2000
DOI: 10.1177/002215540004801112
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A Highly Sensitive Quantitative Cytosensor Technique for the Identification of Receptor Ligands in Tissue Extracts

Abstract: S U M M A R Y Because G-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… Show more

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Cited by 29 publications
(32 citation statements)
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“…N1E-115 differentiation was induced with 1.5% FCS and 1% Me 2 SO for 48 h (28). Transfection of N1E-115 and CHP 212 cell lines with pNT1-EGFP, a plasmid expressing rat NT1 receptor fused at the C terminus with the EGFP coding sequence, was performed using the calcium phosphate coprecipitation method for 16 h at 37°C (29,30). After transfection, the media were removed, and fresh medium was added for 24 h. Stable transfectants were selected with G418 (1 mg/ml), and colonies were screened for EGFP expression using a Nikon Diaphot inverted fluorescence microscope at a ϫ40 magnification and by 125 I-NT binding assays.…”
Section: Methodsmentioning
confidence: 99%
See 1 more Smart Citation
“…N1E-115 differentiation was induced with 1.5% FCS and 1% Me 2 SO for 48 h (28). Transfection of N1E-115 and CHP 212 cell lines with pNT1-EGFP, a plasmid expressing rat NT1 receptor fused at the C terminus with the EGFP coding sequence, was performed using the calcium phosphate coprecipitation method for 16 h at 37°C (29,30). After transfection, the media were removed, and fresh medium was added for 24 h. Stable transfectants were selected with G418 (1 mg/ml), and colonies were screened for EGFP expression using a Nikon Diaphot inverted fluorescence microscope at a ϫ40 magnification and by 125 I-NT binding assays.…”
Section: Methodsmentioning
confidence: 99%
“…The agonist-induced intracellular trafficking of NT1 receptor was studied in CHO cells stably transfected with a plasmid expressing the rat NT1 receptor fused at the C terminus with the EGFP coding sequence (29). Binding characteristics were studied on the chosen clone, CHO-NT1-EGFP.…”
Section: Upon Exposure To a High Dose Of Agonist Nt1 Receptor And Ntmentioning
confidence: 99%
“…Image Quantification-Confocal images were used to quantify the subcellular distribution and translocation of CB1R (11,16). In-housedeveloped macro algorithms (available on request), written for the public domain Object Image software (available on the World Wide Web at simon.bio.uva.nl/object-image.html), were used to measure the subcellular distribution of the CB1Rs in HEK-293 cells.…”
Section: The Nucleotide Sequence(s) Reported In This Paper Has Been Smentioning
confidence: 99%
“…The internalization assay was performed on cycloheximide-treated CHO cells stably expressing the rat apelin-receptor-EGFP (9) cultured at a density of 2 ϫ 10 5 cells per well as described in ref. 24. Cells were incubated in the presence or absence of pE13F or R10F or individual rat hypothalamic (5 l) or plasma (45 l) fractions were diluted in complemented Earle's buffer in a final volume of 50 l. Internalization was examined with a Leica TCS SP 2 confocal laser scanning microscope (Leica Microsystems, Heidelberg), and quantification of apelin receptor-EGFP internalization was performed as described in ref.…”
mentioning
confidence: 99%
“…Cells were incubated in the presence or absence of pE13F or R10F or individual rat hypothalamic (5 l) or plasma (45 l) fractions were diluted in complemented Earle's buffer in a final volume of 50 l. Internalization was examined with a Leica TCS SP 2 confocal laser scanning microscope (Leica Microsystems, Heidelberg), and quantification of apelin receptor-EGFP internalization was performed as described in ref. 24.…”
mentioning
confidence: 99%