Hall R A, Hansen A, Andersen P H, Soderling T R
Vollum Institute, Oregon Health Sciences University, Portland 97201, USA.
J Neurochem. 1997 Feb;68(2):625-30. doi: 10.1046/j.1471-4159.1997.68020625.x.
The surface expression of the alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)-type glutamate receptor (GluR) subunits GluR1, GluR2, and GluR4 was studied in cultures of stably transfected baby hamster kidney (BHK)-570 cells. Two methods were used to quantify surface expression: cross-linking with the membrane-impermeant reagent bis (sulfosuccinimidyl) suberate (BS3) and labeling of surface receptors with the membrane-impermeant biotinylating reagent sulfosuccinimidyl 2-(biotinamido)ethyl-1,3-dithiopropionate (NHS-ss-biotin) followed by precipitation with neutravidin beads. Western blot analyses of control versus treated cultures revealed that, for all three GluR subunits examined, 25-40% of the total GluR population is located in the plasma membrane of the BHK-570 cells. This finding was corroborated by analyses of the surface expression of [3H]AMPA binding sites in the GluR-expressing BHK-570 cells performed via the biotinylation/precipitation method; these studies revealed that 30-40% of the total binding site population is found in the plasma membrane. Analyses of combinations of the subunits, both GluR1 + GluR2 and GluR2 + GluR4, revealed that heteromeric combinations of the subunits are not trafficked to the surface more efficiently than homomeric receptors. For each of the three subunits, western blots revealed two distinct bands; removal of surface receptors reduced immunoreactivity for the upper band of each subunit by > 90%, whereas immunoreactivity for the lower band was reduced by only 10-20%. Treatment of extracts from the various cell lines with glycopeptidase F resulted in the collapse of the two bands into a single band of lower molecular weight, suggesting that the two original bands represent differentially glycosylated forms of the same polypeptides. These data indicate that the majority of the stably expressed GluR subunits in these cell lines are incompletely glycosylated and that complete glycosylation is associated with trafficking of the GluR subunits to the cell surface.
在稳定转染的幼仓鼠肾（BHK）-570细胞培养物中，研究了α-氨基-3-羟基-5-甲基异恶唑-4-丙酸（AMPA）型谷氨酸受体（GluR）亚基GluR1、GluR2和GluR4的表面表达。使用两种方法定量表面表达：用膜不透性试剂双（磺基琥珀酰亚胺基）辛二酸酯（BS3）进行交联，以及用膜不透性生物素化试剂磺基琥珀酰亚胺基2-（生物素酰胺基）乙基-1,3-二硫代丙酸酯（NHS-ss-生物素）标记表面受体，随后用中性抗生物素蛋白珠沉淀。对照培养物与处理后培养物的蛋白质免疫印迹分析表明，对于所检测的所有三种GluR亚基，25%-40%的总GluR群体位于BHK-570细胞的质膜中。通过生物素化/沉淀法对表达GluR的BHK-570细胞中[3H]AMPA结合位点的表面表达进行分析，证实了这一发现；这些研究表明，30%-40%的总结合位点群体存在于质膜中。对亚基组合GluR1 + GluR2和GluR2 + GluR4的分析表明，亚基的异源组合并不比同源受体更有效地转运到表面。对于这三种亚基中的每一种，蛋白质免疫印迹都显示出两条不同的条带；去除表面受体后，每个亚基上带的免疫反应性降低>90%，而下带的免疫反应性仅降低10%-20%。用糖肽酶F处理各种细胞系的提取物后，两条带合并为一条分子量较低的单带，这表明最初的两条带代表同一多肽的不同糖基化形式。这些数据表明，这些细胞系中稳定表达的GluR亚基大多数是不完全糖基化的，并且完全糖基化与GluR亚基向细胞表面的转运有关。
