Lyn Oligino

Nonphosphorylated Peptide Ligands for the Grb2 Src Homology 2 Domain

Critical intracellular signals in normal and malignant cells are transmitted by the adaptor protein Grb2 by means of its Src homology 2 (SH2) domain, which binds to phosphotyrosyl (pTyr) residues generated by the activation of tyrosine kinases. To understand this important control point and to design inhibitors, previous investigations have focused on the molecular mechanisms by which the Grb2 SH2 domain selectively binds pTyr containing peptides. In the current study, we demonstrate that the Grb2 SH2 domain can also bind in a pTyr independent manner. Using phage display, an 11-amino acid cyclic peptide, G1, has been identified that binds to the Grb2 SH2 domain but not the src SH2 domain. Synthetic G1 peptide blocks Grb2 SH2 domain association (IC50 10–25 μm) with a 9-amino acid pTyr-containing peptide derived from the SHC protein (pTyr317). These data and amino acid substitution analysis indicate that G1 interacts in the phosphopeptide binding site. G1 peptide requires a YXN sequence similar to that found in natural pTyr-containing ligands, and phosphorylation of the tyrosine increases G1 inhibitory activity. G1 also requires an internal disulfide bond to maintain the active binding conformation. Since the G1 peptide does not contain pTyr, it defines a new type of SH2 domain binding motif that may advance the design of Grb2 antagonists.

Identification of novel non-phosphorylated ligands, which bind selectively to the SH2 domain of Grb7.

Grb7 is an adapter-type signaling protein, which is recruited via its SH2 domain to a variety of receptor tyrosine kinases (RTKs), including ErbB2 and ErbB3. It is overexpressed in breast, esophageal, and gastric cancers, and may contribute to the invasive potential of cancer cells. Molecular interactions involving Grb7 therefore provide attractive targets for therapeutic intervention. We have utilized phage display random peptide libraries as a source of small peptide ligands to the SH2 domain of Grb7. Screening these libraries against purified Grb7 SH2 resulted in the identification of Grb7-binding peptide phage clones that contained a non-phosphorylated Tyr-X-Asn (YXN) motif. The tyrosine-phosphorylated form of this motif is characteristic of Grb7 SH2 domain binding sites identified in RTKs and other signaling proteins such as Shc. Peptides that are non-phosphorylated have greater potential in the development of therapeutics because of the instability of a phosphate group in vivo. Using a biased library approach with this conserved YXN motif, we identified seven different peptide phage clones, which bind specifically to the SH2 domain of Grb7. These peptides did not bind to the SH2 domain of Grb2 (which also selects for Asn at pY+2) or Grb14, a closely related family member. The cyclic structure of the peptides was required to bind to the Grb7 SH2 domain. Importantly, the synthetic Grb7-binding peptide G7-18 in cell lysates was able to specifically inhibit the association of Grb7 with the ErbB family of RTKs, in particular ErbB3, in a dose-dependent manner. These peptides will be useful in the development of targeted molecular therapeutics for cancers overexpressing Grb7 and in the development of Grb7-specific inhibitors to gain a complete understanding of the physiological role of Grb7.