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WO1995026401A1 - Procede pour l'expression permanente de recepteurs du glutamate - Google Patents

Procede pour l'expression permanente de recepteurs du glutamate Download PDF

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Publication number
WO1995026401A1
WO1995026401A1 PCT/EP1995/001029 EP9501029W WO9526401A1 WO 1995026401 A1 WO1995026401 A1 WO 1995026401A1 EP 9501029 W EP9501029 W EP 9501029W WO 9526401 A1 WO9526401 A1 WO 9526401A1
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WO
WIPO (PCT)
Prior art keywords
glutamate
cell lines
cells
glutamate receptors
receptors
Prior art date
Application number
PCT/EP1995/001029
Other languages
German (de)
English (en)
Inventor
Sylvia Sterrer
Andreas Ultsch
Thomas Höger
Hans-Georg Lemaire
Alfred Bach
Original Assignee
Basf Aktiengesellschaft
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Basf Aktiengesellschaft filed Critical Basf Aktiengesellschaft
Priority to EP95928871A priority Critical patent/EP0753064A1/fr
Priority to JP7524940A priority patent/JPH09510870A/ja
Publication of WO1995026401A1 publication Critical patent/WO1995026401A1/fr
Priority to MXPA/A/1996/004382A priority patent/MXPA96004382A/xx

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Classifications

    • 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

Definitions

  • the invention relates to the permanent ectopic expression of glutamate receptors in eukaryotic cells: the production of suitable recombinant cell lines with the abovementioned. Properties and their use. 0
  • Glutamate the most important excitatory neurotransmitter in the central nervous system (Trends in Pharmacological Sciences 11, 1990, 126-132; Pharmacological Reviews 40, 1989, 143-210; Trends in Pharmacological Sciences 13, 1992, 291-296) is in numerous 5 pathophysiological processes such as Epilepsy, schizophrenia, ischemia involved.
  • Receptors for glutamate are therefore potential targets for pharmaceuticals to treat these diseases. 0
  • the invention relates only to the class of ionotropic glutamate receptors.
  • RNA editing For GluR-B from mouse and rat, "RNA editing" has additionally been shown, which relates to the so-called Q / R site of the second transmembrane membrane. These two GluR-B variants differ 40 considerably in their electrophysiological properties (Cell 67, 1991, 11-19; Neuron 8, 1992, 189-198). The human cDNA's for GluR-Aflip and GluR-Aflop have also been published (PNAS USA 88, 1991, 7557-7561; PNAS USA 89, 1992, 1443-1447).
  • AMPA receptor subunits can form both homo- and heteromeric channels.
  • NMDA receptors can form heteromeric structures which consist of an NR1 subunit (Nature 354, 3 (1991)) and one of four NR2 subunits (2A, 2B, 2C, 2D) (Science 256, 1217 (1992); Nature 358 , 36 (1992); Nature 357, 70 (1992); FEBS Lett. 313, 34 (1992); J. Biol. Chem. 268, 2836 (1993)).
  • NMDA channels have slower kinetics than AMPA channels, but have a high Ca 2+ permeability, have a voltage-dependent Mg 2+ block and require glycine as a coagonist.
  • Functional Kaina receptors can be derived from the subunits GluR5, GluR6, GluR7 (Neuron 5, 583 (1990); Nature 351, 745 (1991); EMBO J. 11, 1651 (1992); Neuron 8, 257 (1992); FEBS Lett . 307, 139 (1992)) and KAI and KA2 (Nature 351, 742 (1991); Neuron 8, 267 and 775 (1992)). These channels are characterized by very rapid kinetics and the activation of very rapidly desensitizing currents by AMPA and Kainat.
  • transiently expressing cells are, however, not very suitable as test cells for the identification of glutamate receptor antagonists, since they cannot be produced in a completely reproducible manner and, as a result, the results obtained therefrom cannot be compared or can only be compared with great difficulty.
  • the object was therefore to provide a method for producing cell lines expressing eukaryotic permanent ectopic glutamate receptors.
  • the invention relates to a process for the production of eukaryotic cell lines expressing permanently ectopic glutamate receptors by transforming cells with a nucleic acid coding for glutamate receptors, characterized in that at least one of the following culture conditions is maintained when establishing the cell lines: a) culturing the transformed cells in a culture medium which contains a glutamate precursor,
  • Eukaryotic cells can generally be used as cells which are suitable for the method according to the invention.
  • cells from lower eukaryotes such as yeasts and fungi, or insect cells can be used.
  • Cells from mammals are preferably used, for example HEK 293, BHK, COS 7.
  • These cells are transformed with one or more nucleic acids which code for a glutamate receptor.
  • the method according to the invention is suitable both for the permanent expression of individual glutamate receptor subunits and for combinations of several subunits. These can be naturally occurring combinations of subunits as well as new, not naturally occurring combinations of subunits. It is also possible to recombine subunits of different species. The combination of glutamate receptor subunits from different subclasses (for example from AMPA, Kainat, NMDA) is also possible.
  • the individual subunits or combinations of individual subunits of the human glutamate receptors are preferably used.
  • the nucleic acids used for the transformation are generally used linked to an expression vector.
  • the choice of a suitable expression vector depends, among other things, on the cell to be transformed. This should ensure that the regulatory signals of the expression vector are also recognized by the cell.
  • a large number of vectors can be used for expression in eukaryotic cells, wherein vectors with cytomegalovirus promoters show particularly good expression.
  • the expression constructs can be introduced into the cells by means of various methods, for example electroporation, calcium phosphate precipitation or liposome-mediated.
  • Eukaryotic expression systems have the advantage of expressing the corresponding expression products effectively and mostly in native form and also of modifying them post-translationally.
  • At least one of the culture conditions described under a) b) and c) must be fulfilled.
  • a culture medium is used for cell growth which does not contain glutamic acid or its salts (glutamates).
  • the culture medium contains one or more glutamate precursors which are converted into glutamate in the cells.
  • Suitable glutamate precursors are compounds which are taken up as such by the cell and from which intracellularly releases glutamine, which is subsequently converted into glutamate by metabolic reactions.
  • Such compounds are, for example, glutamine-containing oligopeptides or oligopeptide derivatives such as the corresponding esters or amides.
  • Dipeptides composed of an apolar amino acid and glutamine are particularly suitable.
  • Such media containing glutamate precursors are also commercially available, e.g. the Gluta ax medium from Gibco BRL, which contains the dipeptide L-alanyl-L-glutamine as the glutamate precursor.
  • the glutamate precursors are generally added to the culture medium in a final concentration in the ⁇ M range, preferably from 1 to 100 ⁇ M.
  • Another possible culture condition for the method according to the invention is the culturing of the transformed cells mentioned under b) in the presence of a glutamate receptor antagonist.
  • Suitable glutamate receptor antagonists are, for example, NBQX or CNQX.
  • the final concentrations of these compounds in the culture medium are usually in the ⁇ M range.
  • Another possible culture condition is the temporary repression of glutamate receptor expression mentioned under c).
  • An inducible expression system is used for this, which is switched on or off depending on the culture conditions.
  • culture conditions are selected in which the glutamate receptor expression is repressed.
  • the repression is then released, which causes a receptor expression.
  • Preferred inducible expression systems are those which are controlled by means of the tetracycline operon.
  • the invention further relates to the cell lines which permanently express ectopic glutamate receptors and which can be prepared using the method described above.
  • Soche cell lines are particularly suitable for the identification of functional ligands of glutamate receptors.
  • Receptor-expressing cell lines are an important tool in the screening for specific receptor ligands.
  • membranes of the cell lines can, for example, be used in receptor binding tests.
  • reporter systems are those in which a promoter which is regulated by compounds of the signal transduction pathway (second messenger) is functionally linked to a gene for an easily detectable product such as luciferase.
  • Such reporter systems are, for example, from Science 252, 1424 (1991); Proc. Natl. Acad. Be. USA 88, 5061 (1991) or J. Rec. Res. 13, 79 (1993).
  • a suitable promoter, which is regulated, for example, by the intracellular Ca 2+ concentration, is that of the fos gene.
  • the me- Tallothionein promoter which can be stimulated by Zn 2+ , is also suitable.
  • the change in the intracellular ion concentration caused by the binding of a ligand to a receptor can be determined via fluorescent dyes (for example FURA 2AM, sodium green, calcium green, aequorin (Analyt. Biochem. 209, 343 (1993)) or via chemical detection reactions (such as precipitation by ions (Neuron 7, 509 (1991)).
  • fluorescent dyes for example FURA 2AM, sodium green, calcium green, aequorin (Analyt. Biochem. 209, 343 (1993)
  • chemical detection reactions such as precipitation by ions (Neuron 7, 509 (1991)).
  • the current flow through the cell membrane depending on the ligand binding can also be measured.
  • the expressed receptor proteins can also serve as antigens for generating polyclonal or monoclonal antibodies which are used for diagnostic purposes or as aids for rational drug design.
  • the pure polypeptide can also be used to elucidate the spatial structure of the receptor and the ligand binding site.
  • the cell lines according to the invention can also be implanted in recipient systems such as transgenic animals or host organisms in order to influence the signal transduction or to analyze the ligand concentration.
  • the corresponding glutamate receptor cDNA molecules (WO 93/23536; Science 249, 556-560, 1990; J. Biol. Chem. 268, 3728-3733, 1993; WO 91/06648) were converted into the eukaryotic expression vector pcDNA3 (Fa. Invitrogen) cloned.
  • pcDNA3 Fa. Invitrogen
  • These expression constructs were introduced individually or in combination in HEK 293 cells according to the following protocol by electroporation: For the electroporation, 10 7 cells in 0.8 ml PBS with 20 ⁇ g of the expression construct with an electroporator (BTX, electro cell manipulator 600, 3 mF, 130 V, 72 Ohm) transfected.
  • BTX electroporator
  • the cells were incubated for 24-36 h in culture medium and then transferred to selection medium (culture medium with 600-800 ⁇ g / ml G418 sulfate, geneticin or 50 ⁇ g / ml hygromycin). Stable genetic or hygromycin-resistant cell clones were isolated after 10-12 days via single cell deposition, expanded and analyzed by means of a membrane binding assay.
  • the recombinant cell lines were cultivated as described in Example 1.
  • Membrane preparation cells were scraped off in 2-3 ml of 5 mM Tris pH 7.4 / 10 cm dish and centrifuged (1200 rpm, 4 ° C.). The pellet was resuspended in 1 ml of ice-cold Tris (5 mM, pH 7.4), incubated for 15 min on ice and then centrifuged at 11500 rpm, 4 ° C. The pellet was resuspended in 1.5 ml binding buffer / 10 cm dish (30 mM Tris pH 7.2 at 10 ° C.), 2.5 mM CaCl, 100 mM KSCN), homogenized, centrifuged (11500 rpm, 30 min , 4 ° C). The supernatant was homogenized again and centrifuged. The membrane pellet is resuspended in 150 ⁇ l / 10 cm dish of binding buffer and used for the binding assay.
  • Binding batch 50 ⁇ l membranes in binding buffer with 149 ⁇ l binding buffer and 1 ⁇ l 3 H-AMPA (600 nM) were incubated for 1 hour on ice, filtered through GFC filters, seen with binding buffer and counted.
  • 50 ⁇ l membranes were incubated in binding buffer with 147 ⁇ l binding buffer, 2 ⁇ l glutamate (100 mM) and 1 ⁇ l 3 H-AMPA (600 nM) and the procedure was the same as for the binding approach.
  • Reporter systems with the aid of which the influencing of the signal transduction path (second messenger) caused by the binding of a ligand can be detected.
  • the cells were cultivated and transfected as described in Example 1.
  • the cell clones were tested for inducible expression.
  • the recombinant cell lines were cultivated as described in Example 1.
  • FURA 2 - labeling of cells cells were detached with trypsin or EDTA and washed with labeling buffer (120 mM NaCl, 5 mM KCl, 1.5 mM MgCl 2 , ImM CaCl, 25 mM HEPES, 10 mM Glucose). The cells (2 ⁇ 10 6 per ml) in labeling buffer were labeled with 2 ⁇ M FURA-2-pentaacetoxymethyl ester for 15 min at 37 ° C. and then washed with labeling buffer. The marked cells were kept on ice and used for the measurements within 3 h.
  • labeling buffer 120 mM NaCl, 5 mM KCl, 1.5 mM MgCl 2 , ImM CaCl, 25 mM HEPES, 10 mM Glucose

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Cell Biology (AREA)
  • General Health & Medical Sciences (AREA)
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  • Gastroenterology & Hepatology (AREA)
  • Biomedical Technology (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Neurology (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Engineering & Computer Science (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

L'invention concerne un procédé de préparation de lignées cellulaires eucaryotes pour l'expression ectopique permanente de récepteurs du glutamate, par transformation des cellules avec des acides nucléiques codant pour des récepteurs du glutamate, caractérisé en ce que, lors de l'établissement des lignées cellulaires, on observe au moins l'une des conditions de culture ci-après: a) culture des cellules transformées dans un milieu de culture renfermant un précurseur du glutamate; b) culture des cellules transformées en présence d'un antagoniste du récepteur du glutamate; c) culture des cellules transformées dans une première phase, dans les conditions pour lesquelles l'expression du récepteur du glutamate est réprimée et, dans une seconde phase, dans les conditions pour lesquelles la répression est de nouveau interrompue. L'invention concerne également les lignées cellulaires obtenues et leur utilisation.
PCT/EP1995/001029 1994-03-29 1995-03-20 Procede pour l'expression permanente de recepteurs du glutamate WO1995026401A1 (fr)

Priority Applications (3)

Application Number Priority Date Filing Date Title
EP95928871A EP0753064A1 (fr) 1994-03-29 1995-03-20 Procede pour l'expression permanente de recepteurs du glutamate
JP7524940A JPH09510870A (ja) 1994-03-29 1995-03-20 グルタメート受容体を恒常的に発現する方法
MXPA/A/1996/004382A MXPA96004382A (en) 1994-03-29 1996-09-27 Method for the permanent expression of glutamate receptors

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DEP4410882.6 1994-03-29
DE19944410882 DE4410882A1 (de) 1994-03-29 1994-03-29 Verfahren zur permanenten Expression von Glutamatrezeptoren

Publications (1)

Publication Number Publication Date
WO1995026401A1 true WO1995026401A1 (fr) 1995-10-05

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PCT/EP1995/001029 WO1995026401A1 (fr) 1994-03-29 1995-03-20 Procede pour l'expression permanente de recepteurs du glutamate

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EP (1) EP0753064A1 (fr)
JP (1) JPH09510870A (fr)
CA (1) CA2186788A1 (fr)
DE (1) DE4410882A1 (fr)
WO (1) WO1995026401A1 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5849895A (en) * 1993-04-20 1998-12-15 Sibia Neurosciences, Inc. Human N-methyl-D-aspartate receptor subunits, nucleic acids encoding same and uses therefor
US7579471B2 (en) 2002-04-08 2009-08-25 Pfizer, Inc. Tropane derivatives useful in therapy

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU4251300A (en) 1999-05-07 2000-11-21 University Of Virginia Patent Foundation Biological production of stable glutamine, poly-glutamine derivatives in transgenic organisms and their use for therapeutic purposes
SG187885A1 (en) * 2010-08-31 2013-03-28 Friesland Brands Bv Culture medium for eukaryotic cells

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992010583A1 (fr) * 1990-12-12 1992-06-25 Zymogenetics, Inc. Recepteurs de glutamate couples a une proteine g
WO1993024629A1 (fr) * 1992-05-25 1993-12-09 Novo Nordisk A/S Cellule exprimant un recepteur de glutamate

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992010583A1 (fr) * 1990-12-12 1992-06-25 Zymogenetics, Inc. Recepteurs de glutamate couples a une proteine g
WO1993024629A1 (fr) * 1992-05-25 1993-12-09 Novo Nordisk A/S Cellule exprimant un recepteur de glutamate

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
TYGESEN C. K. ET AL.: "Stable expression of a fumctional GluR6 homomeric glutamate receptor channel in mammalian cells", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF USA, vol. 91, December 1994 (1994-12-01), WASHINGTON US, pages 13018 - 13022 *

Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5849895A (en) * 1993-04-20 1998-12-15 Sibia Neurosciences, Inc. Human N-methyl-D-aspartate receptor subunits, nucleic acids encoding same and uses therefor
US5985586A (en) * 1993-04-20 1999-11-16 Sibia Neurosciences, Inc. Methods for identifying compounds that modulate the activity of human N-methyl-D-aspartate receptors
US6033865A (en) * 1993-04-20 2000-03-07 Sibia Neurosciences, Inc. Human n-methyl-d-aspartate receptor type 1 subunits, DNA encoding same and uses therefor
US6111091A (en) * 1993-04-20 2000-08-29 Merck & Co., Inc. Human N-methyl-D-aspartate receptor subunits, nucleic acids encoding same and uses therefore
US6316611B1 (en) 1993-04-20 2001-11-13 Merck & Co., Inc. Human N-methyl-D-aspartate receptor subunits, nucleic acids encoding same and uses therefor
US6376660B1 (en) 1993-04-20 2002-04-23 Merck & Co., Inc. Human N-methyl-D-aspartate receptor subunits, nucleic acids encoding same and uses therefor
US6469142B1 (en) 1993-04-20 2002-10-22 Merck & Co., Inc. Human N-methyl-D-aspartate receptor subunits, nucleic acids encoding same and uses therefor
US6521413B1 (en) 1993-04-20 2003-02-18 Merck & Co., Inc. Human N-methyl-D-aspartate receptor subnits, nucleic acids encoding same and uses therefor
US6825322B2 (en) 1993-04-20 2004-11-30 Merck & Co., Inc. Human N-methyl-D-aspartate receptor subunits, nucleic acids encoding same and uses therefor
US6864358B2 (en) 1993-04-20 2005-03-08 Merck & Co., Inc. Human n-methyl-d-aspartate receptor subunits, nucleic acids encoding same and uses therefor
US6956102B2 (en) 1993-04-20 2005-10-18 Merck & Co., Inc. Human N-methyl-D-aspartate receptor subunits nucleic acids encoding same and uses therefor
US7579471B2 (en) 2002-04-08 2009-08-25 Pfizer, Inc. Tropane derivatives useful in therapy

Also Published As

Publication number Publication date
MX9604382A (es) 1997-10-31
EP0753064A1 (fr) 1997-01-15
CA2186788A1 (fr) 1995-10-05
JPH09510870A (ja) 1997-11-04
DE4410882A1 (de) 1995-10-05

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