Muscarinic cholinergic receptors have served as prototypical models for understanding signaling and allostery in GPCRs. However, aspects of signaling related to the role of oligomers on the interaction between the receptor and G proteins and of the receptor and allosteric ligands were largely unexplored. Monomers and oligomers of the M2 muscarinic receptor therefore have been compared to identify properties that are gained in oligomers. Allosteric interactions were monitored by means of a FRET-based sensor of conformation at the allosteric site and in pharmacological assays involving mutants engineered to preclude intramolecular effects. Allosteric effects in monomers were exclusively negative and derived primarily from intramolecular electrostatic repulsion between the allosteric and orthosteric ligands. Allosteric effects in oligomers could be positive or negative, depending upon the allosteric-orthosteric pair, and they arose from interactions within and between the constituent protomers. The size of the oligomer was determined by single-particle photobleaching of immobilized complexes of eGFP-tagged M2 muscarinic receptor complexes. The method was calibrated using multiplexed controls comprising 1–4 copies of fused eGFPs. The patterns indicated that the receptor was a tetramer and the structural feasibility of a tetrameric complex was demonstrated in molecular dynamics (MD) simulations. Such MD simulations were also used to examine electrostatic, steric, and conformational determinants of allostery at the atomic level. The complex pharmacological behavior observed for oligomers and various engineered oligomers is characteristic of muscarinic receptors measured in binding assays from myocardial preparations from porcine tissue.

Allosteric regulation and oligomerization of muscarinic cholinergic receptors

254th National Meeting (2017)

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.