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.
References
[1]
Nadler Y, Gonzalez AM, Camp RL, Rimm DL, Kluger HM, et al. (2010) Growth factor receptor-bound protein-7 (Grb7) as a prognostic marker and therapeutic target in breast cancer. Ann Oncol 21: 466–473.
[2]
Stein D, Wu J, Fuqua SA, Roonprapunt C, Yajnik V, et al. (1994) The SH2 domain protein GRB-7 is co-amplified, overexpressed and in a tight complex with HER2 in breast cancer. EMBO J 13: 1331–1340.
[3]
Tanaka S, Pero SC, Taguchi K, Shimada M, Mori M, et al. (2006) Specific peptide ligand for Grb7 signal transduction protein and pancreatic cancer metastasis. J Natl Cancer Inst 98: 491–498.
[4]
Haran M, Chebatco S, Flaishon L, Lantner F, Harpaz N, et al. (2004) Grb7 expression and cellular migration in chronic lymphocytic leukemia: a comparative study of early and advanced stage disease. Leukemia 18: 1948–1950.
[5]
Kasus-Jacobi A, Bereziat V, Perdereau D, Girard J, Burnol AF (2000) Evidence for an interaction between the insulin receptor and Grb7. A role for two of its binding domains, PIR and SH2. Oncogene 19: 2052–2059.
[6]
Fiddes RJ, Campbell DH, Janes PW, Sivertsen SP, Sasaki H, et al. (1998) Analysis of Grb7 recruitment by heregulin-activated erbB receptors reveals a novel target selectivity for erbB3. J Biol Chem 273: 7717–7724.
[7]
Han DC, Guan JL (1999) Association of focal adhesion kinase with Grb7 and its role in cell migration. J Biol Chem 274: 24425–24430.
[8]
Thommes K, Lennartsson J, Carlberg M, Ronnstrand L (1999) Identification of Tyr-703 and Tyr-936 as the primary association sites for Grb2 and Grb7 in the c-Kit/stem cell factor receptor. Biochem J 341: 211–216.
[9]
Cailliau K, Le Marcis V, Bereziat V, Perdereau D, Cariou B, et al. (2003) Inhibition of FGF receptor signalling in Xenopus oocytes: differential effect of Grb7, Grb10 and Grb14. FEBS Lett 548: 43–48.
[10]
Liu BA, Jablonowski K, Raina M, Arce M, Pawson T, et al. (2006) The human and mouse complement of SH2 domain proteins-establishing the boundaries of phosphotyrosine signaling. Mol Cell 22: 851–868.
[11]
Overduin M, Rios CB, Mayer BJ, Baltimore D, Cowburn D (1992) Three-dimensional solution structure of the src homology 2 domain of c-abl. Cell 70: 697–704.
[12]
Bradshaw JM, Waksman G (2002) Molecular recognition by SH2 domains. Adv Protein Chem 61: 161–210.
[13]
Koch CA, Moran MF, Anderson D, Liu XQ, Mbamalu G, et al. (1992) Multiple SH2-mediated interactions in v-src-transformed cells. Mol Cell Biol 12: 1366–1374.
[14]
Huang H, Li L, Wu C, Schibli D, Colwill K, et al. (2008) Defining the specificity space of the human SRC homology 2 domain. Mol Cell Proteomics 7: 768–784.
[15]
Liu BA, Jablonowski K, Shah EE, Engelmann BW, Jones RB, et al. (2010) SH2 domains recognize contextual peptide sequence information to determine selectivity. Mol Cell Proteomics 9: 2391–2404.
[16]
Hwang PM, Li C, Morra M, Lillywhite J, Muhandiram DR, et al. (2002) A “three-pronged” binding mechanism for the SAP/SH2D1A SH2 domain: structural basis and relevance to the XLP syndrome. EMBO J 21: 314–323.
[17]
Muller AJ, Pendergast AM, Havlik MH, Puil L, Pawson T, et al. (1992) A limited set of SH2 domains binds BCR through a high-affinity phosphotyrosine-independent interaction. Mol Cell Biol 12: 5087–5093.
[18]
Oligino L, Lung FD, Sastry L, Bigelow J, Cao T, et al. (1997) Nonphosphorylated peptide ligands for the Grb2 Src homology 2 domain. J Biol Chem 272: 29046–29052.
[19]
Vayssiere B, Zalcman G, Mahe Y, Mirey G, Ligensa T, et al. (2000) Interaction of the Grb7 adapter protein with Rnd1, a new member of the Rho family. FEBS Lett 467: 91–96.
[20]
Liao YC, Si L, deVere White RW, Lo SH (2007) The phosphotyrosine-independent interaction of DLC-1 and the SH2 domain of cten regulates focal adhesion localization and growth suppression activity of DLC-1. J Cell Biol 176: 43–49.
[21]
Ooi J, Yajnik V, Immanuel D, Gordon M, Moskow JJ, et al. (1995) The cloning of Grb10 reveals a new family of SH2 domain proteins. Oncogene 10: 1621–1630.
[22]
Daly RJ, Sanderson GM, Janes PW, Sutherland RL (1996) Cloning and characterization of GRB14, a novel member of the GRB7 gene family. J Biol Chem 271: 12502–12510.
[23]
Lucas-Fernandez E, Garcia-Palmero I, Villalobo A (2008) Genomic organization and control of the grb7 gene family. Curr Genomics 9: 60–68.
[24]
Porter CJ, Matthews JM, Mackay JP, Pursglove SE, Schmidberger JW, et al. (2007) Grb7 SH2 domain structure and interactions with a cyclic peptide inhibitor of cancer cell migration and proliferation. BMC Struct Biol 7: 58.
[25]
Porter CJ, Wilce MC, Mackay JP, Leedman P, Wilce JA (2005) Grb7-SH2 domain dimerisation is affected by a single point mutation. Eur Biophys J 34: 454–460.
[26]
Pias S, Peterson TA, Johnson DL, Lyons BA (2010) The intertwining of structure and function: proposed helix-swapping of the SH2 domain of Grb7, a regulatory protein implicated in cancer progression and inflammation. Crit Rev Immunol 30: 299–304.
[27]
Spuches AM, Argiros HJ, Lee KH, Haas LL, Pero SC, et al. (2007) Calorimetric investigation of phosphorylated and non-phosphorylated peptide ligand binding to the human Grb7-SH2 domain. J Mol Recognit 20: 245–252.
[28]
Pero SC, Oligino L, Daly RJ, Soden AL, Liu C, et al. (2002) Identification of novel non-phosphorylated ligands, which bind selectively to the SH2 domain of Grb7. J Biol Chem 277: 11918–11926.
[29]
Pero SC, Shukla GS, Cookson MM, Flemer SJ, Krag DN (2007) Combination treatment with Grb7 peptide and Doxorubicin or Trastuzumab (Herceptin) results in cooperative cell growth inhibition in breast cancer cells. Br J Cancer 96: 1520–1525.
[30]
Ambaye ND, Gunzburg MJ, Lim RC, Price JT, Wilce MC, et al. (2011) Benzopyrazine derivatives: A novel class of growth factor receptor bound protein 7 antagonists. Bioorg Med Chem 19: 693–701.
[31]
Ivancic M, Daly RJ, Lyons BA (2003) Solution structure of the human Grb7-SH2 domain/erbB2 peptide complex and structural basis for Grb7 binding to ErbB2. J Biomol NMR 27: 205–219.
[32]
Stein D, Wu J, Fuqua SA, Roonprapunt C, Yajnik V, et al. (1994) The SH2 domain protein GRB-7 is co-amplified, overexpressed and in a tight complex with HER2 in breast cancer. EMBO J 13: 1331–1340.
[33]
Li SC, Gish G, Yang D, Coffey AJ, Forman-Kay JD, et al. (1999) Novel mode of ligand binding by the SH2 domain of the human XLP disease gene product SAP/SH2D1A. Curr Biol 9: 1355–1362.
[34]
Bandyopadhyay S, Chiang CY, Srivastava J, Gersten M, White S, et al. (2010) A human MAP kinase interactome. Nat Methods 7: 801–805.
[35]
Song E, Gao S, Tian R, Ma S, Huang H, et al. (2006) A high efficiency strategy for binding property characterization of peptide-binding domains. Mol Cell Proteomics 5: 1368–1381.
[36]
Xiao YF, Wright SN, Wang GK, Morgan JP, Leaf A (1998) Fatty acids suppress voltage-gated Na+ currents in HEK293t cells transfected with the alpha-subunit of the human cardiac Na+ channel. Proc Natl Acad Sci USA 95: 2680–2685.
[37]
Meyer dos Santos S, Klinkhardt U, Schneppenheim R, Harder S (2010) Using Image J for the quantitative analysis of flow-based adhesion assays in real-time under physiologic flow conditions. Platelets 21: 60–66.
[38]
Xu Y, Piston DW, Johnson CH (1999) A bioluminescence resonance energy transfer (BRET) system: application to interacting circadian clock proteins. Proc Natl Acad Sci USA 96: 151–156.
[39]
Keane MM, Lowrey GA, Ettenberg SA, Dayton MA, Lipkowitz S (1996) The protein tyrosine phosphatase DEP-1 is induced during differentiation and inhibits growth of breast cancer cells. Cancer Res 56: 4236–4243.
