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A Homogeneous Enzyme Fragment Complementation-Based β-Arrestin Translocation Assay for High-Throughput Screening of G-Protein-Coupled Receptors

Lead Discovery Department, Chemistry Research and Discovery, Amgen, Inc., South San Francisco, California, xiaoning{at}amgen.com

Adrie Jones

Lead Discovery Department, Chemistry Research and Discovery, Amgen, Inc., South San Francisco, California

Keith R. Olson

DiscoveRx, Inc., Fremont, California

Kun Peng

DiscoveRx, Inc., Fremont, California

Tom Wehrman

DiscoveRx, Inc., Fremont, California

Adam Park

Lead Discovery Department, Chemistry Research and Discovery, Amgen, Inc., South San Francisco, California

Rommel Mallari

Lead Discovery Department, Chemistry Research and Discovery, Amgen, Inc., South San Francisco, California

Danilo Nebalasca

Lead Discovery Department, Chemistry Research and Discovery, Amgen, Inc., South San Francisco, California

Stephen W. Young

Lead Discovery Department, Chemistry Research and Discovery, Amgen, Inc., South San Francisco, California

Shou-Hua Xiao

Lead Discovery Department, Chemistry Research and Discovery, Amgen, Inc., South San Francisco, California, Josh.Xiao{at}amgen.com

G-protein-coupled receptors (GPCRs) represent one of the largest gene families in the human genome and have long been regarded as valuable targets for small-molecule drugs. The authors describe a new functional assay that directly monitors GPCR activation. It is based on the interaction between β-arrestin and ligand-activated GPCRs and uses enzyme fragment complementation technology. In this format, a GPCR of interest is fused to a small (~4 kDa), optimized fragment peptide (termed ProLink^TM) derived from β-galactosidase, and β-arrestin is fused to an N-terminal deletion mutant of β-galactosidase (termed the enzyme acceptor [EA]). Upon activation of the receptor, the β-arrestin-EA fusion protein binds the activated GPCR. This interaction drives enzyme fragment complementation, resulting in an active β-galactosidase enzyme, and thus GPCR activation can be determined by quantifying β-galactosidase activity. In this report, the authors demonstrate the utility of this technology to monitor GPCR activation and validate the approach using a G_i-coupled GPCR, somatostatin receptor 2. Potential application to high-throughput screens in both agonist and antagonist screening modes is exemplified. (Journal of Biomolecular Screening 2008:737-747)

Key Words: high-throughput screening • arrestin translocation assay • luminescence assay • cell-based assays • G-protein-coupled receptors (GPCRs)

This version was published on September 1, 2008

Journal of Biomolecular Screening, Vol. 13, No. 8, 737-747 (2008)
DOI: 10.1177/1087057108321531


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