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Fluorescence resonance energy transfer to probe human M1 muscarinic receptor structure and drug binding properties
Author(s) -
Ilien Brigitte,
Franchet Christelle,
Bernard Philippe,
Morisset Séverine,
Odile Weill Claire,
Bourguig JeanJacques,
Hibert Marcel,
Galzi JeanLuc
Publication year - 2003
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1046/j.1471-4159.2003.01717.x
Subject(s) - förster resonance energy transfer , muscarinic acetylcholine receptor , biophysics , transmembrane domain , fluorescence , receptor , chemistry , g protein coupled receptor , muscarinic acetylcholine receptor m1 , pirenzepine , green fluorescent protein , ligand (biochemistry) , muscarinic acetylcholine receptor m3 , biochemistry , biology , gene , physics , quantum mechanics
Human M1 muscarinic receptor chimeras were designed (i) to allow detection of their interaction with the fluorescent antagonist pirenzepine labelled with Bodipy [558/568], through fluorescence resonance energy transfer, (ii) to investigate the structure of the N‐terminal extracellular moiety of the receptor and (iii) to set up a fluorescence‐based assay to identify new muscarinic ligands. Enhanced green (or yellow) fluorescent protein (EGFP or EYFP) was fused, through a linker, to a receptor N‐terminus of variable length so that the GFP barrel was separated from the receptor first transmembrane domain by six to 33 amino‐acids. Five fluorescent constructs exhibit high expression levels as well as pharmacological and functional properties superimposable on those of the native receptor. Bodipy‐pirenzepine binds to the chimeras with similar kinetics and affinities, indicating a similar mode of interaction of the ligand with all of them. From the variation in energy transfer efficiencies determined for four different receptor‐ligand complexes, relative donor (EGFP)‐acceptor (Bodipy) distances were estimated. They suggest a compact architecture for the muscarinic M1 receptor amino‐terminal domain which may fold in a manner similar to that of rhodopsin. Finally, this fluorescence‐based assay, prone to miniaturization, allows reliable detection of unlabelled competitors.