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 Ib (NMDAR1b), its corresponding cDNA was extended by codons for six h istidine residues at the 3'-end, cloned into a baculovirus transfer ve ctor and integrated into the viral genome. Infection of Trichoplusia n i insect cells (High Five(TM) cells) with recombinant baculovirus resu lted in the production of 126- and 105-kDa NR1b proteins in the cell m embrane fraction. Enzymatic deglycosylation with PNGase F as well as i nfection of the insect cells in the presence of tunicamycin revealed t hat the two proteins represented the N-glycosylated and non-glycosylat ed forms of NMDAR1b, respectively. The recombinant NR1b protein was al so identified with immunocytochemical methods employing a monoclonal a ntibody which recognized the six histidine residues. The affinity of t his 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-H-3]dichl orokynurenate (DCKA). The observed binding characteristics were simila r to those obtained for native NMDA receptors. Whereas in electrophysi ological 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.