N-linked oligosaccharide chains of the insulin receptor beta subunit are essential for transmembrane signaling.
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.
Leconte
I
I
Institut National de la Santé et de la Recherche Médicale U36, Chaire de Médecine Expérimentale, Collège de France, Paris.
Auzan
C
C
Debant
A
A
Rossi
B
B
Clauser
E
E
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D001483:Base Sequence; D016466:CHO Cells; D002462:Cell Membrane / Q000502:physiology*; D006224:Cricetinae; D003545:Cysteine / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D006031:Glycosylation; D006801:Humans; D007328:Insulin / Q000031:analogs & derivatives / Q000378:metabolism; D007457:Iodine Radioisotopes / Q000378:metabolism; D007700:Kinetics; D046911:Macromolecular Substances; D008715:Methionine / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D009844:Oligosaccharides / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000235:genetics / Q000378:metabolism; D011972:Receptor, Insulin / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D015183:Restriction Mapping; D015398:Signal Transduction; D013462:Sulfur Radioisotopes; D014162:Transfection
D000911:Antibodies, Monoclonal; D007328:Insulin; D007457:Iodine Radioisotopes; D046911:Macromolecular Substances; D009838:Oligodeoxyribonucleotides; D009844:Oligosaccharides; D013462:Sulfur Radioisotopes; C008147:insulin, iodo-; D008715:Methionine; D011505:Protein-Tyrosine Kinases; D011972:Receptor, Insulin; D003545:Cysteine