Characterization of an insulin receptor mutant lacking the subunit processing site.
An insulin receptor mutant was constructed utilizing site-directed mutagenesis to delete the Arg-Lys-Arg-Arg basic amino acid cleavage site (positions 720-723) from the cDNA encoding the human insulin proreceptor. This mutant was transfected into Chinese hamster ovary cells. Immunoprecipitation of metabolically labeled cells revealed a 205-kDa proreceptor which bound to wheat germ agglutinin. Processed 130-kDa alpha and 95-kDa beta subunits were also observed and contained approximately 20% as much protein as the proreceptor on a molar basis. Trypsin digestion of intact metabolically labeled cells decreased the proreceptor band by 80%. Pulse-chase studies revealed a half-life of 28 h for the proreceptor. When cells were photolabeled with 125I-B2(2-nitro-4-azidophenylacetyl)-des-PheB1 (NAPA)-insulin, the proreceptor incorporated 10% as much label as the 130-kDa alpha subunit in spite of a 5-fold molar excess. Incubation of NAPA-labeled cells at 37 degrees C for 20 min resulted in 60% of the labeled subunits, but little labeled proreceptor, becoming resistant to trypsin degradation. Immunoprecipitation of NAPA-insulin-stimulated cells with anti-phosphotyrosine antibodies revealed that 62% of the processed labeled receptors, but very little proreceptor, contained phosphotyrosine. Thus, this mutant receptor is synthesized, glycosylated, and expressed on the cell surface as uncleaved proreceptor, although some processing to alpha and beta subunits still occurs. It exhibits a markedly decreased affinity for insulin, and when insulin is bound to, demonstrates defective internalization, down-regulation, and autophosphorylation. These data suggest that cleavage of the mutant proreceptor into subunits is required not only for the development of high affinity binding sites, but also for normal transduction of the signal which activates the beta subunit tyrosine kinase.
Department of Medicine, University of California, San Diego 92092.
J F
JF
Williams
D A
DA
McClain
T J
TJ
Dull
A
A
Ullrich
J M
JM
Olefsky
D000345:Affinity Labels / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001386:Azides / Q000378:metabolism; D001483:Base Sequence; D002460:Cell Line; D002872:Chromosome Deletion; D002999:Clone Cells; D006801:Humans; D007328:Insulin / Q000031:analogs & derivatives / Q000378:metabolism; D007700:Kinetics; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D009154:Mutation; D015345:Oligonucleotide Probes; D010766:Phosphorylation; D011972:Receptor, Insulin / Q000235:genetics* / Q000378:metabolism; D014162:Transfection
D000345:Affinity Labels; D001386:Azides; D007328:Insulin; D046911:Macromolecular Substances; D015345:Oligonucleotide Probes; C035459:insulin, (2-nitro-4-azidophenylacetyl)(B2)-des-Phe(B1)-; D011972:Receptor, Insulin