairpin (FlAsH), respectively. To obtain a high-yield of fluorescently labelled receptors, detergent-based preparations of M2R were expressed and purified from suspension-based Expi-CHO cells. Immunoblotting analysis of purified M2R under non-denaturing conditions revealed that the receptor preparations are predominantly oligomeric in nature. Labelling of M2R with FlAsH and CoA-AF647 was confirmed using a series of steady-state and time-resolved fluorescence-based assays. Using the designed sensor, smFRET measurements were performed on monomeric M2 controls and on different oligomeric samples in the presence/absence of allosteric modulators. The data confirms the presence of allosteric interactions between constituent protomers within oligomers of M2, both in vitro and in live cells.