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

作者：Ikeda Yuichi^*; Kumagai Hidetoshi; Okazaki Hiroaki; Fujishiro Mitsuhiro; Motozawa Yoshihiro; Nomura Seitaro; Takeda Norifumi; Toko Haruhiro; Takimoto Eiki; Akazawa Hiroshi; Morita Hiroyuki; Suzuki Jun ichi; Yamazaki Tsutomu; Komuro Issei; Yanagisawa Masashi

来源：PLos One, 2015, 10(6): e0127445.

DOI：10.1371/journal.pone.0127445

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 beta-arrestins. As such, monitoring beta-arrestin signaling instead of G protein signaling will increase the likelihood of identifying currently unknown ligands, including beta-arrestin-biased agonists. Here, we developed a cell-based assay for monitoring ligand-dependent GPCR-beta-arrestin interaction via beta-lactamase enzyme fragment complementation. Inter alia, beta-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 beta-lactamase (alpha and omega) were fused to beta-arrestin 2 and GPCR, respectively. Ligand stimulation initiated the interaction of these chimeric proteins (beta-arrestin-alpha and GPCR-omega), and this inducible interaction was measured through reconstituted beta-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.

• 出版日期2015-6-1