Abstract
The Grb7 (growth factor receptor-bound 7) protein, a member of the Grb7 protein family, is found to be highly expressed in such metastatic tumors as breast cancer, esophageal cancer, liver cancer, etc. The src-homology 2 (SH2) domain in the C-terminus is reported to be mainly involved in Grb7 signaling pathways. Using the random peptide library, we identified a series of Grb7 SH2 domain-binding nonphosphorylated peptides in the yeast two-hybrid system. These peptides have a conserved GIPT/K/N sequence at the N-terminus and G/WD/IP at the C-terminus, and the region between the N-and C-terminus contains fifteen amino acids enriched with serines, threonines and prolines. The association between the nonphosphorylated peptides and the Grb7 SH2 domain occurred in vitro and ex vivo. When competing for binding to the Grb7 SH2 domain in a complex, one synthesized nonphosphorylated ligand, containing the twenty-two amino acid-motif sequence, showed at least comparable affinity to the phosphorylated ligand of ErbB3 in vitro, and its overexpression inhibited the proliferation of SK-BR-3 cells. Such nonphosphorylated peptides may be useful for rational design of drugs targeted against cancers that express high levels of Grb7 protein.
Highlights
Grb7 protein is associated with amplification and invasion of many solid cancers, including those of the breast [1], esophagus [2], pancreas [3] and lymphocytic leukemia [4]
Novel nonphosphorylated twenty-two amino residue peptides harboring conserved sequences at both ends selectively bind to the Grb7 src-homology 2 (SH2) domain
To see whether the conserved residues were required for association with the Grb7 SH2 domain, the peptide 10 was used as the mutation template to design three mutated peptides: the NGIPT deletion mutation (10AM), the C-GIP deletion mutation (10BM) and the both N-GIPT and C-GIP deletion mutation (10CM)
Summary
Grb protein is associated with amplification and invasion of many solid cancers, including those of the breast [1], esophagus [2], pancreas [3] and lymphocytic leukemia [4]. Via the SH2 domain, Grb protein participates in many signaling pathways, such as those associated with insulin [5], ErbB2, ErbB3 and ErbB4 [6], FAK [7], c-Kit/SCFR [8] and FGFR [9]. The SH2 domain plays vital roles in signaling pathways by acting as adaptors, kinases or scaffolds [10]. The SH2 domain contains a central anti-parallel b sheet surrounded by two a helices [11]. It has a positively charged binding cavity [12] that promotes its association with phosphotyrosine motifs [13]. Different SH2 domains prefer distinct phosphorylated motifs [14], certain SH2 domains are more flexible in their motif preference [15]
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