Mutational analysis of the N-linked glycosylation sites of the human insulin receptor

Abstract

Site-directed mutagenesis has been used to remove 15 of the 18 potential N-linked glycosylation sites, in 16 combinations, from the human exon 11-minus receptor isoform. The three glycosylation sites not mutated were asparagine residues 25, 397 and 894, which are known to be important in receptor biosynthesis or function. The effects of these mutations on proreceptor processing into α and β subunits, cell-surface expression, insulin binding and receptor autophosphorylation were assessed in Chinese hamster ovary cells. The double mutants 16j78, 16j111, 16j215, 16j255, 337j418, the triple mutants 295j337j418, 295j418j514, 337j418j514 and 730j 743j881 and the quadruple mutants 606j730j743j881 and 671j730j743j881 seemed normal by all criteria examined. The triple mutant 16j215j255 showed only low levels of correctly processed receptor on the cell surface, this processed receptor being autophosphorylated in response to insulin. The quadruple mutant 624j730j743j881 showed normal pro-

Dimeric Fragment of the Insulin Receptor α-Subunit Binds Insulin with Full Holoreceptor Affinity

“…However, this affinity is considerably lower than that typically found with the full-length holoreceptor (hIR) (18,(21)(22)(23). In addition to its high affinity for insulin, hIR exhibits nonclassical receptor binding properties suggestive of negative cooperativity or site-site interactions between the two receptor halves, namely accelerated dissociation of labeled insulin in the presence of unlabeled insulin and curvilinear Scatchard plots (24).…”

MuSK Glycosylation Restrains MuSK Activation and Acetylcholine Receptor Clustering

“…N-Glycosylation of TrkA (34), insulin receptor (35), and epidermal growth factor receptor (36) suppresses ligandindependent activation of these RTKs. In TrkA, even partial deglycosylation is sufficient to cause ligand-independent TrkA phosphorylation, resulting in the recruitment of Shc and phospholipase C-␥ (34).…”

Down-Regulation of Cell Surface Insulin Receptor and Insulin Receptor Substrate-1 Phosphorylation by Inhibitor of 90-kDa Heat-Shock Protein Family: Endoplasmic Reticulum Retention of Monomeric Insulin Receptor Precursor with Calnexin in Adrenal Chromaffin Cells

“…IR precursor undergoes cotranslational glycosylation, intrachain disulfide-bond formation/isomerization (rearrangement), and disulfidelinked homodimerization at the endoplasmic reticulum (ER). The homodimeric IR precursor is proteolytically processed at the trans-Golgi network (TGN) into the disulfide-linked ␣ 2 ␤ 2 complex, which is transported to plasma membrane via as yet unidentified mechanisms (Ronnett et al, 1984;Arakaki et al, 1987;Olson et al, 1988;Caro et al, 1994;Cheatham and Kahn, 1995;Bass et al, 1998;Elleman et al, 2000).…”