N‐Linked Glycosylation of the α‐Amino‐3‐Hydroxy‐5‐Methylisoxazole‐4‐Propionate(AMPA)‐Selective Glutamate Receptor Channel α2 Subunit Is Essential for the Acquisition of Ligand‐Binding Activity

The N‐linked glycosylation of the α2 subunit of the mouse α‐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 [ 35 S]methionine/[ 35 S]cysteine metabolic labeling, western blot analysis, immunocytochemical detection, and [ 3 H]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 ∼102 kDa and a minor species of ∼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 GluRα2 subunit protein in an active conformation for ligand binding. Possible roles of N ‐glycosylation of GluRα2 subunit protein are discussed.