Human insulin-like growth factor I receptor internalization. Role of the juxtamembrane domain.
Cytoplasmic submembrane domains of the insulin-like growth factor I (IGF-1) receptor ranging from glycine 940 to proline 959 were investigated for their role in endocytosis of the IGF-1 ligand in rat pituitary GC cells stably expressing mutant human IGF-1 receptors. Replacement of each of three tyrosine residues within the juxtamembrane domain reduced the internalization rate (Ke) by 33% (943Y-->A), 47% (950Y-->A), and 41% (957Y-->A), respectively. Other substitutions within the submembrane region variably retarded receptor-mediated IGF-1 ligand endocytosis. Thus, each amino acid appears to independently contribute to the overall ligand-stimulated IGF-1 receptor internalization rate. The effect of receptor occupancy on IGF-1 receptor endocytosis was also tested. The rate of 125I-labeled IGF-1 internalization by transfectants overexpressing the wild type IGF-1 receptor was rapid but decreased markedly in the presence of increasing unlabeled IGF-1 (ED50 = 44,000 receptors/cell). Substitution by alanine for any of the three submembrane tyrosines increased the ED50 to > 56,000 receptors/cell, decreased the slope (Kic), and had a variable effect on Vmax in the presence of increasing unlabeled IGF-1. In contrast, 125I-labeled IGF-1 endocytosis by the mutant kinase-deficient 952STOP cells was slow and not further retarded by unlabeled IGF-1. These results suggest that ligand-mediated internalization of the human IGF-I is consistent with saturable interactions between the IGF-I receptor juxtamembrane region (glycine 940-tyrosine 957) and components of the endocytic apparatus.
Prager
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Department of Medicine, Cedars-Sinai Medical Center, University of California, Los Angeles School of Medicine 90048.
Li
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HL
Yamasaki
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H
Melmed
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D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002462:Cell Membrane / Q000378:metabolism*; D004705:Endocytosis; D006801:Humans; D007334:Insulin-Like Growth Factor I / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D010902:Pituitary Gland / Q000378:metabolism*; D051381:Rats; D017526:Receptor, IGF Type 1 / Q000096:biosynthesis / Q000378:metabolism*; D015183:Restriction Mapping; D014162:Transfection
D009838:Oligodeoxyribonucleotides; D007334:Insulin-Like Growth Factor I; D017526:Receptor, IGF Type 1