IndraLab
Statements
sparser
"We provide proof of concept for the detection of protein proximity pairs (β-Catenin:E-Cadherin and EGFR:GRB2), and confirm assay specificity through technical controls involving reagent omission experiments, and biologically by treatment with small molecule kinase inhibitors that interrupt kinase:adaptor complexes."
sparser
"We evaluated whether desensitization of EGF-induced STAT3 phosphorylation could be associated with GRB2 up-regulation and/or increased association of Grb2 to the EGFR in the transgenic mice overexpressing GH, but no significant differences in GRB2 protein content or GRB2 association to EGFR between normal and transgenic mice were found ( xref )."
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"For instance, increased binding of GRB2 and SHC1 to EGFR was detected as early as 30 s, peaked at 2 min, and maintained with high intensity until 2 h (> 60% of the maximal binding); in contrast, SOS1 and MAP4K3, which were also maximally detected at 2 min, were quickly dissociated as their amounts were decreased to < 5% of the maxima by 30 min (XREF_FIG)."
"Erbb1 recruites grb2 through sh2 domain;from these studies, we concluded that grb2 binds directly to the egfr at y-1068, to a lesser extent at y-1086, and indirectly at y-1173. Egfr has six binding sites for the adapter protein grb2, and erbb4 has five, each with different binding strength several tyrosine-based motifs recruit a number of signal transducers to the phosphorylated form of erbb1 such as the adaptor proteins growth-factor-receptor bound-2 (grb2) and src-homology-2-containing (shc)."
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"The model represents ErbB1 synthesis and degradation; EGF ligand binding to ErbB1; receptor dimerization and activation; activation of the signaling pathways associated with the binary interactions between ErbB1 + Shc, Shc + Grb2, ErbB1 + Shc-Grb2, ErbB1 + Grb2, and Grb2 + Sos; deactivation of ErbB1 following PTP + ErbB1 interactions; and dissociation of active Shc from ErbB1."
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"The importance of the kinase domain of HER3 (not removed by the truncation) for EGFR dependent Grb2 interaction is in agreement with the recent structural study showing that although the HER3 kinase domain is not functional in terms of kinase activity, it can activate the EGFR kinase domain by formation of the asymmetric dimer XREF_BIBR."
sparser
"The importance of the kinase domain of HER3 (not removed by the truncation) for EGFR-dependent Grb2 interaction is in agreement with the recent structural study showing that although the HER3 kinase domain is not functional in terms of kinase activity, it can activate the EGFR kinase domain by formation of the asymmetric dimer xref ."
sparser
"These discoveries were extended to mammalian systems, where it was shown that the Grb2 SH2 domain could inducibly bind the tyrosine phosphorylated epidermal growth factor receptor (EGFR) while bound constitutively to son of sevenless (SOS), a guanine nucleotide exchange factor for Ras."
"We determined interaction partners to all cytosolic tyrosine residues of the four members of the ErbB-receptor family in an unbiased fashion by quantitative proteomics using pull-down experiments with pairs of phosphorylated and nonphosphorylated synthetic peptides. Each receptor had characteristic preferences for interacting proteins and most interaction partners had multiple binding sites on each receptor. EGFR and ErbB4 had several docking sites for Grb2, while ErbB3 was characterized by six binding sites for PI3K."
sparser
"However, this notion was challenged by the demonstration that the majority of active EGF receptors and associated signaling molecules localize to early endosomes shortly after ligand stimulation [ xref – xref ], and that active EGF receptor interacts with Grb2 [ xref ] and engages the major signaling pathways required for cell survival and mitogenesis after endocytosis [ xref , xref ]."
"In this study, we describe the specific interaction of PAK1 with the Grb2 adapter protein both in vitro and in vivo. We identify the site of this interaction as the second proline-rich SH3 binding domain of PAK1. Stimulation of the epidermal growth factor receptor (EGFR) in HaCaT cells enhances the level of EGFR-associated PAK1 and Grb2, although the PAK1-Grb2 association is itself independent of this stimulation. "
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"It appears that there are several redundant and possibly interdependent mechanisms, which involve : (a) EGFR binding to the clathrin endocytosis protein complex AP2, and to the phospho-tyrosine binding adaptor protein growth factor receptor bound protein 2 (Grb2); (b) ubiquitination of 15 lysines in the EGFR kinase domain by the E3 ubiquitin ligase Casitas B-lineage lymphoma (Cbl), and of 6 other lysines in the carboxy-terminus region; (c) acetylation of 3 of the latter lysines [XREF_BIBR]."
isi
"For instance, increased binding of GRB2 and SHC1 to EGFR was detected as early as 30 s, peaked at 2 min, and maintained with high intensity until 2 h (>60% of the maximal binding); in contrast, SOS1 and MAP4K3, which were also maximally detected at 2 min, were quickly dissociated as their amounts were decreased to <5% of the maxima by 30 min (Figure 4B)."
sparser
"Work done by Gay et al. using CGP78850 (compound 41 , Fig. xref ), a selective Grb2 SH2 domain-binding inhibitor having a mixed phosphoramidite/phenyl ester – protected phosphonic acid group, showed that this compound could block the association of Grb2 with activated epidermal growth factor receptor (EGFR) in living cells and inhibit the growth of cells driven by PTK signaling through Ras (Gay et al., xref )."
sparser
"For the TIR-FRET screening of larger cell collectives, we performed three separate steps: (1) setting up of a membrane associated test system for probing the interaction between the epidermal growth factor receptor (EGFR) and the growth factor receptor-bound protein 2; (2) use of the Epac-SH188 sensor for quantitative evaluation under the microscope; and (3) application of a TIR fluorescence reader to probe the interaction of GFP with Nile Red."
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"The phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR) pathway is activated by various receptor tyrosine kinases (RTKs), such as EGFR, which binds the adaptor protein growth factor receptor bound protein 2 (GRB2) and GRB2 associated binding protein 1 (GAB1) to recruit and activate PI3K at the cell membrane."
sparser
"Competition experiments with synthetic phosphopeptides corresponding to known autophosphorylation sites on the EGFR demonstrated that phosphopeptides containing Y-1068, and to a lesser extent Y-1086, were able to inhibit the binding of Grb2 to the EGFR, while a Y-1173 peptide did not."
sparser
"Besides
the Ras-Raf-MEK signaling, the PI3K-Akt pathway is the
second major EGFR-mediated signal transduction pathway, involving
binding of Grb2 to the EGFR and subsequent association with Gab1 (Grb2-associated-binding
protein 1; predominantly via the C-terminal SH3 domain) and the p85
subunit of PI3K (phosphoinositide 3-kinases; via pTyr residues of
Gab1), resulting in the production of phosphatidylinositol (3,4,5)-triphosphate
(PIP3) and activation of Akt. xref Like SOS1,
Gab1 forms a constitutive complex with Grb2, xref whereas the association between Grb2:Gab1 and PI3K-p85 can be enhanced
by EGF addition xref ( xref E–H)."
sparser
"The Src homology 2 domain-containing protein Grb2 binds to the tyrosine-phosphorylated ErbB1 molecule and activates at least three signalling pathways, Ras-Raf-MEK-ERK, phosphoinositide 3-kinase (PI3K)-AKT, and JAK-STAT signalling pathways, for cell proliferation, survival, and migration, and their continuous abnormal activation eventually leads to cancer cell tumourigenesis, invasiveness, and metastasis xref – xref , xref – xref ."
sparser
"EGFR is able to activate PI3K through a series of reactions: active EGFR binds adaptor protein Grb2, which recruits the docking protein Gab1, which contains pYXXM motifs to which SH2 domains on the regulatory subunit of PI3K (p85) bind, thereby bringing the catalytic subunit of PI3K (p110) in proximity to its membrane-bound lipid substrates [ xref ]."
sparser
"The results of the intracellular domain of EGFR and Grb2 interaction showed that our Gγ recruitment systems could be exploited as a convenient heterologous system to discern the strong binders to the phosphotyrosines in the intracellular domain of EGFR, and therefore would provide the basis for studying other receptor tyrosine kinases as well."
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"Similarly, we recently isolated UbVs that bound specifically to the human epidermal growth factor receptor 3 (HER3) and the growth factor receptor-bound protein 2 (Grb2) and showed that UbVs acted as potent antagonists of Grb2-mediated cell signalling (Leung, Jarvik, & Sidhu, 2017)."
"The dimerization of two EGFR family molecules leads to phosphorylation of C-terminal tyrosine residues either by autophosphorylation or by a SRC-related kinase followed by binding of GRB2, a RAS adaptor protein, at the SH2 domain of the receptor; binding of the proline-rich C-terminus of SOS, a guanine nucleotide exchange factor, to the SH3 domain of the receptor; exchange of a GTP from the receptor complex for a GDP from the RAS protein to form activated RAS; activation of phosphatidylinositol-3-kinase (PI-3 kinase) to initiate signaling by the biochemical pathways that are triggered by diacylglycerol production and protein kinase C activation; and activation of the series of cytoplasmic serine and threonine kinases beginning with RAF to initiate signaling by the biochemical pathways that are triggered by members of the MAPK family (e.g. ERK 1 and 2). EGFR signaling also activates other pathways in the cell including the Janus kinase (JAK)/signal transduction activators of transcription (STAT) pathways [80, 81]."
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"The average value of 4 +/- 1 EGFR 's per EGFR and Grb2 complex from the FRET-FLIM-ICS experiments is in excellent agreement with the model prediction that the EGFR tetramer is the dominant Grb2 bound form, whereas the dominant Grb2-free form is the monomer, which also agrees well with the cluster size estimate of 1 +/- 0.3 EGFR 's from the cell averaged data."
sparser
"Secondly, these GPCRs may not be recruiting Grb2 as part of their activation process or, alternatively, there is an association but it is different in nature to that generated by the AT 1 R and V 1b R. Indeed, for a number of the GPCRs that did not show enhanced EGFR-Grb2 association, we actually observed decreased BRET ratios following ligand stimulation."
sparser
"The model is an extension of our earlier model xref and now includes EGFR–Grb2 binding in addition to
the processes considered in the original model (i.e., ligand–receptor
binding, self-interactions capable of mediating receptor oligomerization,
and receptor autophosphorylation)."
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"Epidermal growth factor receptor pathway substrate 8 (EPS8)/related to the N-terminus of tre oncogene (RN-TRE) and growth-factor receptor bound protein 2 (GRB2)/Ras and Rab interactor 1 (RIN1) have been reported to coordinate the function of Rab5 GEFs and GTPase activating proteins (GAPs) for the maintenance of normal trafficking of cell membrane receptors."
sparser
"In two series of patients with lung adenocarcinoma, the PLA to detect EGFR-GRB2 interaction was independently associated with longer survival (hazard ratio = 0.46, 95% confidence interval: 0.2-0.78, p = 0.0085 and hazard ratio = 0.48, 95% confidence interval: 0.2-0.85, p = 0.017)."
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"As shown in Figure XREF_FIG, GSTP1 and GRB2 could be detected in the immunoprecipitation complex of phospho-EGFR antibody in 30 ng/mL EGF stimulated CNE2 cells, and could not be detected by the pretreatment of the cells with 1 mum EGFR inhibitor PD153035, which indicates that GSTP1 and GRB2 can interact with phospho-EGFR, are downstream targets of EGFR signaling pathway."
sparser
"It could be reasonably argued that this reflects dynamic changes in an equilibrium between association, dissociation and reconfiguration of putative EGFR-Grb2 complexes – where the balance may favor dissociation or reconfiguration that leads to an increased distance between the donor and acceptor moieties."
sparser
"In contrast, after EGF addition, Grb2-rsTagRFP photoswitching resulted in a striking and reversible ~20% modulation of the EGFR-EYFP fluorescence both at the plasma membrane and on endosomes, strongly suggesting the occurrence of pcFRET due to the EGFR-Grb2 interactions at these locations."
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"Of greater importance, we further show that RhoU is also capable of synergizing with activated EGFR in JNK and AP1 mediated transcriptional activity in a manner dependent on SH3 binding, as the DM RhoU, which is physically uncoupled from GRB2 and EGFR complex, is unable to synergize with activated EGFR in either JNK and AP1 activation or increased cell motility."
sparser
"Therefore, our data with AG-1478 clearly demonstrate that (i) EGFR homomers are constitutively interacting with Grb2 in HEK293FT cells, which may be due to some constitutive activity of the receptor, at least with regard to the Grb2 pathway in this cell line, (ii) EGF- as well as HRG-induced Grb2 recruitment strictly depends on receptor activation (constitutive or ligand-induced), and (iii) the recruitment of Grb2 to HER3 is observed only when HER3 is engaged in a heteromer complex with another receptor subtype such as EGFR."
sparser
"The highest photo-cross-linking
yield for Grb2-D104mPyTK was observed when cells were stimulated with
EGF for 15 min (Figure S11B in), indicating
that the Grb2–EGFR interaction is transient and dynamic, resembling
some other known EGF-dependent protein–protein interactions. xref "
trips
"Competition experiments with synthetic phosphopeptides corresponding to known autophosphorylation sites on the EGFR demonstrated that phosphopeptides containing Y-1068, and to a lesser extent Y-1086, were able to inhibit the binding of Grb2 to the EGFR, while a Y-1173 peptide did not."
sparser
"This reduced EGFR kinase-dependence of the peroxide-induced R4 response was correlated to a significant reduction in the degree of peroxide-induced EGFR tyrosine phosphorylation ( xref ), attenuated peroxide-induced association of EGFR with Grb2 ( xref ) and a strong redistribution of EGFR away from the plasma membrane to the cytosol ( xref )."
sparser
"Both CME and several NCE pathways are involved in EGFR internalization. [ xref ] The ubiquitination of EGFR is necessary for all internalization pathways, and the ubiquitin ligase responsible for EGFR ubiquitination is Cbl, a ring‐finger domain E3 ubiquitin ligase. [ xref ] Cbl can directly bind to EGFR, or indirectly bind to EGFR via the adaptor protein Grb2. [ xref , xref ] The EGFR‐Grb2‐Cbl complex is necessary for NCE, with previous studies showing that EGFR mutants lacking Grb2 binding sites are completely defective for NCE. [ xref , xref ] Our results indicated that SGCE knockdown caused induction of EGFR internalization, and increased the interaction between EGFR and c‐Cbl but not that between EGFR and Grb2."
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"EGFR is able to activate PI3K through a series of reactions : active EGFR binds adaptor protein Grb2, which recruits the docking protein Gab1, which contains pYXXM motifs to which SH2 domains on the regulatory subunit of PI3K (p85) bind, thereby bringing the catalytic subunit of PI3K (p110) in proximity to its membrane bound lipid substrates [XREF_BIBR]."
sparser
"This reduced EGFR kinase dependence of the peroxide-induced R4 response was correlated to a significant reduction in the degree of peroxide-induced EGFR tyrosine phosphorylation ( xref ), attenuated peroxide-induced association of EGFR with Grb2 (Figures xref and xref ), and a strong redistribution of EGFR away from the plasma membrane to the cytosol (Figures xref and xref )."
sparser
"Recent studies also found that PLA signals corresponding to EGFR-related interactions such as those between EGFR and GRB2 or between nonphosphorylated and phosphorylated forms of EGFR were higher in NSCLC cell lines positive for EGFR mutations than in those WT for EGFR [ xref – xref ]."
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"We developed a PLA to detect EGFR and GRB2 complexes that could be quantified using the AQUA (R) method of automated quantitative immunofluorescence (QIF) 18, eliminating any subjective interpretation of the results and allowing the objective and reproducible measurement of the obtained signal."
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"A plausible explanation for cooperativity is that when Cbl and Grb2 are simultaneously bound to EGFR (via pY1045 and pY1068 and pY1086, respectively), they are in a state of enforced proximity, which increases the likelihood of the three species (EGFR, Cbl and Grb2) of binding to each other XREF_BIBR."
sparser
"Phosphorylated EGF-R bound the adaptor protein, Grb2, and the EGF-R-selective tyrphostin, AG1478, blocked the C5b-9-induced ERK2 phosphorylation, [(3)H]AA release, and PGE(2) production by 45 to 65%, supporting a functional role for EGF-R kinase in mediating the activation of these pathways."
sparser
"Ang II also increased the association of the adaptor protein Grb2 with the EGF-R. These findings indicate that Src and Pyk2 act upstream of the EGF-R and that the majority of Ang II-induced ERK phosphorylation is dependent on trans-activation of the EGF-R. Ang II-induced ERK activation in C9 cells is initiated by a PKCdelta-dependent but Ca(2+)-independent mechanism and is mediated by the Src/Pyk2 complex through trans-activation of the EGF-R."
sparser
"To analyze the spatiotemporal regulation of EGFR-Grb2 interactions in living cells, we have combined imaging microscopy with a modified method of measuring fluorescence resonance energy transfer (FRET) on a pixel-by-pixel basis using EGFR fused to cyan fluorescent protein (CFP) and Grb2 fused to yellow fluorescent protein (YFP)."
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"Many of the model predictions exhibited a potential scale reduction factor of below 1.2 immediately after the " learning " period (e.g., ErbB1 expression, phosphorylated ErbB1, Grb2 bound to ErbB1, and phosphorylated Shc) while other predictions were unable to converge for the entire prediction window (e.g., phosphorylated PLC-gamma at times greater than 2 minutes)."
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"To assay the interaction between EGFR and Grb2 in yeast, we used the intracellular domain of EGFR with L834R mutation as the membrane protein by fusing several types of lipidation motifs at both the N-terminus (Gpa1p motif; Gpa1N) and the C-terminus (Ras1p motif; Ras1C and Ste18p motif; Ste18C)."
sparser
"Foremost among these is the Ras/Raf/mitogen-activated protein kinase (MAPK) pathway, in which the adaptor protein Grb2 binds to phosphorylated tyrosine residues of EGFR, thus activating the Son of sevenless protein. xref This protein in turn activates the G-protein Ras, which initiates a cascade of phosphorylation of MAPKs, which are specific serine/threonine kinases."
sparser
"In this study, we generated fusion proteins containing one or two Src-homology 2 (SH2) domains of growth factor receptor bound protein 2 (Grb2), which bind to phosphorylated EGFR, added with HIV-1 transactivating transcription for cell membrane penetration (termed TSF and TSSF, respectively)."
sparser
"Cbl was basally associated with the adaptor protein growth factor receptor-binding protein 2 (Grb2), and this interaction was further enhanced by EGF stimulation; however, the interaction was entirely mediated via the Grb2 Src homology 3 (SH3) domains, suggesting that binding of Grb2 SH2 domain to EGF receptor provides one mechanism of Cbl's association with the EGF receptor."
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"The results of the intracellular domain of EGFR and Grb2 interaction showed that our Ggamma recruitment systems could be exploited as a convenient heterologous system to discern the strong binders to the phosphotyrosines in the intracellular domain of EGFR, and therefore would provide the basis for studying other receptor tyrosine kinases as well."
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"The binding of Cbl and Grb2 to EGFR depended on protein concentrations and binding affinities : protein concentrations were carefully measured, while binding affinities were indirectly determined by fitting ubiquitination dose response curves under various experimental conditions, within well defined experimental constrains taken from published studies (XREF_SUPPLEMENTARY)."
sparser
"To assay the interaction between EGFR and Grb2 in yeast, we used the intracellular domain of EGFR with L834R mutation (EGFR L834R,cyto ; that is constitutively dimerized and activated even in the absence of EGF xref xref ) as the membrane protein by fusing several types of lipidation motifs at both the N-terminus (Gpa1p motif; Gpa1N) and the C-terminus (Ras1p motif; Ras1C and Ste18p motif; Ste18C)."
sparser
"Unlike the tyrosine phosphorylation and Grb2 interaction with the EGFR, repeated peroxide exposure (at R4 response point) did not significantly affect Pyk2 Tyr-881 phosphorylation (Figures xref and xref ) or the Grb2 content of the Pyk2 immunoprecipitates (Figures xref and xref )."