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.
A novel intersubunit repair mechanism in the tryptophan synthase alpha 2 beta 2 complex. Critical role of the beta subunit lysine 167 in intersubunit communication.