Abstract
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.
Highlights
Individual SH2 domain binding sites have been identified and their specificity requirements determined by (i) isolation of specific phosphopeptides after receptor activation, (ii) site-specific mutagenesis, (iii) synthetic peptide binding studies, or (iv) analysis of degenerate phosphopeptide libraries [9, 10]
Synthesis of Peptides—The 256 peptides synthesized corresponded to the sequences centered around all of the tyrosine residues in rat insulin receptor substrate-1 (IRS-1) [20] and the cytoplasmic domains of the human insulin receptor [21], human EGFR [22], mouse PDGFR [23], and chicken basic fibroblast growth factor receptors [24]
Phosphopeptide Design and Binding Assay—Phosphopeptides accounting for all the Tyr-containing sequences in IR, EGFR, PDGFR, and bFGFR (Fig. 1) and IRS-1 were synthesized as 11-mers with the Tyr or Tyr(P) residue in the sixth position
Summary
Individual SH2 domain binding sites have been identified and their specificity requirements determined by (i) isolation of specific phosphopeptides after receptor activation, (ii) site-specific mutagenesis, (iii) synthetic peptide binding studies (see Refs. 2 and 8), or (iv) analysis of degenerate phosphopeptide libraries [9, 10]. In this paper we describe a systematic approach to identify potential binding sites for SH2 domains on cytoplasmic proteins or transmembrane receptors that includes an analysis of every tyrosine-containing peptide. This approach is rapid and sensitive and complements the studies on direct identification of binding sites by mutagenesis and competition binding or specificity determination with degenerate peptide libraries [9, 10]. It confirms many of the sites established by alternative methods and reveals new potential binding sites for Grb at Tyr-1110 in IR, Tyr-1114 in EGFR, Tyr-743 in PDGFR, and Tyr-47, Tyr-727, and Tyr-939 in IRS-1 and for Shc at Tyr-960 in IR, Tyr-703 and Tyr-789 in EGFR, and Tyr-557 in PDGFR
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