Claire Weill

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

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.

A practical approach for intracellular protein delivery

Protein delivery represents a powerful tool for experiments in live cells including studies of protein-protein interactions, protein interference with blocking antibodies, intracellular trafficking and protein or peptide biological functions. Most available reagents dedicated to the protein delivery allow efficient crossing of the plasma membrane. Nevertheless, the major disadvantage for these reagents is a weak release of the delivered protein into the cytoplasm. In this publication we demonstrate efficient protein delivery with a non-peptide based reagent, in human epithelial carcinoma HeLa cells and primary human skin fibroblasts. Using a fluorescent protein in combination with fluorescence microscopy and fluorescence-assisted cell sorting analysis, we show that the delivered protein is indeed released effectively in the cytoplasm, as expected for a dedicated carrier. Furthermore, we present a step-by-step method to optimize conditions for successful intracellular protein delivery.

Functional Characterization and Potential Applications for Enhanced Green Fluorescent Protein‐ and Epitope‐Fused Human M1 Muscarinic Receptors

Abstract : Four recombinant human M1 (hM1) muscarinic acetylcholine receptors (mAChRs) combining several modifications were designed and overexpressed in HEK293 cells. Three different fluorescent chimera were obtained through fusion of the receptor N terminus with enhanced green fluorescent protein (EGFP), potential glycosylation sites and a large part of the third intracellular (i3) loop were deleted, a hexahistidine tag sequence was introduced at the receptor C terminus, and, finally, a FLAG epitope was either fused at the receptor N terminus or inserted into its shortened i3 loop. High expression levels and ligand binding properties similar to those of the wild‐type hM1 receptor together with confocal microscopy imaging demonstrated that the recombinant proteins were correctly folded and targeted to the plasma membrane, provided that a signal peptide was added to the N‐terminal domain of the fusion proteins. Their functional properties were examined through McN‐A‐343‐evoked Ca2+ release. Despite the numerous modifications introduced within the hM1 sequence, all receptors retained nearly normal abilities (EC50 values) to mediate the Ca2+ response, although reduced amplitudes (Emax values) were obtained for the i3‐shortened constructs. Owing to the bright intrinsic fluorescence of the EGFP‐fused receptors, their detection, quantitation, and visualization as well as the selection of cells with highest expression were straightforward. Moreover, the presence of the different epitopes was confirmed by immunocytochemistry. Altogether, this work demonstrates that these EGFP‐ and epitope‐fused hM1 receptors are valuable tools for further functional, biochemical, and structural studies of muscarinic receptors.

Cationic lipid-mediated intracellular delivery of antibodies into live cells

The ability to introduce antibodies to live cells opens new insights to a wide range of applications, such as protein intracellular trafficking studies, protein interference studies with blocking antibodies, and live immunolabeling or protein phosphorylation states studies. Apart from single-chain format variable (scFv) antibodies, DNA transfection of eukaryotic cells is rarely used to produce antibodies in situ, mainly due to inappropriate folding of the antibody in the cytoplasm. Thus, the development of dedicated carriers is needed since antibodies, which are large, unable to cross the plasma membrane and effective release of the antibody in the cytoplasm need to be overcome. We studied these two crucial steps using a dedicated delivery reagent in live cells and compared the results with immunocytochemistry experiments in fixed cells.

Fluorescent Muscarinic EGFP-hM1 Chimeric Receptors: Design, Ligand Binding and Functional Properties

We describe the construction, expression and characterization of recombinant proteins comprising the enhanced green fluorescent protein (EGFP) fused to the amino-terminal part of the muscarinic hM1 receptor together or not with an additional hexahistidine tag placed at the C-terminal end of the receptor. Expression of the fluorescent proteins reaches levels identical to those of the wt hM1 receptor, provided that fusion takes place at the very N-terminal end of the receptor. Also correct protein folding and targeting to plasma membrane is obtained upon addition of a signal peptide promoting amino-terminal domain translocation through the membrane. Ligand binding properties of--and activation of the calcium release response by--the fusion proteins are almost identical to those of the wild-type muscarinic receptor, indicating that such fluorescently-labelled receptors are valuable model systems for further functional, biochemical and structural studies.

Pharmacological and structural integrity of muscarinic M2 acetylcholine receptors produced in Sf9 insect cells.

Muscarinic acetylcholine receptors (human m2 subtype), expressed in Sf9 cells, using the baculovirus system, were purified and found to display the expected ligand binding properties, whether membrane-bound or affinity-purified. The purified recombinant receptors were specifically photolabelled with p-N,N-[3H]dimethylamino and p-N,N-[3H]dibutylamino benzene diazonium derivatives. Electrophoretic patterns for covalent radioactive incorporation of the probes were essentially similar to those for [3H]propylbenzilylcholine mustard-labelled receptor sites but were dependent on the infection time of Sf9 cells. Pharmacological properties of the recombinant receptors being unaltered did not reflect structural integrity of the protein as substantial proteolytic fragmentation was detected at a prolonged infection time, i.e., at the highest level of expression. Selection of overexpression conditions, as illustrated here for muscarinic receptors, thus requires not only pharmacological controls, but also analysis of the covalently labelled protein under strongly dissociating conditions.

Intracellular Peptide Delivery Using Amphiphilic Lipid-Based Formulations

Peptides, highly diverse by their nature, are important biochemical and pharmaceutical tools: ligands for cellular receptors, transcription factors, immunosuppressants, vaccines, etc. As the majority of their targets are intracellular, peptides need to cross the plasma membrane and gain access to the cytoplasm. However, due to their physicochemical properties, most peptides need to be entrapped by a molecular vehicle to be able to reach the cytosol compartment. In this study, we present new biological tools to enhance intracellular peptides delivery. Based on electrostatic interactions, two complementary types of amphiphilic molecules have been designed as delivery vehicles. A diverse set of fluorescently labeled peptides have successfully been delivered. This opens the avenue for the use of peptides combined to delivery vehicles as therapeutic aids. Biotechnol. Bioeng. 2011;108: 2477–2487.

Covalent labeling of muscarinic acetylcholine receptors by tritiated aryldiazonium photoprobes.

p-dimethylamino (A) and p-dibutylamino (B) benzenediazonium salts, previously characterized as efficient labels of membrane-bound and solubilized muscarinic receptor sites, are endowed with overall interesting photochemical and alkylating properties that allow their use as structural probes of the muscarinic ligand binding domain to be considered. Under reversible binding conditions, these antagonists display no binding selectivity towards the 5 muscarinic acetylcholine receptor (mAChR) subtypes. They were used here, in a tritiated form, as photoaffinity labels of purified muscarinic receptors from porcine striatum, and their irreversible binding was assessed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) analysis. When irradiated under energy transfer conditions, [3H]A and [3H]B were both found to covalently label purified muscarinic receptor sites in a light-dependent and atropine-protectable manner. The electrophoretic migration properties of the alkylated sites were similar to those of [3H]propylbenzilylcholine mustard (PrBCM)-labeled mAChRs. Specific radioactive incorporation showed a clear dependency on probe concentration. Labeling efficiency was rather high, with up to 30% and even 60% of the receptor population being photolabeled by [3H]A and [3H]B, respectively. These two photoactivatable ligands have proven to be powerful tools for the structural analysis of other cholinergic targets (acetylcholinesterase and the nicotinic acetylcholine receptor) by allowing the characterization of a number of different residues belonging to their acetylcholine-binding domain. Altogether, these results reinforce the interest of our site-directed labeling approach because [3H]A- and [3H]B-alkylated mAChRs may now be considered as suitable materials to investigate the muscarinic receptor-binding pocket through peptide mapping, sequence analyses, and identification of radiolabeled amino acid residues.