Fluorescent labeling of NK2 receptor at specific sites in vivo and fluorescence energy transfer analysis of NK2 ligand-receptor complexes

A fluorescent unnatural amino acid was introduced biosynthetically at known sites into the G protein-coupled neurokinin (tachykinin) NK2 receptor by suppression of UAG nonsense codons with the aid of a chem. misacylated synthetic tRNA specifically designed for the incorporation of unnatural amino acids during heterologous expression in Xenopus oocytes. A systematic UAG-scanning mutagenesis in NK2 extra- or intracellular loops and proximal transmembrane domains established that read-through at some UAG sites may represent a limitation to the range of applicability of the nonsense suppression methodol. Fluorescence-labeled NK2 mutants contg. an unique fluorescent nitrobenzoxadiazoyl-diaminopropionic acid residue at known sites were shown to be functionally active. Intermol. distances were detd. by measuring the fluorescence resonance energy transfer (FRET) between the fluorescent unnatural amino acid and a fluorescently labeled NK2 heptapeptide antagonist in a native membrane environment. These distances confirmed the seven transmembrane topol. for G protein-coupled receptors and detd. a structural model for NK2 ligand-receptor interactions. The peptide is inserted between the fifth and sixth transmembrane domains, thus suggesting that antagonism may be caused by preventing correct packing of the helixes required for receptor function. [on SciFinder (R)]

Probing the structure and function of the tachykinin neurokinin-2 receptor through biosynthetic incorporation of fluorescent amino acids at specific sites

Jonathan Knowles, Gerardo Turcatti, Horst Vogel

A general method for understanding the mechanisms of ligand recognition and activation of G protein-coupled receptors has been developed. A study of ligand-receptor interactions in the prototypic seven-transmembrane neurokinin-2 receptor (NK2) using this fluorescence-based approach is presented. A fluorescent unnatural amino acid was introduced at known sites into NK2 by suppression of UAG nonsense codons with the aid of a chem. misacylated synthetic tRNA specifically designed for the incorporation of unnatural amino acids during heterologous expression in Xenopus oocytes. Fluorescence-labeled NK2 mutants contg. an unique 3-N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-2,3-diaminopropionic acid (NBD-Dap) residue at either site 103, in the first extracellular loop, or 248, in the third cytoplasmic loop, were functionally active. The fluorescent NK2 mutants were investigated by microspectrofluorimetry in a native membrane environment. Intermol. distances were detd. by measuring the fluorescence resonance energy transfer (FRET) between the fluorescent unnatural amino acid and a fluorescently labeled NK2 heptapeptide antagonist. These distances, calcd. by the theory of Forster, permit to fix the ligand in space and define the structure of the receptor in a mol. model for NK2 ligand-receptor interactions. Our data are the first report of the incorporation of a fluorescent unnatural amino acid into a membrane protein in intact cells by the method of nonsense codon suppression, as well as the first measurement of exptl. distances between a G protein-coupled receptor and its ligand by FRET. The method presented here can be generally applied to the anal. of spatial relationships in integral membrane proteins such as receptors or channels. [on SciFinder (R)]

1996

Investigation of the neurokinin-1 receptor by fluorescence techniques

Bruno Meyer

G protein-coupled receptors (GPCR) constitute by far the largest family of transmembrane cell-surface proteins involved in signal transduction and are the most important targets for the development of novel therapeutic compounds. Many processes mediated by GPCR signal transduction are still not well understood at the molecular level and novel methods for their investigation are of general interest. The thesis presented here describes investigations on the neurokinin-1 receptor (NK1R), a member of the GPCR family, using fluorescence techniques. Fluorescence resonance energy transfer (FRET), the technique of preference in this work, is used to measure ligand-protein and protein-protein interactions in living cells. In order to reach this goal, the NK1R has been labelled with either fluorescent proteins or by using the novel post-translational ACP labelling technique. A FRET-based method for monitoring specific ligand binding to NK1R is described. In this context, a fluorescent agonist for NK1R was synthesized and characterized. The ACP labelling technique was shown to be superior for this kind of experiments compared to labelling with fluorescent proteins. The same ACP labelling approach was shown to be ideally suited to study oligomerization of membrane proteins by FRET. It was shown, that the NK1R does not form homo-dimers, as many other GPCRs do. From these experiments we furthermore conclude, that the NK1R is present in small microdomains in the membrane. Interactions between the NK1R and the different subunits of the heterotrimeric Gαqβ1γ2-complex were investigated by FRET using different instrumentation. Proximity of the NK1R and the G protein could be detected, but no change in FRET was observed after activation of the receptor. Total internal reflection fluorescence (TIRF), was used to develop an assay for studying ligand binding in vitro. Here, the NK1R was immobilized directly on a surface using a cell membrane preparation. This method was shown to be highly sensitive.

EPFL2005

Fluorescence Resonance Energy Transfer Shows a Close Helix-Helix Distance in the Transmembrane M13 Procoat Protein

Serge Cattarinussi, Horst Vogel

During the membrane insertion process the major coat protein of bacteriophage M13 assumes a conformation in which two transmembrane helixes corresponding to the leader sequence and the anchor region in the mature part of the protein coming into close contact with each other. Previous studies on the mol. mechanism of membrane insertion of M13 procoat protein have shown that this interaction between the two helixes might drive the actual translocation process. We investigated the intramol. distance between the two helixes of the transmembrane procoat protein by measuring fluorescence resonance energy transfer (FRET) between the donor (Tyr) placed in one helix and the acceptor (Trp) placed in the other helix. Various mutant procoat proteins with differently positioned donor-acceptor pairs were generated, purified, and reconstituted into artificial lipid bilayers. The results obtained from the FRET measurements, combined with mol. modeling, show that the transmembrane helixes are in close contact on the order of 1-1.5 nm. The present approach might be of general interest for detg. the topol. and the folding of membrane proteins. [on SciFinder (R)]

2001

Investigation of the neurokinin-1 receptor by fluorescence techniques

Bruno Meyer

EPFL2005