beta-Arrestin recruitment assay for the identification of agonists of the sphingosine 1-phosphate receptor EDG1.

beta-Arrestin recruitment assays provide a generic assay platform for drug discovery on G-protein-coupled receptors (GPCRs). The PathHunter assay technology developed by DiscoveRx (Fremont, CA) uses enzyme fragment complementation of beta-galactosidase to measure receptor-beta-arrestin proximity by chemiluminescence. This study describes an agonistic screen on the human endothelial differentiation sphingolipid GPCR 1 (EDG1), also known as S1P1, using PathHunter beta-arrestin recruitment technology. Screening of a collection of 345,052 compounds yielded 2157 agonistic hits. Only 10 of these compounds showed beta-arrestin recruitment activity on a nonrelated receptor, indicating high accuracy and specificity of the assay. The authors show that receptor activation with reference agonists can be detected within the same EDG1 PathHunter cell line at the level of beta-arrestin recruitment, G(i/o) protein-mediated inhibition of cyclic adenosine monophosphate (cAMP), and activation of downstream phosphorylation of extracellular signal-regulated protein kinases. The degree of beta-arrestin recruitment was largely unaffected upon blockade of G(i/o) protein signaling with pertussis toxin, whereas kinetic studies demonstrated a lower rate of beta-arrestin-receptor association. In contrast, inhibition of cAMP and phosphorylation of extracellular signal-regulated protein kinases were fully G(i/o) protein regulated. The data indicate that the beta-arrestin enzyme fragment complementation cell line can be used not only for agonistic screening of GPCRs but also for the identification of "biased ligands" (i.e., compounds that differ in G-protein coupling and beta-arrestin-mediated cellular effects).