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

Amgen Inc

^* To whom correspondence should be addressed. E-mail: xiaoning{at}amgen.com.

	Abstract

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 -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 -galactosidase, and -arrestin is fused to an N-terminal deletion mutant of -galactosidase (termed the enzyme acceptor [EA]). Upon activation of the receptor, the -arrestin-EA fusion protein binds the activated GPCR. This interaction drives enzyme fragment complementation, resulting in an active -galactosidase enzyme, and thus GPCR activation can be determined by quantifying -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 XXXX:xx-xx)

First published on July 25, 2008, doi:10.1177/1087057108321531

Journal of Biomolecular Screening 2008;13:737.

A more recent version of this article appeared on September 1, 2008


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