title: Monitoring β-arrestin recruitment via β-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 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.
authors.affiliation: Department of Cardiovascular Medicine, University of Tokyo, Tokyo, Japan; Department of Molecular Genetics, Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, Texas, United States of America.
authors.forename: Yuichi
authors.initials: Y
authors.lastname: Ikeda
authors.affiliation: Department of Cardiovascular Medicine, University of Tokyo, Tokyo, Japan; Department of Molecular Genetics, Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, Texas, United States of America; Department of Advanced Clinical Science and Therapeutics, University of Tokyo, Tokyo, Japan.
authors.forename: Hidetoshi
authors.initials: H
authors.lastname: Kumagai
authors.affiliation: Department of Diabetes and Metabolic Diseases, University of Tokyo, Tokyo, Japan.
authors.forename: Hiroaki
authors.initials: H
authors.lastname: Okazaki
authors.affiliation: Department of Gastroenterology, University of Tokyo, Tokyo, Japan.
authors.forename: Mitsuhiro
authors.initials: M
authors.lastname: Fujishiro
authors.affiliation: Department of Cardiovascular Medicine, University of Tokyo, Tokyo, Japan.
authors.forename: Yoshihiro
authors.initials: Y
authors.lastname: Motozawa
authors.affiliation: Department of Cardiovascular Medicine, University of Tokyo, Tokyo, Japan.
authors.forename: Seitaro
authors.initials: S
authors.lastname: Nomura
authors.affiliation: Department of Cardiovascular Medicine, University of Tokyo, Tokyo, Japan.
authors.forename: Norifumi
authors.initials: N
authors.lastname: Takeda
authors.affiliation: Department of Cardiovascular Medicine, University of Tokyo, Tokyo, Japan.
authors.forename: Haruhiro
authors.initials: H
authors.lastname: Toko
authors.affiliation: Department of Cardiovascular Medicine, University of Tokyo, Tokyo, Japan.
authors.forename: Eiki
authors.initials: E
authors.lastname: Takimoto
authors.affiliation: Department of Cardiovascular Medicine, University of Tokyo, Tokyo, Japan.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Akazawa
authors.affiliation: Department of Cardiovascular Medicine, University of Tokyo, Tokyo, Japan.
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Morita
authors.affiliation: Department of Advanced Clinical Science and Therapeutics, University of Tokyo, Tokyo, Japan.
authors.forename: Jun-ichi
authors.initials: J
authors.lastname: Suzuki
authors.affiliation: Clinical Research Support Center, University of Tokyo, Tokyo, Japan.
authors.forename: Tsutomu
authors.initials: T
authors.lastname: Yamazaki
authors.affiliation: Department of Cardiovascular Medicine, University of Tokyo, Tokyo, Japan.
authors.forename: Issei
authors.initials: I
authors.lastname: Komuro
authors.affiliation: Department of Molecular Genetics, Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, Texas, United States of America; International Institute for Integrative Sleep Medicine (WPI-IIIS), University of Tsukuba, Tsukuba, Japan.
authors.forename: Masashi
authors.initials: M
authors.lastname: Yanagisawa
mesh_terms: D000311:Adrenal Glands / Q000187:drug effects / Q000378:metabolism; D000818:Animals; D019390:Arrestins / Q000378:metabolism*; D016466:CHO Cells; D002417:Cattle; D003412:Cricetulus; D004745:Enkephalins / Q000494:pharmacology*; D011485:Protein Binding; D018004:Receptors, Bombesin / Q000378:metabolism*; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism; D013329:Structure-Activity Relationship; D000071560:beta-Arrestin 2; D000071557:beta-Arrestins; D001618:beta-Lactamases / Q000378:metabolism*
chemical_list: C000605254:ARRB2 protein, human; D019390:Arrestins; D004745:Enkephalins; D018004:Receptors, Bombesin; D043562:Receptors, G-Protein-Coupled; D000071560:beta-Arrestin 2; D000071557:beta-Arrestins; C039933:peptide E (adrenal medulla); D001618:beta-Lactamases