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.     

Structural and biophysical investigation of the interaction of a mutant Grb2 SH2 domain (W121G) with its cognate phosphopeptide

The adaptor protein Grb2 is a key element of mitogenetically important signaling pathways. With its SH2 domain it binds to upstream targets while its SH3 domains bind to downstream proteins thereby relaying signals from the cell membranes to the nucleus. The Grb2 SH2 domain binds to its targets by recognizing a phosphotyrosine (pY) in a pYxNx peptide motif, requiring an Asn at the +2 position C‐terminal to the pY with the residue either side of this Asn being hydrophobic. Structural analysis of the Grb2 SH2 domain in complex with its cognate peptide has shown that the peptide adopts a unique β‐turn conformation, unlike the extended conformation that phosphopeptides adopt when bound to other SH2 domains. TrpEF1 (W121) is believed to force the peptide into this unusual conformation conferring this unique specificity to the Grb2 SH2 domain. Using X‐ray crystallography, electron paramagnetic resonance (EPR) spectroscopy, and isothermal titration calorimetry (ITC), we describe here a series of experiments that explore the role of TrpEF1 in determining the specificity of the Grb2 SH2 domain. Our results demonstrate that the ligand does not adopt a pre‐organized structure before binding to the SH2 domain, rather it is the interaction between the two that imposes the hairpin loop to the peptide. Furthermore, we find that the peptide adopts a similar structure when bound to both the wild‐type Grb2 SH2 domain and a TrpEF1Gly mutant. This suggests that TrpEF1 is not the determining factor for the conformation of the phosphopeptide.     

Conformation of an Shc-derived phosphotyrosine-containing peptide complexed with the Grb2 SH2 domain

We have determined the structure of an Shc-derivedphosphotyrosine-containing peptide complexed with Grb2 SH2 based on intra-and intermolecular NOE correlations observed by a series of isotope-filteredNMR experiments using a PFG z-filter. In contrast to an extendedconformation of phosphotyrosine-containing peptides bound to Src, Syp andPLC SH2s, the Shc-derived peptide formed a turn at the +1 and +2positions next to the phosphotyrosine residue. Trp¹²¹, locatedat the EF1 site of Grb2 SH2, blocked the peptide binding in an extendedconformation. The present study confirms that eachphosphotyrosine-containing peptide binds to the cognate SH2 with a specificconformation, which gives the structural basis for the binding specificitybetween SH2s and target proteins.     

Grb10, a Positive, Stimulatory Signaling Adapter in Platelet-Derived Growth Factor BB-, Insulin-Like Growth Factor I-, and Insulin-Mediated Mitogenesis

Grb10 has been described as a cellular partner of several receptor tyrosine kinases, including the insulin receptor (IR) and the insulin-like growth factor I (IGF-I) receptor (IGF-IR). Its cellular role is still unclear and a positive as well as an inhibitory role in mitogenesis depending on the cell context has been implicated. We have tested other mitogenic receptor tyrosine kinases as putative Grb10 partners and have identified the activated forms of platelet-derived growth factor (PDGF) receptor beta (PDGFRbeta), hepatocyte growth factor receptor (Met), and fibroblast growth factor receptor as candidates. We have mapped Y771 as a PDFGRbeta site that is involved in the association with Grb10 via its SH2 domain. We have further investigated the putative role of Grb10 in mitogenesis with four independent experimental strategies and found that all consistently suggested a role as a positive, stimulatory signaling adaptor in normal fibroblasts. (i) Complete Grb10 expression from cDNA with an ecdysone-regulated transient expression system stimulated PDGF-BB-, IGF-I, and insulin- but not epidermal growth factor (EGF)-induced DNA synthesis in an ecdysone dose-responsive fashion. (ii) Microinjection of the (dominant-negative) Grb10 SH2 domain interfered with PDGF-BB- and insulin-induced DNA synthesis. (iii) Alternative experiments were based on cell-permeable fusion peptides with the Drosophila antennapedia homeodomain which effectively traverse the plasma membrane of cultured cells. A cell-permeable Grb10 SH2 domain similarly interfered with PDGF-BB-, IGF-I-, and insulin-induced DNA synthesis. In contrast, a cell-permeable Grb10 Pro-rich putative SH3 domain binding region interfered with IGF-I- and insulin- but not with PDGF-BB- or EGF-induced DNA synthesis. (iv) Transient overexpression of complete Grb10 increased whereas cell-permeable Grb10 SH2 domain fusion peptides substantially decreased the cell proliferation rate (as measured by cell numbers) in normal fibroblasts. These experimental strategies independently suggest that Grb10 functions as a positive, stimulatory, mitogenic signaling adapter in PDGF-BB, IGF-I, and insulin action. This function appears to involve the Grb10 SH2 domain, a novel sequence termed BPS, and the Pro-rich putative SH3 domain binding region in IGF-I- and insulin-mediated mitogenesis. In contrast, PDGF-BB-mediated mitogenesis appears to depend on the SH2 but not on the Pro-rich region and may involve other, unidentified Grb10 domains. Distinct protein domains may help to define specific Grb10 functions in different signaling pathways.     

Development of binding assays for the SH2 domain of Grb7 and Grb2 using fluorescence polarization

Adaptor proteins Grb7 and Grb2 have been implicated as being 2 potential therapeutic targets in several human cancers, especially those that overexpress ErbB2. These 2 proteins contain both a SH2 domain (Src homology 2) that binds to phosphorylated tyrosine residues contained within ErbB2 and other specific protein targets. Two assays based on enzyme-linked immunosorbent assay and fluorescence polarization methods have been developed and validated to find and rank inhibitors for both proteins binding to the pY(1139). Fluorescence polarization assays allowed the authors to determine quickly and reproducibly affinities of peptides from low nanomolar to high micromolar range and to compare them directly for Grb7 and Grb2. As a result, the assays have identified a known peptidomimetic Grb2 SH2 inhibitor (mAZ-pTyr-(alphaMe)pTyr-Asn-NH(2)) that exhibits the most potent affinity for the Grb7 SH2 domain described to date.     

Prediction of binding sites of peptide recognition domains: an application on Grb2 and SAP SH2 domains

Determination of the binding motif and identification of interaction partners of the modular domains such as SH2 domains can enhance our understanding of the regulatory mechanism of protein-protein interactions. We propose here a new computational method to achieve this goal by integrating the orthogonal information obtained from binding free energy estimation and peptide sequence analysis. We performed a proof-of-concept study on the SH2 domains of SAP and Grb2 proteins. The method involves the following steps: (1) estimating the binding free energy of a set of randomly selected peptides along with a sample of known binders; (2) clustering all these peptides using sequence and energy characteristics; (3) extracting a sequence motif, which is represented by a hidden Markov model (HMM), from the cluster of peptides containing the sample of known binders; and (4) scanning the human proteome to identify binding sites of the domain. The binding motifs of the SAP and Grb2 SH2 domains derived by the method agree well with those determined through experimental studies. Using the derived binding motifs, we have predicted new possible interaction partners for the Grb2 and SAP SH2 domains as well as possible interaction sites for interaction partners already known. We also suggested novel roles for the proteins by reviewing their top interaction candidates.     

Binding of the Grb2 SH2 domain to phosphotyrosine motifs does not change the affinity of its SH3 domains for Sos proline-rich motifs.

Phosphotyrosine peptide binding to Grb2 induces tryptophan fluorescence changes in the Src homology 2 (SH2) domain. Affinities are in the nanomolar range, the Shc peptide having the highest affinity, followed by peptides mimicking Grb2 binding sites on EGF and HGF receptors, the putative sites on insulin and IGF-1 receptors having much lower affinities. Proline-rich peptide binding to the SH3 domains induces fluorescence changes mainly in the C-terminal SH3. Affinities are in the micromolar range, the highest affinity peptides mimicking the first proline-rich motif of the Sos C-terminus. Additional residues before this PVPPPVPP motif provide a minor contribution to the binding, but the two residues after this motif are important and may contribute to specificity. The affinity of each SH3 for each proline-rich motif is too low to account for the high stability of the Grb2-Sos complex, suggesting that Grb2 recognizes other structural features in the Sos C-terminus. Binding of a phosphotyrosine peptide to the SH2 has no effect on the SH3s. Thus the binding of Grb2 to a receptor or to an associated protein phosphorylated on tyrosines is unlikely to activate the exchange factor activity of Sos through a conformational change transmitted from the SH2 to the SH3 domains.     

Interaction of the non-phosphorylated peptide G7-18NATE with Grb7-SH2 domain requires phosphate for enhanced affinity and specificity

Src‐homology (SH2) domains are an attractive target for the inhibition of specific signalling pathways but pose the challenge of developing a truly specific inhibitor. The G7‐18NATE cyclic peptide is reported to specifically inhibit the growth factor receptor bound protein 7 (Grb7) adapter protein, implicated in the progression of several cancer types, via interactions with its SH2 domain. G7‐18NATE effectively inhibits the interaction of Grb7 with ErbB3 and focal adhesion kinase in cell lysates and, with the addition of a cell permeability sequence, inhibits the growth and migration of a number of breast cancer cell lines. It is thus a promising lead in the development of therapeutics targeted to Grb7. Here we investigate the degree to which G7‐18NATE is specific for the Grb7‐SH2 domain compared with closely related SH2 domains including those of Grb10, Grb14, and Grb2 using surface plasmon resonance. We demonstrate that G7‐18NATE binds with micromolar binding affinity to Grb7‐SH2 domain (K_D = 4–6 μm ) compared with 50–200 times lower affinity for Grb10, Grb14, and Grb2 but that this specificity depends critically on the presence of phosphate in millimolar concentrations. Other differences in buffer composition, including use of Tris or 2‐(N‐Morpholino)ethanesulfonic acid or varying the pH, do not impact on the interaction. This suggests that under cellular conditions, G7‐18NATE binds with highest affinity to Grb7. In addition, our findings demonstrate that the basis of specificity of G7‐18NATE binding to the Grb7‐SH2 domain is via other than intrinsic structural features of the protein, representing an unexpected mode of molecular recognition. Copyright © 2011 John Wiley & Sons, Ltd.     

Characterization of endocytic uptake of MK2‐inhibitor peptides

Cell penetrating peptides (CPP) have been widely used to increase the cellular delivery of their associated cargo. Multiple modes of uptake have been identified; however, they cannot be predicted a priori. Elucidating these mechanisms is important for understanding peptide function as well as further optimizing cellular delivery. We have developed a class of mitogen activated protein kinase activated protein kinase 2 (MK2) inhibitor peptides, named FAK and YARA that utilize CPP domains to gain cellular access. In this study, we investigate the mechanism of endocytosis of these MK2 inhibitors by examining the uptake of fluorescently labeled peptide in human monocyte (THP‐1) and mesothelial cells, and looking for colocalization with known markers of endocytosis. Our results indicate that uptake of the MK2 inhibitors was minimally enhanced by the addition of the fluorescent label, and that the type of endocytosis used by the inhibitor depends on several factors including concentration, cell type, and which CPP was used. We found that in THP‐1 cells, the uptake of YARA occurred primarily via macropinocytosis, whereas FAK entered via all three mechanisms of endocytosis examined in this study. In mesothelial cells, uptake of YARA occurred via caveolae‐mediated endocytosis, but became less specific at higher concentrations; whereas uptake of FAK occurred through clathrin‐mediated endocytosis. In all cases, the delivery resulted in active inhibition of MK2. In summary, the results support endocytic uptake of fluorescently labeled FAK and YARA in two different cell lines, with the mechanism of uptake dependent on extracellular concentration, cell type, and choice of CPP. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

Novel Src homology 3 domain-binding motifs identified from proteomic screen of a Pro-rich region

The Src homology (SH) 3 domain has been shown recently to bind peptide sequences that lack the canonical PXXP motif. The diverse specificity in ligand recognition for a group of 15 SH3 domains has now been investigated using arrays of peptides derived from the proline-rich region of the SH2 domain-containing leukocyte protein of 76 kDa (SLP-76). A screen of the peptide arrays using individual or mixed SH3 domains has allowed the identification of a number of candidate SH3-binding peptides. Although some peptides contain the conventional PXXP motif, most are devoid of such a motif and are instead enriched in basic residues. Fluorescent polarization measurements using soluble peptides and purified SH3 domains demonstrated that several SH3 domains, including those from growth factor receptor-bound protein 2 (Grb2), NCK, and phospholipase C (PLC)-gamma1, bound with moderate affinities (10-100 microm) to a group of non-conventional peptides. Of particular interest, the PLC-gamma1 SH3 domain was found to associate with SLP-76 through at least three distinct sites, two of which bore a novel KKPP motif and the other contained the classic PXXP sequence. Intriguingly mutation of critical residues for the three sites not only affected binding of SLP-76 to the PLC-gamma1 SH3 domain but also to the Grb2 C-terminal SH3 domain, indicating that the binding sites in SLP-76 for the two SH3 domains are overlapped. Our studies suggest that the SH3 domain is an inherently promiscuous interaction module capable of binding to peptides that may or may not contain a PXXP motif. Furthermore the identification of numerous non-conventional SH3-binding peptides in SLP-76 implies that the global ligand pool for SH3 domains in a mammalian proteome may be significantly greater than previously acknowledged.     

Solution conformations of some azetidine cyclic peptides

The ¹H-nmr spectra (270 MHz) of cyclic di- and tri-L -azatidine-2-carboxylic acid [cy-clo(L -Aze)₂ and cyclo(L -Aze)³] were determined in CDCl₃ and D₂O and computer simulated. The spectral results were compared with those obtained with cyclo (L -Pro)₂ and cyclo (L, -Pro)₃. In CDCl₃ and D₂O solution, the four membered ring of cyclo (L -Aze)₂ is puckered with the α-proton in a pseudo-axial position, and the ? angle is smaller in absolute value than ?60°, as found for cyclo (L -Pro)_2,. The puckering of the four-membered ring of cyclo(L, -Aze)₃ in CDCl₃ has the α-proton in a pseudo–equatorial position and ? angle larger in absolute value than ?60°, in agreement with cyclo(L -Pro)₃. In D₂O, cyclo(L -Aze)₃ was found to interconvert rapidly between different conformers. In the azetidine cyclic peptides studied, the range of values found for the ? angles was smaller than in the related proline cyclic peptides, indicating greater rigidity in the four-membered ring.     

Solution structure of the Grb2 SH2 domain complexed with a high-affinity inhibitor

The solution structure of the growth factor receptor-bound protein 2 (Grb2) SH2 domain complexed with a high-affinity inhibitor containing a non-phosphorus phosphate mimetic within a macrocyclic platform was determined by nuclear magnetic resonance (NMR) spectroscopy. Unambiguous assignments of the bound inhibitor and intermolecular NOEs between the Grb2 SH2 domain and the inhibitor was accomplished using perdeuterated Grb2 SH2 protein. The well-defined solution structure of the complex was obtained and compared to those by X-ray crystallography. Since the crystal structure of the Grb2 SH2 domain formed a domain-swapped dimer and several inhibitors were bound to a hinge region, there were appreciable differences between the solution and crystal structures. Based on the binding interactions between the inhibitor and the Grb2 SH2 domain in solution, we proposed a design of second-generation inhibitors that could be expected to have higher affinity.     

Diverse recognition of non-PxxP peptide ligands by the SH3 domains from p67(phox), Grb2 and Pex13p

The basic function of the Src homology 3 (SH3) domain is considered to be binding to proline-rich sequences containing a PxxP motif. Recently, many SH3 domains, including those from Grb2 and Pex13p, were reported to bind sequences lacking a PxxP motif. We report here that the 22 residue peptide lacking a PxxP motif, derived from p47(phox), binds to the C-terminal SH3 domain from p67(phox). We applied the NMR cross-saturation method to locate the interaction sites for the non-PxxP peptides on their cognate SH3 domains from p67(phox), Grb2 and Pex13p. The binding site of the Grb2 SH3 partially overlapped the conventional PxxP-binding site, whereas those of p67(phox) and Pex13p SH3s are located in different surface regions. The non-PxxP peptide from p47(phox) binds to the p67(phox) SH3 more tightly when it extends to the N-terminus to include a typical PxxP motif, which enabled the structure determination of the complex, to reveal that the non-PxxP peptide segment interacted with the p67(phox) SH3 in a compact helix-turn-helix structure (PDB entry 1K4U).     

Global optimization of conformational constraint on non-phosphorylated cyclic peptide antagonists of the Grb2-SH2 domain

Following our earlier work on a phage library derived non-phosphorylated thioether-cyclized peptide inhibitor of Grb2 SH2 domain, a series of small peptide analogues with various cyclization linkage or various ring size were designed and synthesized and evaluated to investigate the optimal conformational constraint for this novel Grb2-SH2 blocker. Our previous SAR studies have indicated that constrained conformation as well as all amino acids except Leu(2) and Gly(7) in this lead peptide, cyclo(CH(2)CO-Glu(1)-Leu-Tyr-Glu-Asn-Val-Gly-Met-Tyr-Cys(10))-amide (termed G1TE), was necessary for sustenance of the biological activity. In this study, in an effort to derive potent and bioavailable Grb2-SH2 inhibitor with minimal sequence, we undertook a systematic conformational study on this non-phosphorylated cyclic ligand by optimizing the ring linkage, ring configuration and ring size. The polarity and configuration of the cyclization linkage were implicated important in assuming the active conformation. Changing the flexible thioether linkage in G1TE into the relatively rigid sulfoxide linkage secured a 4-fold increase in potency (4, IC(50)=6.5 microM). However, open chain, shortening or expanding the ring size led to a marked loss of inhibitory activity. Significantly, the introduction of omega-amino carboxylic acid linker in place of three C-terminal amino acids in G1TE can remarkably recover the apparently favorable conformation, which is otherwise lost because of the reduced ring size. This modification, combined with favorable substitutions of Gla for Glu(1) and Adi for Glu(4) in the resulting six-residue cyclic peptide, afforded peptide 19, with an almost equal potency (19, IC(50)=23.3 microM) relative to G1TE. Moreover, the lipophilic chain in omega-amino carboxylic acid may confer better cell membrane permeability to 19. These newly developed G1TE analogues with smaller ring size and less peptide character but equal potency can serve as templates to derive potent and specific non-phosphorylated Grb2-SH2 antagonists.     

Grb2 dominantly associates with dynamin II in human hepatocellular carcinoma HepG2 cells

The two SH3 domains and one SH2 domain containing adaptor protein Grb2 is an essential element of the Ras signaling pathway in multiple systems. The SH2 domain of Grb2 recognizes and interacts with phosphotyrosine residues on activated tyrosine kinases, whereas the SH3 domains bind to several proline‐rich domain‐containing proteins such as Sos1. To define the difference in Grb2‐associated proteins in hepatocarcinoma cells, we performed coprecipitation analysis using recombinant GST‐Grb2 fusion proteins and found that several protein components (p170, p125, p100, and p80) differently associated with GST‐Grb2 proteins in human Chang liver and hepatocarcinoma HepG2 cells. Sos1 and p80 proteins dominantly bind to Grb2 fusion proteins in Chang liver, whereas p100 remarkably associate with Grb2 in HepG2 cells. Also GST‐Grb2 SH2 proteins exclusively bound to the p46^Shc, p52^Shc, and p66^Shc are important adaptors of the Ras pathway in HepG2 cells. The p100 protein has been identified as dynamin II. We observed that the N‐SH3 and C‐SH3 domains of Grb2 fusion proteins coprecipitated with dynamin II besides Sos1. These results suggest that dynamin II may be a functional molecule involved in Grb2‐mediated signaling pathway on Ras activation for tumor progression and differentiation of hepatocarcinoma cells. J. Cell. Biochem. 84: 150–155, 2002. © 2001 Wiley‐Liss, Inc.     

Enhanced intracellular peptide delivery by multivalent cell-penetrating peptide with bioreducible linkage

Multivalent cell-penetrating peptides (CPPs) have been reported to show enhancement in cellular uptake and endosomolytic activity. However, its application was limited to trans-delivery of cargo which is lower in cellular uptake efficiency of cargo than cis-delivery. Here, we tried the cis-delivery of cargo with multivalent CPP by preparing bioreducible dimeric CPP–cargo with apoptotic activity using TatBim peptide, a fusion of Tat CPP and Bim peptide derived from Bim apoptosis-inducing protein. Dimeric TatBim was almost twice as highly internalized by cells and significantly induced apoptosis compared to monomeric TatBim. Contribution of bioreducible linkage of dimeric TatBim towards apoptotic activity was also confirmed.     

Development of Grb2 SH2 Domain Signaling Antagonists: A Potential New Class of Antiproliferative Agents

Aberrant signaling through protein-tyrosine kinase (PTK)-dependent pathways is associated with several proliferative diseases. Accordingly, PTK inhibitors are being developed as new approaches for the treatment of certain cancers. Growth factor receptor bound protein 2 (Grb2) is an important downstream mediator of PTK signaling that serves obligatory roles in many pathogenic processes. One of the primary functions of Grb2 is to bind to specific phosphotyrosyl (pTyr)-containing sequences through its Src homology 2 (SH2) domain. Agents that bind to the Grb2 SH2 domain and prevent its normal function could disrupt associated PTK signaling and serve as alternatives to kinase-directed inhibitors. Starting from the X-ray crystal structure of a lead peptide bound to the Grb2 SH2 domain, this review will summarize important contributions to these efforts. The presentation will be thematically arranged according to the region of peptide modified, proceeding from the N-terminus to the C-terminus, with a special section devoted to aspects of conformational constraint.     

Grb2 SH2 domain-binding peptide analogs as potential anticancer agents

The growth factor receptor-bound protein 2 (Grb2) plays an important role in the Ras signaling pathway. Several proteins were found to be overexpressed by oncogenes in the Ras signaling pathway, rendering Grb2 a potential target for the design of antitumor agents. Blocking the interaction between the phosphotyrosine-containing activated receptor and the Src-homology 2 (SH2) domain of Grb2 thus constitutes an important strategy for the development of potential anticancer agents. X-ray, NMR structural investigations, and molecular modeling studies have provided the target structure of Grb2 SH2 domain-alone or complexed with a phosphotyrosine-containing peptide-which is useful for the structure-based design of peptides or peptidomimetics with high affinity for the Grb2 SH2 domain. We review here the variety of approaches to Grb2 SH2 pepide inhibitors developed with the aim of interrupting Grb2 recognition. Inhibitory effects of peptide analogs on the Grb2 SH2 domain and their binding affinities for Grb2 SH2 were determined by ELISA, cell-based assays, or Surface Plasman Resonance (SPR) technology. Results of theses studies provide important information for further modifications of lead peptides, and should lead to the discovery of potent peptides as anticancer agents.