A highly sensitive quantitative cytosensor technique for the identification of receptor ligands in tissue extracts

被引:24
作者
Lenkei, Z
Beaudet, A
Chartrel, N
De Mota, N
Irinopoulou, T
Braun, B
Vaudry, H
Llorens-Cortes, C
机构
[1] Coll France, INSERM U36, F-75005 Paris, France
[2] McGill Univ, Montreal Neurol Inst, Montreal, PQ, Canada
[3] Univ Rouen, INSERM U413, IFRMP 23, Mt St Aignan, France
[4] INSERM U430, Paris, France
关键词
neurotensin; green fluorescent protein; internalization; cell-based assay; orphan receptor;
D O I
10.1177/002215540004801112
中图分类号
Q2 [细胞生物学];
学科分类号
071009 ; 090102 ;
摘要
Because C-protein-coupled receptors (GPCRs) constitute excellent putative therapeutic targets, functional characterization of orphan GPCRs through identification of their endogenous ligands has great potential for drug discovery. We propose here a novel single cell-based assay for identification of these ligands. This assay involves (a) fluorescent tagging of the GPCR, (b) expression of the tagged receptor in a heterologous expression system, (c) incubation of the transfected cells with fractions purified from tissue extracts, and (d) imaging of ligand-induced receptor internalization by confocal microscopy coupled to digital image quantification. We tested this approach in CHO cells stably expressing the NT1 neurotensin receptor fused to EGFP (enhanced green fluorescent protein), in which neurotensin promoted internalization of the NT1-EGFP receptor in a dose-dependent fashion (EC50 = 0.98 nM). Similarly, four of 120 consecutive reversed-phase HPLC fractions of frog brain extracts promoted internalization of the NT1-EG FP receptor. The same four fractions selectively contained neurotensin, an endogenous ligand of the NT1 receptor, as detected by radioimmunoassay and inositol phosphate production. The present internalization assay provides a highly specific quantitative cytosensor technique with sensitivity in the nanomolar range that should prove useful for the identification of putative natural and synthetic ligands for GPCRs.
引用
收藏
页码:1553 / 1563
页数:11
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