N-linked glycosylation of the alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA)-selective glutamate receptor channel alpha 2 subunit is essential for the acquisition of ligand-binding activity.
The N-linked glycosylation of the alpha 2 subunit of the mouse alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA)-selective glutamate receptor (GluR) channel was characterized. The receptor subunit protein has five putative N-glycosylation sites. The recombinant receptor proteins were identified by [35S]methionine/[35S]cysteine metabolic labeling, western blot analysis, immunocytochemical detection, and [3H]AMPA binding experiments when expressed in insect Spodoptera frugiperda cells using a baculovirus system. The effect of tunicamycin on the metabolic labeling and immunoblots suggested that the two products, a major protein species of approximately 102 kDa and a minor species of approximately 98 kDa, correspond to glycosylated and unglycosylated forms, respectively, which was also supported by the enzymic deglycosylation experiments. Immunofluorescence staining of tunicamycin-treated cells expressing only the unglycosylated form differed little from that of tunicamycin-nontreated cells expressing both glycosylated and unglycosylated forms. The lack of AMPA-binding activity of the unglycosylated form expressed in the presence of tunicamycin suggested that N-glycosylation is required, directly or indirectly, for functional expression in insect cells for ligand binding. These results demonstrate that occupancy of at least one N-glycosylation site is required for the formation and maintenance of the GluR alpha 2 subunit protein in an active conformation for ligand binding. Possible roles of N-glycosylation of GluR alpha 2 subunit protein are discussed.
Kawamoto
S
S
Department of Bacteriology, Yokohama City University School of Medicine, Japan.
Hattori
S
S
Sakimura
K
K
Mishina
M
M
Okuda
K
K
D000818:Animals; D005260:Female; D006031:Glycosylation / Q000187:drug effects; D066298:In Vitro Techniques; D015640:Ion Channel Gating / Q000187:drug effects; D051379:Mice; D008807:Mice, Inbred BALB C; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D018091:Receptors, AMPA / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins; D018411:Spodoptera; D014415:Tunicamycin / Q000494:pharmacology; D018350:alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid / Q000494:pharmacology*
D018091:Receptors, AMPA; D011994:Recombinant Proteins; D014415:Tunicamycin; D018350:alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid