Ligand-binding properties and N-glycosylation of α1 subunit of the α-amino-3-hydroxy-5-methyl-4-isoxazole-propionate(AMPA)-selective glutamate receptor channel expressed in a baculovirus system.

The αl subunit of the mouse α-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 α-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 α-amino-3-hydroxy-5-methyl-4-isoxazole-propionate binding showed a single binding site with Kd 30 nM and a Bmax value of 2.6x10⁵ binding sites/cell or 1.5 pmol/mg protein in the total particulate fraction. Among the compounds tested in the competition studies. β-(3.5-dioxo-1.2.4-oxadiazolidin-2-yl)-L-alanine (quisqualate) was the most potent inhibitor of the ³H-labeled α-amino-3-hydroxy-5-methyl-4-isoxazole-propionate binding (IC50 = 30 nM), followed in decreasing order by α-amino-3-hydroxy-5-metbyl-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 α-amino-3-hydroxy-5-methyl-4-isoxazole-propionate-binding activity of the homomeric (single subunit) glutamate receptor channel of mouse al subunit and discuss possible roles of N-glycosylation of the glutamate receptor channel αl subunit. [ABSTRACT FROM AUTHOR]

Copyright of European Journal of Biochemistry is the property of Wiley-Blackwell and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)