In vitro association of the phosphatidylinositol 3‐kinase regulatory subunit (p85) with the human insulin receptor


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The insulin receptor, as a consequence of ligand binding, undergoes autophosphorylation of critical tyrosyl residues within the cytoplasmic portion of its β‐subunit. The 85 kDa regulatory subunit of phosphatidylinositol (PI) 3‐kinase (p85), an SH2 domain protein, has been implicated as a regulatory molecule in the insulin signal transduction pathway. For the present study, glutathione S‐transferase (GST) fusion proteins of p85 SH2 domains were used to determine if such motifs associate directly with the autophosphorylated human insulin receptor. The p85 N + C (amino plus carboxyl) SH2 domains were demonstrated to associate with the autophosphorylated β‐subunit, while neither the GTPase activator protein (GAP) N SH2 domain nor the phospholipase C‐γ1 (PLCγ1) N + C SH2 domains exhibited measurable affinity for the activated receptor. The p85 N SH2 domain demonstrated weak association with the insulin receptor, while the p85 C SH2 domain alone formed no detectable complexes with the insulin receptor. The association of p85 N + C SH2 domains with the autophosphorylated receptor was competed efficiently by a 15‐residue tyrosine‐phos‐phorylated peptide corresponding to the carboxyl‐terminal region of the insulin receptor, but not by phos‐phopeptides of similar length derived from the juxtamembrane or regulatory regions. The insulin receptor C domain phosphopeptide inhibited the p85 N i C SH2 domain‐insulin receptor complex with an IC0.5 of 2.3 ± 0.35 μM, whereas a 10‐residue phosphopeptide derived from the insulin receptor substrate 1 (IRS‐1) competed with an IC0.5 of 0.54 ± 0.10 μM. These results demonstrate that, in vitro, there is an association between the p85 regulatory protein and the carboxyl‐terminal region of the activated insulin receptor that requires the presence of both the N and C SH2 domains. Furthermore, formation of the p85/insulin receptor complex may lead to signaling pathways independent of IRS‐1. © Munksgaard 1995.

Original languageEnglish (US)
Pages (from-to)346-353
Number of pages8
JournalInternational Journal of Peptide and Protein Research
Issue number5
StatePublished - Nov 1995


  • SH2 domains
  • insulin receptor
  • insulin receptor substrate‐1
  • phosphatidylinositol 3‐kinase
  • tyrosine phosphopeptides


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