Fluorescence resonance energy transfer to probe human M1 muscarinic receptor structure and drug binding properties
2003
Human M1
muscarinicreceptor chimeras were designed (i) to allow detection of their interaction with the fluorescent antagonist
pirenzepinelabelled 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
muscarinicligands. 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 domainby 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-
pirenzepinebinds 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
muscarinicM1 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.
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