The isoform A of the insulin receptor (IR) (IR-A) is a bifunctional receptor, because it binds both insulin and IGF-II. IR-A activation by IGF-II plays a role in development, but its physiological role in adults is unknown. IGF-II signaling through IR-A is deregulated in cancer and favors tumor progression. We hypothesized that IGF-II binding to the IR-A elicits a unique signaling pathway. In order to obtain an unbiased evaluation of IR-A substrates differentially involved after IGF-II and insulin stimulation, we performed quantitative proteomics of IR-A substrates recruited to ty-rosine-phosphorylated protein complexes using stable isotope labeling with amino acids in cell culture in combination with antiphosphotyrosine antibody pull down and mass spectrometry. Using cells expressing only the human IR-A and lacking the IGF-I receptor, we identified 38 IR-A substrates. Only 10 were known IR mediators, whereas 28 substrates were not previously related to IR signaling. Eleven substrates were recruited by stimulation with both ligands: two equally recruited by IGF-II and insulin, three more strongly recruited by IGF-II, and six more strongly recruited by insulin. Moreover, 14 substrates were recruited solely by IGF-II and 13 solely by insulin stimulation. Interestingly, discoidin domain receptors, involved in cell migration and tumor metastasis, and ephrin receptor B4, involved in bidirectional signaling upon cell-cell contact, were predominantly activated by IGF-II. These findings indicate that IR-A activation by IGF-II elicits a unique signaling pathway that may play a distinct role in physiology and in disease. © 2011 by The Endocrine Society.

Research resource: New and diverse substrates for the insulin receptor isoform a revealed by quantitative proteomics after stimulation with IGF-II or insulin

Pandini, Giuseppe;Vigneri, Riccardo;Belfiore, Antonino
2011

Abstract

The isoform A of the insulin receptor (IR) (IR-A) is a bifunctional receptor, because it binds both insulin and IGF-II. IR-A activation by IGF-II plays a role in development, but its physiological role in adults is unknown. IGF-II signaling through IR-A is deregulated in cancer and favors tumor progression. We hypothesized that IGF-II binding to the IR-A elicits a unique signaling pathway. In order to obtain an unbiased evaluation of IR-A substrates differentially involved after IGF-II and insulin stimulation, we performed quantitative proteomics of IR-A substrates recruited to ty-rosine-phosphorylated protein complexes using stable isotope labeling with amino acids in cell culture in combination with antiphosphotyrosine antibody pull down and mass spectrometry. Using cells expressing only the human IR-A and lacking the IGF-I receptor, we identified 38 IR-A substrates. Only 10 were known IR mediators, whereas 28 substrates were not previously related to IR signaling. Eleven substrates were recruited by stimulation with both ligands: two equally recruited by IGF-II and insulin, three more strongly recruited by IGF-II, and six more strongly recruited by insulin. Moreover, 14 substrates were recruited solely by IGF-II and 13 solely by insulin stimulation. Interestingly, discoidin domain receptors, involved in cell migration and tumor metastasis, and ephrin receptor B4, involved in bidirectional signaling upon cell-cell contact, were predominantly activated by IGF-II. These findings indicate that IR-A activation by IGF-II elicits a unique signaling pathway that may play a distinct role in physiology and in disease. © 2011 by The Endocrine Society.
Animals; Blotting, Western; Cell Line; Humans; Insulin; Insulin-Like Growth Factor II; Intracellular Space; Isotope Labeling; Mice; Models, Biological; Protein Isoforms; Proteomics; Receptor, Insulin; Reproducibility of Results; Signal Transduction; Substrate Specificity; Time Factors; Molecular Biology; Endocrinology
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/20.500.11769/318449
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