Monitoring ?-arrestin recruitment via ?-lactamase enzyme fragment complementation: purification of peptide E as a low-affinity ligand for mammalian bombesin receptors.

ABSTRACT: Identification of cognate ligands for G protein-coupled receptors (GPCRs) provides a starting point for understanding novel regulatory mechanisms. Although GPCR ligands have typically been evaluated through the activation of heterotrimeric G proteins, recent studies have shown that GPCRs signal not only through G proteins but also through ?-arrestins. As such, monitoring ?-arrestin signaling instead of G protein signaling will increase the likelihood of identifying currently unknown ligands, including ?-arrestin-biased agonists. Here, we developed a cell-based assay for monitoring ligand-dependent GPCR-?-arrestin interaction via ?-lactamase enzyme fragment complementation. Inter alia, ?-lactamase is a superior reporter enzyme because of its cell-permeable fluorescent substrate. This substrate makes the assay non-destructive and compatible with fluorescence-activated cell sorting (FACS). In a reporter cell, complementary fragments of ?-lactamase (? and ?) were fused to ?-arrestin 2 and GPCR, respectively. Ligand stimulation initiated the interaction of these chimeric proteins (?-arrestin-? and GPCR-?), and this inducible interaction was measured through reconstituted ?-lactamase activity. Utilizing this system, we screened various mammalian tissue extracts for agonistic activities on human bombesin receptor subtype 3 (hBRS3). We purified peptide E as a low-affinity ligand for hBRS3, which was also found to be an agonist for the other two mammalian bombesin receptors such as gastrin-releasing peptide receptor (GRPR) and neuromedin B receptor (NMBR). Successful purification of peptide E has validated the robustness of this assay. We conclude that our newly developed system will facilitate the discovery of GPCR ligands.

SUBMITTER: Ikeda Y 

PROVIDER: S-EPMC4452343 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature