IntroductIonG -protein-coupled receptors (Gpcrs) are the largest superfamily of cell surface signal transduction molecules. they are important targets in drug discovery, with an estimated 25% of prescription drugs exerting their effects through them. 1 Gpcrs exert many of their effects by activation of heterotrimeric G proteins (Gi, Gs, Gq). Following activation, G proteins divide into respective α and βγ subunits, both of which have the ability to activate or inhibit effector molecules, such as adenylate cyclase and phospholipase c. the receptor then becomes phosphorylated on intracellular serine and threonine residues by Gpcr kinases (GrKs). 2,3 the phosphorylated receptor is then bound by β-arrestin, which sterically prevents further G-protein interaction and recruits other proteins such as clathrin, which are involved in receptor internalization. in addition, β-arrestin has been shown to function as a signaling intermediate and, for instance, is involved in the activation of the MApK pathway. 4,5 Historically, within the drug discovery environment, Gpcr agonists have been discovered through traditional second messenger assay formats, which measure the activation of G proteins (e.g., [ 35 s]Gtpγs binding) and subsequent signaling events (e.g., inhibition of forskolin-stimulated cAMp production). However, in recent years, pressure to develop more robust and high-throughput assays, with higher Z′ values and the discovery of non-G-protein-mediated signaling through Gpcrs, has led to the development of alternative assay formats, such as β-arrestin recruitment. in particular, the discoverx pathHunter assay technology used in this report uses an enzyme complementation system to detect the interaction between a tagged Gpcr and tagged β-arrestin. 6,7 Although this assay is being used with increasing frequency, the pharmacologic values generated have not been thoroughly investigated and put into context with traditional pharmacological models.Agonists have two properties: affinity (K A ), which is a purely drug-dependent parameter and determines how well the agonist binds to the receptor (and therefore should be equivalent for all functional endpoints), and efficacy (tau), which measures the efficiency of the transduction of binding into a response and is dependent on the ligand and the tissue/signaling pathway involved (and therefore is likely to differ for different functional endpoints). the translation of occupancy into effect is what is measured in the form of an agonist-concentration/effect ([A]/e) curve. the ability to discriminate between agonists with different efficacies is of critical importance in drug discovery. However, overexpressing recombinant systems can lead to the overestimation of the potency and efficacy of compounds, and in particular, 1 discovery Biology, pfizer inc., Kent, uK. the correct interpretation of data is fundamental to the study of G-protein-coupled receptor pharmacology. often, new assay technologies are assimilated into the drug discovery environment without full consideration of t...