Document Type
Article
Publication Date
5-2014
Abstract
Introduction
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).
Results and discussion
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.
Recommended Citation
Ehlert, F.J., Griffin, M.T., 2014. Estimation of ligand affinity constants for receptor states in functional studies involving the allosteric modulation of G protein-coupled receptors: Implications for ligand bias. Journal of Pharmacological and Toxicological Methods 69, 253–279. doi:10.1016/j.vascn.2014.01.002
Copyright
Elsevier
Creative Commons License
This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 4.0 License.
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Comments
NOTICE: this is the author’s version of a work that was accepted for publication in Journal of Pharmacological and Toxicological Methods. Changes resulting from the publishing process, such as peer review, editing, corrections, structural formatting, and other quality control mechanisms may not be reflected in this document. Changes may have been made to this work since it was submitted for publication. A definitive version was subsequently published in Journal of Pharmacological and Toxicological Methods, volume 69, in 2014. DOI: 10.1016/j.vascn.2014.01.002
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