title: Estimation of ligand affinity constants for receptor states in functional studies involving the allosteric modulation of G protein-coupled receptors: implications for ligand bias.
abstract: BACKGROUND
  The affinity constants of a ligand for active and inactive states of a receptor ultimately determine its capacity to activate downstream signaling events. In this report, we describe a reverse-engineering strategy for estimating these microscopic constants.
  
  
  METHODS
  Our approach involves analyzing responses measured downstream in the signaling pathway of a G protein-coupled receptor under conditions of allosteric modulation and reduced receptor expression or partial receptor inactivation. 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).
  
  
  CONCLUSIONS
  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. Knowing the isomerization constant and sensitivity constant of a signaling pathway in a given cell line or tissue preparation enables future investigators to estimate the affinity constants of agonists for receptor states simply through analysis of their concentration-response curves. Our approach also provides a means of validating in silico estimates of ligand affinity for crystal structures of active and inactive states of the receptor.
authors.affiliation: Department of Pharmacology, School of Medicine, University of California, Irvine, Irvine, CA 92617-4625, United States; Crean School of Health and Life Sciences, Schmid College of Science and Technology, Chapman University, Orange, CA, United States. Electronic address: fjehlert@uci.edu.
authors.forename: Frederick J
authors.initials: FJ
authors.lastname: Ehlert
authors.affiliation: Department of Pharmacology, School of Medicine, University of California, Irvine, Irvine, CA 92617-4625, United States; Crean School of Health and Life Sciences, Schmid College of Science and Technology, Chapman University, Orange, CA, United States.
authors.forename: Michael T
authors.initials: MT
authors.lastname: Griffin
mesh_terms: D000494:Allosteric Regulation; D003198:Computer Simulation; D054314:Drug Inverse Agonism; D006801:Humans; D008024:Ligands; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology*
chemical_list: D008024:Ligands; D043562:Receptors, G-Protein-Coupled
keywords: Affinity modulation; Agonist-bias; Allosterism; Constitutive activity; Efficacy modulation; Ligand-bias; Monod-Wyman-Changeux; Muscarinic receptor; Probe-dependence; Receptor states