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

Yuichi Ikeda, Hidetoshi Kumagai, Hiroaki Okazaki, Mitsuhiro Fujishiro, Yoshihiro Motozawa, Seitaro Nomura, Norifumi Takeda, Haruhiro Toko, Eiki Takimoto, Hiroshi Akazawa, Hiroyuki Morita, Jun Ichi Suzuki, Tsutomu Yamazaki, Issei Komuro, Masashi Yanagisawa

Research output: Contribution to journalArticle

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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 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.

Original languageEnglish (US)
Article numbere0127445
JournalPLoS One
Volume10
Issue number6
DOIs
StatePublished - Jun 1 2015

Fingerprint

arrestins
Bombesin Receptors
Arrestin
G-Protein-Coupled Receptors
Purification
peptides
Ligands
Monitoring
monitoring
Enzymes
enzymes
G-proteins
Assays
GTP-Binding Proteins
agonists
CCR10 Receptors
assays
Arrestins
recombinant fusion proteins
Heterotrimeric GTP-Binding Proteins

ASJC Scopus subject areas

  • Agricultural and Biological Sciences(all)
  • Biochemistry, Genetics and Molecular Biology(all)
  • Medicine(all)

Cite this

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

In: PLoS One, Vol. 10, No. 6, e0127445, 01.06.2015.

Research output: Contribution to journalArticle

Ikeda, Y, Kumagai, H, Okazaki, H, Fujishiro, M, Motozawa, Y, Nomura, S, Takeda, N, Toko, H, Takimoto, E, Akazawa, H, Morita, H, Suzuki, JI, Yamazaki, T, Komuro, I & Yanagisawa, M 2015, 'Monitoring β-arrestin recruitment via β-lactamase enzyme fragment complementation: Purification of peptide E as a low-affinity ligand for mammalian bombesin receptors', PLoS One, vol. 10, no. 6, e0127445. https://doi.org/10.1371/journal.pone.0127445
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. / Monitoring β-arrestin recruitment via β-lactamase enzyme fragment complementation : Purification of peptide E as a low-affinity ligand for mammalian bombesin receptors. In: PLoS One. 2015 ; Vol. 10, No. 6.
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AU - Kumagai, Hidetoshi

AU - Okazaki, Hiroaki

AU - Fujishiro, Mitsuhiro

AU - Motozawa, Yoshihiro

AU - Nomura, Seitaro

AU - Takeda, Norifumi

AU - Toko, Haruhiro

AU - Takimoto, Eiki

AU - Akazawa, Hiroshi

AU - Morita, Hiroyuki

AU - Suzuki, Jun Ichi

AU - Yamazaki, Tsutomu

AU - Komuro, Issei

AU - Yanagisawa, Masashi

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