A single-vector EYFP reporter gene assay for G protein-coupled receptors

Anal Biochem. 2015 May 1:476:40-4. doi: 10.1016/j.ab.2015.02.003. Epub 2015 Feb 11.

Abstract

We here present an improved and simplified assay to study signal transduction of the Gs class of G protein-coupled receptors (GPCRs). The assay is based on a single plasmid combining the genes for any Gs protein-coupled GPCR and the cAMP response element-related expression of enhanced yellow fluorescent protein. On transfection, stable human embryonic kidney 293 (HEK293) cell lines presented high assay sensitivity and an unprecedented signal-to-noise ratio of up to 300, even in the absence of trichostatin A. The robustness of the assay was demonstrated through the cloning of reporter gene cell lines with melanocortin 4 receptor (MC4R), the human type I pituitary adenylate cyclase-activating polypeptide receptor (hPAC1), and the two vasoactive intestinal peptide receptors (VPAC1 and VPAC2).

Keywords: Cell Image Analyzer software; EYFP; G protein-coupled receptors; Monoclonal GPCR assay cells; Reporter gene assay.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism*
  • Genes, Reporter / genetics*
  • HEK293 Cells
  • Humans
  • Luminescent Proteins / genetics
  • Luminescent Proteins / metabolism*
  • Receptors, G-Protein-Coupled / genetics
  • Receptors, G-Protein-Coupled / metabolism*
  • Software

Substances

  • Bacterial Proteins
  • Luminescent Proteins
  • Receptors, G-Protein-Coupled
  • yellow fluorescent protein, Bacteria