Specific glycosylation site mutations of the insulin receptor alpha subunit impair intracellular transport.
The insulin receptor is a transmembrane protein found on multiple cell types. This receptor is synthesized as a 190-kDa proreceptor which is cleaved to produce mature alpha and beta subunits. The proreceptor contains 18 potential sites for N-linked glycosylation: 14 on the alpha subunit and 4 on the beta subunit. The codons for asparagine in the first four sites at the amino terminus of the alpha subunit were mutated to code for glutamine. This mutant receptor cDNA was stably transfected into NIH 3T3 cells. The insulin receptor produced in these cells remained in the proreceptor form; no mature alpha and beta subunits were produced. The proreceptor was slightly smaller on SDS-PAGE gels than the wild-type proreceptor and contained four less oligosaccharide chains by tryptic peptide mapping. The carbohydrate chains on the mutant proreceptor remained endoglycosidase H sensitive. However, in the presence of brefeldin A, these oligosaccharide chains could be processed to endoglycosidase H resistant chains. By immunofluorescence, the mutant proreceptor was shown to be localized to the endoplasmic reticulum. No insulin receptors could be found on the cell-surface either with cell surface labeling with biotin or with 125I-insulin binding. Thus, glycosylation of the first four N-linked glycosylation sites of the insulin receptor is necessary for the proper processing and intracellular transport of the receptor. This is in contrast to glycosylation at the four sites on the beta subunit which appear not to be important for processing but necessary for signal transduction. Therefore, N-linked glycosylation of the insulin receptor at specific sites has multiple distinctive roles.
Collier
E
E
Diabetes Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892.
Carpentier
J L
JL
Beitz
L
L
Carol
H
H
Taylor
S I
SI
Gorden
P
P
D016475:3T3 Cells; D000818:Animals; D001216:Asparagine / Q000378:metabolism; D001692:Biological Transport; D004247:DNA; D004591:Electrophoresis, Polyacrylamide Gel; D005455:Fluorescent Antibody Technique; D005973:Glutamine / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D007328:Insulin / Q000378:metabolism; D051379:Mice; D016297:Mutagenesis, Site-Directed; D010449:Peptide Mapping; D011972:Receptor, Insulin / Q000235:genetics / Q000378:metabolism*; D014162:Transfection
D007328:Insulin; D005973:Glutamine; D001216:Asparagine; D004247:DNA; D011972:Receptor, Insulin