WO2004013178A1 - Proteines hybrides entre une proteine fluorescente et un recepteur ionotropique et utilisations associees - Google Patents

Proteines hybrides entre une proteine fluorescente et un recepteur ionotropique et utilisations associees Download PDF

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WO2004013178A1
WO2004013178A1 PCT/EP2003/008490 EP0308490W WO2004013178A1 WO 2004013178 A1 WO2004013178 A1 WO 2004013178A1 EP 0308490 W EP0308490 W EP 0308490W WO 2004013178 A1 WO2004013178 A1 WO 2004013178A1
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receptor
fluorescent
protein
fluorescent protein
subunit
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PCT/EP2003/008490
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Régis GRAILHE
Lia Prado De Carvalho
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Centre National De La Recherche Scientifique
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70571—Receptors; Cell surface antigens; Cell surface determinants for neuromediators, e.g. serotonin receptor, dopamine receptor
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the present invention relates to fusion proteins between a fluorescent protein and an ionotropic receptor, preferably a subunit of a nicotinic or serotoninergic ionotropic receptor. It also relates to the uses of these fusion proteins, in particular in a process for the detection and quantification of non-covalent interactions between such a fusion protein and a ligand said receptor.
  • LGIC pentameric ligand-gated ion channels
  • GFP Green Fluorescent Protein
  • the first group belongs to the anionic receptor family. This is the case for the receptor GABA-A and Glycinergic type 1 (Kittler et al, 2000). These fluorescent anionic receptors were grafted with fluorescent proteins were found to be functional and were used to study their cellular compartmentalisation.
  • Cationic receptors which are members of the superfamily ligand-gated ion channels receptors, namely the serotoninergic 5HT and ,the cationic heteromeric nicotinic ⁇ x ⁇ y receptors are major neurotransmitter receptors in the mammalian brain, and play a critical role in the mediation and the modulation of chemical interneuronal communications in the central and peripheral nervous systems.
  • the serotoninergic 5- HT3 and nicotinic ⁇ x ⁇ y nACh receptors are respectively organized as functional homopentamers and heteropentamers.
  • TM subunit topology is dictated by a large N-terminal extracellular domain, followed by four transmembrane segments, and a large cytoplasmic region which connects TM3 and TM4 (Fig. IB).
  • High densities of 5HT 3 A are found in the central nervous system (Barnes et al, 1990; Gehlert et al, 1991), whereas the ⁇ 3 ⁇ 4nAChR is mainly distributed in the periphery, where it contributes to synaptic input in autonomic ganglia (Stollberg & Berg, 1987).
  • GFP green fluorescent protein
  • the grafted receptors were not functional when grafted in position N-terminal.
  • the receptors electrophysiological properties were altered by the presence of the GFP (Palma et al, 2002).
  • the aim of the present invention is to provide new fusion proteins between fluorescent protein variants and the N-terminus of receptor subunit, in particular the 5HT A and 3, ⁇ 4-nACh and ⁇ 4, ⁇ 2-nACh subunits without affecting the functionality and targeting thereof.
  • the present invention relates to a fusion protein between:
  • fluorescent protein chosen from fluorescent proteins obtained or derived from autofluorescent proteins, this protein being chosen in particular from:
  • GFP green fluorescent protein
  • the fluorescent protein (FP) variants such as the cyan fluorescent protein (CFP) and the yellow fluorescent protein (YFP), which are known to have distinct spectral properties, may be fused to the N-terminus of the ligand-gated cationic pentameric channels, in particular, the 5HT 3 A and ⁇ 3, ⁇ 4-nACh and ⁇ 4, ⁇ 2-nACh subunits without affecting the functionality and targeting thereof.
  • fusion at the N-terminal position did not impair the receptor-channel activity and enabled to follow the location or targeting of the receptors inside the cell and at the cell surface.
  • GFP green fluorescent protein
  • CFP - cyan fluorescent protein
  • BFP blue fluorescent protein
  • optimal codons indicates the replacement of codons of the wild-type protein with the host organism's preferred homologues thereof, without changing the code and thus without changing the protein sequence.
  • the green fluorescent protein (GFP) is decribed in Ward et al. (1980, Photochem. Photobiol. 31:611-615) and in Chalfie et al. (1995, Photochem. Photobiol. 62:651-656) and EGFP is decribed in Heim & Tsien (Current Biology, 1996, vol. No. 6, pp. 178- 182) and inMiyawaki et al (Nature 1997, vol-388, pp. 882-887).
  • the cyan fluorescent protein (CFP or ECFP) is described in Heim & Tsien (Current Biology, 1996, vol. No. 6, pp. 178-182) and in Miyawaki et al. (Nature 1997, vol. 388, pp. 882-887).
  • the yellow fluorescent protein (YFP or EYFP) is decribed in Cormack et al. (1995, Gene 173:33-38), in Heim, Cubitt and Tsien (1995, Nature), in Ehrig et al. (1995, FEBS Lett. 367:163-166) and in Miyawaki et al. (Nature 1997, vol. 388, pp. 882-887).
  • the GFPUV exhibiting the following mutations: F99S, M153T, V163A with excitation and emission wavelengths of 395 and 510, respectively, is described in Crameri et al. 1996 Nature Biotechnol. 14:315-319, or with the mutation T203I and the excitation and emission wavelengths of 400 and 512, respectively, is described in Ehrig et al. 1995 FEBS Lett. 367:163-166.
  • EGFP has the following mutations:
  • EYFP has the following mutations:
  • ECFP has the following mutations:
  • EBFP has the following mutations:
  • the present invention also relates to a fusion protein such as defined above, characterized in that the fluorescent protein is bound to the N-terminal or C-terminal part of the receptor subunit via a peptide of formula Pro- Ala- Ala- Ala or an analogous derived sequence.
  • the present invention also relates to a fusion protein such as defined above, characterized in that it is preceded by a signal peptide such as the signal peptide of the subunit alphal glycine receptor from zebrafish ( ⁇ ZIL), said signal peptide being eventually bound via a linker such as the dipeptide AV or an analogous sequence to said fusion protein.
  • a signal peptide such as the signal peptide of the subunit alphal glycine receptor from zebrafish ( ⁇ ZIL)
  • ⁇ ZIL subunit alphal glycine receptor from zebrafish
  • the fusion protein of the invention is characterized in that the subunit of the nicotinic receptors is chosen among the ⁇ and ⁇ subunits of the neuronal nitcotinic receptors family, particularly among the ⁇ 3, 4, ⁇ 2 and ⁇ 4 subunits.
  • a preferred fusion protein of the invention is a fusion protein such as defined above, wherein the fluorescent protein is YFP and the subunit of a nicotinic or serotoninergic ionotropic receptor is the ⁇ 3 nicotinic receptor subunit of the rat nicotinic acetylcholine receptor represented by SEQ ID NO : 1.
  • This preferred fusion protein is called YFP- ⁇ 3.
  • a preferred fusion protein of the invention is a fusion protein such as defined above, wherein the fluorescent protein is CFP and the subunit of a nicotinic or serotoninergic ionotropic receptor is the ⁇ 4 nicotinic receptor subunit of the rat nicotinic acetylcholine receptor represented by SEQ ID NO : 2.
  • This preferred fusion protein is called CFP- ⁇ 4.
  • the present invention also relates to a fusion protein such as defined above, characterized in that the subunit of the serotoninergic receptor is the subunit 5-HT 3 A.
  • a preferred fusion protein of the invention is a fusion protein such as defined above, wherein the fluorescent protein is YFP and the subunit of a nicotinic or serotoninergic ionotropic receptor is the mouse 5-HT 3 A subunit represented by SEQ ID NO : 3.
  • the present also relates to a fusion protein such as defined above, characterized in that it is in the form of a pentamer ' of identical or different subunits of a nicotinic or serotoninergic ionotropic receptor, one at least of these subunits being bound to a fluorescent protein.
  • the present invention also relates to a fusion protein between ⁇ and ⁇ subunits of nicotinic receptors and a fluorescent protein such as defined above, characterized in that the pentamer comprises a mixture of ⁇ 3 and ⁇ 4 subunits, or of ⁇ 4 and ⁇ 2 subunits, in particular at the ratio of about 2 ⁇ subunits for 3 ⁇ subunits, or inversely in particular at the ratio of about 3 ⁇ subunits for 2 ⁇ subunits.
  • a fusion protein of the invention is characterized in that only the ⁇ subunits are bound to the fluorescent protein, or inversely only the ⁇ subunits are bound to the fluorescent protein.
  • the present invention also relates to a fusion protein such as defined above, characterized in that it comprises a mixture of subunits ⁇ 3 and ⁇ 4, wherein only the ⁇ 3 subunits are bound to the fluorescent protein, which is preferably YFP, or only the j subunits ⁇ 4 are bound to the fluorescent protein, which is preferably YFP.
  • the present invention also relates to a nucleotide sequence coding for a fusion protein such as defined above.
  • the present invention also relates to a vector, particularly a plasmid, containing a nucleotide sequence such as defined above.
  • the present invention also relates to a host cell chosen among any type of cells, and being in particular Human Embryonic Kidney Cells (HEK-293), transformed with a vector such as defined above.
  • HEK-293 Human Embryonic Kidney Cells
  • the present invention also relates to a process for the preparation of a fusion protein such a defined above, characterized in that it comprises a step of culture of host cells such as defined previously transformed with a vector such as defined previously, and the recovering, eventually after a purification, of the fusion proteins such as produced by said cells.
  • the present invention also relates to a process such as defined above, for the preparation of fusion proteins between subunits of nicotinic receptors and a fluorescent protein in the form of a pentamer such as defined previously, characterized in that it comprises a step of mixing the different subunits of the nicotinic receptors, advantageously in equivalent proportions.
  • the present invention also relates to the use of fusion proteins such as defined above, for the implementation of a process of targeting in vitro of the nicotinic and/or serotoninergic receptors at the surface of the cells.
  • the present invention also relates to the use of fusion proteins such as defined above, for the implementation of a process of screening in vitro of ligands of the nicotinic and/or serotoninergic receptors or of inhibitors of such ligands.
  • the present invention also relates to the use of fusion proteins such as defined above, for the implementation of a process for the detection and quantification of non- covalent interactions between a fusion protein such as defined above and a ligand of the subunit of a nicotinic or serotoninergic ionotropic receptor comprised in said fusion protein, said ligand being labelled with a label consisting:
  • the fluorescent substance being such that either it is excitable at the emission wavelength of the above- mentioned fluorescent protein or of one of the above-mentioned variants, or of one of the above-mentioned fragments, or it emits at the excitation wavelength of the above- mentioned fluorescent protein, or of one of the above-mentioned variants, or of one of the above-mentioned fragments, or
  • ligand refers to any molecule which interacts non-covale ⁇ tly and reversibly with another molecule.
  • the present invention also relates to the use of fusion proteins such as defined above, for the detection and quantification of non-covalent interactions between a fusion protein such as defined above and a ligand of the subunit of a nicotinic or serotoninergic ionotropic receptor, said receptor subunit being comprised in said fusion protein, said ligand being labelled
  • the fluorescent substance then being a chemical compound
  • the fluorescent substance then being a fluorescent peptide or protein which can be chosen in particular from the fluorescent proteins obtained or derived from autofluorescent proteins, this fluorescent substance being chosen in particular from:
  • GFP green fluorescent protein
  • Acid Violet group [Acid Violet 5, CAS 10130-48-0; Acid Violet 1, CAS 4321-69-1; Acid Violet 17, CAS 4129- 84-4], the Acid Red group [Acid Red 1, CAS 3734-67-6; Acid Red 8, CAS 4787-93-3; Acid Red 37, CAS 6360-07-2; Acid Red 40, CAS 12167-45-2; Acid Red 106, CAS 6844-74-2; Acid Red 114, CAS 6459-94-5], alizarins, aluminon, azocarmine B [CAS 25360-72-9], basic fuschin [Basic Red 9, CAS 569-61-9], Bordeaux R [Acid Red 17, CAS 5858-33-3] and Carmine [CAS 1390-65-4].
  • the present invention also relates to a process for detecting and quantifying non- covalent interactions between a fusion protein such as defined above, and a ligand of the subunit of a nicotinic or serotoninergic ionotropic receptor said receptor subunit being comprised in said fusion protein, characterized in that: - cells or cell fragments are prepared containing a DNA sequence comprising the gene coding for a fluorescent protein comprised in the above-mentioned fusion protein, fused with the gene coding for a subunit of a nicotinic or serotoninergic ionotropic receptor comprised in the above-mentioned fusion protein, the fusion between the gene coding for the fluorescent protein and the gene coding for the above-mentioned. receptor being such that the properties of the above-mentioned receptor subunit are not modified by the presence of the above-mentioned fluorescent protein, namely:
  • the fluorescent protein being chosen from the fluorescent proteins obtained or derived from autofluorescent proteins, this protein being chosen in particular from:
  • GFP green fluorescent protein
  • the above-mentioned cells or the above-mentioned cell fragments are placed in contact with a ligand for the above-mentioned receptor subunit, said ligand being labelled with a label consisting:
  • the fluorescent protein being the fluorescence energy donor and the label being the fluorescence energy acceptor, or the fluorescent protein being the fluorescence energy acceptor and the label being a fluorescent substance which is a fluorescence energy donor, and
  • - irradiation is carried out at a wavelength which makes it possible either to excite the fluorescent protein or to excite the fluorescent substance
  • the above-mentioned steps of placing in contact and irradiation to be carried out either simultaneously or one after the other, or - the above-mentioned cells or the above-mentioned cell fragments are placed in contact with a ligand for the above-mentioned protein labelled with a label, the cells or the ligand having been irradiated before being placed in contact,
  • the present invention also relates to a process for detecting and quantifying non- covalent interactions between a fusion protein such as defined above, and a ligand of the subunit of a nicotinic or serotoninergic ionotropic receptor, said receptor subunit being comprised in said fusion protein, characterized in that:
  • a fluorescent protein comprised in the above-mentioned fusion protein, fused with a subunit of a nicotinic or serotoninergic ionotropic receptor, comprised in the above-mentioned fusion protein, the fusion between the fluorescent protein and the above-mentioned receptor subunit being such that the properties of the receptor subunit are not modified by the presence of the fluorescent protein, namely:
  • the fluorescent protein being chosen from the fluorescent proteins obtained or derived from autofluorescent proteins, this protein being chosen in particular from:
  • GFP green fluorescent protein
  • the above-mentioned fluorescent protein fused with the receptor subunit is placed in contact with a ligand for the * above-mentioned receptor subunit, this ligand being labelled with a label consisting:
  • the fluorescent protein being a fluorescence energy donor and the label being a fluorescence energy acceptor, or the fluorescent protein being a fluorescence energy acceptor and the label being a fluorescent substance which is a fluorescence energy donor, and
  • - irradiation is carried out at a wavelength which makes it possible either to excite the fluorescent protein or to excite the fluorescent substance
  • the present invention also relates to a process such as defined above, in which the fluorescent protein is CFP or YFP, and in which:
  • CFP or YFP is a fluorescence energy donor and the label is a fluorescence energy acceptor and is chosen from substances whose excitation spectrum overlaps the emission spectrum of CFP or YFP, and in particular, when the label is a fluorescent substance, it is chosen from: 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (Bodipy), eosin, erythrosin, tetramethylrhodamine, sulphorhodamine 101 sold by Molecular Probe under the name Texas Red, and derivatives thereof which, on the one hand, allow grafting, and, on the other hand, have an excitation spectrum which overlaps the emission spectrum of CFP or YFP, and, when the label is not a fluorescent substance, it is chosen from the Acid Violet group [Acid Violet 5, CAS 10130-48-0; Acid Violet 7, CAS 4321-69-1; Acid Violet 17, CAS 4129-84-4], the Acid Red group [Acid Red 1,
  • the CFP or YFP is a fluorescence energy acceptor and the fluorescent substance is a fluorescence energy donor and is chosen from substances whose emission spectrum overlaps the excitation spectrum of CFP or YFP, and in particular from: coumarins, fluorescamine, 6-(N-methylanilino)naphthalene, (mansyl) and derivatives thereof which, on the one hand, allow grafting, and, on the other hand, have an excitation spectrum which overlaps the emission spectrum of CFP or YFP.
  • the present invention also relates to a method for identifying a ligand for a serotoninergic or nicotinic receptor comprising:
  • a second recombinant gene encoding a polypeptide to be tested, liable to be a ligand of said receptor, said polypeptide being labelled with a label such as defined above, wherein said receptor is expressed on the cell membrane of said cell such that FRET signal is modulated by interaction between the fluorescent protein of the receptor and the label of the polypeptide to be tested; wherein said second recombinant gene is expressed and the polypeptide to be tested is transported to a location allowing interaction with the receptor expressed on the cell membrane, and wherein collectively the mixture of cells expresses a library of said polypeptides to be tested, said library capable of being expressed at a sufficient level such that modulation of the targeting of the receptor protein by a polypeptide to be tested within the library provides a detectable signal;
  • the present invention relates to functional fluorescent cationic receptors of the superfamily of pentameric ligand-gated ion channels, such as serotoninergic and nicotinic receptors, and the preparation thereof.
  • GFP green fluorescent protein
  • fluorescent proteins which were isolated from various organisms were shown to fold as a fluorescent protein in the absence of additional reagent and to be fluorescent in a specific range of the spectra such as the red for the Ds Red isolated from Discosoma striata or the HcRed isolated from the reef coral, Heteractis crispa.
  • the structural conformation of all these fluorescent proteins is very similar, since the structure of these proteins is based on beta strand which forms a barrel.
  • the fluorescent label is a biologically inert participant that is used merely as a visible marker. Thanks to the barrel-like structures the chromophore is shielded from the external environment.
  • the fluorescent receptor channels belonging to the cationic receptor family are fully functional.
  • the poly-amino acid sequence (amino acids 1-751) of the YFP- ⁇ 3 nicotinic subunit receptor is composed from position 1 to 29 by the poly- amino acid sequence of the zebrafish alphal glycinergic receptor, from position 30 to 31 by a linker (AV), from position 32 to 268 by the fluorescent protein YFP, from position 269 to 277 by a linker (PMASPMPP), and from position 278 to 751 by the rat alpha3 nicotinic subunit amino sequence.
  • AV linker
  • PMASPMPP linker
  • the CFP- ⁇ 4 nicotinic subunit receptor (amino acids 1-752; SEQ ID NO : 2) of the present invention can be further characterized as having the same amino acid sequence as amino acids 1 to 31 and 269 to 277, as set forth in SEQ ID NO : 1 (for the YFP- ⁇ 3 nicotinic subunit receptor) and from amino acids from position 32 to 268 by the fluorescent protein CFP, from position 269 to 277, and from position 278 to 752 by the rat beta4 nicotinic subunit amino sequence.
  • the YFP-5HT3A nicotinic subunit receptor (amino acids 1-740; SEQ ID NO : 3) of the present invention can be further characterized as having the same amino acid sequence as amino acids 1 to 268, as set forth in SEQ ID NO : 1, from position 269 to 272 by a linker (PAAA) and from position 278 to 740 by the mouse 5HT3A nicotinic subunit receptor.
  • amino acids 1-740 amino acids 1-740; SEQ ID NO : 3
  • PAAA linker
  • fluorescent protein (FP) variants such as the cyan fluorescent protein (CFP) and the yellow fluorescent protein (YFP), known to have distinct spectral properties, were fused to the N-terminus of the 5HT A and ⁇ 3, ⁇ 4-nACh subunits. Their electrophysiological properties were found to be similar to those of non-tagged receptors in whole-cell patch-clamp recordings performed in transiently transfected HEK-293. Fusion in position N-terminal contrary to the ⁇ or ⁇ subunits of nicotinic muscular receptor did not impair the receptor-channel activity.
  • CFP cyan fluorescent protein
  • YFP yellow fluorescent protein
  • Preferred cationic pentameric ligand-gated ion channels include heteromeric nicotinic acetylcholine subunits receptors (AChR) 3, ⁇ 4, ⁇ 5, 6, ⁇ 2, ⁇ 3, ⁇ 4 and serotoninergic subunits receptors 5-HT3A, 5-HT3B and 5-HT3C.
  • AChR heteromeric nicotinic acetylcholine subunits receptors
  • the present invention also provides for the use of the functional fluorescent cationic serotoninergic and nicotinic fluorescent receptors for the characterization of their targeting and regulation of such targeting within the cells in vivo by extracellular agents. .
  • the present invention thus provides for a reliable and effective assay for screening and identifying pharmaceutically" effective compounds that specifically interact with and modulate the targeting of the cation channel serotoninergic and nicotinic receptors.
  • the subject assay enables rapid screening of large numbers of polypeptides in a library in order to identify those polypeptides which inhibit or foster receptor targeting and bioactivity in the cells or in vivo.
  • the assay is characterized by the use of a library of recombinant cells, each cell of which include (i) the serotoninergic or nicotinic receptors whose targeting can be modulated by interaction with an exogenous polypeptide, and (ii) an expressible recombinant gene encoding said exogenous polypeptide to be tested from a polypeptide library.
  • a gene library By the use of a gene library, the mixture of cells collectively expresses a population of test polypeptides.
  • fluorescent functional receptors are useful to be used to screen for ligands that affect the cellular distribution thereof when expressed in cells.
  • the ability of particular constituents of the peptide library to modulate the targeting of the serotoninergic receptor can be scored, hi any event, a statistically significant change in the detection signal can be used to facilitate isolation of those cells from the mixture which contain a nucleic acid encoding a polypeptide to be tested which is an effector of the target receptor.
  • polypeptides to be tested which induce or block receptors targeting in specific compartment in the cells can be identified, allowing the understanding of the mechanisms of such polypeptides.
  • the reagent cells express the serotoninergic or nicotinic receptor endogenously.
  • the cells are engineered to express a heterologous target receptor protein.
  • a heterologous receptor in which a heterologous receptor is provided utilize host cells in which the gene for the homologous receptor has been inactivated.
  • the present invention further provides for a dynamic assay for screening and identifying pharmacological compounds that specifically interact and modulate the activity of the serotoninergic or nicotinic receptor.
  • the method uses the co-expression of functional recombinant subunits of the serotoninergic or nicotinic receptors grafted with two different fluorophores in order to follow by FRET the motion of the subunits inside the receptors which was activated by pharmacological compounds.
  • Fluorescence Resonance Energy Transfer in Living Cells should enable to study ligand-receptor, and protein-protein interactions in living cells.
  • This method involves two fluorophores defined as a pair: a donor fluorophore, the emission spectrum of which overlaps with the excitation spectrum of the other fluorophore, called acceptor fluorophore.
  • acceptor fluorophore the emission spectrum of which overlaps with the excitation spectrum of the other fluorophore
  • the transferred energy amount is proportional to inverse 6th power of the distance between the two fluorophores.
  • the method can imply the labelling of two proteins with a fluorophore pair (respectively with the donor and with the acceptor).
  • a fluorophore pair When excited at the excitation wavelength of the donor fluorophore, if FRET occurs, light emission is observed at the emission wavelength of the acceptor.
  • An alternative fluorescence imaging method is Fluorescence lifetime imaging (FLDvf).
  • FLDvf Fluorescence lifetime imaging
  • the fluorescence decay time the average time that the fluorophore (donor) resides in the excited state before emitting a fluorescent photon, is employed for imaging purposes.
  • the fluorescence lifetime is independent of fluorescent concentration, excitation intensity, and fading due to photobleaching.
  • FLIM may yield direct information about the local chemical environment and the state of the fluorescent molecules in the specimen.
  • the fluorescence lifetime of a donor position at proximity of an acceptor, in a FRET situation is reduced.
  • the fluorescence lifetime of the donor can be easily related to the FRET efficiency, i conclusion both methods (measurement of the fluorescence intensity or the fluorescence lifetime) are sensitive to study inter- and intra-molecular interactions (detects distances variations between the fluorophore in the range of 50-100 angstroms).
  • these receptors belong to the family of cationic pentameric ionotropic receptor specific for the acetylcholine and the serotonin, which are the natural neurotransmitters.
  • These fluorescent functional receptors are useful to be used to screen for chemical compounds and proteins that non-covalently interact therewith.
  • the fluorescent resonance energy transfer between subunits can be used to follow the conformation changes of the subunits in presence or absence of drugs.
  • the subunits will be grafted with two different fluorescents protein adapted for the fluorescent resonance energy transfer measurement
  • the invention finally concerns, the use of these fluorescents proteins for screening by fluorescent resonance energy transfer the protein partners that are associated with these receptors in the central nervous system.
  • the present invention thus provides for a dynamic assay for screening and identifying extracellular or intracellular partners by measuring the fluorescence signal and/or by FRET, and more particularly for the screening of protein partner genes of the cationic receptors, and the screening of new chemicals and drugs useful for example in the frame of the treatment of central nervous system diseases, and more particularly myasthenia gravis, Parkinson disease, epilepsy, nervous breakdown.
  • FIGS 1 and IB Schematic representation of fluorescent subunits.
  • FIG. 1A represents a schematic representation of the fusion proteins of the invention.
  • the fluorescent protein (FP; dotted box) was inserted between a common leader peptide (LP) and the first amino acid of the mature rat ⁇ 3 and ⁇ 4 nicotinic receptor subunits (white box) and the mouse "5HT 3A receptor subunit (grey box).
  • the numbered black boxes correspond to transmembrane domains.
  • Figure IB corresponds to a proposed transmembrane (TM) topology of FP- ⁇ 3 tagged subunit (left), and a top view of FP- ⁇ 3 ⁇ 4 pentameric organization (right).
  • TM transmembrane
  • Figures 2A-2F Functional expression of FP-tagged ⁇ 3 ⁇ 4-nAChR and 5HT 3A R.
  • Ach-evoked currents in HEK-293 cells expressing wild type and fluorescently tagged ⁇ 3 ⁇ 4 and 5HT 3A receptors ⁇ 3 ⁇ 4 ( Figure 2A), YFP- ⁇ 3 ⁇ 4 ( Figure 2B), ⁇ 3CFP- ⁇ 4 ( Figure 2C), YFP- ⁇ 3CFP- ⁇ 4 ( Figure 2D), and YFP-5HT 3A ( Figure 2F).
  • Horizontal bar indicate the time of ACh (acetylcholine) or 5HT applications, at concentrations indicated in the figures.
  • Figures 3 -3D Fluorescence signal from YFP-5HT 3A R, and ⁇ 3CFP- ⁇ 4 nAChR, in living HEK-293 cells.
  • Figures 3A and 3B show HEK-293 cells expressing the YFP-5HT 3A receptor.
  • Figures 3C and 3D show HEK-293 cells expressing the ⁇ 3CFP- ⁇ 4 receptor.
  • Figures 4A-4C Subcellular localization of YFP-5HT 3A receptor in HEK-293 cells and hippocampal neuron.
  • Figures 3 A and 3B correspond to the expression of YFP-5HT 3 A receptor in HEK- 293 cell and Figure 3C corresponds to the expression of YFP-5HT 3 A receptor in hippocampal single neuron. It should be noted that in HEK-293 a subcellular localization of the YFP-5HT 3 A receptor in several micropodia. The YFP-5HT 3 A receptor is localized in micropodia end points and spine-like formations of the hippocampal neurons. Scale: A, lO ⁇ m; B, 2 ⁇ m; C, 30 ⁇ m.
  • Figures 5 and 5B Probing subunit-subunit interaction by measurement of lifetime values of fluorescent receptors.
  • Figure 5A corresponds to an illustration for the FRET-Fluorescence resonance energy transfer (arrows) between fluorescent proteins CFP (donor) to YFP (acceptor). Note that the co-expression of non-fluorescent subunit ⁇ should theoretically decrease the FRET efficiency between the fluorescents subunits CFP- ⁇ 2 and YFP- ⁇ 2 as illustrated by the number of arrows (schematic representation of the FRET).
  • Figure 2B corresponds to fluorescence lifetime images of CFP- ⁇ 2 measured alone (2.6 ns ⁇ 0.021).
  • the fluorescence lifetime images of cells coexpressing CFP- ⁇ 2 and YFP- ⁇ 2 clearly display a decrease of the CFP- ⁇ 2 lifetime.
  • the existence of this low CFP- ⁇ 2 lifetime (FLDVl data: 2.37 ns ⁇ 0.031) suggests auto-association between fluorescent ⁇ 2 subunit.
  • the increase of CFP- ⁇ 2 lifetime (FLTM data: 2.49 ns ⁇ 0.029) in presence of non fluorescent 4 subunit demonstrate that the subunit is interacting with the ⁇ 2 subunit.
  • Example 1 DNA constructs and transfection of HEK-293 cells
  • the fluorescent-tagged nicotinic ⁇ 3 and ⁇ 4 and serotoninergic 5HT 3A subunits were generated using a strategy analogous to that previously reported for the zebrafish Glycine subunit (GlyRzl) (David-Watine et al, 1999).
  • Rat ⁇ 3, ⁇ 4 nicotinic subunits and mouse 5HT 3A long splicing form sequences were amplified by polymerase chain reaction (PCR) from the codon encoding the first amino-acid to the stop codon.
  • PCR products were digested with proper restriction enzymes and ligated into a pMT 3 based vector (Swick et al, 1992) containing, in frame, the peptide signal from the GlyRzl, a linker (AV), the enhanced cyan or yellow fluorescent proteins (CFP, YFP; Ozyme, France) and a linker.
  • This linker was PAAA and PAAASPMPP for the 5-HT3 receptor and the x ⁇ y nicotinic receptors, respectively. All expression plasmid constructs were checked by restriction mapping and nucleotide sequencing.
  • HEK-293 cell transfection of the constructions was carried out using the LipofectAMINE Reagent PLUS procedure (Gibco BRL) and visualized 2 to 7 days afterwards.
  • Fusing a fluorescent protein to one subunit (YFP- 3 ⁇ 4, ⁇ 3CFP- ⁇ 4) or to both nAChR subunits (YFP- 3CFP- ⁇ 4) still allowed the expression of functional receptors, hi cells transfected either with the FP-label fused to one subunit or with both FP-labeled subunits, ACh elicited currents displaying time course and current-voltage relationships typical of those obtained with their wild counterparts (Fig. 2B-E). The proportion of responding cells were also similar, however, the current amplitudes seemed to be smaller ⁇ vith FP-grafted receptors.
  • Example 3 Demonstration of the targeting of the fluorescent receptor by digital-imaging fluorescence microscopy
  • Fluorescence images were acquired using a Zeiss Axiovert fluorescent microscope that was fitted with a Sensicam charge-coupled device camera (12 bit) and controlled by MetaVue software package (Universal Imaging Corporation, USA). Filter cube specifications for the fluorescence channels were as follows (for excitation, emission, and main dichroic beam splitter, respectively): CFP (440 ⁇ 21nm, 480 ⁇ 30nm, 455nm); YFP (500 ⁇ 25nm, 530nm LP, 525nm), (Chroma, USA).
  • Example 4 Demonstration of the targeting of the fluorescent receptors in rat hippocampal primary culture and transfection
  • Hippocampal neurons were prepared from 17 days-old rat embryos. Briefly, dissected hippocampi were mechanically and chemically dissociated in single-cell suspension with trypsin (0.25 mg/ml) (Brewer et al, 1993). The cells were centiifuged and suspended in Neurobasal medium (GibcoBRL) supplemented with Glutamax I (GibcoBRL), B27 (GibcoBRL), 100 U/ml penicillin, and 100 ⁇ g/ml streptomycin (DMEM medium; GibcoBRL). The dissociated cells were plated onto 0.18 mm thick glass bottom dishes pre-coated with 0.5 mg/ml poly(L-ornithine) (Sigma).
  • the cDNAs encoding for the CFP and YFP were introduced as described in Fig. 1.
  • the corresponding fluorescently tagged subunits (FP -tagged) are termed YFP- ⁇ 3, CFP- ⁇ 4 and YFP-5HT 3A .
  • FP-tagged receptor subunits form functional channels
  • FP-tagged subunits designed here assemble into fully functional receptor channels
  • their electrophysiological properties were compared to those of non-tagged receptors in .whole-cell patch-clamp recordings performed in transiently transfected HEK-293 cells.
  • the co-expression of ⁇ 3 and ⁇ 4 nAChR subunits is required to form functional hetero-pentameric channels (Duvoisin et al, 1989). ha fifty percents of the cells transfected with wild-type ⁇ 3 ⁇ 4 or fluorescent YFP- ⁇ 3 ⁇ 4, ⁇ 3CFP- ⁇ 4 nAChRs, ACh elicited inward currents at a holding potential of 100 mV.
  • Example 6 Demonstration of differential expression of 5HT 3A Rs and ⁇ 3 ⁇ 4 nAChRs at the surface of HEK-293 cells
  • HEK-293 cells were transiently transfected with various tagged subunits.
  • the co-expression of YFP- ⁇ 3 with wild-type ⁇ 4 and the co-expression of CFP- ⁇ 4 with wild-type ⁇ 3 were investigated.
  • 5HT 3 A subunits are known to form functional homo-oligomers, thus the YFP-5HT 3A fusion was transfected alone (Maricq er a/., 1991).
  • HEK-293 cells Two to seven days after transfection, HEK-293 cells were visualized by epifluorescence (Fig. 3A) and confocal microscopy (Fig. 3G-L). Nicotinic and serotoninergic receptors displayed specific fluorescence distributions. For the YFP- ⁇ 3 ⁇ 4 and ⁇ 3CFP- ⁇ 4 subunit co-expressions, the fluorescence was distributed within cytoplasmic compartments of the cell (Fig. 3D). Expression of YFP-5HT 3A R yielded fluorescence both in intracellular compartments and near or within the cytoplasmic membrane (see arrows in Fig. 3B).
  • live imaging enables visualization of the micro- extensions of the plasma membrane that are otherwise easily damaged by fixation procedures or mechanical force during specimen preparation. Indeed, more detailed analysis of images shows that the 5HT 3 AR is specifically targeted to the tips of micropodia (Fig. 4B).
  • Example 7 Demonstration of the differential the intracellular targeting of 3 ⁇ 4 nAChRs and 5HT 3A Rs in the endoplasmic reticulum
  • YFP-tagged subunits were co-transfected with the endoplasmic reticulum fluorescent marker ER-CFP.
  • YFP- ⁇ 3 ⁇ 4 expressed in HEK-293 cells along with ER-CFP protein marker revealed a strong co-localization of the two labels, indicating that the 3 ⁇ 4 receptor is mainly located in the ER.
  • YFP-5HT 3A R was found in intracellular vesicles, and to a lesser extent in the ER. Time imaging sequence of the vesicles expressing the YFP-5HT 3 AR follows linear trajectory consistent with a microtubule-mediated movement.
  • Example 8 Demonstration of the differential targeting of 5HT 3A R and ⁇ 3 ⁇ 4 nAChR to the plasmalemma in hippocampal neurons
  • the fluorescence signals of ⁇ 3CFP- ⁇ 4 receptors and YFP-5HT 3A receptors were normalized with respect to each other by matching the fluorescence in the cell body region.
  • Two-channel, quantitative analysis of normalized fluorescent intensities indicated that unlike the ⁇ 3CFP- ⁇ 4 which remained in the cell body, the YFP-5HT 3A is highly localized in the dendrites.
  • Example 9 Demonstration of subimit-subunit interaction by measurement of lifetime fluorescence for Fluorescent nicotinic receptor expressed in HEK-293 cells.
  • nicotinic ⁇ 2 subunit was grafted in N-terminal with YFP or CFP fluorescent protein using the same strategy as developed as for the example 1.
  • the expressing vectors encoding for the subunits' were transfected in HEK-293 cell lines using Lipofectamine method and tested after 48 hours.
  • Fluorescence Resonance Energy Transfer in. Living Cells should enable to study protein-protein interactions in living cells. If the two fluorophores, the donor (CFP) and the acceptor (YFP) are close enough, part of the energy absorbed by the donor is transferred to the acceptor. In this respect, we have used fluorescence life time imaging techniques to probe the fluorescence lifetime of CFP- ⁇ 2 fluorescent nicotinic subunit to measure the respective putative proximity of the YFP- ⁇ 2 nicotinic subunit. When the donor is close ( ⁇ 100 A) of an acceptor, in a FRET situation, the lifetime is reduced.
  • LIMO High Speed Lifetime Module
  • fluorescents subunits we were able to monitor protein-protein interaction by fluorescence resonance energy transfer (FLIM-TREF) between subunits belonging to the cationic super-family of pentameric ligand-gated ion channels.
  • FLIM-TREF fluorescence resonance energy transfer

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Abstract

La présente invention concerne une protéine hybride entre:- une sous-unité d'un récepteur nicotinique neuronal hétéromère ou d'un récepteur ionotropique sérotoninergique, et, - une protéine fluorescente choisie parmi des protéines fluorescentes obtenues ou dérivées à partir de protéines autofluorescentes, ladite protéine étant choisie en particulier parmi: - une protéine verte fluorescente (GFP), ou des variants dérivés de GFP par addition, délétion ou substitution d'un ou de plusieurs acides aminés, à condition que lesdits variants conservent la capacité de fluorescence, - ou des fragments de GFP, ou des fragments des variants susmentionnés, à condition que lesdits fragments conservent la capacité de fluorescence, - ou des protéines autofluorescentes qui ont été isolées à partir d'autres organismes que l'Aequorea Victoria.
PCT/EP2003/008490 2002-07-31 2003-07-31 Proteines hybrides entre une proteine fluorescente et un recepteur ionotropique et utilisations associees WO2004013178A1 (fr)

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* Cited by examiner, † Cited by third party
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DE102010009996A1 (de) * 2010-03-02 2011-09-08 Jacobs University Bremen Ggmbh Verfahren zum Herstellen einer ersten codierenden Sequenz für Peptid, Protein zur Translationsverstärkung, codierende Sequenz, Expressionsvektor

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WO2000075311A1 (fr) * 1999-06-09 2000-12-14 Medical Research Council Recepteur nicotinique de recombinaison du nematode et ses utilisations
WO2002036629A2 (fr) * 2000-10-31 2002-05-10 Bayer Aktiengesellschaft Regulation du precurseur humain des recepteurs de la serotonine

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WO2000075311A1 (fr) * 1999-06-09 2000-12-14 Medical Research Council Recepteur nicotinique de recombinaison du nematode et ses utilisations
WO2002036629A2 (fr) * 2000-10-31 2002-05-10 Bayer Aktiengesellschaft Regulation du precurseur humain des recepteurs de la serotonine

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DUTTON J L ET AL: "P2X1 receptor membrane redistribution and down-regulation visualized by using receptor-coupled green fluorescent protein chimeras", NEUROPHARMACOLOGY, vol. 39, no. 11, 23 August 2000 (2000-08-23), pages 2054 - 2066, XP002264728, ISSN: 0028-3908 *
GENSLER SVEN ET AL: "Assembly and clustering of acetylcholine receptors containing GFP-tagged epsilon or gamma subunits: Selective targeting to the neuromuscular junction in vivo", EUROPEAN JOURNAL OF BIOCHEMISTRY, vol. 268, no. 8, April 2001 (2001-04-01), pages 2209 - 2217, XP002264725, ISSN: 0014-2956 *
MARSHALL J ET AL: "THE JELLYFISH GREEN FLUORESCENT PROTEIN: A NEW TOOL FOR STUDYING ION CHANNEL EXPRESSION AND FUNCTION", NEURON, CAMBRIDGE, MA, US, vol. 14, no. 2, 1 February 1995 (1995-02-01), pages 211 - 215, XP000645993 *
PALMA ELEONORA ET AL: "Some properties of human neuronal alpha7 nicotinic acetylcholine receptors fused to the green fluorescent protein", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES, vol. 99, no. 6, 19 March 2002 (2002-03-19), March 19, 2002, pages 3950 - 3955, XP002264726, ISSN: 0027-8424 *
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE102010009996A1 (de) * 2010-03-02 2011-09-08 Jacobs University Bremen Ggmbh Verfahren zum Herstellen einer ersten codierenden Sequenz für Peptid, Protein zur Translationsverstärkung, codierende Sequenz, Expressionsvektor

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