title: A single-vector EYFP reporter gene assay for G protein-coupled receptors.
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).
authors: Center for Evolutionary Chemical Biology, Department of Chemistry, University of Copenhagen, DK-2100 Copenhagen, Denmark.
authors: Helle
authors: H
authors: Hald
authors: Carlsberg Laboratory, DK-2500 Copenhagen, Valby, Denmark.
authors: Boqian
authors: B
authors: Wu
authors: Novartis Pharma, CH-4002 Basel, Switzerland.
authors: Lamine
authors: L
authors: Bouakaz
authors: Center for Evolutionary Chemical Biology, Department of Chemistry, University of Copenhagen, DK-2100 Copenhagen, Denmark. Electronic address: meldal@nano.ku.dk.
authors: Morten
authors: M
authors: Meldal
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D017930:Genes, Reporter / Q000235:genetics*; D057809:HEK293 Cells; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism*; D043562:Receptors, G-Protein-Coupled / Q000235:genetics / Q000378:metabolism*; D012984:Software
chemical_list: D001426:Bacterial Proteins; D008164:Luminescent Proteins; D043562:Receptors, G-Protein-Coupled; C054562:yellow fluorescent protein, Bacteria
keywords: Cell Image Analyzer software; EYFP; G protein-coupled receptors; Monoclonal GPCR assay cells; Reporter gene assay