Fluorescence resonance energy transfer to probe human M1 muscarinic receptor structure and drug binding properties

Abstract

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.

Fluorescence/Bioluminescence Resonance Energy Transfer Techniques to Study G-Protein-Coupled Receptor Activation and Signaling

“…Dynamic FRET measurements were also reported for the muscarinic M 1 -acetylcholine receptor (Ilien et al, 2003). This report described the use of N-terminally GFPor YFP-tagged M 1 -acetylcholine receptors in combination with Bodipy(558/568)-labeled pirenzepine and used the FRET signal as readout for ligand binding studies.…”

Molecular Mechanisms of Methoctramine Binding and Selectivity at Muscarinic Acetylcholine Receptors

“…The DNA sequence of chicken a 7 nicotinic signal peptide (a peptide 31 amino acids long with extracellular protease cleavage site included; Ilien et al, 2003) was fused directly to stop-codon-free GFP (Invitrogen) at its 59 end to ensure extracellular localization of fluorescent protein. cDNA for the hM 2 receptor (cloned into pcDNA3.11 vector) were mutated (QuikChange II Site-Directed Mutagenesis Kit) to obtain AgeI restriction site at the 59 cDNA end.…”

Exploration of the Orthosteric/Allosteric Interface in Human M1 Muscarinic Receptors by Bitopic Fluorescent Ligands

“…Lew and Angus (1995) as well as Arunlakshana and Schild (1959) analyses (Fig. 2, right) (Ilien et al, 2003(Ilien et al, , 2009Tahtaoui et al, 2004). Figure 3 depicts real-time recordings of association and dissociation processes for BoPz tracers and the impact of allosteric modulators (brucine and gallamine) on their kinetics.…”

“…The human M1 muscarinic receptor (hM1) with a truncated N terminus (deletion of 17 amino acids) fused to EGFP is the construct of reference, defined as EGFP(D17)hM1 (Ilien et al, 2003). The corresponding cDNA served as a template in polymerase chain reaction to get single-point-mutated M1 receptors at tryptophan residues 400 and 405.…”

“…Fluorescence data were acquired from living cells suspended in Hepes buffer (1-3 10 6 cells/ml, depending on receptor expression) and kept at 20°C in a thermostated quartz cuvette under magnetic stirring. The interaction of BoPz tracers with chimeric M1 receptors was followed as a variation in cell fluorescence intensity (recorded at 510 nm) due to resonance energy transfer from the EGFP donor (excited at 470 nm) toward the ligand Bodipy acceptor species (Ilien et al, 2003(Ilien et al, , 2009.…”