title: Microbial fluorescence sensing for human neurotensin receptor type 1 using Gα-engineered yeast cells.
abstract: Neurotensin receptor type-1 (NTSR1) is a member of the G-protein-coupled receptor (GPCR) family. The natural ligand of NTSR1 is neurotensin (NT), a neuromodulator of the central nervous system. Because NT is also involved in many oncogenic actions, the signaling mediator NTSR1 is a significant molecular target in medicinal and therapeutic fields. In the current study, we constructed a fluorescence-based microbial yeast biosensor that can monitor the activation of human NTSR1 signaling responding to its agonist. To increase the sensitivity of the biosensor, a yeast strain with the green fluorescent protein (GFP) reporter gene was genetically engineered to enhance binding with human NTSR1 expressed on the membrane. Following previous reports, the 5 carboxy-terminal amino acid residues of the guanine nucleotide binding protein α-subunit (Gα) in yeast Gpa1p were substituted with the equivalent human Gα(q) sequences (Gpa1/Gα(q) transplant). After optimizing the assay conditions, the Gα-engineered yeast demonstrated significantly improved sensing for NTSR1 signaling. Because detection using a GFP fluorescence reporter considerably simplifies the measurement procedure, this microbial fluorescence sensor holds promise for use in the diagnosis of NTSR1-associated diseases and the development of agonists.
authors.affiliation: Organization of Advanced Science and Technology, Kobe University, Nada, Kobe 657-8501, Japan.
authors.forename: Jun
authors.initials: J
authors.lastname: Ishii
authors.affiliation: Department of Chemical Science and Engineering, Graduate School of Engineering, Kobe University, Nada, Kobe 657-8501, Japan.
authors.forename: Asami
authors.initials: A
authors.lastname: Oda
authors.affiliation: Department of Chemical Science and Engineering, Graduate School of Engineering, Kobe University, Nada, Kobe 657-8501, Japan.
authors.forename: Shota
authors.initials: S
authors.lastname: Togawa
authors.affiliation: Laboratory of Hygienic Chemistry, Graduate School of Pharmaceutical Sciences, Nagoya City University, Mizuho-ku, Nagoya 467-8603, Japan.
authors.forename: Akira
authors.initials: A
authors.lastname: Fukao
authors.affiliation: Laboratory of Hygienic Chemistry, Graduate School of Pharmaceutical Sciences, Nagoya City University, Mizuho-ku, Nagoya 467-8603, Japan.
authors.forename: Toshinobu
authors.initials: T
authors.lastname: Fujiwara
authors.affiliation: Department of Chemical Science and Engineering, Graduate School of Engineering, Kobe University, Nada, Kobe 657-8501, Japan.
authors.forename: Chiaki
authors.initials: C
authors.lastname: Ogino
authors.affiliation: Department of Chemical Science and Engineering, Graduate School of Engineering, Kobe University, Nada, Kobe 657-8501, Japan. Electronic address: akondo@kobe-u.ac.jp.
authors.forename: Akihiko
authors.initials: A
authors.lastname: Kondo
mesh_terms: D015374:Biosensing Techniques / Q000379:methods*; D044385:GTP-Binding Protein alpha Subunits / Q000235:genetics*; D005818:Genetic Engineering; D006801:Humans; D006863:Hydrogen-Ion Concentration; D018028:Receptors, Neurotensin / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000166:cytology*; D013050:Spectrometry, Fluorescence; D013997:Time Factors
chemical_list: D044385:GTP-Binding Protein alpha Subunits; D018028:Receptors, Neurotensin; C114036:neurotensin type 1 receptor
keywords: G-protein-coupled receptor; Green fluorescent protein; Neurotensin; Neurotensin receptor; Yeast