Localization of the insulin-like growth factor I receptor binding sites for the SH2 domain proteins p85, Syp, and GTPase activating protein.
Potential signaling substrates for the insulin-like growth factor I (IGF-I) receptor are SH2 domain proteins including the p85 subunit of phosphatidylinositol 3-kinase, the tyrosine phosphatase Syp, GTPase activating protein (GAP), and phospholipase C-gamma (PLC-gamma). In this study, we demonstrate an association between the IGF-I receptor and p85, Syp, and GAP, but not with PLC-gamma in lysates of cells overexpressing the human IGF-I receptor. We further investigated these interactions using glutathione S-transferase (GST) fusion proteins containing the amino-terminal SH2 domains of p85 or GAP, or both SH2 domains of Syp or PLC-gamma to precipitate the IGF-I receptor from purified receptor preparations and from whole cell lysates. p85-, Syp-, and GAP-GSTs precipitated the IGF-I receptor, whereas the PLC-gamma-GST did not. Using phosphopeptides corresponding to IGF-I receptor phosphorylation sites, we determined that the p85- and Syp-GST association with the IGF-I receptor could be inhibited by a carboxyl-terminal peptide containing pY1316 and that the GAP-GST association could be inhibited by a NPXY domain peptide. The GAP-GST binding site was confirmed by showing that a mutant IGF-I receptor with a deletion of the NPXY domain including tyrosine 950 was poorly precipitated by the GAP-GST. We conclude that p85 and Syp may bind directly to the IGF-I receptor at tyrosine 1316, and that GAP may bind to the IGF-I receptor at and PLC-gamma was not evident. p85, Syp, and GAP are potential modulators of IGF-I receptor signal transduction.
Seely
B L
BL
Department of Medicine, University of California at San Diego, La Jolla 92093, USA.
Reichart
D R
DR
Staubs
P A
PA
Jhun
B H
BH
Hsu
D
D
Maegawa
H
H
Milarski
K L
KL
Saltiel
A R
AR
Olefsky
J M
JM
D000595:Amino Acid Sequence; D000818:Animals; D001152:Arsenicals / Q000494:pharmacology; D001665:Binding Sites; D016466:CHO Cells; D006224:Cricetinae; D020690:GTPase-Activating Proteins; D007334:Insulin-Like Growth Factor I / Q000494:pharmacology; D047908:Intracellular Signaling Peptides and Proteins; D008969:Molecular Sequence Data; D019869:Phosphatidylinositol 3-Kinases; D010766:Phosphorylation; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000378:metabolism*; D054562:Protein Tyrosine Phosphatase, Non-Receptor Type 1; 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*; D017526:Receptor, IGF Type 1 / Q000378:metabolism*; D054593:SH2 Domain-Containing Protein Tyrosine Phosphatases; D010738:Type C Phospholipases / Q000378:metabolism; D014443:Tyrosine / Q000378:metabolism
D001152:Arsenicals; D020690:GTPase-Activating Proteins; D047908:Intracellular Signaling Peptides and Proteins; D011506:Proteins; C029341:oxophenylarsine; D014443:Tyrosine; D007334:Insulin-Like Growth Factor I; D019869:Phosphatidylinositol 3-Kinases; D017853:Phosphotransferases (Alcohol Group Acceptor); D017526:Receptor, IGF Type 1; C516724:PTPN11 protein, human; C509249:PTPN6 protein, human; D054562:Protein Tyrosine Phosphatase, Non-Receptor Type 1; D054592:Protein Tyrosine Phosphatase, Non-Receptor Type 11; D053824:Protein Tyrosine Phosphatase, Non-Receptor Type 6; D017027:Protein Tyrosine Phosphatases; D054593:SH2 Domain-Containing Protein Tyrosine Phosphatases; D010738:Type C Phospholipases