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.
机构:
Natl Childrens Med Res Ctr, Dept Mol & Cell Pharmacol, Setagaya Ku, Tokyo 154, JapanNatl Childrens Med Res Ctr, Dept Mol & Cell Pharmacol, Setagaya Ku, Tokyo 154, Japan
Awaji, T
;
Hirasawa, A
论文数: 0引用数: 0
h-index: 0
机构:
Natl Childrens Med Res Ctr, Dept Mol & Cell Pharmacol, Setagaya Ku, Tokyo 154, JapanNatl Childrens Med Res Ctr, Dept Mol & Cell Pharmacol, Setagaya Ku, Tokyo 154, Japan
Hirasawa, A
;
Kataoka, M
论文数: 0引用数: 0
h-index: 0
机构:
Natl Childrens Med Res Ctr, Dept Mol & Cell Pharmacol, Setagaya Ku, Tokyo 154, JapanNatl Childrens Med Res Ctr, Dept Mol & Cell Pharmacol, Setagaya Ku, Tokyo 154, Japan
Kataoka, M
;
Shinoura, H
论文数: 0引用数: 0
h-index: 0
机构:
Natl Childrens Med Res Ctr, Dept Mol & Cell Pharmacol, Setagaya Ku, Tokyo 154, JapanNatl Childrens Med Res Ctr, Dept Mol & Cell Pharmacol, Setagaya Ku, Tokyo 154, Japan
Shinoura, H
;
Nakayama, Y
论文数: 0引用数: 0
h-index: 0
机构:
Natl Childrens Med Res Ctr, Dept Mol & Cell Pharmacol, Setagaya Ku, Tokyo 154, JapanNatl Childrens Med Res Ctr, Dept Mol & Cell Pharmacol, Setagaya Ku, Tokyo 154, Japan
Nakayama, Y
;
Sugawara, T
论文数: 0引用数: 0
h-index: 0
机构:
Natl Childrens Med Res Ctr, Dept Mol & Cell Pharmacol, Setagaya Ku, Tokyo 154, JapanNatl Childrens Med Res Ctr, Dept Mol & Cell Pharmacol, Setagaya Ku, Tokyo 154, Japan
Sugawara, T
;
Izumi, S
论文数: 0引用数: 0
h-index: 0
机构:
Natl Childrens Med Res Ctr, Dept Mol & Cell Pharmacol, Setagaya Ku, Tokyo 154, JapanNatl Childrens Med Res Ctr, Dept Mol & Cell Pharmacol, Setagaya Ku, Tokyo 154, Japan
Izumi, S
;
Tsujimoto, G
论文数: 0引用数: 0
h-index: 0
机构:
Natl Childrens Med Res Ctr, Dept Mol & Cell Pharmacol, Setagaya Ku, Tokyo 154, JapanNatl Childrens Med Res Ctr, Dept Mol & Cell Pharmacol, Setagaya Ku, Tokyo 154, Japan
机构:
Natl Childrens Med Res Ctr, Dept Mol & Cell Pharmacol, Setagaya Ku, Tokyo 154, JapanNatl Childrens Med Res Ctr, Dept Mol & Cell Pharmacol, Setagaya Ku, Tokyo 154, Japan
Awaji, T
;
Hirasawa, A
论文数: 0引用数: 0
h-index: 0
机构:
Natl Childrens Med Res Ctr, Dept Mol & Cell Pharmacol, Setagaya Ku, Tokyo 154, JapanNatl Childrens Med Res Ctr, Dept Mol & Cell Pharmacol, Setagaya Ku, Tokyo 154, Japan
Hirasawa, A
;
Kataoka, M
论文数: 0引用数: 0
h-index: 0
机构:
Natl Childrens Med Res Ctr, Dept Mol & Cell Pharmacol, Setagaya Ku, Tokyo 154, JapanNatl Childrens Med Res Ctr, Dept Mol & Cell Pharmacol, Setagaya Ku, Tokyo 154, Japan
Kataoka, M
;
Shinoura, H
论文数: 0引用数: 0
h-index: 0
机构:
Natl Childrens Med Res Ctr, Dept Mol & Cell Pharmacol, Setagaya Ku, Tokyo 154, JapanNatl Childrens Med Res Ctr, Dept Mol & Cell Pharmacol, Setagaya Ku, Tokyo 154, Japan
Shinoura, H
;
Nakayama, Y
论文数: 0引用数: 0
h-index: 0
机构:
Natl Childrens Med Res Ctr, Dept Mol & Cell Pharmacol, Setagaya Ku, Tokyo 154, JapanNatl Childrens Med Res Ctr, Dept Mol & Cell Pharmacol, Setagaya Ku, Tokyo 154, Japan
Nakayama, Y
;
Sugawara, T
论文数: 0引用数: 0
h-index: 0
机构:
Natl Childrens Med Res Ctr, Dept Mol & Cell Pharmacol, Setagaya Ku, Tokyo 154, JapanNatl Childrens Med Res Ctr, Dept Mol & Cell Pharmacol, Setagaya Ku, Tokyo 154, Japan
Sugawara, T
;
Izumi, S
论文数: 0引用数: 0
h-index: 0
机构:
Natl Childrens Med Res Ctr, Dept Mol & Cell Pharmacol, Setagaya Ku, Tokyo 154, JapanNatl Childrens Med Res Ctr, Dept Mol & Cell Pharmacol, Setagaya Ku, Tokyo 154, Japan
Izumi, S
;
Tsujimoto, G
论文数: 0引用数: 0
h-index: 0
机构:
Natl Childrens Med Res Ctr, Dept Mol & Cell Pharmacol, Setagaya Ku, Tokyo 154, JapanNatl Childrens Med Res Ctr, Dept Mol & Cell Pharmacol, Setagaya Ku, Tokyo 154, Japan