|
|
|
|
 |
|||
|
|||
Synthesis of a biologically active fluorescent probe for labeling neurotensin receptors
MP Faure, P Gaudreau, I Shaw, NR Cashman and A Beaudet
Neurobiology group, Montreal Neurological Institute, Quebec, Canada.
We synthesized a fluorescent derivative of the tridecapeptide neurotensin (NT), with the aim of providing a new tool for the pharmacological characterization and anatomic localization of NT receptors in mammalian brain. Fluoresceinylated NT (N alpha- fluoresceinyl thiocarbamyl (FTC)-[Glu1]NT; fluo-NT) was synthesized using solid-phase methodology and purified to 99% homogeneity by preparative high-pressure liquid chromatography (HPLC). Analytical HPLC, acidic and carboxypeptidase Y hydrolysis, and fast atom bombardment-mass spectroscopy confirmed that the purified compound was selectively labeled on the [Glu1] terminus and that a single FTC moiety was coupled to each molecule of [Glu1]NT. Flow cytometric analysis of the binding of fluo-NT to SN17 septal neuroblastoma cells indicated that the fluorescent derivative bound neural NT receptors with an affinity comparable to that of monoiodinated NT([125I]-NT). Competition experiments on mouse brain membrane preparations showed fluo-NT to inhibit specific [125I]-NT binding with a coefficient of inhibition (KI) virtually identical to that of the native peptide (0.67 vs 0.55 nM). Conventional epifluorescence and confocal microscopic analysis of specific fluo-NT binding to sections of the rat midbrain revealed a topographic distribution of the bound fluorescent ligand similar to that previously observed with autoradiography using [125I]-NT. However, fluo-NT provided markedly higher cell resolution and enabled, in particular, the detection of hitherto unnoted intracytoplasmic receptor clusters. Binding of fluo-NT to live SN17 hybrid cells indicated that the fluorescent ligand had retained its ability to internalize in vivo and confirmed that this internalization process was both time- and temperature-dependent. In sum, the present study demonstrates that fluo- NT is applicable to both the pharmacological study of NT binding sites using flow cytometry and to the regional and cellular localization of these sites by conventional epifluorescence and confocal microscopy.
Volume 42,
Issue 6,
pp. 755-763,
06/01/1994
Copyright © 1994 by The Histochemical Society
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?
This article has been cited by other articles:
|
|
 |
|
  F Vandenbulcke, D Nouel, J. Vincent, J Mazella, and A Beaudet Ligand-induced internalization of neurotensin in transfected COS-7 cells: differential intracellular trafficking of ligand and receptor J. Cell Sci., January 9, 2000; 113(17): 2963 - 2975. [Abstract] [PDF]
|
|
|
|
 |
|
 F. Souaze, W. Rostene, and P. Forgez Neurotensin Agonist Induces Differential Regulation of Neurotensin Receptor mRNA. IDENTIFICATION OF DISTINCT TRANSCRIPTIONAL AND POST-TRANSCRIPTIONAL MECHANISMS J. Biol. Chem., April 11, 1997; 272(15): 10087 - 10094. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. Nouel, M.-P. Faure, J.-A. St. Pierre, R. Alonso, R. Quirion, and A. Beaudet Differential Binding Profile and Internalization Process of Neurotensin via Neuronal and Glial Receptors J. Neurosci., March 1, 1997; 17(5): 1795 - 1803. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J.ėl. Chabry, J.-M. Botto, D. Nouel, A. Beaudet, J.-P. Vincent, and J. Mazella Thr-422 and Tyr-424 Residues in the Carboxyl Terminus Are Critical for the Internalization of the Rat Neurotensin Receptor J. Biol. Chem., February 10, 1995; 270(6): 2439 - 2442. [Abstract] [Full Text] [PDF]
|
|
| Guidelines | Subscriptions | About | exPRESS - Current - Archive | Business Information | Contact |