Mutational analysis of the NH2-terminal glycosylation sites of the insulin receptor alpha-subunit.
The insulin receptor is synthesized as a single chain of 190 kiloDaltons, which is processed to disulfide-linked mature alpha- and beta- subunits, containing N- and O-linked oligosaccharides and fatty acids. Previously (Collier E, Carpentier J-L, Beitz L, Caro LHP, Taylor SI, Gorden P: Biochemistry 32:7818-23, 1993), site directed mutagenesis of the asparagine in the first four sites of N-linked glycosylation to glutamine resulted in a receptor that was retained in the endoplasmic reticulum and not processed past the proreceptor form. In this study, mutation of these sites individually and in various combinations is studied. Mutation in the first or second glycosylation site does not significantly impair processing of the receptor; the receptor is found on the cell surface and binds insulin normally. If both the first and second sites are mutated, a significant reduction occurs in the amount of receptor found on the cell surface and in insulin binding. There is some processing of the receptor in cells expressing this mutant compared with the four-part mutant. If only the third and fourth sites are mutated, processing is impaired less than in the mutant with the first and second sites mutated. However, the amount of receptor found on the cell surface is less than in the mutant of only the first or only the second site. In all of these glycosylation mutants, the amount of receptor on the cell surface correlates with the level of 125I-labeled insulin binding on the cell surface.(ABSTRACT TRUNCATED AT 250 WORDS)
Caro
L H
LH
Diabetes Branch, National Institute of Diabetes, Digestive, and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892.
Ohali
A
A
Gorden
P
P
Collier
E
E
D016475:3T3 Cells; D000818:Animals; D001216:Asparagine; D001665:Binding Sites; D002462:Cell Membrane / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D004252:DNA Mutational Analysis; D005973:Glutamine; D006031:Glycosylation; D006801:Humans; D007328:Insulin / Q000378:metabolism; D046911:Macromolecular Substances; D051379:Mice; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000302:isolation & purification; D010449:Peptide Mapping; D010766:Phosphorylation; D011972:Receptor, Insulin / Q000096:biosynthesis / Q000378:metabolism*; D015183:Restriction Mapping; D014357:Trypsin
D007328:Insulin; D046911:Macromolecular Substances; D010446:Peptide Fragments; D005973:Glutamine; D001216:Asparagine; D011972:Receptor, Insulin; D014357:Trypsin