Systematic mapping of potential binding sites for Shc and Grb2 SH2 domains on insulin receptor substrate-1 and the receptors for insulin, epidermal growth factor, platelet-derived growth factor, and fibroblast growth factor.
Multipin peptide synthesis has been employed to produce biotinylated 11-mer phosphopeptides that account for every tyrosine residue in insulin receptor substrate-1 (IRS-1) and the cytoplasmic domains of the insulin-, epidermal growth factor-, platelet-derived growth factor- and basic fibroblast growth factor receptors. These phosphopeptides have been screened for their capacity to bind to the SH2 domains of Shc and Grb in a solution phase enzyme-linked immunosorbent assay. The data revealed new potential Grb2 binding sites at Tyr-1114 (epidermal growth factor receptor (EGFR) C-tail); Tyr-743 (platelet-derived growth factor receptor (PDGFR) insert region), Tyr-1110 from the E-helix of the catalytic domain of insulin receptor (IR), and Tyr-47, Tyr-939, and Tyr-727 in IRS-1. None of the phosphopeptides from the juxtamembrane or C-tail regions of IR bound Grb2 significantly, and only one phosphopeptide from the basic fibroblast growth factor receptor (Tyr-556) bound Grb2 but with medium strength. Tyr-1068 and -1086 from the C-tail of EGFR, Tyr-684 from the kinase insert region of PDGFR, and Tyr-895 from IRS-1 were confirmed as major binding sites for the Grb2 SH2 domain. With regard to Shc binding, the data revealed new potential binding sites at Tyr-703 and Tyr-789 from the catalytic domain of EGFR and at Tyr-557 in the juxtamembrane region of PDGFR. It also identified new potential Shc binding sites at Tyr-764, in the C-tail of basic fibroblast growth factor receptor, and Tyr-960, in the juxtamembrane of IR, a residue previously known to be required for Shc phosphorylation in response to insulin. The study confirmed the previous identification of Tyr-992 and Tyr-1173 in the C-tail of EGFR and several phosphopeptides from the PDGFR as medium strength binding sites for the SH2 domain of Shc. None of the 34 phosphopeptides from IRS-1 bound Shc strongly, although Tyr-690 showed medium strength binding. The specificity characteristics of the SH2 domains of Grb2 and Shc are discussed. This systematic peptide mapping strategy provides a way of rapidly scanning candidate proteins for potential SH2 binding sites as a first step to establishing their involvement in kinase-mediated signaling pathways.
Ward
C W
CW
CSIRO, Division of Biomolecular Engineering, Parkville, Victoria 3052, Australia.
Gough
K H
KH
Rashke
M
M
Wan
S S
SS
Tribbick
G
G
Wang
J
J
D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D001665:Binding Sites; D003593:Cytoplasm / Q000378:metabolism; D066246:ErbB Receptors / Q000737:chemistry / Q000378:metabolism*; D016222:Fibroblast Growth Factor 2 / Q000378:metabolism; D051380:GRB2 Adaptor Protein; D006801:Humans; D055504:Insulin Receptor Substrate Proteins; D051379:Mice; D009842:Oligopeptides / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism; D010750:Phosphoproteins / Q000737:chemistry / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism*; D011972:Receptor, Insulin / Q000737:chemistry / Q000378:metabolism*; D017468:Receptors, Fibroblast Growth Factor / Q000737:chemistry / Q000378:metabolism*; D017479:Receptors, Platelet-Derived Growth Factor / Q000737:chemistry / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D018909:src Homology Domains
D048868:Adaptor Proteins, Signal Transducing; D051380:GRB2 Adaptor Protein; C495948:GRB2 protein, human; C495949:Grb2 protein, mouse; C526292:IRS1 protein, human; D055504:Insulin Receptor Substrate Proteins; C526295:Irs1 protein, mouse; D009842:Oligopeptides; D010750:Phosphoproteins; D011506:Proteins; D017468:Receptors, Fibroblast Growth Factor; D016222:Fibroblast Growth Factor 2; D066246:ErbB Receptors; D011972:Receptor, Insulin; D017479:Receptors, Platelet-Derived Growth Factor