Kawamoto S, Hattori S, Oiji I, Hamajima K, Mishina M, Okuda K
Department of Bacteriology, Yokohama City University School of Medicine, Japan.
Eur J Biochem. 1994 Jul 15;223(2):665-73. doi: 10.1111/j.1432-1033.1994.tb19039.x.
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.
利用杆状病毒系统，已在昆虫草地贪夜蛾细胞中表达了小鼠α-氨基-3-羟基-5-甲基-4-异恶唑丙酸（AMPA）选择性谷氨酸受体通道的α1亚基。通过免疫细胞化学检测、蛋白质免疫印迹分析以及[35S]甲硫氨酸/[35S]半胱氨酸代谢标记实验鉴定了重组受体蛋白。衣霉素对代谢标记和免疫印迹的影响表明，两种产物，一种约104 kDa的主要蛋白和一种约100 kDa的次要蛋白，分别对应糖基化和非N-糖基化形式，酶促去糖基化实验也支持了这一点。在衣霉素存在下表达的非N-糖基化谷氨酸受体缺乏α-氨基-3-羟基-5-甲基-4-异恶唑丙酸结合活性，这表明N-糖基化直接或间接是昆虫细胞中配体结合功能表达所必需的。对α-氨基-3-羟基-5-甲基-4-异恶唑丙酸结合的Scatchard分析显示，在总颗粒部分中有一个单一结合位点，Kd为30 nM，Bmax值为2.6×10(5)个结合位点/细胞或1.5 pmol/mg蛋白质。在竞争研究中测试的化合物中，β-(3,5-二氧代-1,2,4-恶二唑烷-2-基)-L-丙氨酸（quisqualate）是3H标记的α-氨基-3-羟基-5-甲基-4-异恶唑丙酸结合的最有效抑制剂（IC50 = 30 nM），其次依次为α-氨基-3-羟基-5-甲基-4-异恶唑丙酸、L-谷氨酸、6,7-二硝基喹喔啉-2,3-二酮、6-氰基-7-硝基喹喔啉-2,3-二酮和2-羧基-4-(1-甲基乙烯基)-3-吡咯烷乙酸（海人藻酸）。因此，在本研究中，我们对小鼠α1亚基的同聚体（单个亚基）谷氨酸受体通道的α-氨基-3-羟基-5-甲基-4-异恶唑丙酸结合活性进行了详细分析，并讨论了谷氨酸受体通道α1亚基N-糖基化的可能作用。
