Estimation of Ligand Affinity for Receptor States in Functional Studies on G Protein‐Coupled Receptors

We developed a method for estimating drug affinity for active and inactive receptor states in functional studies on heterologously expressed G protein‐coupled receptors (GPCRs). Our protocol involves measuring agonist‐induced signaling responses through a given GPCR and a constitutively active mutant of it (CAM). Agonist responses are measured in the absence and presence of either partial receptor inactivation or reduced receptor expression. Global nonlinear regression analysis of the data yields estimates of 1) the active ( K act ) and inactive ( K inact ) receptor‐state affinity constants, 2) the isomerization constant of the unoccupied receptor ( K q‐obs ), and 3) the sensitivity constant of the signaling pathway ( K E‐obs ). The latter two parameters define the output response of the receptor. Hence, if the cellular system is reasonably stable, it can be used subsequently to estimate the K act and K inact values of additional agonists. For this latter analysis, all that is required are the corresponding concentration‐response curves for the wild type receptor–no additional experiments with the CAM are needed. Our analysis also yields 1) an absolute estimate of agonist efficacy and 2) an estimate of the constitutive activity of the wild type receptor even if constitutive activity is undetectable. We used an analytical proof to validate that our protocol yields sufficient information to estimate K act , K inact , K q‐obs and K E‐obs . We also validated our method using computer simulation and the analysis of previously published data. Our approach has several advantages over conventional methods for analyzing agonist concentration‐response curves. It provides 1) a more meaningful approach to structure‐activity relationships, 2) a means of validating in silico docking experiments on active and inactive receptor structures and 3) a method for quantifying agonist bias that does not depend on expressing agonist activity relative to that of a standard agonist. Future studies could lead to the establishment of a database of K q‐obs and K E‐obs values for various defined cells expressing specific GPCRs. Such a database would enable investigators to estimate the K act and K inact values of agonists from their concentration‐response curves on GPCR‐expressing cells for which K q‐obs and K E‐obs values are known.Parameters describing agonist‐induced GPCR activation and signaling