Bright fluorescence monitoring system utilizing Zoanthus sp. green fluorescent protein (ZsGreen) for human G-protein-coupled receptor signaling in microbial yeast cells

PLoS One. 2013 Dec 5;8(12):e82237. doi: 10.1371/journal.pone.0082237. eCollection 2013.

Abstract

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).

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Anemone / genetics*
  • Animals
  • Green Fluorescent Proteins* / biosynthesis
  • Green Fluorescent Proteins* / genetics
  • Humans
  • Receptors, Neurotensin* / biosynthesis
  • Receptors, Neurotensin* / genetics
  • Receptors, Somatostatin* / biosynthesis
  • Receptors, Somatostatin* / genetics
  • Recombinant Fusion Proteins* / biosynthesis
  • Recombinant Fusion Proteins* / genetics
  • Saccharomyces cerevisiae* / genetics
  • Saccharomyces cerevisiae* / metabolism
  • Signal Transduction / genetics*

Substances

  • Receptors, Neurotensin
  • Receptors, Somatostatin
  • Recombinant Fusion Proteins
  • Green Fluorescent Proteins

Grant support

This work was supported in part by a Research Fellowship for Young Scientists from the Japan Society for the Promotion of Science, the Naito Foundation, and Special Coordination Funds for Promoting Science and Technology, Creation of Innovation Centers for Advanced Interdisciplinary Research Areas (Innovative Bioproduction Kobe; iBioK) from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.