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

• Published in: PloS one 2015-06, Vol.10 (6), p.e0127445-e0127445
• Main Authors: 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
• Format: Article
• Language: English
• Subjects: Adrenal Glands - drug effects; Adrenal Glands - metabolism; Affinity; Animals; Arrestin; Arrestins - metabolism; Assaying; beta-Arrestin 2; beta-Arrestins; beta-Lactamases - metabolism; Bombesin; Cattle; CHO Cells; Cricetulus; Enkephalins - pharmacology; Enzymes; Flow cytometry; Fluorescence; Fragmentation; G protein-coupled receptors; Gastrin; Gastrin-releasing peptide; Ligands; Mammals; Medicine; Narcotics; Neuromedin; Peptides; Physiology; Plasmids; Protein Binding; Proteins; Purification; Receptors; Receptors, Bombesin - metabolism; Receptors, G-Protein-Coupled - metabolism; Regulatory mechanisms (biology); Science; Signal monitoring; Signal transduction; Signaling; Structure-Activity Relationship; Substrates
• ISSN: 1932-6203; 1932-6203
• 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 β-arrestins. As such, monitoring β-arrestin signaling instead of G protein signaling will increase the likelihood of identifying currently unknown ligands, including β-arrestin-biased agonists. Here, we developed a cell-based assay for monitoring ligand-dependent GPCR-β-arrestin interaction via β-lactamase enzyme fragment complementation. Inter alia, β-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 β-lactamase (α and ω) were fused to β-arrestin 2 and GPCR, respectively. Ligand stimulation initiated the interaction of these chimeric proteins (β-arrestin-α and GPCR-ω), and this inducible interaction was measured through reconstituted β-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.