THE FUNCTIONS OF THE HUMAN INSULIN-RECEPTOR ARE AFFECTED IN DIFFERENTWAYS BY MUTATION OF EACH OF THE 4 N-GLYCOSYLATION SITES IN THE BETA-SUBUNIT

The functional role of the oligosaccharide chains linked to the insuli n receptor (IR) beta subunit was investigated by site directed mutagen esis of each of the 4 acceptor asparagines (N1 to N4 from the amino to the carboxyl terminus) and stable expression of the receptors in CHO cells. All mutant receptors are expressed normally at the cell surface , bind insulin with similar affinity, but have a beta subunit of small er molecular mass, and a defect in ligand-induced internalization as c ompared to wild type receptor. In terms of receptor activation and sig nal transduction, the N1 and N2 mutants function normally, whereas the N4 mutant exhibits major alterations in in vitro tyrosine kinase acti vity and autophosphorylation and is unable to transduce the signal for either glycogen or DNA synthesis. By contrast, in vivo autophosphoryl ation and IRS-1 phosphorylation appear quantitatively normal, and only partial alterations of phosphatidylinositol 3-kinase and mitogen-acti vated protein kinase activation are observed. Mutation of the N3 site results in partial defect of IR activation. These data provide evidenc e for (i) glycosylation of each N-linked glycosylation site of the IR beta subunit, (ii) absence of correlation between internalization and transmembrane signaling, and (iii) a major role for oligosaccharide si de chain(s) located close to the cell membrane in IR activation and tr ansmembrane signaling.