Expression and characterization of the zeta 1 subunit of the N-methyl-D-aspartate (NMDA) receptor channel in a baculovirus system.
Using a baculovirus expression vector system, the zeta 1 subunit of the mouse N-methyl-D-aspartate (NMDA) receptor channel was expressed in Spodoptera frugiperda insect cells. The peptide corresponding to the C-terminus of the zeta 1 subunit was synthesized by using the multiple antigen peptide (MAP) system, and an antibody to the synthetic peptide was produced. Immunoblotting using the newly developed antibody revealed the major 122-kDa and the minor 104-kDa protein bands. The effect of tunicamycin on the immunoblots and [35S]methionine/[35S]cysteine metabolic radiolabeling suggested that the two bands corresponded to glycosylated and non-N-glycosylated forms, respectively. Membranes prepared from insect cells infected with the recombinant virus had the binding activity of antagonist ligand 5,7-[3-3H]dichlorokynurenate (DCKA) of a glycine recognition domain of the receptor. Both immunofluorescence labeling and the [3H]DCKA binding assays also showed a greater level of expression (Bmax = 51 pmol/mg protein) in the insect cells. The ligand binding characteristics of the receptors expressed in insect cells suggested that the single zeta 1 subunit protein has glycine antagonist binding properties comparable to those of the native NMDA receptor channels. The lack of DCKA-binding activity of the non-N-glycosylated NMDA receptor expressed in the presence of tunicamycin suggested that N-linked oligosaccharide is essentially required for expression of a functional receptor in insect cells. This is the first report describing the importance of N-glycosylation for the acquisition of ligand binding to NMDA receptor channel subunit protein.
Kawamoto
S
S
Department of Bacteriology, Yokohama City University School of Medicine, Japan.
Uchino
S
S
Hattori
S
S
Hamajima
K
K
Mishina
M
M
Nakajima-Iijima
S
S
Okuda
K
K
D000818:Animals; D000906:Antibodies / Q000276:immunology; D016367:Baculoviridae / Q000235:genetics* / Q000378:metabolism; D015153:Blotting, Western; D002478:Cells, Cultured; D004586:Electrophoresis; D005455:Fluorescent Antibody Technique; D015870:Gene Expression; D007700:Kinetics; D011817:Rabbits; D017470:Receptors, Glutamate / Q000235:genetics; D016194:Receptors, N-Methyl-D-Aspartate / Q000096:biosynthesis* / Q000235:genetics; D018411:Spodoptera; D014415:Tunicamycin / Q000494:pharmacology
D000906:Antibodies; D017470:Receptors, Glutamate; D016194:Receptors, N-Methyl-D-Aspartate; D014415:Tunicamycin