β-Arrestin GPCR cell lines built on the principles of EFC are engineered to co-express the ProLink™ (PK) tagged GPCR and the Enzyme Acceptor (EA) tagged β-Arrestin. Activation of the GPCR-PK induces β-Arrestin-EA recruitment, forcing complementation of the two β-galactosidase enzyme fragments (EA and PK). The resulting functional enzyme hydrolyzes substrate to generate chemiluminescent signal.
PathHunter β-Arrestin Principle for GPCR Applications
PathHunter GPCR β-Arrestin assays take advantage of DiscoverX’s proprietary Enzyme Fragment Complementation technology. The GPCR is fused in frame with a small enzyme donor fragment ProLink™ (PK) and co-expressed in cells stably expressing a fusion protein of β-arrestin and the larger, N-terminal deletion mutant of β-galactosidase (called enzyme acceptor or EA). Activation of the GPCR stimulates binding of β-arrestin to the PK-tagged GPCR and forces complementation of the two enzyme fragments, resulting in the formation of an active β-galactosidase enzyme. This interaction leads to an increase in enzyme activity that can be measured using chemiluminescent PathHunter Detection Reagents.
