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β-Arrestin Recruitment Assay for the Identification of Agonists of the Sphingosine 1-Phosphate Receptor EDG1

Schering-Plough Research Institute, Oss, the Netherlands

Maaike Bras

Schering-Plough Research Institute, Oss, the Netherlands

Chris J. van Koppen

Schering-Plough Research Institute, Oss, the Netherlands

Guido J.R. Zaman

Schering-Plough Research Institute, Oss, the Netherlands, guido.zaman{at}spcorp.com

β-Arrestin recruitment assays provide a generic assay platform for drug discovery on G-protein-coupled receptors (GPCRs). The PathHunter^TM assay technology developed by DiscoveRx (Fremont, CA) uses enzyme fragment complementation of β-galactosidase to measure receptor-β-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^TM β-arrestin recruitment technology. Screening of a collection of 345,052 compounds yielded 2157 agonistic hits. Only 10 of these compounds showed β-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^TM cell line at the level of β-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 β-arrestin recruitment was largely unaffected upon blockade of G_i/o protein signaling with pertussis toxin, whereas kinetic studies demonstrated a lower rate of β-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 β-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 β-arrestin-mediated cellular effects). (Journal of Biomolecular Screening 2008:986-998)

Key Words: G-protein-coupled receptor • S1P1 • EDG1 • β-arrestin • β-galactosidase • enzyme fragment complementation

This version was published on December 1, 2008

Journal of Biomolecular Screening, Vol. 13, No. 10, 986-998 (2008)
DOI: 10.1177/1087057108326144


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