A homogeneous enzyme fragment complementation-based beta-arrestin translocation assay for high-throughput screening of G-protein-coupled receptors.

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 beta-arrestin and ligand-activated GPCRs and uses enzyme fragment complementation technology. In this format, a GPCR of interest is fused to a small (approximately 4 kDa), optimized alpha fragment peptide (termed ProLink) derived from beta-galactosidase, and beta-arrestin is fused to an N-terminal deletion mutant of beta-galactosidase (termed the enzyme acceptor [EA]). Upon activation of the receptor, the beta-arrestin-EA fusion protein binds the activated GPCR. This interaction drives enzyme fragment complementation, resulting in an active beta-galactosidase enzyme, and thus GPCR activation can be determined by quantifying beta-galactosidase activity. In this report, the authors demonstrate the utility of this technology to monitor GPCR activation and validate the approach using a Galphai-coupled GPCR, somatostatin receptor 2. Potential application to high-throughput screens in both agonist and antagonist screening modes is exemplified.