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.
Pero
Stephanie C
SC
Department of Surgery and the Vermont Cancer Center, University of Vermont School of Medicine, Burlington, Vermont 05405, USA.
Oligino
Lyn
L
Daly
Roger J
RJ
Soden
Amy L
AL
Liu
Chen
C
Roller
Peter P
PP
Li
Peng
P
Krag
David N
DN
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry; D001667:Binding, Competitive; D015153:Blotting, Western; D004305:Dose-Response Relationship, Drug; D004797:Enzyme-Linked Immunosorbent Assay; D051382:GRB7 Adaptor Protein; D015723:Gene Library; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D008024:Ligands; D008969:Molecular Sequence Data; D019151:Peptide Library; D010455:Peptides / Q000737:chemistry; D010766:Phosphorylation; D011233:Precipitin Tests; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014443:Tyrosine / Q000378:metabolism; D018909:src Homology Domains
D000596:Amino Acids; C495955:GRB7 protein, human; D008024:Ligands; D019151:Peptide Library; D010455:Peptides; D011506:Proteins; D011993:Recombinant Fusion Proteins; D051382:GRB7 Adaptor Protein; D014443:Tyrosine; D005982:Glutathione Transferase