Ligand-binding properties and N-glycosylation of alpha 1 subunit of the alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionate(AMPA)-selective glutamate receptor channel expressed in a baculovirus system

Abstract

The alpha 1 subunit of the mouse alpha-amino-3-hydroxy-5-methyl-4-isoxazole- propionate(AMPA)-selective glutamate receptor channel has been expressed in insect Spodoptera frugiperda cells using a baculovirus system. The recombinant receptor proteins were identified by immunocytochemical detection, Western-blot analysis, and [35S]methionine/[35S]cysteine metabolic labeling experiments. The effect of tunicamycin on the metabolic labeling and immunoblots suggested that the two products, a major protein species of approximately 104 kDa and a minor species of approximately 100 kDa, correspond to glycosylated and non-N-glycosylated forms, respectively, which was also supported by the enzymic deglycosylation experiments. The lack of alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionate-binding activity of non-N-glycosylated glutamate receptor expressed in the presence of tunicamycin suggested that N-glycosylation is required, directly or indirectly, for functional expression in insect cells for ligand binding. Scatchard analysis of alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionate binding showed a single binding site with Kd 30 nM and a Bmax value of 2.6 x 10(5) binding sites/cell or 1.5 pmol/mg protein in the total particulate fraction. Among the compounds tested in the competition studies, beta-(3,5-dioxo-1,2,4-oxadiazolidin-2-yl)-L-alanine (quisqualate) was the most potent inhibitor of the 3H-labeled alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionate binding (IC50 = 30 nM), followed in decreasing order by alpha-amino-3-hydroxy-5- methyl-4-isoxazole propionate, L-glutamate, 6,7-dinitroquinoxaline-2,3-dione, 6-cyano-7-nitroquinoxaline-2,3-dione, and 2-carboxy-4-(1-methylethenyl)-3-pyrrolidineacetate (kainate). Thus, in this study we present detailed analysis of alpha-amino-3-hydroxy-5-methyl-4- isoxazole-propionate-binding activity of the homomeric (single subunit) glutamate receptor channel of mouse alpha 1 subunit and discuss possible roles of N-glycosylation of the glutamate receptor channel alpha 1 subunit.