Effect of alternative glycosylation on insulin receptor processing
Effect of alternative glycosylation on insulin receptor processing
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DOI：
10.1074/jbc.274.32.22813
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发表时间：
1999-08-06
影响因子：
4.8
通讯作者：
Frost, SC
中科院分区：
文献类型：
--
作者：
Hwang, JB;Frost, SC
成熟胰岛素受体是由两个α-和两个P-亚基组成的细胞表面异源四聚体糖蛋白。在3T3-L1脂肪细胞中，如同在其他细胞类型中一样，受体被合成为由未切割的α-和β-亚基组成的单一多肽，作为190-kDa糖蛋白迁移。为了检查N-连接的糖基化对胰岛素受体加工的重要性，我们使用葡萄糖剥夺作为改变蛋白质糖基化的工具，蛋白质印迹分析表明，葡萄糖剥夺导致170 kDa的替代前受体在亚细胞部分中的时间依赖性积累，与内质网定位一致，共沉淀试验提供的证据表明，替代前受体结合GRP78，内质网分子伴侣。N-糖苷酶F处理表明，替代前受体含有N-连接的寡糖。然而，内切糖苷酶H不敏感性表明异常的寡糖结构。使用脉冲追踪方法，我们表明，正常和替代前受体的合成率是相似的。然而，正常的前受体被加工成α和β亚基（t（1/2）= 1.3 +/-0.6 h），而替代前受体被降解（t（1/2）= 5.1 +/-0.6 h）。在重新喂养最初被剥夺葡萄糖的细胞时，替代前受体被加工成更高分子量的形式，并获得对内切糖苷酶H的敏感性。这种"中间"形式的前受体也被降解，尽管一小部分逃脱了降解，导致切割成α-和β-亚基。这些数据首次提供了证据，即葡萄糖剥夺导致替代前受体的积累，其可以在诱导加速降解和成熟的葡萄糖的再加入下进行后糖基化。
The mature insulin receptor is a cell surface heterotetrameric glycoprotein composed of two alpha- and two P-subunits. In 3T3-L1 adipocytes as in other cell types, the receptor is synthesized as a single polypeptide consisting of uncleaved alpha- and beta-subunits, migrating as a 190-kDa glycoprotein, To examine the importance of N-linked glycosylation on insulin receptor processing, we have used glucose deprivation as a tool to alter protein glycosylation, Western blot analysis shows that glucose deprivation led to a time-dependent accumulation of an alternative proreceptor of 170 kDa in a subcellular fraction consistent with endoplasmic reticulum localization, Co-precipitation assays provide evidence that the alternative proreceptor bound GRP78, an endoplasmic reticulum molecular chaperone. N-Glycosidase F treatment shows that the alternative proreceptor contained N-linked oligosaccharides. Yet, endoglycosidase H insensitivity indicates an aberrant oligosaccharide structure. Using pulse-chase methodology, we show that the synthetic rate was similar between the normal and alternative proreceptor. However, the normal proreceptor was processed into alpha- and beta-subunits (t(1/2) = 1.3 +/- 0.6 h), while the alternative proreceptor was degraded (t(1/2) = 5.1 +/- 0.6 h). Upon refeeding cells that were initially deprived of glucose, the alternative proreceptor was processed to a higher molecular weight form and gained sensitivity to endoglycosidase H. This "intermediate" form of the proreceptor was also degraded, although a small fraction escaped degradation, resulting in cleavage to the alpha- and beta-subunits, These data provide evidence for the first time that glucose deprivation leads to the accumulation of an alternative proreceptor, which can be post-translationally glycosylated with the readdition of glucose inducing both accelerated degradation and maturation.