Localization of the insulin receptor binding sites for the SH2 domain proteins p85, Syp, and GAP.
The insulin receptor is known to interact with the SH2 domain proteins p85 (the regulatory subunit of phosphatidylinositol 3-kinase), Syp (a tyrosine phosphatase), and GAP (GTPase-activating protein). In this study, we mapped the insulin receptor binding sites for each of these proteins by examining the ability of phosphopeptides, corresponding to insulin receptor phosphorylation sites, and mutant insulin receptors to inhibit an insulin receptor-SH2 domain interaction. Precipitation of partially purified insulin receptors by glutathione S-transferase fusion proteins containing the N-terminal SH2 domains of p85 and GAP and both SH2 domains of Syp was demonstrated. The effect of the addition of each phosphopeptide on insulin receptor precipitation was tested. pY1322, the C-terminal insulin receptor peptide, inhibited insulin receptor precipitation by both p85- and Syp-GST. The NPXY internalization domain peptide inhibited insulin receptor precipitation by GAP-GST. These data were confirmed by mutant insulin receptor experiments. The insulin receptor C-terminal mutants, delta CT and Y/F2, were not precipitated by p85- or Syp-GST and the NPXY mutant insulin receptors, delta Ex16 and HI delta NPEY, were not precipitated by GAP-GST. Therefore, we conclude that p85 and Syp bind to the insulin receptor C terminus at tyrosine 1322 and GAP binds to the insulin receptor NPXY domain at tyrosine 960.
Staubs
P A
PA
Department of Medicine, University of California, San Diego 92093-0673.
Reichart
D R
DR
Saltiel
A R
AR
Milarski
K L
KL
Maegawa
H
H
Berhanu
P
P
Olefsky
J M
JM
Seely
B L
BL
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D020690:GTPase-Activating Proteins; D066298:In Vitro Techniques; D047908:Intracellular Signaling Peptides and Proteins; D008969:Molecular Sequence Data; D019869:Phosphatidylinositol 3-Kinases; D010748:Phosphopeptides / Q000378:metabolism*; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000378:metabolism*; D019000:Phosphotyrosine; D054592:Protein Tyrosine Phosphatase, Non-Receptor Type 11; D053824:Protein Tyrosine Phosphatase, Non-Receptor Type 6; D017027:Protein Tyrosine Phosphatases / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism*; D051381:Rats; D011972:Receptor, Insulin / Q000378:metabolism*; D054593:SH2 Domain-Containing Protein Tyrosine Phosphatases; D014443:Tyrosine / Q000031:analogs & derivatives / Q000378:metabolism
D020690:GTPase-Activating Proteins; D047908:Intracellular Signaling Peptides and Proteins; D010748:Phosphopeptides; D011506:Proteins; D019000:Phosphotyrosine; D014443:Tyrosine; D019869:Phosphatidylinositol 3-Kinases; D017853:Phosphotransferases (Alcohol Group Acceptor); D011972:Receptor, Insulin; D054592:Protein Tyrosine Phosphatase, Non-Receptor Type 11; D053824:Protein Tyrosine Phosphatase, Non-Receptor Type 6; D017027:Protein Tyrosine Phosphatases; C516727:Ptpn11 protein, rat; C509250:Ptpn6 protein, rat; D054593:SH2 Domain-Containing Protein Tyrosine Phosphatases