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

被引：2

作者：

Hald, Helle ^{[1
]}

Wu, Boqian ^{[2
]}

Bouakaz, Lamine ^{[3
]}

Meldal, Morten ^{[1
]}

机构：

[1] Univ Copenhagen, Ctr Evolutionary Chem Biol, Dept Chem, DK-2100 Copenhagen, Denmark

[2] Carlsberg Lab, DK-2500 Copenhagen, Valby, Denmark

[3] Novartis Pharmaceut, CH-4002 Basel, Switzerland

来源：

ANALYTICAL BIOCHEMISTRY | 2015年 / 476卷

基金：

新加坡国家研究基金会;

关键词：

G protein-coupled receptors; Reporter gene assay; EYFP; Monoclonal GPCR assay cells; Cell Image Analyzer software; GREEN-FLUORESCENT-PROTEIN; DRUG TARGETS; CAMP; TRANSCRIPTION; ACETYLATION; EXPRESSION; VARIANTS; CREB;

D O I：

10.1016/j.ab.2015.02.003

中图分类号：

Q5 [生物化学];

学科分类号：

071010 ; 081704 ;

摘要：

We here present an improved and simplified assay to study signal transduction of the G(s) 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). (C) 2015 Elsevier Inc. All rights reserved.

引用

页码：40 / 44

页数：5

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