N-LINKED GLYCOSYLATION OF THE PHA-AMINO-3-HYDROXY-5-METHYLISOXAZOLE-4-PROPIONATE (AMPA)-SELECTIVE GLUTAMATE-RECEPTOR CHANNEL ALPHA-2 SABUNIT IS ESSENTIAL FOR THE ACQUISITION OF LIGAND-BINDING ACTIVITY

The N-linked glycosylation of the alpha 2 subunit of the mouse oxy-5-m ethylisoxazole-4-propionate(AMPA)-selective glutamate receptor (GluR) channel was characterized. The receptor subunit protein has five putat ive N-glycosylation sites. The recombinant receptor proteins were iden tified by [S-35]methionine/[S-35]cysteine metabolic labeling, western blot analysis, immunocytochemical detection, and [3H]AMPA binding expe riments when expressed in insect Spodoptera frugiperda cells using a b aculovirus system. The effect of tunicamycin on the metabolic labeling and immunoblots suggested that the two products, a major protein spec ies of similar to 102 kDa and a minor species of similar to 98 kDa, co rrespond to glycosylated and unglycosylated forms, respectively, which was also supported by the enzymic deglycosylation experiments. Immuno fluorescence staining of tunicamycin-treated cells expressing only the unglycosylated form differed little from that of tunicamycin-nontreat ed cells expressing both glycosylated and unglycosylated forms. The la ck of AMPA-binding activity of the unglycosylated form expressed in th e 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 o ne N-glycosylation site is required for the formation and maintenance of the GluR alpha 2 subunit protein in an active conformation for liga nd binding. Possible roles of N-glycosylation of GluR alpha 2 subunit protein are discussed.