Ligand competition assays indicate allosterism and insufficiency of the ternary complex model

Eyad Qunaibi¹, Roger Barber<sup>2

1</sup>Formerly (during the time of the study): School of Pharmacy, University of Houston, Houston, TX. At present: School of Pharmacy, Applied Sciences University, Amman, Jordan; ²Formerly (during the time of the study): Department of Integrative Biology and Pharmacology, University of Texas/Houston, Houston, TX, USA

Background: Many researchers have tried to correlate characteristics of ligand binding at G-protein–coupled receptor (GPCR) with ligand efficacy. The ternary complex model (TCM) is the traditional model for explaining the equilibrium of agonist-GPCR-G-protein interaction, and the effect of this interaction on agonist efficacy. However, no consistent correlation has been proven for various binding-efficacy data, so several extensions of the model have been proposed. These extensions are of descriptive value but their validity cannot be verified by binding-efficacy correlations. Therefore, we developed a novel approach to validate the TCM and its extensions.

Methods: We simulated the predictions of the TCM for relationships within binding parameters. According to the TCM, an increase in the difference between high and low agonist affinities for a receptor (ie, greater K_L/K_H) should be accompanied by stability or an increase in the fraction of receptors bound to the agonist with high affinity (R_H). To validate these predictions we performed ligand competition experiments for a set of ß₂-adrenergic receptor (AR) agonists and analyzed the resulting binding data as well as data taken from relevant literature.

Results: No smooth relationship exists between R_H and K_L/K_H in our or others’ data, indicating the insufficiency of the TCM and its extensions. We introduce the allosteric modulators model as an alternative.

Conclusions: To our knowledge, this is the first paper in which insufficiency of the TCM and its extensions based on binding data are shown, and the first in which the presence of allosteric modulators of ligand affinity is proven to be a necessity for explaining binding data at GPCRs.

Keywords: G-protein-coupled receptor, ternary complex model, allosteric modulators model, fraction of high affinity receptors, ratio of high and low receptor affinities for agonist, binding-functional correlations