title: In vitro association of the phosphatidylinositol 3-kinase regulatory subunit (p85) with the human insulin receptor.
abstract: The insulin receptor, as a consequence of ligand binding, undergoes autophosphorylation of critical tyrosyl residues within the cytoplasmic portion of its beta-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 beta-subunit, while neither the GTPase activator protein (GAP) N SH2 domain nor the phospholipase C-gamma 1 (PLC gamma 1) 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-phosphorylated peptide corresponding to the carboxyl-terminal region of the insulin receptor, but not by phosphopeptides of similar length derived from the juxtamembrane or regulatory regions. The insulin receptor C domain phosphopeptide inhibited the p85 N + C SH2 domain-insulin receptor complex with an IC0.5 of 2.3 +/- 0.35 microM, whereas a 10-residue phosphopeptide derived from the insulin receptor substrate 1 (IRS-1) competed with an IC0.5 of 0.54 +/- 0.10 microM. 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.
authors.affiliation: Department of Chemistry, University of Minnesota, Minneapolis, USA.
authors.forename: E A
authors.initials: EA
authors.lastname: Ottinger
authors.forename: T Y
authors.initials: TY
authors.lastname: Hui
authors.forename: Z
authors.initials: Z
authors.lastname: Man
authors.forename: G
authors.initials: G
authors.lastname: Barany
authors.forename: D A
authors.initials: DA
authors.lastname: Bernlohr
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D001665:Binding Sites; D001667:Binding, Competitive; D001710:Biotin; D005982:Glutathione Transferase; D006801:Humans; D008969:Molecular Sequence Data; D019869:Phosphatidylinositol 3-Kinases; D010766:Phosphorylation; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000378:metabolism*; D019000:Phosphotyrosine / Q000378:metabolism; D011972:Receptor, Insulin / Q000378:metabolism*; D011993:Recombinant Fusion Proteins; D015398:Signal Transduction; D018909:src Homology Domains
chemical_list: D011993:Recombinant Fusion Proteins; D019000:Phosphotyrosine; D001710:Biotin; D000255:Adenosine Triphosphate; D005982:Glutathione Transferase; D019869:Phosphatidylinositol 3-Kinases; D017853:Phosphotransferases (Alcohol Group Acceptor); D011972:Receptor, Insulin