Collier E, Carpentier J L, Beitz L, Carol H, Taylor S I, Gorden P
Diabetes Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892.
Biochemistry. 1993 Aug 3;32(30):7818-23. doi: 10.1021/bi00081a029.
The insulin receptor is a transmembrane protein found on multiple cell types. This receptor is synthesized as a 190-kDa proreceptor which is cleaved to produce mature alpha and beta subunits. The proreceptor contains 18 potential sites for N-linked glycosylation: 14 on the alpha subunit and 4 on the beta subunit. The codons for asparagine in the first four sites at the amino terminus of the alpha subunit were mutated to code for glutamine. This mutant receptor cDNA was stably transfected into NIH 3T3 cells. The insulin receptor produced in these cells remained in the proreceptor form; no mature alpha and beta subunits were produced. The proreceptor was slightly smaller on SDS-PAGE gels than the wild-type proreceptor and contained four less oligosaccharide chains by tryptic peptide mapping. The carbohydrate chains on the mutant proreceptor remained endoglycosidase H sensitive. However, in the presence of brefeldin A, these oligosaccharide chains could be processed to endoglycosidase H resistant chains. By immunofluorescence, the mutant proreceptor was shown to be localized to the endoplasmic reticulum. No insulin receptors could be found on the cell-surface either with cell surface labeling with biotin or with 125I-insulin binding. Thus, glycosylation of the first four N-linked glycosylation sites of the insulin receptor is necessary for the proper processing and intracellular transport of the receptor. This is in contrast to glycosylation at the four sites on the beta subunit which appear not to be important for processing but necessary for signal transduction. Therefore, N-linked glycosylation of the insulin receptor at specific sites has multiple distinctive roles.
胰岛素受体是一种存在于多种细胞类型上的跨膜蛋白。该受体最初以190 kDa的前体受体形式合成，随后被切割产生成熟的α和β亚基。前体受体含有18个潜在的N-糖基化位点：α亚基上有14个，β亚基上有4个。α亚基氨基末端前四个位点的天冬酰胺密码子被突变为谷氨酰胺密码子。这种突变的受体cDNA被稳定转染到NIH 3T3细胞中。这些细胞中产生的胰岛素受体仍以前体受体形式存在；未产生成熟的α和β亚基。在SDS-PAGE凝胶上，前体受体比野生型前体受体略小，经胰蛋白酶肽图谱分析，其寡糖链少四条。突变前体受体上的糖链对内切糖苷酶H仍敏感。然而，在布雷菲德菌素A存在的情况下，这些寡糖链可被加工成对内切糖苷酶H有抗性的链。通过免疫荧光法显示，突变前体受体定位于内质网。无论是用生物素进行细胞表面标记还是用125I-胰岛素结合，在细胞表面都未发现胰岛素受体。因此，胰岛素受体前四个N-糖基化位点的糖基化对于受体的正确加工和细胞内运输是必需的。这与β亚基上四个位点的糖基化形成对比，后者似乎对加工不重要，但对信号转导是必需的。因此，胰岛素受体在特定位点的N-糖基化具有多种独特作用。
