Haruta T, Sawa T, Takata Y, Imamura T, Takada Y, Morioka H, Yang G H, Kobayashi M
First Department of Medicine, Toyama Medical and Pharmaceutical University, Japan.
Biochem J. 1995 Jan 15;305 ( Pt 2)(Pt 2):599-604. doi: 10.1042/bj3050599.
The extracellular portion of the insulin receptor (IR) beta-subunit has four cysteine and four asparagine residues which are potentially involved in disulphide bond formation between the alpha- and beta-subunits and N-linked glycosylation respectively. However, the function of this portion is not fully understood. In order to investigate the role of the extracellular domain of beta-subunit, we created a deletion mutant of IR cDNA which lacked 47 amino acid residues encoded by 141 bp corresponding to exon 13 of the IR gene. Insulin binding and surface labelling of COS 7 cells transiently expressing the mutant insulin receptors (IR delta Ex13) showed that the mutated receptors were not expressed on the cell surface. However, immunoblot analysis showed that uncleaved form (190 kDa) of the mutant receptors were intracellularly expressed. Deglycosylation with endoglycosidase H showed that the mutant receptors had mainly high-mannose oligosaccharide chains. The mutant IRs bound with high affinity to lentil lectin but with low affinity to wheat germ agglutinin. Therefore, it is suggested that misfolding of the mutant receptors inhibits transport to the Golgi apparatus where processing of oligosaccharide chains, as well as proteolytic cleavage into subunits, takes place. The binding affinity of the mutant receptors for insulin was 50% of normal. Furthermore, insulin-stimulated autophosphorylation of IR delta Ex13 was markedly impaired. These data provide the evidence for a critical role of the extracellular domain of IR beta-subunit for processing and transport as well as the intramolecular signal transduction to activate IR tyrosine kinase.
胰岛素受体（IR）β亚基的细胞外部分有四个半胱氨酸和四个天冬酰胺残基，它们可能分别参与α亚基和β亚基之间二硫键的形成以及N-连接糖基化。然而，这部分的功能尚未完全明了。为了研究β亚基细胞外结构域的作用，我们构建了IR cDNA的缺失突变体，该突变体缺失了由141 bp编码的47个氨基酸残基，这141 bp对应于IR基因的外显子13。对瞬时表达突变型胰岛素受体（IR delta Ex13）的COS 7细胞进行胰岛素结合和表面标记显示，突变型受体未在细胞表面表达。然而，免疫印迹分析表明，突变型受体的未切割形式（190 kDa）在细胞内表达。用内切糖苷酶H进行去糖基化显示，突变型受体主要具有高甘露糖寡糖链。突变型IR与扁豆凝集素具有高亲和力结合，但与麦胚凝集素的亲和力较低。因此，提示突变型受体的错误折叠抑制了向高尔基体的转运，而在高尔基体中会发生寡糖链的加工以及蛋白水解切割成亚基。突变型受体对胰岛素的结合亲和力为正常的50%。此外，胰岛素刺激的IR delta Ex13自身磷酸化明显受损。这些数据为IRβ亚基细胞外结构域在加工、转运以及激活IR酪氨酸激酶的分子内信号转导中起关键作用提供了证据。
