Microbial fluorescence sensing for human neurotensin receptor type 1 using G伪-engineered yeast cells

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• 作者：Jun Ishii ; Asami Oda ; Shota Togawa ; Akira Fukao ; Toshinobu Fujiwara ; Chiaki Ogino ; Akihiko Kondo
• 关键词：Neurotensin ; Neurotensin receptor ; G-protein-coupled receptor ; Yeast ; Green fluorescent protein
• 刊名：Analytical Biochemistry
• 出版年：1 February, 2014
• 年：2014
• 卷：446
• 期：Complete
• 页码：37-43
• 全文大小：1185 K

文摘

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 伪-subunit (G伪) in yeast Gpa1p were substituted with the equivalent human G伪_q sequences (Gpa1/G伪_q transplant). After optimizing the assay conditions, the G伪-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.