Leconte I, Auzan C, Debant A, Rossi B, Clauser E
Institut National de la Santé et de la Recherche Médicale U36, Chaire de Médecine Expérimentale, Collège de France, Paris.
J Biol Chem. 1992 Aug 25;267(24):17415-23.
Insulin receptor (IR) is a glycoprotein possessing N-linked oligosaccharide side chains on both alpha and beta subunits. The present study focuses for the first time on the potential contribution of N-linked oligosaccharides of the beta subunit in the processing, structure, and function of the insulin receptor. To investigate this point, a receptor mutant (IR beta N1234) was obtained by stable transfection into Chinese hamster ovary cells of an IR cDNA modified by site-directed mutagenesis on the four potential N-glycosylation sites (Asn-X-Ser/Thr) of the beta subunit. The mutated receptor presents an alpha subunit of 135 kDa, indistinguishable from the wild type alpha subunit, but the beta subunit has a reduced molecular mass (80 kDa instead of 95 kDa) most likely due to the absence of N-glycosylation. Metabolic labeling experiments indicate a normal processing and maturation of this mutated receptor which is normally expressed at the surface of the cells as demonstrated by indirect immunofluorescence. The affinity of the mutant for insulin (Kd = 0.12 nM) is similar to that of the wild type receptor (Kd = 0.12 nM). However, a major defect of the mutated IR tyrosine kinase was assessed both in vitro and in vivo by (i) the absence of insulin-stimulated phosphorylation of the poly(Glu-Tyr) substrate in vitro; (ii) the reduction of the insulin maximal stimulation of the mutated IR autophosphorylation in vitro (2-fold stimulation for the mutant receptor as compared to a 7-fold stimulation for the wild type); and (iii) a more complex alteration of the mutated receptor tyrosine autophosphorylation in vivo (3-fold increase of the basal phosphorylation and a 4-fold simulation of this phosphorylation as compared to the wild type receptor, the phosphorylation of which is stimulated 14-fold by insulin). The physiological consequences of this defect were tested on three classical insulin cellular actions; in Chinese hamster ovary IR beta N1234, glucose transport, glycogen synthesis, and DNA synthesis were all unable to be stimulated by insulin indicating the absence of insulin transduction through this mutated receptor. These data provide the first direct evidence for a critical role of oligosaccharide side chains of the beta subunit in the molecular events responsible for the IR enzymatic activation and signal transduction.
胰岛素受体（IR）是一种糖蛋白，其α和β亚基上均带有N - 连接寡糖侧链。本研究首次聚焦于β亚基的N - 连接寡糖在胰岛素受体的加工、结构和功能中的潜在作用。为研究这一点，通过将经定点诱变修饰的IR cDNA稳定转染至中国仓鼠卵巢细胞中，该cDNA针对β亚基的四个潜在N - 糖基化位点（天冬酰胺 - X - 丝氨酸/苏氨酸）进行了改造，从而获得了一种受体突变体（IRβN1234）。突变后的受体呈现出一个135 kDa的α亚基，与野生型α亚基无差异，但β亚基的分子量降低（80 kDa而非95 kDa），这很可能是由于缺乏N - 糖基化所致。代谢标记实验表明该突变受体的加工和成熟正常，间接免疫荧光显示其正常表达于细胞表面。突变体对胰岛素的亲和力（Kd = 0.12 nM）与野生型受体相似（Kd = 0.12 nM）。然而，通过以下方式在体外和体内评估了突变的IR酪氨酸激酶的一个主要缺陷：（i）体外缺乏胰岛素刺激的聚（谷氨酸 - 酪氨酸）底物磷酸化；（ii）体外突变IR自身磷酸化的胰岛素最大刺激作用降低（突变受体为2倍刺激，而野生型为7倍刺激）；（iii）体内突变受体酪氨酸自身磷酸化发生更复杂的改变（基础磷酸化增加3倍，与野生型受体相比，该磷酸化的刺激作用为4倍，野生型受体的磷酸化受胰岛素刺激14倍）。在三种经典的胰岛素细胞作用中测试了这一缺陷的生理后果；在中国仓鼠卵巢IRβN1234中，葡萄糖转运、糖原合成和DNA合成均不能被胰岛素刺激，这表明通过该突变受体不存在胰岛素转导。这些数据首次直接证明了β亚基的寡糖侧链在负责IR酶激活和信号转导的分子事件中起关键作用。
