Efficacy as an Intrinsic Property of the M₂ Muscarinic Receptor in Its Tetrameric State

Abstract

Muscarinic and other G protein-coupled receptors exhibit an agonist-specific heterogeneity that tracks efficacy and commonly is attributed to an effect of the G protein on an otherwise homogeneous population of sites. To examine this notion, M2 muscarinic receptors were purified from Sf9 cells as monomers devoid of G protein and reconstituted as tetramers in phospholipid vesicles. In assays with N-[(3)H]methylscopolamine, seven agonists revealed a dispersion of affinities indicative of two or more classes of sites. Unlabeled N-methylscopolamine and the antagonist quinuclidinylbenzilate recognized one class of sites; atropine recognized two classes with a preference that was the opposite of that of agonists, as indicated by the effects of N-ethylmaleimide. The data were inconsistent with an explicit model of constitutive asymmetry within a tetramer, and the fit improved markedly upon the introduction of cooperative interactions (P < 0.00001). Purified monomers appeared to be homogeneous or nearly so to all ligands except the partial agonists pilocarpine and McN-A-343, where heterogeneity emerged from intramolecular cooperativity between the orthosteric site and an allosteric site. The breadth of each dispersion was quantified empirically as the area between the fitted curve for two classes of sites and the theoretical curve for a single class of lower affinity, which approximates the expected effect of GTP if a G protein were present. The areas measured for 10 ligands at reconstituted tetramers correlated with similar measures of heterogeneity and with intrinsic activities reported previously for binding and response in natural membranes (P ≤ 0.00002). The data suggest that the GTP-sensitive heterogeneity typically revealed by agonists at M2 receptors is intrinsic to the receptor in its tetrameric state. It exists independently of the G protein, and it appears to arise at least in part from cooperativity between linked orthosteric sites.

Graded activation and free energy landscapes of a muscarinic G-protein–coupled receptor

“…Binding of ARC leads to significantly increased fluctuations in the ECL2 and orthosteric pocket. This is consistent with the fact that ARC possesses lower affinity than IXO (14,16,20). Thus, weaker interaction is formed between ARC and the receptor.…”

“…The simulations also revealed new low-energy states in the IXO/ARC-bound receptor upon removal the nanobody. Whereas the inverse agonist QNB with high binding affinity (∼0.06 nM) (14) remains tightly bound to the orthosteric site, the full and partial agonists with lower affinities, ∼5 μM for ARC (16,20) and ∼0.01 μM for IXO (14), exhibit significantly higher fluctuations. We have captured both dissociation and binding of an orthosteric ligand in a single all-atom GPCR simulation in the case of ARC binding to the M 2 receptor.…”

“…However, the allosteric site is located in the receptor extracellular vestibule, ∼15Å above the deeply buried orthosteric site. It is also important to note that a direct link between the binding affinity of individual GPCR ligands and their efficacy is still lacking (16,20,24). Whereas ARC exhibits lower affinity than IXO or QNB as investigated in the present study, certain partial agonists are not necessarily weaker binders compared with full or inverse agonists (16,24).…”

“…It is also important to note that a direct link between the binding affinity of individual GPCR ligands and their efficacy is still lacking (16,20,24). Whereas ARC exhibits lower affinity than IXO or QNB as investigated in the present study, certain partial agonists are not necessarily weaker binders compared with full or inverse agonists (16,24). Nevertheless, there appears to be a correlation between efficacy and the breadth of the dispersion of affinities for agonists of the M 2 receptor (16,20,25).…”

“…In addition to binding inverse and full agonists, the M 2 receptor recognizes partial agonists that often possess lower affinities and elicit submaximal activity (16). Coupling of the receptor with the G protein or mimetic nanobody typically increases agonist binding affinities (17).…”