Overexpression of a functional NMDA receptor subunit (NMDAR1) in baculovirus-infected Trichoplusia ni insect cells.
For overexpression of the N-methyl-D-aspartate (NMDA) receptor subunit 1b (NMDAR1b), its corresponding cDNA was extended by codons for six histidine residues at the 3'-end, cloned into a baculovirus transfer vector and integrated into the viral genome. Infection of Trichoplusia ni insect cells (High FiveTM cells) with recombinant baculovirus resulted in the production of 126- and 105-kDa NR 1b proteins in the cell membrane fraction. Enzymatic deglycosylation with PNGase F as well as infection of the insect cells in the presence of tunicamycin revealed that the two proteins represented the N-glycosylated and non-glycosylated forms of NMDAR1b, respectively. The recombinant NR1b protein was also identified with immunocytochemical methods employing a monoclonal antibody which recognized the six histidine residues. The affinity of this histidine tag to nickel ions was used for the purification of the NR1b protein. The glycine binding site of the subunit was successfully identified and analyzed with the specific antagonist 5,7-[3-3H]dichlorokynurenate (DCKA). The observed binding characteristics were similar to those obtained for native NMDA receptors. Whereas in electrophysiological measurements a functional NMDA receptor channel could not be found in infected insect cells, its expression was demonstrated in the Xenopus oocyte system after injection of the NMDAR1b cDNA construct.
Sydow
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Department of Molecular Neuroendocrinology, Max Planck Institute for Experimental Medicine, Göttingen, Germany. sydow@mail.mpiem.gwdg.de
Köpke
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AK
Blank
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Spiess
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D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D002846:Chromatography, Affinity; D018076:DNA, Complementary / Q000235:genetics; D004594:Electrophysiology; D015967:Gene Expression Regulation, Viral; D005822:Genetic Vectors / Q000235:genetics*; D006031:Glycosylation / Q000187:drug effects; D006639:Histidine / Q000378:metabolism; D007736:Kynurenic Acid / Q000031:analogs & derivatives / Q000378:metabolism; D009036:Moths / Q000166:cytology*; D009532:Nickel / Q000378:metabolism; D017924:Nucleopolyhedroviruses / Q000235:genetics*; D009865:Oocytes; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D016194:Receptors, N-Methyl-D-Aspartate / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D014162:Transfection; D014415:Tunicamycin / Q000494:pharmacology; D014982:Xenopus laevis
D018076:DNA, Complementary; D006639:Histidine; D007736:Kynurenic Acid; D009532:Nickel; D016194:Receptors, N-Methyl-D-Aspartate; D011993:Recombinant Fusion Proteins; D014415:Tunicamycin; C066192:5,7-dichlorokynurenic acid