Studying G protein-coupled receptor activation using split-tobacco etch virus assays

Anal Biochem. 2011 May 15;412(2):141-52. doi: 10.1016/j.ab.2011.01.042. Epub 2011 Feb 2.

Abstract

G protein-coupled receptors (GPCRs) constitute the largest receptor family in mammals and represent important drug targets. Signaling through GPCRs mediates physiological effects that are strongly dependent on the cellular context. Therefore, the availability of assays monitoring GPCR activation applicable in different cell types could help to better understand GPCR functions and to realize the potential of known substances as well as novel ones. Here we introduce a split-TEV (tobacco etch virus) assay to monitor GPCR activation through the stimulation-dependent recruitment of β-arrestin 2. Inactive N- and C-terminal fragments of the TEV protease are coupled to a GPCR and β-arrestin 2, respectively. Ligand-dependent interaction of the two fusion proteins leads to functional complementation of the TEV protease, followed by the cleavage of an artificial transcription factor and successive reporter gene activation. The presented split-TEV assay system is highly sensitive and was successfully applied in heterologous cell lines as well as in primary cultured neuronal and glial cells. We show that assay performance strongly depends on the endogenous properties of different cell types. The sensitivity and flexibility make split-TEV assays a valuable tool to analyze GPCR activation in different cell types in a rapid and cost-effective way.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Biological Assay / methods*
  • Endopeptidases / metabolism
  • Humans
  • PC12 Cells
  • Plant Viruses / genetics*
  • Rats
  • Receptors, G-Protein-Coupled / metabolism*
  • Receptors, Vasopressin / metabolism
  • Recombinant Fusion Proteins / metabolism
  • Reproducibility of Results
  • Signal Transduction
  • Tobacco / virology*

Substances

  • Receptors, G-Protein-Coupled
  • Receptors, Vasopressin
  • Recombinant Fusion Proteins
  • Endopeptidases
  • TEV protease