Measuring the Kinetics of G‐protein‐coupled Receptor Signaling and Biased Agonism.

Abstract

Quantifying the time course of G‐protein‐coupled receptor signaling can reveal biological properties of GPCR function such as receptor desensitization, pathway selectivity, and spatiotemporally compartmentalized signaling. Applying signaling kinetics to modern receptor research and drug discovery requires assay modalities that can monitor signaling responses over time, and data analysis methods to extract the kinetic drug parameters. Here we describe a suite of fluorescent biosensors for monitoring multiple pathways of GPCR signaling (Gs, Gi, Gq and arrestin). These biosensors are genetically‐encoded and amenable to kinetic analysis, with continuous “real time” responses detected on plate readers or imaging systems. A kinetic data analysis framework is applied to the time course data using familiar equations on commonly‐used curve‐fitting software (e.g. GraphPad Prism). The analysis yields the initial rate of signaling (kTau), a biologically‐meaningful parameter of ligand efficacy at generating the signal. Biased agonism assessment is simplified using this approach, and demonstrated using arrestin recruitment and second messenger signaling via the AT1 angiotensin and GLP‐1 receptors. Bias is quantified as the ratio of the initial rates of signaling reported by each assay. This platform provides a straightforward method that investigators can use to quantify GPCR receptor pharmacology and biased signaling with the application of kinetic analysis to understanding receptor function and to the development of new therapeutics.Support or Funding InformationThis work was supported by grants from the National Institutes of HealthR44 GM125390‐02 and R44 DA050357‐01