GPCRs are one of the largest family of drug targets. Their activity is mediated by both G proteins and β-arrestins which activate a network of distinct signaling pathways. Drugs that activate GPCRs stabilize the receptor in different conformational states to produce distinct biological responses that represent varying degrees of are bias toward either the G-protein or β-arrestin signaling pathways. Depending on the receptor and cell type, biased drugs could produce either therapeutic or adverse effects by selectively engaging some signals, while minimizing other signals mediated by the same receptor. Despite the potential impact of ligand bias on therapeutic drug development, traditional cell-based assays are poorly suited for detecting bias. Current approaches involve comparing a series of disparate assays for arrestin recruitment, GPCR second messengers and ERK phosphorylation, each using different assay conditions, modalities, and instrumentation and measured in different cell populations.
In this tutorial, Montana Molecular describes a new approach that takes advantage of the dual channel fluorescence detection capability of the BMG CLARIOstar plate reader to introduce an efficient and practical method to simultaneously measure kinetic responses of both arrestin and G-protein second messenger signaling. We will discuss results from assays using agonists to both the Angiotensin and Vasopressin Receptors to illustrate the precision with which agonists that are known to be arrestin biased can be distinguished from unbiased or partially biased agonists in a single experiment.