Analysis of ligand bias in functional studies involving the allosteric modulation of G protein‐coupled receptors (654.6)

The affinity constants of a ligand for active and inactive states of a receptor ultimately determine its ability to activate downstream signaling events. In this report, we describe a reverse‐engineering strategy for estimating these microscopic constants. Our approach involves analyzing responses measured downstream in the signaling pathway of a G protein‐coupled receptor under conditions of partial receptor inactivation and allosteric modulation. The analysis also yields estimates of the isomerization constant of the unoccupied receptor, the sensitivity constant of the signaling pathway, and the more empirical parameters of the receptor population including the observed affinities and efficacies of allosteric and orthosteric ligands ‐ including inverse agonists ‐ and the efficacy of the unoccupied receptor (i.e., constitutive activity). We validate our approach with an analytical proof and by analysis of simulated data. We also use our method to analyze data from the literature. We show that the values of the microscopic constants of orthosteric and allosteric ligands are constant regardless of the allosteric interaction and the nature of the receptor‐signaling pathway as long as the same active state mediates the response. Our analysis is useful for quantifying probe‐dependent allosteric interactions and the selectivity of agonists for different signaling pathways. Grant Funding Source : NIH grant GM 69829