Bright Fluorescence Monitoring System Utilizing Zoanthus sp. Green Fluorescent Protein (ZsGreen) for Human G-Protein-Coupled Receptor Signaling in Microbial Yeast Cells

PLoS One

Yasuyuki Nakamura¹  Akihiko Kondo¹  Jun Ishii² 

[1] Department of Chemical Science and Engineering, Graduate School of Engineering, Kobe University, Kobe, Japan;Organization of Advanced Science and Technology, Kobe University, Kobe, Japan

关键词: Fluorescence microscopy;    Signal peptides;    Green fluorescent protein;    Somatostatin;    G protein coupled receptors;    Plasmid construction;    Yeast;    Flow cytometry;   

DOI  :  10.1371/journal.pone.0082237

学科分类：医学（综合）

来源: Public Library of Science

【 摘 要 】

G-protein-coupled receptors (GPCRs) are currently the most important pharmaceutical targets for drug discovery because they regulate a wide variety of physiological processes. Consequently, simple and convenient detection systems for ligands that regulate the function of GPCR have attracted attention as powerful tools for new drug development. We previously developed a yeast-based fluorescence reporter ligand detection system using flow cytometry. However, using this conventional detection system, fluorescence from a cell expressing GFP and responding to a ligand is weak, making detection of these cells by fluorescence microscopy difficult. We here report improvements to the conventional yeast fluorescence reporter assay system resulting in the development of a new highly-sensitive fluorescence reporter assay system with extremely bright fluorescence and high signal-to-noise (S/N) ratio. This new system allowed the easy detection of GPCR signaling in yeast using fluorescence microscopy. Somatostatin receptor and neurotensin receptor (implicated in Alzheimer’s disease and Parkinson’s disease, respectively) were chosen as human GPCR(s). The facile detection of binding to these receptors by cognate peptide ligands was demonstrated. In addition, we established a highly sensitive ligand detection system using yeast cell surface display technology that is applicable to peptide screening, and demonstrate that the display of various peptide analogs of neurotensin can activate signaling through the neurotensin receptor in yeast cells. Our system could be useful for identifying lead peptides with agonistic activity towards targeted human GPCR(s).

【 授权许可】

CC BY