期刊
ANALYTICAL BIOCHEMISTRY
卷 408, 期 2, 页码 253-262出版社
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.ab.2010.09.030
关键词
G protein-coupled receptor; Growth hormone secretagogue receptor; type 1a; Ghrelin; Tag-lite; HTRF; SNAP-tag; Ligand-binding; TR-FRET
资金
- University of Montpellier 1
- CNRS
- INSERM
- CisBio
- Agence Nationale pour la Recherche [PCV08_323163]
- University of Montpellier 2
The growth hormone secretagogue receptor type la (GHS-R1a) belongs to class A G-protein-coupled receptors (GPCR). This receptor mediates pleiotropic effects of ghrelin and represents a promising target for dysfunctions of growth hormone secretion and energy homeostasis including obesity. Identification of new compounds which bind GHS-R1 a is traditionally achieved using radioactive binding assays. Here we propose a new fluorescence-based assay, called Tag-lite binding assay, based on a fluorescence resonance energy transfer (FRET) process between a terbium cryptate covalently attached to a SNAP-tag fused GHS-R1a (SNAP-GHS-R1a) and a high-affinity red fluorescent ghrelin ligand. The long fluorescence lifetime of the terbium cryptate allows a time-resolved detection of the FREE signal. The assay was made compatible with high-throughput screening by using prelabeled cells in suspension under a 384-well plate format. K-i values for a panel of 14 compounds displaying agonist, antagonist, or inverse agonist properties were determined using both the radioactive and the Tag-lite binding assays performed on the same batches of GHS-R1 a-expressing cells. Compound potencies obtained in the two assays were nicely correlated. This study is the first description of a sensitive and reliable nonradioactive binding assay for GHS-R1a in a format amenable to high-throughput screening. (c) 2010 Elsevier Inc. All rights reserved.
作者
我是这篇论文的作者
点击您的名字以认领此论文并将其添加到您的个人资料中。
推荐
暂无数据