An intact and functional soluble form of the insulin receptor is secreted by cultured cells.
We have recently reported that the insulin receptor (IR), a tetrameric transmembrane protein located on the surface of target cells, is present as a soluble form in human plasma. In the present study we investigated whether human cells in culture release an intact and functional form of the IR. We found that IRs are secreted into the incubation medium by four cell lines (IM-9 human lymphoblasts, MCF-7 human breast cancer cells, HepG2 human hepatoma cells, and 3T3 mouse fibroblasts transfected with human IRs). IR secretion was further characterized in IM-9 cells. IR release was time, temperature, and energy dependent, and enhanced by incubation with insulin. The dilution slope of secreted IRs in a specific IR RIA was parallel to that produced by highly purified human placenta IRs. Ligand binding studies revealed that secreted receptors bound insulin with high affinity, and the Scatchard analysis revealed two orders of binding sites (the high affinity site had a dissociation constant of 0.32 +/- 0.08 nM). Analysis of secreted receptors by sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated a molecular size of 135 kilodaltons for the alpha-subunit and 95 kilodaltons for the beta-subunit. Other experiments indicated that the beta-subunit tyrosine kinase activity of the secreted receptor was stimulated by insulin. These studies indicate, therefore, that a soluble, intact, and functional IR is secreted by cultured cells, and that this soluble protein could be involved in certain insulin-mediated functions, such as receptor down-regulation.
Cattedra di Endocrinologia, Università di Catania, Ospedale Garibaldi, Italy.
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Papa
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Russo
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Gliozzo
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Goldfine
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Vigneri
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Pezzino
D016475:3T3 Cells; D000818:Animals; D001943:Breast Neoplasms; D006528:Carcinoma, Hepatocellular; D002452:Cell Count; D004734:Energy Metabolism; D006801:Humans; D007328:Insulin / Q000378:metabolism / Q000494:pharmacology; D007700:Kinetics; D008113:Liver Neoplasms; D008214:Lymphocytes / Q000378:metabolism; D051379:Mice; D011480:Protease Inhibitors / Q000494:pharmacology; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D017526:Receptor, IGF Type 1 / Q000378:metabolism; D011972:Receptor, Insulin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013439:Sulfhydryl Reagents / Q000494:pharmacology; D013696:Temperature; D014162:Transfection; D014407:Tumor Cells, Cultured
D007328:Insulin; D011480:Protease Inhibitors; D013439:Sulfhydryl Reagents; D011505:Protein-Tyrosine Kinases; D017526:Receptor, IGF Type 1; D011972:Receptor, Insulin