grb2 sequence  (Addgene inc)

Journal: The Journal of Biological Chemistry

Article Title: Measurement of FGFR3 signaling at the cell membrane via total internal reflection fluorescence microscopy to compare the activation of FGFR3 mutants

doi: 10.1016/j.jbc.2022.102832

Figure Lengend Snippet: Experimental design and proof of principle. A and B, antibody patterns are used to enrich and immobilize mGFP-FGFR3 at specific sites (“ON” regions) in the plasma membrane of HeLa cells, leaving other regions depleted of mGFP-FGFR3 (“OFF”). Colocalization of the adaptor protein GRB2-mScarlet to mGFP-FGFR3 patterns reports on the activation state of FGFR3, with no or little copatterning observable in the nonactivated state ( A ) and a high degree of copatterning for the activated receptor after addition of the ligand fgf1 ( B ). TIRF illumination is used to specifically detect membrane-proximal protein. C, the fluorescence contrast of GRB2-mScarlet (C mScarlet ) relates the fluorescence intensity within ON (I ON,mGFP ) and OFF (I OFF,mGFP ) areas of FGFR3-enriched regions and serves to quantify the extent of colocalization. Each dot represents one cell. C mScarlet data for the WT receptor, a kinase-dead mutant (K508M) and an mGFP-FGFR3-mScarlet fusion protein as positive control is shown ( p value annotation legend: ∗0.01 ≤ p ≤ 0.05; ∗∗0.001 ≤ p ≤ 0.01; ∗∗∗0.0001 ≤ p ≤ 0.001; and ∗∗∗∗ p ≤ 0.0001). D and E, correlation between the receptor’s intensity in ON ( D ) and OFF ( E ) regions and the GRB2-mScarlet contrast for the WT receptor. Data in the absence ( black ) and presence ( orange ) of fgf1 are shown. The gray box indicates the cell population with C mScarlet <0.2, which likely represents nonactivated cells. F and G, correlation between GRB2-mScarlet contrast and mGFP-FGFR3 intensity in ON ( F ) and OFF ( G ) regions for K508M. All correlation coefficients can be found in Table S1 . FGFR3, fibroblast growth factor receptor 3; GRB2, growth factor receptor–bound 2; mGFP, monomeric GFP; TIRF, total internal reflection fluorescence.

Article Snippet: To obtain the GRB2-mScarlet plasmid, we carried out PCR to amplify the mScarlet sequence from the ITPKA-mScarlet plasmid (Addgene) as well as the GRB2 sequence from GRB2-YFP (gift from J. Weghuber) with >15 nt overhangs complementary to adjacent regions on the target plasmid.

Techniques: Clinical Proteomics, Membrane, Activation Assay, Fluorescence, Mutagenesis, Positive Control

Journal: The Journal of Biological Chemistry

Article Title: Measurement of FGFR3 signaling at the cell membrane via total internal reflection fluorescence microscopy to compare the activation of FGFR3 mutants

doi: 10.1016/j.jbc.2022.102832

Figure Lengend Snippet: Effect of the ligands fgf1 and fgf2 on receptor activation. A, comparison of GRB2 contrast (C mScarlet ) determined for the WT and mutant forms of FGFR3 in the absence and presence of fgf2. The p value annotation legend is ∗0.01 ≤ p ≤ 0.05; a full list can be found in Table S3 . B, normalization of mean C mScarlet values to the nonactivated WT FGFR3. Data are shown as mean ± standard error. FGFR3, fibroblast growth factor receptor 3; GRB2, growth factor receptor–bound 2.

Article Snippet: To obtain the GRB2-mScarlet plasmid, we carried out PCR to amplify the mScarlet sequence from the ITPKA-mScarlet plasmid (Addgene) as well as the GRB2 sequence from GRB2-YFP (gift from J. Weghuber) with >15 nt overhangs complementary to adjacent regions on the target plasmid.

Techniques: Activation Assay, Comparison, Mutagenesis

Journal: Journal of Cellular and Molecular Medicine

Article Title: Integrated transcriptomic and proteomic analyses reveal ɑ‐lipoic acid‐regulated cell proliferation via Grb2‐mediated signalling in hepatic cancer cells

doi: 10.1111/jcmm.13447

Figure Lengend Snippet: Protein–protein interaction network of DEPs. ( A ) Venn diagram showing the overlapping DEGs (blue) and DEPs (yellow). ( B ) Heatmaps showing the expression variation at the mRNA level of the 72 DEGs/DEPs. ( C ) Protein–protein interactions among these 72 DEGs/DEPs according to the STRING database ( http://string-db.org/ ). Line segments indicate protein–protein interactions, red indicates up‐regulated DEGs/DEPs, and green indicates down‐regulated DEGs/DEPs. ( D ) Grb2‐centred protein–protein interaction network. The network was constructed with the BioGRID database ( https://thebiogrid.org/ ) and used to predict the protein network related to Grb2.

Article Snippet: For the overexpression studies, the full‐length Grb2 sequence was purchased from Vigene (Rockville, MD, USA) and subcloned into the pENTR expression vectors.

Techniques: Expressing, Protein-Protein interactions, Construct

Journal: Journal of Cellular and Molecular Medicine

Article Title: Integrated transcriptomic and proteomic analyses reveal ɑ‐lipoic acid‐regulated cell proliferation via Grb2‐mediated signalling in hepatic cancer cells

doi: 10.1111/jcmm.13447

Figure Lengend Snippet: Grb2 mediates the ɑ‐LA‐induced reduction in cell proliferation. ( A ) HepG2 cell proliferation was measured through a CCK‐8 assay at the indicated times. ( B and C ) Cell cycle regulatory protein expression in HepG2 cells after treatment with 2.0 mM ɑ‐LA for 24 hrs was assessed via Western blotting. ( D ) Grb2 levels were measured via real‐time PCR and Western blotting in HepG2 cells after treatment with 1.0 mM ɑ‐LA for 12 and 24 hrs (upper panel). HepG2 cells were transfected with siRNA against Grb2, and 24 hrs after transfection, the cells were seeded into 96‐well plates for CCK‐8 assays at the indicated times (middle panel). After transfection with Grb2 overexpression plasmids, cell proliferation was measured at the indicated times through a CCK‐8 assay (lower panel). ( E ) A Western blotting assay was performed to assess the levels of phosphorylated EGFR, Met, ERK and Ras in HepG2 cells after treatment with 2.0 mM ɑ‐LA for 6 and 24 hrs. ( F ) HepG2 cells were transfected with 50 nM scramble siRNA or Grb2 siRNA (siGrb2) for 48 hrs and then treated with ɑ‐LA for 24 hrs, and the levels of phosphorylated EGFR and Met were analysed via Western blotting.

Article Snippet: For the overexpression studies, the full‐length Grb2 sequence was purchased from Vigene (Rockville, MD, USA) and subcloned into the pENTR expression vectors.

Techniques: CCK-8 Assay, Expressing, Western Blot, Real-time Polymerase Chain Reaction, Transfection, Over Expression

Journal: Journal of Cellular and Molecular Medicine

Article Title: Integrated transcriptomic and proteomic analyses reveal ɑ‐lipoic acid‐regulated cell proliferation via Grb2‐mediated signalling in hepatic cancer cells

doi: 10.1111/jcmm.13447

Figure Lengend Snippet: Correlations of EGFR, Met and Grb2 in human HCC specimens. ( A ) Grb2 expression in normal tissue downloaded from the GeneCards website. ( B ) Grb2 expression in various cancers queried from the cBioPortal database. ( C ) The expression of Grb2 in HCC and adjacent tissue (NCBI GEO accession number 20140) was analysed. ( D ) Kaplan–Meier curves showing survival times according to the Grb2 signature in HCC patients. The patients with expression above the median are shown in red, and the patients with expression below the median are shown in blue. ( E ) A Pearson's correlation analysis of Met/Grb2 and EGFR/Grb2 expression from microarrays of human hepatic malignant lesions tissue (NCBI GEO accession number 20140), which provided data from 287 specimens, including a training cohort (80 tumour and 82 non‐tumour liver samples) and a validation cohort (225 non‐tumour liver tissue samples surgically resected from patients with HCC), was performed.

Article Snippet: For the overexpression studies, the full‐length Grb2 sequence was purchased from Vigene (Rockville, MD, USA) and subcloned into the pENTR expression vectors.

Techniques: Expressing, Biomarker Discovery

Journal: Journal of Cellular and Molecular Medicine

Article Title: Integrated transcriptomic and proteomic analyses reveal ɑ‐lipoic acid‐regulated cell proliferation via Grb2‐mediated signalling in hepatic cancer cells

doi: 10.1111/jcmm.13447

Figure Lengend Snippet: Schematic diagram illustrating how ɑ‐LA, via regulation of Grb2 expression, modulates different signalling pathways that participate in proliferation‐related stress responses. Growth factors (EGF/HGF) in the extracellular matrix bind to RTKs on the plasma membrane and subsequently phosphorylate the docking site and recruit effector molecules [Grb2, Grb2‐associated‐binding protein 1 (Gab 1), SRC homology 2 domain‐containing phosphatase 2 (SHP2), Son of Sevenless (Sos) and sarcoma non‐receptor tyrosine kinase (SRC)]. ɑ‐LA, by down‐regulating Grb2 expression, impairs the phosphorylation of the docking site of effector molecules and then subsequently attenuates the downstream ERK/MAPK pathway and the PI3K‐AKT pathway, leading to decreased ERK1/2 and/or mTOR translocation across the nuclear membrane and/or inhibition of the activation of transcription factors (TFs) and gene expression. The signals affect gene expression and repress cell proliferation and survival, resulting in the arrest of cancer growth and progression.

Article Snippet: For the overexpression studies, the full‐length Grb2 sequence was purchased from Vigene (Rockville, MD, USA) and subcloned into the pENTR expression vectors.

Techniques: Expressing, Clinical Proteomics, Membrane, Binding Assay, Phospho-proteomics, Translocation Assay, Inhibition, Activation Assay, Gene Expression

Journal: Journal of Cellular and Molecular Medicine

Article Title: Integrated transcriptomic and proteomic analyses reveal ɑ‐lipoic acid‐regulated cell proliferation via Grb2‐mediated signalling in hepatic cancer cells

doi: 10.1111/jcmm.13447

Figure Lengend Snippet: List of overlapping DEGs/DEPs

Article Snippet: For the overexpression studies, the full‐length Grb2 sequence was purchased from Vigene (Rockville, MD, USA) and subcloned into the pENTR expression vectors.

Techniques: Ubiquitin Proteomics

Journal: The Journal of Biological Chemistry

Article Title: Membrane Recruitment of the Non-receptor Protein GIV/Girdin (Gα-interacting, Vesicle-associated Protein/Girdin) Is Sufficient for Activating Heterotrimeric G Protein Signaling *

doi: 10.1074/jbc.M116.764431

Figure Lengend Snippet: Recruitment of the GBA motif of GIV to activated EGFR is sufficient to induce G protein activation. A, diagram depicting how Grb2 fused to the GBA motif (aa 1660–1705) of GIV (Grb2-GBA) is recruited from the cytosol to tyrosine phosphorylated EGFR at the plasma membrane upon activation. Grb2-mediated binding to EGFR brings the GBA motif close to membrane-bound Gi3. B and C, HEK293T cells were transfected with plasmids for all the components required BRET-based G protein activity measurements as described for Fig. 3, Grb2-GBA (WT or FA) and EGFR. EGF (50 ng/μl) was added at the indicated time (arrow in B). BRET results (B) are the averages of three or four independent experiments, and the error bars are the S.E. (shown only at 5-s intervals for clarity). Representative immunoblots of the cells used in these experiments are shown in C.

Article Snippet: Grb2-GBA was generated by replacing the mRFP and FKBP sequences of FKBP-GIV-GBA (1660–1705) with the human Grb2 sequence (amplified from Addgene plasmid catalog no. 70383) preceded by a Myc tag (NheI/NruI sites). pcDNA6A-EGFR was obtained from Addgene (catalog no. 42665).

Techniques: Activation Assay, Clinical Proteomics, Membrane, Binding Assay, Transfection, Activity Assay, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Membrane Recruitment of the Non-receptor Protein GIV/Girdin (Gα-interacting, Vesicle-associated Protein/Girdin) Is Sufficient for Activating Heterotrimeric G Protein Signaling *

doi: 10.1074/jbc.M116.764431

Figure Lengend Snippet: Model depicting the parallelism between the mechanisms of activation of Gαi and Ras by their respective cytoplasmic GEFs, GIV, and SOS, upon RTK stimulation. A, under resting conditions, SOS is primarily located in the cytosol along with Grb2, whereas its substrate G protein Ras is constitutively anchored to the plasma membrane, thereby precluding SOS action. Upon RTK stimulation, Grb2-SOS complexes translocate to the plasma membrane via binding of Grb2 SH2 domains to tyrosine phosphorylated EGFR. This change of localization brings SOS in physical proximity to Ras, thereby promoting G protein activation. B, under resting conditions, GIV is primarily located in the cytosol, whereas its substrate G protein Gαi is constitutively anchored to the plasma membrane, thereby precluding GIV action. Upon RTK stimulation, GIV translocates to the plasma membrane via binding of its SH2-like domain to tyrosine phosphorylated EGFR. This change of localization brings GIV in physical proximity to Gαi, thereby promoting G protein activation.

Article Snippet: Grb2-GBA was generated by replacing the mRFP and FKBP sequences of FKBP-GIV-GBA (1660–1705) with the human Grb2 sequence (amplified from Addgene plasmid catalog no. 70383) preceded by a Myc tag (NheI/NruI sites). pcDNA6A-EGFR was obtained from Addgene (catalog no. 42665).

Techniques: Activation Assay, Clinical Proteomics, Membrane, Binding Assay